Activation of MNK2/eIF4E Pathway by the Splicing Factor SRSF1 Supports Pancreatic Cancer Cell Survival to Genotoxic Stress
L. Adesso,1,2 S. Calabretta,1,2 F. Barbagallo,2 Pilozzi,3 G. Capurso,1 R. Geremia,2 G. Delle Fave,1 C. Sette,21Digestive and Liver Disease and 2Pathology Units II School of Medicine, University of Rome "La Sapienza" 3Departmet of Public Health and CellBiology, University of "Tor Vergata" Rome, Italy
Context: eIF4E is a key regulator of translation. Increased eIF4E levels are observed in cancer and its overexpression elicits cell transformation by enhancing cell cycle progression and survival. Phosphorylation of eIF4E by the MNK kinases seems required for its oncogenic potential. The MNK family comprises two proteins, MNK1 and 2, whose mRNAs are alternatively spliced in two variants, a and b.
Aim: We have investigated the role of eIF4E phosphorylation in pancreatic cancer cells concentrating on eIF4E modulation and discriminating between MNK1 and MNK2 activity during chemotherapy.
Methods: WB analysis to analyze phosphorylation status of eIF4E under gemcitabine exposure. Immunofluorescence against capsase3 and clonogenic assay to analyze apoptotic rate and toxicity. RNAi to obtain MNK1/2 or SRSF1 gene silencing and RT-PCR to analyze splicing variants of MNK2. IHC for phosphorylated eIF4E.
Results: We found that eIF4E phosphorylation is stimulated in response to gemcitabine treatment of pancreatic cancer cells, in concomitance with the promotion of MNK2b variant that overrides upstream regulatory pathways. The modulation of MNK2 splicing was exerted by upregulation of the oncogenic SRSF1 splicing factor. Preventing eIF4E phosphorylation by pharmacological inhibition of MNKs, depletion of endogenous MNK2 or SRSF1 synergistically enhanced the cytotoxic effects of gemcitabine. Moreover, IHC of patients' specimens indicated that eIF4E phosphorylation represents an independent negative prognostic factor for pancreatic cancer.
Conclusions: Treatment of pancreatic cancer cells with gemcitabine triggers a positive feedback through SRSF1- mediated splicing of MNK2b and phosphorylation of eIF4E, which protects cancer cells from drug-induced genotoxic stress. These results suggest that inhibition of MNKs activity might represent a novel therapeutic strategy for pancreatic cancer.
Adrenomedullin: A Mediator of Pancreatic Cancer-Associated Diabetes
G. Aggarwal,1 G. Klee,2 E.W. Klee,4 T. Smyrk,3 W. Bamlet,4 J.J. Han,2 M. de Andrade,4 G.M. Petersen,4 M.E. Fernandez-Zapico,1,2 S.T. Chari,11Division of Gastroenterology; 2Schulze Center for Novel Therapeutics, 3Department of Pathology; 4Department of Health Sciences Research, Mayo Clinic, Rochester, Minnesota
Background: New onset diabetes (DM) in pancreatic cancer (PaC) is likely a paraneoplastic phenomenon caused by tumor-secreted products. Our aim was to identify diabetogenic secretory product(s) of PaC.
Methods: We identified human PaC cell lines whose supernatants impaired insulin signaling in a rat beta cell line (INS1). Using microarray, we found that the adrenomedullin (AM) gene was up-regulated in such cell lines. We performed quantitative RT -PCR and tissue immunohistochemistry (IHC) on human PaC and control pancreas to determine AM gene and protein expression, respectively. We studied the effects of conditioned media from PANC1 cells on insulin secretion from INS1 cells, before and after shRNA-mediated AM knockdown. We measured plasma AM levels in 61 subjects with resectable PaC (31 with normal fasting glucose and 30 with new onset DM) and 55 controls (28 healthy controls with normal fasting glucose and 27 with new onset type 2 DM).
Results: Quantitative RT-PCR and IHC confirmed AM gene and protein-overexpression, respectively, in human PaC compared to control pancreas. Both AM and conditioned media from PaC cell lines (including PANC1) inhibited glucose-stimulated insulin secretion from INS1 cells, the effect of conditioned media from PANC1 cells was ameliorated by shRNA-mediated knockdown of AM. Mean plasma AM levels (fmol/l) were higher in PaC compared to non-PaC controls (20.5±9.2 vs 13.8 ±8.8, p =0.0001), especially in PaCDM compared to type 2 DM (22.9±10.7 vs 14.8 ±10.7, p =0.006).
Conclusion: AM is overexpressed in human PaC and impairs insulin release from beta cell lines. AM is a strong candidate for being the mediator of PaC-induced diabetes.
Cathepsin D Activates Cathepsin B and is Therefore Required for Trypsin Activation in Pancreatic Acinar Cells
A. Aghdassi,1 M. Sendler,1 C. Storck,1 T. Wartmann,2 W. Halangk,2 F.U. Weiss,1 M.M. Lerch,1 J. Mayerle,11Department of Medicine A, University of Greifswald, 2Division of Experimental Surgery, University of Magdeburg, Germany
Background: Acute pancreatitis is initiated by premature zymogen activation and trypsinogen activation is one of the earliest steps. Trypsinogen itself is activated by cathepsin B whereas it is degraded by Cathepsin L.
Aim: We investigated the role of cathepsin D (CTSD), another lysosomal protease, regarding trypsin activation in isolated acini.
Methods: Pancreatic tissue and juice were examined for cathepsin D expression by western blot and immunohistochemistry. Trypsin(ogen) was incubated with purified cathepsin D in-vitro. Cathepsin B was precipitated from pancreatic tissues and incubated with cathepsin D. Pancreatic acinar cells were isolated from wild type and cathepsin D knock-out mice and expression of digestive enzymes was assessed. Acini were subjected to supramaximal CCK stimulation and trypsin, elastase and cathepsin B as well as necrosis were measured by fluorogenic substrates.
Results: Cathepsin D is expressed in pancreatic tissue and secreted into pancreatic juice. Unstimulated acini did not differ in the basal expression of digestive enzymes. After supramaximal stimulation CTSD-/- acini showed significantly less trypsin and elastase activity as well as less necrosis. Concomitantly cathepsin B activity was decreased. Neither trypsinogen nor trypsin were processed by cathepsin D, however CTSD activates cathepsin B in-vitro.
Conclusion: Cathepsin D is highly expressed in the pancreas and is involved in trypsinogen activation by regulating cathepsin B. It therefore acts to be a master regulator for early zymogen activation.
Efficacy of a Blake DrainR on Pancreatic Fistula after Pancreaticoduodenectomy
T. Aimoto, E. Uchida, Y. Nakamura, K. Yamahatsu, A. Matsushita, A. Katsuno, K. Cho, M. Kawamoto Department of Surgery, Nippon Medical School, Tokyo, Japan
Background: Pancreatic fistula (PF) is the most prevalent cause of morbidity after pancreaticoduodenectomy (PD). The most crucial management for PF is adequate drainage of the amylase-rich fluid by peripancreatic drains.
Aim: The aim of this study was to evaluate the efficacy of the fluted drain with continuous suction for the management of PF in comparison with the conventional drain.
Methods: Our study consisted of two parts: a basic experiment to investigate the effects of the fluted drain on the management of PF in an animal model, and a retrospective review of 42 patients with PF after PD.
Results: In the basic experiment, Blake drains could accomplish complete drainage of fluids through channels along the sides while holes of Duple drains could not remove fluids collection completely. In an animal model, no collections of fluid were detected around the Blake drains. When leakage occurred, it did not lead to abdominal abscess, and a "drain canal" formation linking the anastomosis with the extracorporeal orifice was demonstrated all along the drainage route. In the clinical study, 28 patients received Blake drains (B-group) and 14 received Duple drains (D-group). Grade C fistulas with abdominal bleeding developed in only 2 patients in the B-group. All the patients in the B-group healed with conservative treatment (P<0.01), and none of them required percutaneous drainage or reoperation (P<0.05).
Conclusions: Blake drains may be efficient therapeutic tools in patients with grade B fistulas. The basic experiment affirms that Blake drains provide excellent drainage and contribute to the formation of "drain canals" effective in localizing and controlling PF.
Middle Pancreatectomy: Our Clinical Experiences of Eight Patients
Y. Akiyama, N. Minagawa, K. Yamaguchi Department of Surgery 1, School of Medicine, University of Occupational and Environmental Health, Japan
Introduction: Middle pancreatectomy is a possible limited pancreatic surgery for benign or low grade malignant lesions.
Method: We retrospectively reviewed our experiences of eight patients who underwent middle pancreatectomy in our hospital from April 2008 to June 2011.
Result: They were four men and four women, with a mean age of 74.5 years (68-89). The mean operating time was 329.75 minutes (260-405). The pathologic diagnosis were intraductal papillary-mucinous adenoma in three, invasive ductal carcinoma in one, metastasis of renal carcinoma in one, foregut cyst in one and autoimmune pancreatitis in one, and retention cyst in the other. Postoperatively, two patients developed intraabdominal abscess and one of them underwent abscess drainage. All the patients were discharged on a mean postoperative day of 28.4 days.
Conclusions: Middle pancreatectomy can be safely done in patients with benign or borderline malignant diseases.
Acute Pancreatitis is Less Severe Among Patients with Underlying Chronic Pancreatitis
V. Akshintala,2 S. Hutfless,2 M. Khashab,2 A.M. Lennon,2 M. Makary,1,3 K. Hirose,1,3 D. Andersen,1,3 A. Kalloo,1,2 V. Singh,1,21Pancreatitis Center, 2Division of Gastroenterology, 3Department of Surgery, Johns Hopkins Hospital, Baltimore, MD
Background: Little is known about the severity of acute pancreatitis among patients with underlying chronic pancreatitis (CP).
Aim: To evaluate the severity of AP in patients with (ACP) and without (AP) underlying CP.
Methods: The Maryland Health Services Cost Review Commission database was queried for all adult patient admissions with a primary diagnosis of AP between 1994-2010. ACP was defined as AP with secondary diagnosis of CP. All diagnoses and severity markers were defined by ICD-9 codes. The Elixhauser method was used to tabulate comorbidity.
Results: There were 70,944 admissions for AP of which 9,747 (14%) were for ACP. The number of ACP admissions doubled from 8.8 to 17.6% over the 16 year period (p<0.0001 for trend). Patients with ACP were more likely to be younger (mean age 47.3 vs 52.1), male (59.7 vs 50.7%), African American (52.2 vs 40%) and had higher rates of alcohol (49.2 vs 28.6%) & drug abuse (18.1 vs 8.5%) compared to AP (p<0.0001 for all comparisons). ACP patients had less mortality (0.4 vs 1.2%), multiorgan failure (0.7 vs 1.5%), need for mechanical ventilation (0.7 vs 1.8%) & ICU (3.5 vs 4.7%) compared to AP (p<0.0001). Patients with ACP were 55% less likely to die after adjusting for all risk factors for mortality (OR 0.45, p<0.0001). Weight loss (OR 2.2) & comorbidity (OR 3.6) were predictors of mortality, organ failure & need for ICU in patients with ACP after adjusting for age, gender, race, alcohol abuse, & pseudocyst (both p<0.0001).
Conclusions: 1) Nearly 1 in 7 patents admitted for AP have ACP. 2) Patients with ACP have less disease severity compared to AP. 3) Weight loss and comorbidity are predictors of severity in ACP.
Primary and Secondary Sarcoma of the Pancreas. Is Radical Surgery Justified?
A. Alexander, A. Rehders, M. Ghadimi, K. Cupisti, J. Schulte am Esch, W.T. Knoefel Dept. of Surgery, Heinrich-Heine-University Düsseldorf, Germany
Aim: Few reports exist on sarcomas of the pancreas. The result of these case reports is that complete resection ist the best option since alternatives are lacking. We investigated what factors predispose to failure.
Methods: All patients with sarcomas at this institution were documented prospectively since 9/2003. Perioperative complications and long term follow-up were our primary targets.
Results: 8 patients qualified. Six suffered from primary tumors, two from metastatic lesions. Of the primary tumors 2 had metastasized at the time of diagnosis, 2 died of multiorgan failure after 4 and 5 months, 2 required resection of a recurrence or metastases. One after 16 and 21 months. This patient died 6 months after the third resection of progressive disease. The other 5 months after distal pancreatecomy is now disease free. Of these 9 resections 8 were R0. 3 patients are recurrence free at a 3, 5, and 96 months.
Both patients with metastases to the pancreas required multiple operations (3 and 6) involving multiple organs over a period of 7 and 4 years, and are currently scheduled for another operation for recurrence (lung and diaphragm). Both achieved excellent quality of live after each operation.
Discussion: This is the largest single center series of primary and secondary sarcoma of the pancreas. Both carry a better prognosis than adenocarcinoma of the pancreas and should be resected if an R0 status can be achieved. Oncologic failures occurred in all secondary tumors to the pancreas. Redo surgery provides long term survival in these cases and justifies resection. Oncological failure after primary sarcoma is rare after R0 resection. For extended multiorgan resections the risk of perioperative mortality is substantial and patient selection remains demanding. Neoadjuvant strategies are urgently needed.
In Vivo Sensitization of Pancreatic Tumors to Gemcitabine and Erlotinib by Isoflavone
S. Ali,1 A. Aboukameel,1 S. Banerjee,2 B. Bao,2 P.A. Philip,1 F.H. Sarkar,21Department of Oncology; 2Department of Pathology. Karmanos Cancer Institute, Wayne State University, Detroit, Michigan
Background: Inactivation of multiple pathways is required for the treatment of advanced pancreatic cancer (PC) as suggested by many clinical trials. Isoflavone is a well known chemopreventive agent, which targets multiple signaling pathways; therefore, isofalvone treatment concurrently with erlotinib would likely potentiate the anti-tumor effects of cytotoxic drug gemcitabine and would thereby prolong survival of mice in a PC-xenograft model.
Methods: The effect of isoflavone, erlotinib and gemcitabine combinations were determined using AsPC-1 cells derived tumors in SCID mice. Mice were randomized into 7 groups with 7 animals in each group. 1 mg/mouse of isoflavone, and 1 mg/ mouse of erlotinib was administered daily by gavage and I.P, respectively for a total of 14 days, while 50 mg/kg of gemcitabine was given I.V. every other day for a total of three injection. The experiment was terminated when the tumors in each group approached about 2,000 mg.
Results: Our in vivo studies showed that isoflavone in combination with erlotinib and gemcitabine was significantly more effective than individual agents or even the dual combinations. We also found a significant increase in survival in mice with triple combination treatment compared to mice treated with single agent or dual combination.
Conclusion: Based on our pre-clinical in vivo results, we conclude that the isoflavone sensitized PC cells derived tumors to conventional therapeutics and that the triple combination was superior in prolonging survival, suggesting that this strategy could be useful for the treatment of patients diagnosed with PC.
GLI Proteins Orchestrate the Cytokine Milieu in Pancreatic Tumor Microenvironment
L.L. Almada, S.F. Elsawa, M.G. Fernandez-Barrena, V. Ellenrieder, M.E. Fernandez-Zapico Schulze Center for Novel Therapeutics, Mayo Clinic, Rochester, MN, USA
Pancreatic cancer (PaCa) has one of the poorest prognoses among all neoplasms. Despite advances in our understanding of the pathogenesis of PaCa, the molecular mechanisms involved in the maintenance of the transformed phenotype remain elusive. In recent years, attention has turned to the desmoplastic reaction, a typical feature of the active tumor microenvironment present in most pancreatic cancers. This reaction is characterized by abundant extracellular matrix deposition, which infiltrates and envelops the tumoral cell compartments, influencing the growth and survival of these cells. Recent studies have demonstrated that interaction between PaCa and stellate cells modulates the genesis and maintenance of this desmoplastic reaction; however, the molecular mechanisms by which these cells contribute to the formation of this component of the active microenvironment are still unknown. Here, using a combination of co-culture models and biochemical assays we describe a novel signaling mechanism initiated by cancer cells leading to the upregulation of known regulators of stellate cell function, namely transforming growth factor (TGF)-β and platelet-derived growth factor (PDGF). Analysis of the mechanism reveals GLI proteins (GLI1 and GLI2) are required to modulate TGF-β and PDGF expression in cancer cells. Luciferase reporter and expression assays show that GLI proteins are able to induce TGF-β and PDGF in these cells. Knockdown of these transcription factors using RNA interference decreases the promoter and expression of these ligands in cancer cells as well as diminish the activation of stellate cells and activity of TGF-β and PDGF pathways in these cells. Interestingly, this effect was rescue by the exogenous addition of TGF-β and PGDF ligands. We hope that this knowledge of the role of GLI function in the PaCa stromal microenvironment will help us to understand the complex signaling network implicated in pancreatic tumorigenesis and serve as a foundation for the development of new diagnostic and therapeutic approaches.
Prevention of Post-ERCP Acute Pancreatitis: Complete Systematic Review
K. Altaf, M.A. Javed, F. Wright, R. Sutton Liverpool NIHR Pancreas Biomedical Research Unit, RLBUHT and University of Liverpool
Background: Post-ERCP acute pancreatitis (post-ERCP-AP) occurs in ∼5% of patients undergoing ERCP, severe in ∼1%. Despite multiple trials, optimal prophylaxis remains undetermined. We sought to clarify the effectiveness of prophylactic interventions for post-ERCP AP through multiple meta-analyses of randomised clinical trials (RCTs).
Methods: MEDLINE, EMBASE and the Cochrane Library were searched by two independent reviewers to identify all RCTs that tested treatments to reduce post-ERCP AP. Data were extracted to permit Jadad scoring, grouping of RCTs by therapeutic mechanism and separate meta-analysis of each group. The main outcome measure was post-ERCP AP, defined as amylase elevated to >3× upper limit of normal with >24 h abdominal pain.
Results: 49 high quality RCTs (Jadad ≥3) were identified. Pancreatic stents (trials (T) - 3; patients (P) - 294; RR 0.21; 95% CI 0.09-0.50) were most effective; significant reductions in post-ERCP AP resulted from non-steroidal anti-inflammatory drugs (NSAIDs; T - 8; P - 1901; RR 0.59; CI 0.38-0.92), secretion inhibitors (T - 12; P - 4178; RR 0.61; CI 0.43-0.88), protease inhibitors (T - 6; P - 3296; RR 0.59; CI 0.38-0.93) and smooth muscle relaxants (T - 6; P - 1903; RR 0.68; CI 0.48-0.97). Anti-oxidants (T - 5; P - 2100; RR 0.90; CI 0.54-1.50), anti-coagulants (T - 2; P - 553; RR 0.94; CI 0.54-1.65), non-ionic (vs ionic) contrast agents (T - 3; P - 2155; RR 1.12; CI 0.86-1.46) and steroids (T - 4; P - 2039; RR 1.18; CI 0.93-1.50) did not reduce post-ERCP AP; low quality RCT inclusion did not alter these findings.
Conclusion: Pancreatic stents, NSAIDs, inhibitors of secretion and of proteases or smooth muscle relaxants reduce the risk of post-ERCP AP. Large well-designed RCTs of combination versus single agent prophylaxis are required.
A Population-Based Evaluation of Severity among Transferred Patients with Acute Pancreatitis
G. Anand,1 S. Hutfless,1 M. Khashab,1 A.M. Lennon,1 M. Makary,2 K. Hirose,2 D. Andersen,2 A. Kalloo,1 V. Singh,1Pancreatitis Center, 1Division of Gastroenterology, 2Department of Surgery, Johns Hopkins Hospital, Baltimore, MD
Background: Current guidelines recommend transferring patients with severe acute pancreatitis (AP) to referral centers but there is limited data on severity among transferred patients.
Aim: To evaluate severity of AP among transferred patients and factors which lead to being transferred.
Methods: The Maryland Health Services Cost Review Commission database was queried for adult patient admissions with a primary diagnosis of AP between 1/1/1994-12/31/2010. All diagnoses and interventions were defined by ICD-9-CM coding. The presence of organ failure in ≥2 systems was defined as multisystem organ failure (MS).
Results: There were 71,305 admissions for AP at 48 hospitals. 1,657 (2.3%) patients were transferred. Multivariate analysis revealed that younger patients (OR 0.99), African American patients (OR 0.55) or the uninsured (OR 0.46) were less likely to be transferred, whereas those with MS (OR 3.5), need for ICU (OR 2.3) or mechanical ventilation (MV) (OR 2.1) were more likely to be transferred (p<0.01 for all comparisons). Transferred patients had higher mortality (6.1 vs 1.1%), MS (5.6 vs 1.2%), as well as need for MV (13.1 vs 1.4%), hemodialysis (4.2 vs 2.7%), and ICU (22.8 vs 4.3%) compared to nontransferred patients (p<0.0001 for all comparisons). Among the subgroup of patients with MS, need for MV and ICU, mortality was similar between transferred and nontransferred patients (48.6 vs 49.8%, p=NS).
Conclusions: Severity of AP is increased among transferred patients. Patients with severe AP are the most likely to be transferred but have a similar mortality compared to matched nontransferred patients.
Targeting Gemcitabine Resistance in Pancreatic Cancer - Basal and Acquired
T. Arumugam,1 V. Ramachandran,1 T. Fujii,1 H. Haojie,1 R.F. Hwang,2 H. Wang,3 W. Choi,4 D.J. McConkey,4 C.D. Logsdon,1Dept. of 1Cancer Biology, 2Surgical Oncology, 3Pathology, 4Urology, UT MD Anderson Cancer Center, Houston, TX
Background: The clinical standard of care for pancreatic cancer (PC) is treatment with gemcitabine (Gem), but gem has meager benefits. In order to identify resistance mechanisms, we investigated the effects of gem on PC cell gene expression.
Methods: Basal gem sensitivity/resistance pattern in 28 PC cells lines was determined and mRNA expression was analyzed using Illumina microarray. Acquired gem resistance was obtained by treating in vitro 12 PC cell lines with gem 10μM for 24hrs before mRNA expression analysis. Apoptosis and cell cycle was analyzed by FACS. IHC, WB and ELISA were used to quantify protein expression. SiRNA and ShRNA mediated silencing was utilized to study the therapeutic value of this targets.
Results-Basal Resistance: mRNA analysis showed that S100A4 expression correlated with basal gem resistance and silencing of S100A4 sensitized all the gem resistance cells. IHC using human tissue array revealed association between S100A4 and poor outcome. Orthotopic in vivo model demonstrated that stable silencing of S100A4 sensitized the tumors to gem and reduced metastasis.
Acquired Resistance: Gem treatment altered gene expression in all cells. One among the highly induced genes was p21Cip/Waf. IHC and ELISA confirmed gem induction of p21 expression both in vitro and in vivo in mouse xenograft models and pretreated human PDAC tissues. p21 induction caused all PC cells to accumulate in S-phase thus becoming cytostatic and resistant. SiRNA against p21 sensitized resistant PC cells to gem both in vitro and in vivo.
Conclusion: Strategies aimed at inhibiting the actions of S100A4 and p21 may be useful for overcoming gem resistance.
Hepatocyte Growth Factor: A Potential Therapeutic Target in Pancreatic Cancer
S.J. Arun, Z. Xu, E. Fiala-Beer, L. Yang, P.A. Phillips, D. Goldstein, A. Biankin,2 R. Pirola, J.S. Wilson, M.V. Apte Pancreatic Research Group, University of New South Wales, 2Garvan Institute of Medical Research, Sydney, Australia
Pancreatic stellate cells (PSCs) produce the desmoplasia of pancreatic cancer (PC). Moreover, they interact with PC cells to potentiate PC progression. Hepatocyte growth factor (HGF) may mediate this interaction. High serum HGF levels in PC patients correlate with poor outcome. However, little is known about the role of HGF in PC.
Aims: To i) determine whether human PSCs and PC cells express HGF; and ii) assess the effects of HGF inhibition on PC progression in an orthotopic mouse model.
Methods: 1) HGF expression in PSCs and PC cells was assessed by RT-PCR, immunoblotting/immunocytochemistry. 2) Orthotopic model: AsPC-1 (human PC cell line) ± human PSCs were implanted into the pancreas. One week later, mice were divided into groups (n=8/group) and treated with: a fully-human, anti-HGF monoclonal antibody Rilotumumab (AMG102) [(Amgen Inc.), 300 or 600μg IP biweekly] or isotype IgG (600 μg IP biweekly). Seven weeks later, tumour size and metastasis were assessed.
Results: 1) PSCs express HGF at both mRNA and protein levels while PC cells (MiaPaCa2, Panc-1, AsPC-1) express negligible HGF mRNA. 2) Orthotopic model: (A) IgG treated AsPC-1+PSC mice showed larger tumours than mice injected with AsPC-1 alone [tumour volume (mm3), 1312.63±175.27 vs 667.94±93.93mm3; p<0.001]; (B) Compared to IgG treatment, 300 and 600μg AMG102 i) reduced tumour volume in AsPC-1+PSC mice (1312.63 ±175.27 mm3 vs 532.47±91.83* and 382.85±42.70* respectively, *p<0.001), but ii) did not reduce tumour size in the AsPC-1 alone group (667.94±93.93 vs 517.07±68.95 and 415.39±61.80 mm3 respectively). (C) Compared to IgG treatment, 600μg AMG102 treatment significantly inhibited distant metastasis in AsPC-1+PSC mice, (p<0.05).
Conclusions: We have shown for the first time that i) human PSCs produce HGF; ii) HGF inhibition reduces growth and metastasis of tumours representative of human PC i.e. exhibiting both tumour elements and a stromal reaction, but not of the clinically non-representative cancers formed by PC cells alone.
Implication: Targeting the stromal reaction with relevant specific inhibitors may represent a novel therapeutic approach in pancreatic cancer.
Drug-eluting Microparticles for the Treatment of Pancreatic Cancer: Preliminary in vivo Results
A. Asgharpour,1 M. Ganesh,2 A. Gooding,1,3 A. Stanek,4 S. Andrawes,1,3 I. Danishpajooh,1,3 R. Gross,2 F. Gress,1,3 L. Martello-Rooney,1,31SUNY Downstate Medical Center, Dept of Medicine, Brooklyn,NY; 2NYU Polytechnic Institute,Broolyn,NY; 3Division of Gastroenterology & Hepatology; 4Dept of Surgery
Pancreatic cancer is the fourth leading cause of cancer death in the U.S. Current treatment regimens have had a minimal impact on altering the course of the disease, establishing the need for alternative modalities of therapy. One obstacle with systemic chemotherapy is in vivo data suggesting compromised blood flow to the pancreatic tumor. Here we explore the feasibility of direct injection of biodegradable polymer-based microparticles(MPs) into the tail portion of the mouse pancreas. A laparotomy was performed on C57BL/6 mice to expose the pancreas followed by injection of 50μl PBS, 50μl MPs/PBS or 25μl MPs/PBS into the pancreatic tail using a 29-gauge needle. The mice were sacrificed at the following post-op. timepoints: 24 hrs, 3 days and 7 days. The mice were weighed daily and blood drawn pre- and post-op. for pancreatic enzyme testing. Mouse tissue samples of the pancreas, liver, spleen and duodenum were placed in formalin upon sacrifice. All of the mice survived the surgery and exhibited minimal wt loss, which was reversed by day 7. Lipase and amylase levels were mildly elevated after 24 hrs, but returned to pre-bleed levels by day 3. The analysis of the pancreatic tissue sections disclosed acinar cell damage only in the area surrounding the injection site. There was no evidence of pancreatitis. No indication of MP migration was observed in sections of the liver, spleen or duodenum. These findings establish that direct injection of MPs into the mouse pancreas is feasible and safe and support proceeding to the next phase of using drug-eluting MPs in a mouse model of pancreatic cancer.
Novel Small Molecule CRM-1 Inhibitor for Pancreatic Cancer Therapy
A.S. Azmi,1 M. Kauffman,2 D. McCauley,2 S. Shacham,2 R.M. Mohammad,31Department of Pathology, Wayne State University School of Medicine, 2Karyopharm Therapeutics, MA, 3Department of Oncology, Karmanos Cancer Institute, Detroit MI, USA
Aim and Background: Pancreatic cancer (PC) is a deadly disease that lacks druggable target rendering effective clinical outcome unmanageable. CRM-1 is an exportin protein in humans that is coded by XPO1 gene. The protein encoded by this gene mediates leucine-rich nuclear export signal (NES)-dependent protein export. CRM1 regulates key cellular processes by controlling the localization of critical tumor suppressors and growth regulatory molecules. High CRM-1 expression has also been correlated with poor prognosis in PC making it an attractive therapeutic target.
Methods: Using structure based drug design; we have identified novel small molecule inhibitors of CRM-1 that irreversibly lock target proteins in the nucleus leading to effective apoptosis. The drugs selectively kill cancer cells with minimal toxicity to normal tissue and possess clinically acceptable pharmacokinetic parameters. In this report, using multiple molecular biology techniques, we have evaluated the role of CRM-1 inhibition on nuclear localization and apoptosis by prostate apoptosis response protein (PAR-4, established by our lab as a potential therapeutic target in PC).
Results: Most potent CRM-1 inhibitor (KPT-185) induced growth inhibition and apoptosis in a panel of PC cell lines with an IC50 ∼150 nM. Western blot and confocal microscopy analysis demonstrated that KPT-185 treatment resulted in PAR-4 nuclear localization (a pre-requisite for PAR-4 mediated apoptosis). Most significantly, siRNA knockdown of PAR-4 abrogated the apoptotic potential of KPT-185, confirming that this was indeed a PAR-4 dependent apoptotic mechanism. KPT-185 showed synergistically enhanced apoptosis when combined with oxaliplatin. Animal xenograft studies are currently being performed.
Conclusions: This is the first report demonstrating CRM-1 as a potential therapeutic target in PC and the drug KPT-185 warrants further clinical investigations for this deadly malignancy.
Pancreatic Castleman's Disease: Studies of Three Cases and a Cumulative Review of the Literature
S.R Babu,1 XL Wang,1 L Fu,2 ZL Wang,1 Y Zhang,1 AP Su,1 T Hu,1 BL Tian,1Δ1Department of Hepato-bilio-pancreatic Surgery, 2General Ward of Cancer Centre, West China Hospital, Sichuan University, Chengdu, China
Castleman's disease(CD) is a relatively rare and benign disorder, characterised by giant lymphnode hyperplasia. It was initially described as pathological entity in 1954 and later defined by Castleman in 1956. Histopathologically CD presents with three distinct histologic variants. The hyaline vascular type (HV) also called angiofollicular lymphoid hyperplasia, is the most common histological variant of the CD, accounting for 90% of cases. Second histological variants of CD is plasma cell type (PC) which consist of 10% of cases characterised by large lymphoid follicles surrounded by sheets of plasma cells. The third histological variant of CD is mixed type (MV). Initially several articles reported that mediastinum followed by cervical region was the most common sites of disease. However it can present in non-mediastinal sites wherever lymphnodes are present and the common sites are the pelvic, retroperitonium, and axillary regions. Pancreatic localization of CD is even more rare and is usually indistinguishable from pancreatic neoplasms. To date, only 13 cases of CD associated with pancreas were described in the world literature. We report 3 cases of CD in which pancreas was all involved. One located in the tail of pancreas, who accepted distal pancreatectomy, and the others in the head accepted enucleation. In addition, we review current data on its pathogenesis, imaging findings, diagnosis, differential diagnosis and treatment.
Radioresistance in Pancreatic Cancer: The Role of Autophagy
M.J. Baine,1 S. Kaur,2 C. Lin,3 S. Chen,3 K. Lester,2 F. Sahak,2 S.K. Batra,1,21Eppley Institute for Research in Cancer and Allied Diseases, 2Department of Biochemistry and Molecular Biology, 3Department of Radiation Oncology, University of Nebraska Medical Center, Omaha, NE
Background: Radiation therapy (RT) is a therapeutic staple for all resectable and potentially-resectable pancreatic cancer (PC) patients, though >75% are found to have radioresistant tumors. Recently, multiple studies have suggested a role for autophagy in the treatment resistant phenotype of many malignancies. In this study, we analyzed the roles of autophagy, apoptosis, and necrosis both quantitatively and temporally in the cellular response of PC cells to ionizing radiation (IR) under the hypothesis that "autophagy is a significant contributor to PC radioresistance".
Results: We found that PC cell lines are differentially radioresistant, maintaining between 73% and 93% viability 96h after exposure to 7Gy IR. Using Annexin-V and Propidium Iodide staining, we observed that reduction in cellular viability of differentially radiosensitive cells (Panc-1) within the first 24h was primarily attributed to necrotic cell death (p=0.03), with apoptosis not becoming prominently induced until 72h after IR. Conversely, in differentially radioresistant cells (BxPC3), neither apoptosis nor necrosis was substantially increased with-in the first 96h post-IR. Interestingly, differential radioresistance corresponded with a robust induction of autophagy with-in 24h of IR exposure while radiosensitive cells gradually induced autophagy starting from the 48h time point as evidenced by conversion of LC3-I to LC3-II and confirmed by ATG-7 induction.
Conclusions: Autophagy may play a significant role in PC radioresistance and will be further evaluated as to its direct effect on post-IR cell survival as well as the mediating pathways, such as mTOR, AKT, and ERK, underlying these observations.
Role of C-Reactive Protein, APACHE II and Glasgow Score in Early Prediction of Severity of Acute Pancreatitis
Dj. Bajec,1 D. Radenkovic,1 P. Gregoric,2 N. Ivancevic,2 V. Jeremic,2 B. Karadzic,2 A. Antic,1 I. Pejovic,11Clinic for Digestive Surgery and 2Clinic for Emergency Surgery, Clinical Center of Serbia, Belgrade, Serbia
Background/Aim: The early prediction of the severity of an acute attack of acute pancreatitis (AP) has important implications for management and timely intervention. The aim of this study was to compare the accuracy of Glasgow and Apache II scores and C-reactive protein (CRP) level in prediction of severity of acute pancreatitis.
Methods: Ninety one consecutive patients with acute pancreatitis primarily admitted were prospectrively studied. APACHE II score was recorded on admission, while Glasgow score and CRP levels were determinated 48 hours later. Severity of AP was defined according to Atlanta classification system. Two study groups comprising 33 patients with mild AP (MAP) and 58 patients with severe AP (SAP) were compared.
Results: Mean values of Glasgow and Apache II scores and CRP were 4 and 10 points and 331 mg/L, respectively in SAP group, while in MAP group it were 1 and 4 points and 62 mg/L, respectively. The accuracy of Glasgow and APACHE II scores, and CRP level, in prediction of severity of AP, using cut-off values of 8 and 3 points, 150 mg/l, respectively (derived from ROC curves), were calculated. The sensitivity and specificity of Glasgow and Apache II scores and C-reactive protein were 79% and 81%, 72.7% and 100%, 90.9% and 85.7%, respectively.
Conclusion: The CRP offers little, if any, advantage over the Glasgow and APACHE II score. CRP level and Glasgow score proved to be powerful a prognostic model as the more complicated APACHE II scoring systems, but with the disadvantage of a 24-hour delay.
Maximizing Islet Yield from Pancreata with Chronic Pancreatitis for Use in Islet Auto-Transplantation Requires a Modified Strategy from Islet Allograft Preparations
A.N. Balamurugan,1,2 M.D. Bellin,1 K.K. Papas,1,2 T.B. Dunn,2 S.M. Vickers,2 S. Chinnakotla,2 T.L. Pruett,2 G.J. Beilman,2 B.J. Hering,1,2 D.E.R. Sutherland,1,21Schulze Diabetes Institute, 2Department of Surgery, University of Minnesota, Minneapolis, MN, USA
Background: Islet auto-transplantation (IAT) has been successfully performed at our institution since 1977 (n=400). Isolating islets from pancreata with chronic pancreatitis (CP) poses multiple challenges. Alterations in duct structure and accumulation of fibrotic bundles in the pancreatic parenchyma can affect enzymatic digestion and make it difficult to obtain high islet yield. We have developed new approaches to maximize the islet yield from pancreata with CP.
Methods: We applied a modified strategy to isolate islets from pancreata of 55 CP patients undergoing IAT. Key steps include: (a) effective delivery of enzyme throughout the pancreas by combined ductal and parenchymal injection, (b) a new enzyme mixture (NEM) composed of intact C1 and C2 collagenase with neutral protease from C. histolyticum, (c) enzyme dosing according to the severity of fibrosis and age of the donor pancreas, and (d) high density purification to reduce transplantable tissue volume.
Results: Our method consistently recovered a high islet yield, averaging 340,622±182,798 IEQ/pancreas and 4,783±2,627 IEQ/g pancreas. In 43 of 55 (78%) pancreata with CP, >200,000 total IEQ were recovered (>500,000 IEQ's in 9 [16%] cases, >400,000 IEQ's in 10 [18%] cases, and >300,000 IEQ's in 11 [20%] cases). Higher islet yield was obtained from pancreata with "minimal change" CP (4,265 IEQ/g, n=30) compared to normal allograft pancreata (4,072 IEQ/g, n=46) (p=0.75). This method of islet isolation reduced the undigested tissue volume (average 20% vs 40% in historical cases). Purification of islets using a denser solution in the COBE process effectively reduced the tissue volume from an average of 30 ± 10.5 to 11 ± 9.2 ml (63%) while maintaining high post-purification islet recovery (84 ± 29.2%) and superior islet morphology. After purification, most of the islets were distributed in fractions 1-12 in contrast to islet allograft purification where most of the islets commonly sediment in layers 1-8.
Conclusion: This modified islet isolation strategy results in increased islet yield and superior islet morphology when compared with standard techniques used in allo-islet isolation. IAT is a safe and effective therapy for patients with CP.
Anti-tumor and Chemo-sensitization Efficacy of a Novel Curcumin Analog-CDF against Pancreatic Tumors in Animal Model in vivo
S. Banerjee, S. Ali, B. Bao, Z. Wang, A. Azmi, A. Ahmad, S. Padhye, F. H. Sarkar Department of Pathology, Karmanos Cancer Institute, Detroit, MI-MI-48201
Purpose: Intrinsic and extrinsic (acquired) resistance to gemcitabine contributes to treatment failure in patients with locally advanced and metastatic pancreatic cancer (PC). For 'proof-of-principle', bioactive agents with multitargeted pleiotropic activity and increased bioavailability could be useful for overcoming resistance of PC cells through sensitization to gemcitabine treatment. We recently reported the pharmacokinetics and tissue distribution of a novel curcumin analog- curcumin-difluorinated (CDF) in mice compared to curcumin. The current study was aimed at assessing the dose dependent antitumor effect as well as its chemo-sensitization potential to gemcitabine in an animal model of human PC in vivo, and further investigated the molecular basis of chemo-sensitization by CDF by evaluating microRNAs (miRNAs) and transcription factors NF-κB and AP-1 status in tumor remnant.
Methods: All studies were performed using female ICR-SCID mice orthotopically implanted with human PC cell line, MiaPaCa-2. For dose dependent antitumor studies, mice were randomized into 3 treatment groups comprising (n=5): (a) untreated control; (b) CDF- 2.5 mg/mice/day orally by gavage for 21 days; and (c) CDF- 5.0 mg/mice/day orally by gavage for 21 days. For chemo-sensitization, mice were randomized into 4 treatment groups comprising (n=5): (a) untreated control; (b) CDF only 5.0 mg/mice/day orally by gavage orally by gavage) for 21 days (c) Gemcitabine-75 mg/kg b.wt/i.v x3 inj; and (c) Gemcitabine + CDF for 21 days.
Results: In our orthotopic mouse model, the administration of CDF showed dose dependent antitumor effect, and resulted in statistically higher chemo-sensitizing activity relative to either CDF or gemcitabine alone treatment. Analysis of average pancreatic tumor weight in different treatment groups revealed significant reduction in tumor weight in CDF+ gemcitabine group relative to untreated control and monotherapy (p<0.05). Additionally, miR-21 expression was reduced whereas miR-200 was significantly elevated. These results suggest that CDF, which functions as a potent NF-κB and AP-1 inhibitor, together with gemcitabine offers promising effective strategy for future translational studies for the treatment of PC.
Conclusion: Our observations clearly suggest that CDF would likely be clinically useful in the future, and thus further development is warranted.
Muc1-c Regulates Cell Survival in Pancreatic Cancer by Preventing Lysosomal Permealization
S. Banerjee, N. Mujumder, V. Sangwan, S. M Vickers, A. Saluja Division of Basic and Translational Research, Department of Surgery, University of Minnesota, Minneapolis, MN
Background: Muc1 is a type I transmembrane glycoprotein aberrantly overexpressed in various cancer cells including pancreatic cancer. Muc1 is translated as a single polypeptide which undergoes post-translational processing to form an extracellular N-terminal protein and a cytosolic C-terminal polypeptide (Muc1-c). The cytosolic Muc1-c is extensively involved in a number of signaling pathways. Muc1-c is reported to inhibit apoptosis in a number of cancer cells but the mechanism of inhibition is unclear. In this study, we have shown that Muc1c interacts with HSP70 and prevents lysosomal permeabilization and thus promotes cell survival in pancreatic cancer.
Method: Expression of Muc1c was studied in the pancreatic cancer cell line Mia-PaCa at the RNA level by using qRTPCR and at protein level by western blotting. Muc1-c expression was inhibited either by siRNA or by a specific peptide inhibitor GO-201. Effect of Muc1c inhibition on viability and proliferation; activation of caspase activity and lysosomal permeabilization were studied. Association of Muc1c with HSP70 was detected by coimmunoprecipitation of Muc1-c and HSP70. Localization of Muc1-c in cellular organelles was monitored by immunofluorescence and by western blotting by Muc1-c antibody after isolating different cellular organelles.
Results: In the current study we show that Muc1-c is mainly cytosolic in localization where it associates with HSP70. However, some Muc1 is also present in the lysosomes. Inhibition of Muc1-c by an inhibitor or siRNA resulted in reduced viability, reduced proliferation and activation of apoptotic genes. Inhibition of Muc1 expression showed an enhanced cathepsin B activity in the cytosol indicating lysosomal permeabilization. Similar activity of cathepsin B was also seen on silencing HSP70 expression.
Discussion: Our results indicate that Muc1-c expression is crucial for cell survival and inhibition of apoptosis in pancreatic cancer. Also, Muc1-c or HSP70 confer protection against lysosomal permeabilization and cell death. It thus seems likely that HSP70, which is constitutively upregulated in pancreatic cancer is involved in transporting Muc1 to the lysosomes, thereby preventing permeabilization and inhibiting apoptosis. In the absence of Muc1 or HSP70, resident proteases in the lysosomes like cathepsins are secreted in the cytosol and result in activation of pro-apoptotic genes and result in cell death.
Triptolide Mediated Downregulation of HSP70 in Pancreatic Cancer Is Modulated By Altered O-GlcNAc Modification of Transcription Factor Sp1
S. Banerjee, V. Sangwan, S. M Vickers, A. Saluja Division of Basic and Translational Research, Department of Surgery, University of Minnesota, Minneapolis, MN
Background: O-GlcNAc transferase (OGT) is involved in modifying a large number of proteins post-translationally thereby regulating a large number of signaling pathways in the cell. OGT is also one of the crucial links responsible for the "Warburg Effect" in cancer cells. Recent studies have shown OGT to be overexpresed in a number of cancers. Our laboratory has previously shown that Triptolide, a diterpene triepoxide, decreases cell viability of human pancreatic cancer cells in vitro and inhibits tumor growth in vivo, by downregulating constitutively active HSP70. However, the mechanism of this downregulation leading to cell death is still unclear. In this study, we show that downregulation of HSP70 expression by triptolide is due to altered O-GlcNAc modification of the transcription factor Sp1.
Methods: Expression of OGT was studied in the pancreatic cancer cell line Mia-PaCa at the RNA level by using qRTPCR and at protein level by western blotting. OGT activity was inhibited either by siRNA or by inhibitors of O-GlcNAc modification. Effect of OGT inhibition on viability and cell death (Caspase activity and Annexin expression) were studied. Activity of transcription factors HSF1 and Sp1 following triptolide treatment, silencing or OGT activity inhibition was studied by reporter assays for the respective genes. O-GlcNAc modification of the transcription factors was detected by immunoprecipitation by specific antibodies followed by western hybridization with O-GlcNAc antibody.
Results: OGT was found to be overexpressed in pancreatic cancer cells. Expression of OGT as well as global O-GlcNAc modification was found to be downregulated by triptolide. Inhibiting OGT activity or decreasing OGT expression resulted in decrease in cell viability to 40% of control cells. Inhibition of OGT by siRNA, specific inhibitors or triptolide also resulted in downregulation of HSP70 and HSF1 protein, RNA and HSF1 activity, though HSF1 itself was not modified by O-GlcNAcylation. Inhibition of OGT by siRNA, inhibitors and triptolide also resulted in blocking nuclear translocation of Sp1 and lowered transcriptional activity. Similarly, triptolide treatment also prevented nuclear translocation of Sp1 and lowered its transcriptional activity, thereby resulting in lowered HSF1 activity and reduced HSP70 expression.
Discussion: Downregulation of OGT activity or expression and treatment of pancreatic cancer cells with triptolide showed comparable effects on cell viability, global O-GlcNAc modification, transcriptional activity of HSF1 / Sp1 and HSP70 expression. This indicated that triptolide mediated downregulation of HSP70 expression was controlled by O-GlcNAc status of the transcription factor Sp1.
Endoscopic Placement of Permanent Indwelling Transmural Stents in Disconnected Pancreatic Duct Syndrome: Does Benefit Outweigh the Risks?
J.Y. Bang, C.M. Wilcox, S. Varadarajulu Division of Gastroenterology-Hepatology, University of Alabama at Birmingham, Birmingham, AL
Background: Recurrence of pancreatic fluid collections (PFCs) is common in patients with walled-off pancreatic necrosis (WOPN) and disconnected pancreatic duct syndrome (DPDS) following initial success with endoscopic transmural drainage. The objective of this study was to evaluate the impact and safety profile of permanent indwelling transmural stents in patients with WOPN in the setting of DPDS.
Methods: This is an observational study of 22 patients with WOPN and DPDS who underwent endoscopic transmural drainage over an 8-year period. Double pigtail plastic stents placed at the site of transmural drainage in patients with WOPN and DPDS were left indefinitely in place. The main outcome measure was to evaluate the rate of PFC recurrence and assess the safety profile of the treatment approach.
Results: Over a median follow-up of 679 days (IQR=318-1141 days), none of the 22 patients with permanent transmural stents developed a PFC recurrence. However, stent migration occurred in 3 of 22 (13.6%; 95% CI: 4.95-33.6) patients at 790, 819 and 1083 days, respectively. Migration occurred within the PFC cavity in one patient and to the distal small intestines in two others causing bowel obstruction. While one patient with small bowel obstruction underwent surgery, two other patients were managed conservatively.
Conclusions: Although permanent transmural stents appear to decrease the rate of PFC recurrence in patients with DPDS, stent migration remains a concern. Better stents designed to overcome this limitation are needed.
Metformin Inhibits Cell Proliferation, Migration and Invasion by Attenuating CSC Function Mediated by Deregulating miRNAs in Pancreatic Cancer Cells
B. Bao,1 S. Ali,2 Z. Wang,1 A. Ahmad,1 A.S. Azmi,1 S.H. Sarkar,1 S. Banerjee,1 D. Kong,1 Y. Li,1 S. Thakur, F.H. Sarkar,1* 1Department of Pathology; 2Department of Oncology Karmanos Cancer Institute, Wayne State University, Detroit, Michigan
Pancreatic cancer (PC) is the fourth leading cause of cancer-related deaths in the United States, which is in part due to intrinsic (de novo) and extrinsic (acquired) resistance to conventional therapeutics, suggesting that innovative treatment strategies are required for overcoming therapeutic resistance in order to improve overall survival. Oral administration of metformin in diabetes mellitus (DM) patients has been reported to be associated with reduced risk of PC and that metformin has been reported to kill cancer stem cells (CSCs); however, the exact molecular mechanism(s) has not been fully elucidated. In the current study, we examined the effect of metformin on cell proliferation, cell migration and invasion, self-renewal capacity of cancer stem-like cells (CSC), and further assessed the expression of CSC marker genes and microRNAs (miRNAs) in human PC cells. We found that metformin significantly decreased cell survival, clonogenicity, wound healing capacity, sphere-forming capacity (pancreatospheres), and increased disintegration of pancreatospheres in both gemcitabine-sensitive and gemcitabine-resistant PC cells. Metformin also decreased the expression of CSC markers, CD44, EpCAM, EZH2, Notch-1, Nanog and Oct4, and caused re-expression of miRNAs (let-7a,b,c, miR-26a, miR-101, and miR-200b,c) that are typically lost in PC and especially in pancreatospheres. We also found that re-expression of miR-26a by transfection led to decreased expression of EZH2 and EpCAM in PC cells. These results clearly suggest that the biological effects of metformin is mediated through re-expression of miRNAs and decreased expression of CSC-specific genes, suggesting that metformin could be useful for overcoming therapeutic resistance of PC cells.
Epithelial-Mesenchymal Transition Mediates the Aggressiveness of Pancreatic Ductal Adenocarcinoma
F. Bednar,1 M. Goto,1 M. Hynes,1 M. Pasca di Magliano,1,2 D.M. Simeone,1,3Departments of 1Surgery, 2Cell and Developmental Biology, and 3Molecular and Integrative Physiology, University of Michigan, Ann Arbor, MI
Epithelial-mesenchymal transition (EMT) is a recognized process in tumor biology, but how EMT relates to cancer stem cell (CSC) biology and tumor aggressiveness in genetically engineered mouse models of pancreatic ductal adenocarcinoma (PDA) has not been determined. We utilized a panel of five murine PDA cell lines (one from the Pdx-Cre, LSL-Kras, p53+/- model, four from the Pdx-Cre, LSL-Kras, Ink4afl/fl model) to interrogate the relationship between EMT, CSC, and PDA biology. FACS profiling revealed a dominant CD24+/CD44+/ESA-/CD133- population in all five cell lines. Two cell lines (one Kras/p53+/-, one Kras/Ink4afl/fl) contained a population of CD24+/CD44+/ESA+/CD133+ cells that comprised 1-15% of the total cells. ALDH+ cells comprised 12% of the Kras/p53+/- line and 40-55% of the Kras/Ink4afl/fl lines and were found in both the ESA+ and ESA- populations. Further analysis of the Kras/p53+/- cell line revealed that the ESA+ cell population gave rise to both ESA+ and ESA- progeny through an EMT-like process. The ESA+ population expressed higher levels of E-cadherin, while the ESA- population expressed N-cadherin, vimentin, and the EMT transcription factors Zeb1 and Zeb2. ∼20% of ESA- cells formed tumorspheres in vitro compared with ∼2% of ESA+ cells. ESA- mesenchymal cells demonstrated faster tumor growth compared with ESA+ epithelial cells in vivo when injected into NOD/SCID mice and formed invasive metastases in the liver, spleen, and peritoneum. Together the data suggest that the ESA- mesenchymal population is more invasive and contains more tumor-initiating cells than the ESA+ epithelial cell population in these murine pancreatic cancer cell line models.
Clinical Relevance of Chymotrypsin C (CTRC) Mutations in Chronic Pancreatitis
S. Beer, M. Sahin-Tóth Department of Molecular and Cell Biology, Boston University, Boston, MA
Background and Aims: Mutations p.A73T, p.R254W, and p.K247_R254del of chymotrypsin C (CTRC) are risk factors for chronic pancreatitis. The mutations cause loss of activity or secretion and thereby decrease the protective function of CTRC in trypsinogen/trypsin degradation. A large number of rare CTRC variants have been found in patients with chronic pancreatitis, however, the clinical significance of these remains unknown. To classify CTRC mutations according to their phenotype, we analyzed secretion and activity of 30 CTRC exonic mutations.
Methods: Secretion was measured from transiently transfected HEK 293T cells using enzyme activity assays and SDS-PAGE. Mutants with reduced activity were purified and their activity was determined on a small peptide substrate, on beta-casein and in degradation of human cationic trypsinogen.
Results: Eighteen of 30 mutants showed normal or nearly normal secretion and activity. Five mutants (p.Q44Q, p.G61R, p.A73T, p.C155Y, and p.L220R) exhibited severely reduced secretion (<20%) and two mutants (p.Q48R and p.G217R) showed moderately reduced secretion (<40%). Mutant p.R254W was secreted at about 60% of wild-type levels. Four mutants (p.Q178R, p.G217S, p.K247_R254del, and p.P249L) showed diminished activity (<5%) but were normally secreted (60-90%). Mutant p.G217R was degraded by trypsin instead of activation.
Conclusion: Although only three CTRC mutations are statistically associated with chronic pancreatitis, here we demonstrate 9 additional mutations cause considerable functional impairment and therefore are likely to be pathogenic. This phenotypic dataset may aid in the classification of the clinical relevance of CTRC mutations identified in patients with chronic pancreatitis.
A Novel Transgenic Zebrafish Model for Studying Secretion in the Exocrine Pancreas
N. Behrendorff,1 J. Behrendorff,2 A. Wall,3 E. Scott,1 P. Thorn,11School of Biomedical Sciences, University of Queensland, 4072 QLD Australia, 2School of Chemistry and Molecular Biosciences, University of Queensland, 4072 QLD Australia, 3Institute for Molecular Biosciences, University of Queensland, 4072 QLD Australia
Here we report on the generation of a dual-fluorescent probe integrated specifically into the exocytotic apparatus of exocrine pancreas of the zebrafish (Danio rerio). As far as we are aware, this fish is the first generated of its kind. The probe is a fusion protein consisting of a pH-sensitive super-elliptic pHluorin (SEP), Vesicle-associated-membrane-protein-3 (VAMP3), and an mCherry (generated by A. Wall). This probe is developed in such a way that the SEP sits inside the granule and the mCherry in the cytosol. This allows quantitative pH measurements of the intra-granular environment, which thus far has been difficult to quantify in vivo (Behrendorff, 2010). This construct was paired with the ElastaseA promoter (courtesy of Z Gong, National University of Singapore, Singapore) which allows exocrine pancreas specific expression in the zebrafish (Wan, 2006) and subcloned into the TOL2 transposon vector (courtesy of E. Scott). The construct was injected into embryos and the progeny screened. Positive expression could be detected at 5dpf in vivo using our custom-made two-photon microscope.
Quality of Life After Pancreatectomy and Islet Autotransplant
M.D. Bellin, D.M. Radosevich, G.J. Beilman, T.B. Dunn, S. Chinnakotla, B. Bland, K.L. Berry, S.J. Schwarzenberg, M. Freeman, T.L. Pruett, D.E.R. Sutherland Depts of Surgery, Pediatrics, Medicine, and the Schulze Diabetes Institute, University of Minnesota, Minneapolis, MN
Background: Early data in children suggest significant improvement in health-related quality of life (HRQoL) after total pancreatectomy and islet autotransplant (TPIAT) for chronic pancreatitis (CP), but large studies are lacking. The objective of this analysis was to review HRQoL in TPIAT recipients at a single center.
Methods: Since 9/2006, patients undergoing TPIAT have completed HRQoL assessments before and at 3 mos, 6 mos, and yearly after TPIAT, including the Medical Outcomes Study 36-item Short Form (SF-36) Health Survey. Statistical analysis was performed using a mixed models method.
Results: 86% (173/201) of eligible patients completed ≥1 survey, for a total of 358 surveys. Median patient age was 37 years (5-66 years); 77% were female. The most common diagnosis was idiopathic CP (37%). Prior to surgery, mean Physical Component Score (PCS) was 27 and Mental Component Score (MCS) was 32 (standardized normal=50, SD=10). Both PCS and MCS significantly improved after surgery (p<0.001). At 1 and 2 years, mean PCS scores were 37 and 42 respectively and MCS scores were 42 and 46, an improvement of >1 SDS. Change in PCS from baseline to 1y was greater for children than adults (+15 v. +10). All 8 subscale scores improved over time (p<0.001), with the greatest effect on bodily pain. Children demonstrated more rapid improvement, particularly for bodily pain and role physical.
Conclusions: These findings suggest meaningful improvement in HRQoL after TPIAT. Although the greatest improvement was in bodily pain, other dimensions of physical, social, and emotional health improved. TPIAT should be considered for patients with CP impacting quality of life.
Lipids of Synthetic Nanoparticles Mimicking Exosome Lipid Composition Induce Death of Pancreatic Human Tumoral Cells
S. Beloribi,1 E. Ristorcelli,1 G. Breuzard,1 J. Bertrand-Michel,2 E. Beraud,1 A. Verine,1 D. Lombardo,11INSERM UMR 911-CRO2; Université de la Méditerranée, Campus Santé Timone, 27 Bld Jean Moulin, 13385 Marseille cedex 05, France. 2INSERM -IFR-BMT-MetaToul, Lipidomic Core Facility, CHU Purpan, 31024 Toulouse cedex 3, France
We reported that exosomes rich in lipid microdomains, secreted by human pancreatic tumor cells, inhibit Notch-1 pathway and exert apoptotic effects on pancreatic tumor cells. We hypothesized that lipid microdomains were privileged sites of interactions between exosomes and cells, and that lipids might be key-elements of cell death.
We designed synthetic exosome-like nanoparticles (SELN) with a composition of lipids forming liquid-ordered phase over lipids forming liquid-disordered phase (Lo/Ld) ratio comparable to that of cancer cell exosomes.
We showed that exosome lipid composition differed from that of parent cells, and was enriched in lipids forming Lo. SELN decreased tumor cell proliferation mimicking exosomes. Higher was the Lo/Ld ratio, greater was the decrease in cell proliferation. This decreased proliferation was due to activation of apoptotic death pathway with inhibition of the Notch-1-Hes-1 axis (assessed by decreased Hes-1 expression). These experiments suggest that fusion between SELN and tumor cells occured at lipid microdomains of plasma and/or endocytic membrane, where the Notch-1 survival pathway matures. Fluorescent SELN collocated with GM1 at plasma membrane lipid microdomains, then with Rab5A, a marker of early endosomes. We thus demonstrated a major role for lipids in interactions between SELN and tumor cells, and in the ensued cell death.
Triptolide Induces Cell Death of Pancreatic Cancer Cells by Inhibition of JAK-2/STAT-3 Signaling
G. Beyer, V. Dudeja, S. Banerjee, V. Sangwan, N. Mujumdar, T.N. Mackenzie, R.K. Dawra, S.M. Vickers, A.K. Saluja Department of Surgery, University of Minnesota, Minneapolis, MN, USA
Introduction: We have previously shown that triptolide, a diterpene tripoxide from the Chinese plant Tripterygium wilfordii, induces caspase dependent apoptotic cell death in pancreatic cancer and markedly reduces the growth and loco-regional spread of pancreatic tumors in orthotopic animal models of pancreatic cancer. The aim of the current study was to evaluate the effect of triptolide on JAK/STAT3 pathway and the role of such an effect in triptolide's action.
Methods: Highly aggressive metastatic pancreatic cancer cell lines (S2013, S2VP10) were treated with triptolide (0-400nM) or WP1066 (0-20μM), an inhibitor of JAK-2/STAT-3 phosphorylation, for 6-48h and the effect on viability (MTT assay), apoptosis (caspase 3 and 9 activity, annexin-V staining), level of phosphorylated and total JAK-2 and STAT3 proteins (western blotting), and on the downstream protein target of JAK-2/STAT-3 pathway like c-myc, cyclin D-1 and survivin (western blotting and qPCR)was evaluated.
Results: Triptolide treatment led to time and dose dependent decrease in the phosphorylation of JAK-2, STAT-3and decrease in the protein and mRNA levels of c-myc, cyclin D-1 and survivin. Direct inhibition of JAK-2/STAT-3 pathway by WP1066 led to dose and time dependent decrease in the viability of pancreatic cancer cells, activation of caspase 3 and 9, and increase in annexin-V staining suggesting activation of caspase dependent apoptosis.
Conclusion: JAK-2/STAT-3 pathway is critical for the survival of pancreatic cancer cells. One of the mechanisms by which Triptolide induces cell death in pancreatic cancer cells is by inhibition of JAK-2/STAT-3 pathway.
The Impact of CFTR Mutations and Genetic Testing on Life Insurance Premiums
A. Brown Department of Medicine, Division of Gastroenterology, The Beth Israel Deaconess Medical Center, Boston, MA 02215
Background: Genetic testing in pancreatitis is a powerful tool. The goal of this study was to determine if and individual is heterozygous for a CFTR mutation will it affect how much they may pay for life insurance premiums (LIP).
Methods: We contacted 10 major US insurance companies (IC's) and asked them the cost to insure a 30 year old non-smoking male for 1 million dollars with a guaranteed 20 year (LIP) (query 1). Five IC's responded. One week later we contacted these IC's again. We asked them for quote on a 20 year LIP for a similar male who had genetic testing showing that he had a single CFTR mutation and a theoretical risk of developing acute pancreatitis (CFTR query). Four IC's responded. Two of the IC's responded to both queries. To preserve confidentiality of the IC's we labeled them A, B, C, D, E, F G. IC's A and B answered both. IC's C and D answered only query 1. IC's E, F and G only answered only the CFTR query. A comparison was made between the difference in costs per IC for a query's 1 and CFTR.
Results: The mean insurance rate in dollars per year for query 1 was 627.8 dollars per year (dpy). The mean insurance rate for all companies that responded to the CFTR query was 2607.5 dpy. Companies A and B were the only two companies that responded to both. Companies A and B quoted LIP's of 759 dpy and 610 dpy (mean= 685 respectively in response to query 1. In response to CFTR query IC's A and B quoted LIP's of 2,760 and 2,980 dpy (mean= 2,870).
Discussion: There was approximately a 400% increase in LIP's reported by all IC's.
Conclusion: This study indicates that patients with pancreatic gene mutations may pay higher (LIP's). Patients should be informed that the results of genetic testing for pancreatic disease may affect the cost of their LIP's.
Meta-Analysis of the Placebo Rate of Abdominal Pain Remission in Clinical Trials of Chronic Pancreatitis
G. Capurso,1* L. Cocomello,1* U. Benedetto,2 C. Cammà,3 G. Delle Fave,1*These authors contributed equally. 1Digestive and Liver Disease Unit and 2Cardiac Surgery Department, II School of Medicine, University of Rome "La Sapienza", 3Gastroenterology Unit, University of Palermo, Italy
Context: Abdominal pain is the main clinical manifestation of chronic pancreatitis. The benefit of currently available therapies for pain management is uncertain. Knowledge of placebo outcomes and understanding the specific study features that influence these outcomes may aid the design of future trials.
Objective: The aim of this study was to investigate the placebo effect on abdominal pain remission rates in patients with chronic pancreatitis, and to identify influencing factors.
Methods: The Medline, Embase and Scopus databases were searched and randomized placebo-controlled trials in chronic pancreatitis patients providing data on abdominal pain remission rates in placebo arms were included. Pooled estimates of the placebo rate were calculated using random-effects logistic regression analysis. Stratum-specific rates for different patient and study-level covariates were calculated to account for heterogeneity.
Results: Six randomized controlled trials (172 placebo-treated patients) met the predefined criteria. The pooled estimate of the placebo rate for abdominal pain remission was 19% (95%CI 8-39%). There was a statistically significant heterogeneity among the studies (I2=78%; p<0.001). A multicenter study design, a run-in period of < 2 weeks, and the absence of a wash-out period in crossover trials were all significant sources of heterogeneity associated with higher placebo responses.
Conclusions: This meta-analysis identifies for the first time the efficacy of placebo for pain in chronic pancreatitis. A multicenter design, the length of the run-in period, and the absence of a wash-out in crossover trials explain the heterogeneity between studies. These data provide a sound basis for designing future placebo-controlled randomized clinical trials for the treatment of pain in chronic pancreatitis.
Value of C-reactive Protein in Predicting Organ Failure, Complications and Mortality in Acute Pancreatitis
F. Cardoso, L. Ricardo, A. Oliveira, C. Rodrigues, D. Horta, A. Figueiredo, J. Deus Department of Gastroenterology, Hospital Fernando Fonseca, Amadora, Universidade Nova, Lisboa, Portugal
Background: C-reactive protein (CRP) is one the most accessible biochemical markers that can be used to identify patients at risk for severe disease early in the course of acute pancreatitis (AP) in order to improve outcome. The aim of this study was to assess the value of CRP in predicting organ failure, pancreatic necrosis and in-hospital mortality in AP.
Methods: CRP determinations at admission, 24 hours, 48 hours and 72 hours of hospital stay from consecutive patients with AP admitted in the emergency department of our secondary care hospital were collected between January 2009 and December 2010. Predictive accuracy of CRP was measured by the area under the receiver-operating curve (AUC).
Results: There were 299 patients with AP, with a median age of 61 years and 52.8% men. Sixteen patients (9%) developed organ failure and were classified as severe AP. Thirty-five (20.7%) developed pancreatic necrosis and 10 (3.3%) died. AUCs for CRP at 48 hours after admission in predicting severity, pancreatic necrosis and in-hospital mortality were 0.83 (95% CI 0.73-0.93), 0.80 (95% CI 0.70-0.91) and 0.86 (95% CI 0.78-0.95), respectively. Specificity for CRP at 48 hours after admission ≥15mg/dl comparing to ≥17mg/dl increased from 52%, 49% and 51%, respectively, to 64%, 58% and 60%, respectively, with equal sensitivity of 100% (p<0.01).
Conclusions: CRP at 48 hours after admission is a simple and accurate method for the early identification of patients at increased risk for organ failure, pancreatic necrosis and in-hospital mortality in AP. The new cut-off level of 17mg/dl proved to be more specific, while equally sensitive.
Downregulation of MUC4 by the Extract and Essential Oil of Holy Basil Leaves Suggests a Potential for Basil as a Novel Therapeutic Adjuvant in Pancreatic Cancer
S. Chakraborty, T. Shimizhu, M.T. Gonzales, J. Souchek, S. Rachagani, M. Macha, A.K. Ganti, E.D. Moore, S.K. Batra University of Nebraska Medical Center, Omaha, NE
Background:Ocimum sanctum (or "Holy basil"), a tropical medicinal herb, has long been used to treat benign ailments. However, its efficacy in cancer is unknown. MUC4 mucin is a novel target for therapy in PC cells. Our objective of this study was to investigate whether ethanolic/aqueous extracts of O.sanctumleaves (EEOL/AEOL) or the essential oil of O.sanctum leaves (EOOS) could downregulate MUC4 expression and inhibit aggressiveness of PC cells.
Methods: Dried basil leaves (n=4 vendors) and EOOS were purchased from separate vendors. PC cells (ASPC-1, MiaPaca, Capan-1 and HPAF/CD18) were treated with EEOL or EOOS diluted in the culture medium. The optimum dose for in vitro studies was determined from a combination of IC-50 values and ability to downregulate MUC4 expression. Effect of EEOL/EOOS on PC cells was assessed through functional assays. AEOLs from 3/4 sources were tested in vivo for anti-tumor activity.
Results: The optimum concentration for EEOL/EOOS that inhibited proliferation of PC cells was 80μg/ml and 0.1% v/v respectively. EOOS/EEOL was more effective than AEOL in downregulating MUC4 expression, with maximum downregulation observed after 48 hours. The downregulation appeared to be primarily transcriptional. Both EOOS and EEOL significantly inhibited cell cycle progression, induced apoptosis and inhibited migration and invasion in PC cells.EEOL/EOOS pre-treatment significantly increased the sensitivity of PC cells to chemotherapy and downregulated the expression of genes regulating proliferation, migration and invasion (ERK-1/2, FAK, p65 and N-cadherin)..Intraperitoneal injections of AEOLs (300mg/kg thrice weekly) from three selected vendors significantly inhibited growth of ASPC-1 cells implanted orthotopically into athymic mice. AEOL treatment significantly upregulated anti-metastatic (E-cadherin) and pro-apoptotic (BAD) genes while downregulating pro-survival (Bcl-2 and Bcl-xL) and chemo/radiation resistance (AURKA, CHK1 and Survivin) genes.
Conclusion: Extracts or essential oil of basil leaves could be a source for novel anti-cancer compounds.
Glycolytic Regulation of Calcium Homeostasis in Pancreatic Cancer
A. Chan,1 A.K. Siriwardena,2 J. Bruce,11Faculty of Life Sciences, University of Manchester, Manchester, UK; 2Department of HPB Surgery, Manchester Royal Infirmary, Oxford Road, Manchester, UK
Background: Pancreatic ductal adenocarcinoma (PDAC) is an insidiously aggressive disease with abysmal survival rates. The Warburg Effect describes a switching in cancer cells of ATP production away from the predominant mitochondrial oxidative phosphorylation and towards cytosolic glycolysis. Inhibiting glycolysis can therefore limit the supply of ATP and affect cancer cells more profoundly. Plasma-membrane calcium ATPase (PMCA) is an ion transporter important in calcium homeostasis, and very sensitive to changes in ATP concentration.
Hypothesis: Inhibiting glycolysis will reduce PMCA activity which in turn will lead to cytosolic Ca2+ overload and cell death.
Methods: Two PDAC cell lines (Panc-1 and Mia-PaCa) were cultured in Dulbecco's modified Eagle's medium (with 10% FBS and 1% penicillin/streptomycin) and treated glycolytic and mitochondrial inhibitors. Cytoscolic Ca2+ was monitored using Fura-2 imaging.
Results: Characterization of Panc-1 cells shows calcium clearance is independent of Na+/Ca2+ exchange and reliant on PMCA. Treatment with glycolytic inhibitors 3-bromopyruvate (500μM) and iodoacetate (5mM) reduced PMCA activity to 71.7% and 65.8% of normal respectively (p<0.05). Mitochondrial inhibitors oligomycin (10μM) and carbonyl cyanide m-chlorophenyl hydrazone (4μM) had no significant effect (113.5% and 97.7% respectively, p>0.5). More profound effects were seen in the Mia-PaCa cell line, whereby 3-bromopyruvate impairs the PMCA's ability to fully restore cytosolic Ca2+ concentration.
Conclusion: Our preliminary data suggests the Warburg Effect is present in pancreatic cancer cells, and glycolysis may be an effective therapeutic target for selectively killing pancreatic cancer cells whilst sparing healthy cells.
Loss of Raf Kinase Inhibitory Protein (RKIP) Enhances Pancreatic Stellate Cell NFκB Activity and EtOH-induced Pancreatic Fibrosis
C. Chao,1 S. Kim,1 K.L. Ives,1 X. Wang,1 J.F. Aronson,2 C. Rastellini,1 M.R. Hellmich,1Departments of 1Surgery and 2Pathology, University of Texas Medical Branch, Galveston, TX
Background: Alcohol (EtOH) is a risk factor for chronic pancreatitis (CP). Aberrant activation of pancreatic stellate cells (PSCs) and NFκB signaling have been implicated in CP. RKIP is an inhibitor of NFκB; however, its function in PSCs and EtOH-induced CP is unknown.
Method: RKIP null (KO) and their mixed strain (129ola/C57BL6) control mice were exposed to EtOH (0.8, 1.6 or 3.2 g/kg) or an equal volume of H2O by daily IP injection for 2, 4, 6 or 8 days. Formalin-fixed, paraffin-embedded pancreata and livers where sectioned, stained, and scored by a pathologist for acute pancreatitis and CP. AP scoring criteria: interacinar edema, acinar necrosis, hemorrhage, fat necrosis, and inflammation. CP scoring criteria: distribution of lesions (% tissue involved), presence of glandular atrophy and pseudotubular complexes, and fibrosis. Livers were evaluated for inflammation/necrosis, fibrosis, and steatosis. Pancreatic stellate cells (PSCs) were isolated using a minced tissue outgrowth procedure on collagen-coated plates. NFκB activity in PSC nuclear extracts was assessed before and after EtOH exposure by Electrophoresis Mobility Shift Assay. Specific NFκB subunits were identified by super shift assay.
Results: EtOH induced a dose- and time-dependent increase in CP scores in KO mice when compared to either H2O-treated KO mice or EtOH- and H2O-treated controls. Although pancreata from EtOH-treated KO and control mice exhibited elevated AP scores compared to H2O-treated mice, serum amylase levels were low and not significantly different between all groups. The livers from both EtOH control and KO mice scored low for inflammation and necrosis but showed no fibrosis or steatosis. PSCs from KO mice exhibited enhanced basal and EtOH-stimulated NFκB activation compared to PSCs from control mice; active subunits included p65 and p50, but not p52.
Conclusion: RKIP inhibits NFκB activity in PSCs; loss of RKIP sensitized the pancreas, but not the liver, to EtOH-induced fibrosis.
Eicosapentaenoic Acid Induces Apoptosis through an Akt-dependent Pathway in Pancreatic Cancer Cells
M.C. Chen, J.L. Park, H. Takahashi, P. Srihari, 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
Background: We previously reported that the omega-3 polyunsaturated fatty acid (PUFA) eicosapentaenoic acid (EPA) exhibited antitumor activities in pancreatic cancer (PaCa) cells in vitro and in vivo through the induction of caspase-dependent apoptosis. However, the exact molecular pathways underlying EPA-induced apoptosis are unclear.
Aim: We tested the hypothesis whether the Akt signaling module, which is constitutively active in PaCa cells and a known pro-survival pathway, mediates the pro-apoptotic effects of EPA in PaCa cells.
Methods and Results: We first confirmed that EPA (0-100 μM) dose-dependently increased apoptotic cell death and stimulated the cleavage of PARP and caspase-7 in BxPC-3 and MIA PaCa-2 cells. This effect was accompanied by an inhibition of Akt phosphorylation at Ser-473. Exposure of PaCa cells to the PI3K inhibitor LY294002 also induced apoptosis and decreased cell growth as measured by Cell Death Elisa and BrdU assay, thereby mimicking the effects of EPA. BxPC-3 cells transfected with constitutively-active Akt (myr-Akt1) exhibited higher total Akt expression and Akt Ser-473 phosphorylation compared to cells transfected with control DNA. EPA decreased Akt Ser-473 phosphorylation in control cells, but this inhibition was abrogated in myr-Akt1 transfected cells. Importantly, EPA-induced apoptosis and growth inhibition was significantly attenuated in myr-Akt1 transfected cells.
Conclusion: Our data strongly indicate that the growth-inhibitory and pro-apoptotic effect of the omega-3 PUFA EPA in pancreatic cancer cells is mediated by inhibition of the Akt signaling module.
Eicosapentaenoic Acid Decreases Fatty Acid Synthase Through an Akt-Dependent Pathway in Pancreatic Cancer Cells
M.C. Chen, S. Esquivel, H.A. Reber, O. J. Hines, V.L.W. Go, G. Eibl UCLA Center of Excellence in Pancreatic Diseases, Department of Surgery, David Geffen School of Medicine at UCLA, Los Angeles, CA
Background: Increased de novo lipogenesis is a hallmark of human cancers. Fatty acid synthase (FASN), a central molecule in de novo lipogenesis, is increasingly recognized as a promising target for cancer prevention and therapy. We have previously reported that the omega-3 polyunsaturated fatty acid eicosapentaenioc acid (EPA) induced apoptosis in pancreatic cancer (PaCa) cells in vitro and in vivo.
Aim: Our aim was to investigate the role of FASN as a potential target of EPA in PaCa cells.
Methods and Results: FASN was expressed in six human PaCa cell lines of varying degrees of differentiation (ASPC-1, BxPC-3, Capan-2, HPAF-II, MIA PaCa-2, Panc-1). Inhibition of FASN by C75, a synthetic FASN inhibitor, reduced cell growth and induced apoptosis, as seen by cleavage of PARP and caspase-3/7. Exposure of cells to EPA (0-100 μM) dose dependently inhibited FASN expression in PaCa cells, which was accompanied by inhibition of cell growth and induction of apoptosis. Exposure of cells to insulin stimulated Akt phosphorylation and increased FASN expression, while the PI3K inhibitor LY294002 decreased FASN levels. Additionally, MIA PaCa-2 cells transfected with constitutively-active Akt (myr-Akt1) exhibited increased FASN expression, indicating the importance of the Akt pathway in baseline FASN expression. Importantly, EPA decreased Akt phosphorylation and FASN expression in control-transfected cells, but this inhibition was abrogated in myr-Akt1 transfected cells.
Conclusion: The omega-3 polyunsaturated fatty acid EPA decreased FASN expression through an Akt-dependent pathway. Our data demonstrated that FASN may be a promising therapeutic target in pancreatic cancer.
Outcomes of a Single-Center Multidisciplinary Management Approach to Pancreatic Necrosectomy for the Treatment of Severe Acute Pancreatitis
J. Chennat, B. Funaki, S. Thomas, S. Vesireddy, K. Wroblewski, E. Choi, I. Waxman, J. Alverdy, J.B. Matthews The University of Chicago Medical Center, Chicago, IL
Background: Minimally invasive necrosectomy approaches have been reported for pancreatic necrosis treatment, combining 1 or 2 surgical, endoscopic, and interventional radiologic techniques. However to date, no clinical algorithms exist which consider a tri-disciplinary management approach for pancreatic necrosis related to severe acute pancreatitis (SAP).
Aim: To report our single tertiary center's outcomes with a multidisciplinary approach to SAP-related pancreatic necrosis management, to identify potential factors that may be associated with our outcomes, and to suggest an algorithm for management.
Methods: We performed an IRB-approved retrospective chart review of all patients with SAP who underwent pancreatic necrosectomy from 2006-2010 at our institution. Descriptive data and location/duration of necrosis were noted. Primary outcomes measured were types/number of interventions, whether necrosis resolved, time to resolution, complications, need for more aggressive interventions, and determination of factors associated with outcomes.
Results: A total of 20 patients (11 male) median age 54 (range 21-75) years were included in this study. Pancreatitis etiology was alcohol (9), gallstones (6), post-ERCP pancreatitis (3) and idiopathic (2). Median APACHE score was 9.5 (range 3-23). Median (range) size of the necroma was 128 (20-200) mm. 13 patients had percutaneous drainage followed by either endoscopic and/or surgical. The other 7 had either solely endoscopic or surgical treatment. 4 patients required eventual open laparotomy for completion necrosectomy. Non-fatal complications occurred in 4 patients. 2 patients died of sepsis before necrosis resolved. Necrosis resolved in 18/20, within a median of 90 days.
Conclusions: A minimally invasive multidisciplinary necrosectomy approach is feasible and appears beneficial for the primary treatment of pancreatic necrosis. The choice of initial mode(s)of therapy should take into account optimal window for necrosis access based on cross-sectional imaging and collaborative discussion. Further long-term prospective assessments of cost-utilization outcomes are required to clarify the role and durability of this "step-up" strategy.
Intraarterial Antibiotics Infusion in the Prophylaxis of Septic Complications in Severe Acute Pancreatitis
S. Chooklin, O. Hranat Department of Surgery, Medical University, Lviv, Ukraine
Introduction: Septic complications are the main cause of death in patients with necrotizing pancreatitis. Due to that the infusions of antibiotics are necessary for these patients. Unfortunately, the results of antibiotics applying in the management of necrotizing pancreatitis are so far from sufficient.
Materials and methods: The aim of this study was to compare the results of intravenous and intraarterial administration of antibiotics in patients with necrotizing pancreatitis. 97 patients with severe acute pancreatitis were studied. The patients were divided into two groups: one received the antibiotic (ertapenem, gatifloxacin) by continuous regional arterial infusion (49 patients) during 12-14 days and the other received antibiotics by intravenous infusion (48 patients). The contamination of necrotic foci was determined using fine needle aspiration.
Results: At the time of admission the Ranson, APACHE II, and Balthazar score was not different in both groups. Contamination of the necrotic focuses was lower in patients of the first group than in second group. The use antibiotics in intraarterially decreases the frequency of infection, limit the destructive process, reduced the number of surgical interventions and mortality. Continuous arterial infusion is effective in preventing bacterial translocation in acute pancreatitis, improves capillary circulation. All patients with infected necrosis were operated. By that, all patients of the second group required the wide laparotomy due to the widespread necrosis. In patients of the first group the operation contented with drain of abscesses.
Conclusion: The intraarterial infusion of antibiotics is effective for the prevention of septic complications in patients with necrotizing pancreatitis.
Modeling Oncogenic Kras Inhibition in Murine Pancreatic Cancer
M.A. Collins,1 F. Bednar,2 Y. Zhang,2 S. Rakshit,2 K.S. Flannagan,2 N. Volkan Adsay,5 M. Pasca di Magliano,1,2,3,41Program in Cellular and Molecular Biology, 2Department of Surgery, 3Department of Cell and Developmental Biology, 4Comprehensive Cancer Center, University of Michigan, Ann Arbor, MI, USA; 5Department of Pathology, Emory University, Atlanta, Georgia
Pancreatic cancer, one of the deadliest human malignancies, is almost invariably associated with a mutation in the KRAS gene, most commonly KrasG12D, that results in a dominant-active form of the KRAS GTPase. However, how the Kras mutation promotes pancreatic carcinogenesis is not fully understood, and, most importantly, the requirement for oncogenic Kras for pancreatic cancer maintenance has not been addressed.
We have generated two new mouse models of inducible, tissue-specific and reversible expression of the oncogenic KrasG12D allele in the pancreas, which we have named iKras* (inducible Kras*) and iKras*-p53 (lacking one copy of the tumor suppressor gene p53). Here, we report that during the earliest stages of carcinogenesis, oncogenic Kras expression reversibly alters epithelial differentiation. Additionally, in established precursor lesions and cancer, Kras* becomes required for tumor cell survival. Strikingly, during all the stages of carcinogenesis, Kras regulated several pathways that mediate the interaction of the epithelial cells with their surrounding microenvironment, thus promoting formation and maintenance of the fibro-inflammatory stroma that is a key characteristic of pancreatic cancer. Therefore, we show that Kras* is a key regulator of all pancreatic tumorigenesis and is essential for tumor maintenance.
Taken together, our data validate the notion of inhibiting Kras, or its downstream effectors, for the treatment of pancreatic cancer.
Qualitative Magnetic Resonance Imaging (MRI/sMRCP) Features for Diagnosis of Chronic Pancreatitis: Covariates for a Clinical Prediction Model
D. L. Conwell,1 N.I. Sainani,2 V. Kadiyala,1 L.S. Lee,1 J. Rosenblum,1 J. Paulo,1 P.A. Banks,1 K. Mortele,21Division of Gastroenterology, Endoscopy and Hepatology; 2Department of Radiology; Brigham and Women's Hospital, Harvard Medical School, Boston MA
Aims: Identify Qualitative MRI/ sMRCP imaging features that are associated with pancreas secretory dysfunction in assessment of chronic pancreatitis (CP).
Methods: Cross-sectional study of subjects with both MRI/MRCP and endoscopic pancreas function test (ePFT) for evaluation of CP. Demographics, qualitative MRI features (parenchymal, ductal, secretory), and peak pancreas fluid [HC03] recorded. Statistical analysis (SPSS v16.0) for association and identification of independent predictors of secretory dysfunction.
Results: Of 109 subjects enrolled 47 (43.1%) had abnormal duct cell secretion during ePFT. Association: 10 radiologic features were identified: MRI parenchymal - atrophy [<0.0001], decreased T1 signal [<0.0001], decreased lobularity [<0.0008], delayed enhancement [0.0013] and heterogenous enhancement [0.021]; MRCP/sMRCP ductal - irregularity [<0.0001], stricture [<0.0001], filling defects [<0.0001], ≥3 visible side branches [0.002] and sMRCP secretory - ductal non-compliance [0.0009].
Logistic regression (controlling for age and gender): 4 radiologic features were identified: decreased parenchymal T1 signal [p=0.0008], glandular atrophy [p<0.0001], ductal irregularity [p<0.0001] and ductal non-compliance [p=0.0009].
- 4 qualitative MRI/sMRCP features were independent predictors of pancreas secretory dysfunction.
- This data can be used as the qualitative imaging component of a clinical prediction model for diagnosing chronic pancreatitis.
- Clinical Implication: MRCP reporting must be standardized to accurately diagnose CP.
Protein C Inhibitor (Serpin A5) in ePFT Collected Pancreas Fluid (PF): A Potential Biomarker of Chronic Pancreatitis (CP)
D.L. Conwell,1 J.A. Paulo,1,2 L.C. Lee,1 P.A. Banks,1 H. Steen,21Division of Gastroenterology, Endoscopy and Hepatology, Brigham and Women's Hospital; 2Department of Pathology, Children's Hospital Boston; Harvard Medical School, Boston MA
Aim: Characterize differential protein expression using proteomic analysis of PF from patients with severe CP and controls.
Methods: PF was collected from CP (n-9) and control (n=9) subjects using ePFT. Proteins precipitated, separated by SDS-PAGE and analyzed by GeLC-MS/MS. Proteins identified and molecular function determined. Spectra count analysis with QSPEC statistical software using Bayesian statistical methods. Protein filtering: Bayes factor>10 and >2-fold difference in protein expression between cohorts was statistically significant (p< 0.05).
Results: The mean peak [HC03] for CP and controls was 37.7 ± 13.3 and 93.4 ± 13.6 mEq/L (p<0.05), respectively. 1392 non-redundant PF proteins were identified. Protein filtering revealed significantly up-regulated proteins [serpin A5, neprilysin, mucin 13, annexin A5 and defensin 5] and down-regulated proteins [trypsin, chymotrypsin, lipase and aminopeptidase]. Proteases were the most common down-regulated proteins, and protein binding the most common up-regulated proteins. Protein C Inhibitor [Serpin A5]; a serine protease inhibitor/"binder" was the top-hit protein in 8/9 CP and 0/9 control PF samples (p=0.00041): Sensitivity (89%), Specificity (100%), PPV (100%), NPV (90%) and diagnostic accuracy of 94.4% [74.2, 99.0].
- 1) Serpin A5 is up-regulated and proteases are down-regulated in PF of patients with severe chronic pancreatitis compared to controls.
- 2) Serpin A5 expression in PF may serve as a biomarker for CP.
Clinical Implication: Further investigation of Serpin A5 in milder forms of chronic pancreatitis is warranted.
Management of Insulinomas: Perioperative and Long-Term Outcomes in 198 Patients
S. Crippa,1,2 A. Zerbi,3 L. Boninsegna,1,2 V. Capitanio,3 Balzano,3 P. Pederzoli,1 V. Di Carlo,3 M. Falconi,11Department of Surgery, University of Verona, Verona, Italy; 2Department of Surgery, Ospedale Sacro Cuore Don Calabria, Negrar (VR), Italy; 3Department of Surgery, San Raffaele Hospital, Milna, Italy
Background: Enucleation represents the procedure of choice for the treatment of insulinomas but pancreatic resections may be required.
Aim: To analyze perioperative and long-term outcomes following surgical treatment of insulinomas.
Methods: 198 patients (58.5% females; median age 48 yrs) with symptoms of hyperinsulinism and positive fasting glucose test were included. Ki67 index was evaluated. WHO classifications were applied.
Results: Most insulinomas were benign (65%) with only seven WDEC (3.5%) and all were G1/G2 NETs. Multiple insulinomas were found in 8% of patients despite 5.5% of patients had a MEN-1 syndrome. Surgical procedures included 106 enucleations (54%) and 92 pancreatic resections (46%). Mortality was nil. Rate of clinically significant pancreatic fistula was 18%. Enucleations had a higher reoperation rate compared to pancreatic resections (8.5% vs 1%, P=0.018). MEN-1 syndrome was significantly (P<0.005) associated with a younger onset-age, higher rate of malignancies and of multiple lesions. Median follow-up was 65 months. Six patients (3 WDEC and 3 uncertain behavior tumor; 5 had G2 NETs) developed tumor-recurrence. Four patients (2%) died of disease. New exocrine (1.5%) and endocrine (4%) insufficiencies were associated with pancreatic resections.
Conclusion: Insulinoma is usually a benign and unifocal tumor. Surgical resection is safe with no mortality and good functional outcomes. Recurrence is uncommon (3%) and is more likely associated with WDEC and G2 NET. Insulinomas in MEN-1 syndrome are at higher risk of being malignant and multifocal, requiring pancreatic resections.
Pancreatic Exocrine Insufficiency in Advanced Pancreatic Cancer: Faecal Elastase-1 (FE-1) Value is a Strong Independent Predictor of Poor Survival
S. Crippa,1,2 S. Partelli,1,2 L. Frulloni,3 L. Benini,3 C. Minniti,1 P. Pederzoli,1 M. Falconi,11Department of Surgery, University of Verona, Verona, Italy; 2Department of Surgery, Ospedale Sacro Cuore Don Calabria, Negrar (VR), Italy; 3Department of Gastroenterology, University of Verona, Verona, Italy
Background: The relationship between prognosis of advanced pancreatic cancer (APC) and exocrine insufficiency (PEI) is unknown.
Aim: To investigate a possible correlation between FE-1 value and survival in APC patients.
Methods: A prospective non-randomized study was carried out between 2007 and 2009. FE-1 was measured at admission. PEI was defined as absent (FE-1: >200 μg/g of stool), moderate (FE-1: 100-200 μg/g), severe (FE-1 <100 and >20 μg/g) and extremely severe (FE-1 ≤20 μg/g). Univariate and multivariable analyses were performed.
Results: 194 patients with APC (metastatic or locally-advanced PDAC) were enrolled. The median FE-1 was 204 μg/g (IQR 19; 489), being normal in 97 patients (50%). Overall, 48 (25%) had an extremely severe PEI, 28 (14%) a severe PEI and 21 (11%) a moderate PEI. Patients with extremely severe PEI had higher incidence of albumin values <40 g/L (44% versus 29% versus 14%, P<0.01), higher pancreatic head localizations (96% versus 73.5% versus 59%, P<0.01), higher rate of jaundice (70% versus 37% versus 34%, P<0.01). The median overall survival was 10.5 months. Patients with FE-1 ≤20 μg/gram had a worse prognosis (median survival: 7 versus 11 months, P=0.031). By multivariable analysis, presence of metastases (HR 1.81, P<0.0001), haemoglobin ≤12 g/L (HR 2.12, P=0.001), albumin ≤40 g/L (HR 1.64, P=0.010) and FE-1 ≤20 μg/g (HR 1.59 P=0.023) resulted as independent predictors of survival in APC patients.
Conclusions: A low value of FE-1 is strongly correlated with a poor survival in patients with APC.
HIF2α, A Key Player in Kras-Mediated Pancreatic Neoplasia
A. Criscimanna, J. Speicher, F. Esni Department of Surgery; University of Pittsburgh, Pittsburgh, PA
While apoptosis is the primary mechanism of programmed cell death, autophagy may be considered as a programmed cell survival mechanism when cells have to respond to stress, such as hypoxia. Many of the cellular responses to hypoxia are meditated through the hypoxia-inducible factors HIF1α and HIF2α. Accumulating data suggest that apoptosis is associated with early tumorigenesis, whereas autophagy is more evident in late-stage PanINs, and also is required for tumor growth in pancreatic cancer.
Given the absence of HIF1α, and the presence of HIF2α in early PanINs, the pro-autophagy nature of HIF1α, and the pro-apoptotic nature of HIF2α, we wished to determine whether loss of HIF2α would shift the balance between apoptosis and autophagy, and thereby influence the progression of Kras-mediated pancreatic neoplasia.
To do so, we used the PtfCre transgenic system to activate KrasG12D and delete Hif2a in all pancreatic epithelium. At 3 months, compared to their age matched PtfCre;KrasG12D cohort, the PtfCre;KrasG12D;Hif2afl/fl mice exhibited almost total loss of acinar tissue, a significant increase in acinar to ductal metaplasia, and desmoplasia. At 9 months, the PtfCre;KrasG12D pancreas displayed back to back PanINs with no stromal elements, whereas in the PtfCre;KrasG12D;Hif2afl/fl pancreas there was a strong stromal proliferation between PanINs. Surprisingly, the PtfCre;KrasG12D;Hif2afl/fl pancreas also showed recovery of acinar tissue. The observed phenotype in PtfCre;KrasG12D;Hif2afl/fl pancreas was associated with compensatory expression of Hif1α in PanINs, accelerated autophagy, and inhibited or delayed apoptosis.
Together, our data suggest that HIF2α and HIF1α may be required at different stages during pancreatic tumorigenesis to maintain the delicate balance between apoptosis and autophagy.
Detection of Pancreatic Cancer Tumors and Precursor Lesions Using Cathepsin E Activity
Z. Cruz-Monserrate,1 W. R. Abd-Elgaliel,2 D. Deng,1 Baoan Ji,1 T. Arumugam,1 C. Tung,2 C. D. Logsdon,11Department of Cancer Biology, UT M.D. Anderson Cancer Center, Houston, TX, USA. 2Department of Radiology, Methodist Hospital Research Institute, Weill Cornell Medical College, Houston, TX, USA
Background: Pancreatic ductal adenocarcinoma (PDAC) is the fourth leading cause of cancer death in the USA. Surgical resection is the only effective treatment; but only 20% of patients are candidates for surgery. The ability to detect early PDAC would increase the availability of surgery and improve patient survival. This study assessed the feasibility of using the enzymatic activity of Cathepsin E (Cath E), a protease highly and specifically expressed in PDAC, as a novel biomarker for the detection of pancreas bearing pancreatic intraepithelial neoplasia (PanIN) lesions and PDAC.
Methods: Pancreas from normal, chronic pancreatitis and PDAC patients were assessed for Cath E expression by quantitative real time PCR (QRT-PCR) and immunohistochemistry (IHC). Human PDAC xenografts and genetic mouse models (GMMs) of PDAC were injected with a Cath E activity selective fluorescent probe and imaged using an IVIS optical imaging system.
Results: Specificity of Cath E expression in PDAC patients and GMMs of pancreatic cancer was confirmed by QRT-PCR and IHC. The novel probe for Cath E activity specifically detected PDAC in both human xenografts and GMM models in vivo. The Cath E sensitive probe was also able to detect pancreas with PanIN lesions in GMMs prior to tumor formation.
Conclusions: The elevated Cath E expression in PanINs and pancreatic tumors allowed in vivo detection of human PDAC xenografts and imaging of pancreas with PanINs and PDAC tumors in GMMs. Our results support the usefulness of Cath E activity as a potential molecular target for PDAC and early detection imaging.
Loss of Expression of the SWI/SNF Chromatin Remodeling Subunit BRG1/SMARCA4 Is Frequently Observed in Intraductal Papillary Mucinous Neoplasms (IPMNs) of the Pancreas
M. Dal Molin,1 S. Hong,1 S. Hebbar,1 R. Sharma,1 F. Scrimieri,1 R.F. de Wilde,1 S.C. Mayo,2 M. Goggins,1,3,4 C.L. Wolfgang,2 R.D. Schulick,2,3 M. Lin,1 J.R. Eshleman,1,3 R.H Hruban,1,3 A. Maitra,1,3 H. Matthaei,1,5The Sol Goldman Pancreatic Cancer Research Center, Departments of 1Pathology, 2Surgery, 3Oncology, 4Medicine, The Johns Hopkins University, Baltimore, USA; 5Department of General, Visceral, Thoracic, and Vascular Surgery, University of Bonn, Germany
Background: A better molecular characterization of Intraductal Papillary Mucinous Neoplasm (IPMN), the most frequent cystic precursor of pancreatic adenocarcinoma, may have a pivotal role in its early detection and in the development of effective therapeutic strategies. BRG1, a central component of the chromatin remodeling complex SWI/SNF regulating transcription, is inactive in several malignancies.
Methods: We evaluated Brg1 expression in IPMN in order to better understand its role in pancreatic carcinogenesis. Tissue microarrays (TMAs) of 66 surgically resected IPMNs were immunolabeled for the Brg1 protein. Expression patterns were correlated with clinicopathologic parameters.
Results: Normal pancreatic epithelium strongly immunolabeled for Brg1. Reduced Brg1 expression was observed in 32 (53.3%) of the 60 evaluable lesions and occurred more frequently in high-grade IPMNs (13 of 17 showed loss; 76%) compared to intermediate-grade (15 of 29 showed loss; 52%) and low-grade (4 of 14 showed loss; 28%) (p=0.03). A complete loss of expression was observed in 5 of the 60 (8.3%) lesions. Finally, a decrease in Brg1 protein expression was found in a low-passage non-invasive IPMN cell line by Western blot analysis.
Conclusion: We provide first evidence that Brg1 expression is lost in IPMN.
Trypsin Activation Is Required for Intra-Acinar Chymotrypsinogen Activation
R. Dawra, R. Sah, A. Bekolay, V. Dudeja, A. Saluja Division of Basic and Translational Research, Department of Surgery, University of Minnesota, Minneapolis, MN
Background and Aims: Significant intra-acinar activation of trypsin and chymotrypsin is observed in early stages of experimental pancreatitis and in-vitro in pancreatic acinar cells as a consequence of pathological insult. It is considered an important initiating event in acinar cell injury. However, it is not clear if trypsin activation is required for chymotrypsinogen activation or if it proceeds independently. Mice with trypsinogen 7 gene deletion lack pathological activation of trypsin. The aim of this study was to define the role of intra-acinar trypsin activity in chymotrypsinogen activation.
Methods: Wild type and trypsinogen T7 gene deleted mice were administered caerulein (i.p. 50μg/kg). Pancreases were collected at different time points. Trypsin and chymotrypsin activities were measured in pancreatic homogenates using their specific substrates by fluoremetric method.
Results: In wild type mice, there was a maximum increase in trypsin activity (7.9±1.03 fold over control) as well as in chymotrypsin activity (68.8±30.1 fold) at 30min after caerulein injection. But in mice with T7 deletion there was no significant increase in trypsin activity (1.45±0.22 fold of control) in response to caerulein at 30 min. There was also no significant increase in chymotrypsin activity (2.2±0.7 fold).
Conclusions: Our data suggest that trypsin activation is required for intra-acinar activation of chymotrypsinogen.
Supported in part by NIH grant RO1DK093047.
Human Pancreatic Acinar Cells Do Not Respond to Direct Stimulation with Caerulein
R. Dawra, L. Rishi, B. Appakalai, B. Hering, A. Saluja Division of Basic and Translational Research and Schulz Diabetes Institute, Department of Surgery, University of Minnesota, Minneapolis, MN
Background and Aims: In rodents cholecystokinin or its analog caerulein cause secretion from pancreas either directly by binding to its receptors on acinar cells or indirectly through its receptors on vagal neurons causing release of acetylcholine which in turn binds to muscainic receptors on acinar cells. However, in human pancreas it is not clear if cholecytokinin induced secretion is direct, indirect or both. There are reports in support as wells as against secretion through direct binding. Main limitation has been non-availability of acinar cells from healthy human pancreas. Aim of the present study was to check if acinar cells prepared from healthy human pancreas respond directly to cholecystokinin analog caerulein.
Methods: Acinar cells prepared from healthy human pancreas by collagenase digestion were stimulated with different concentrations of caerulein, carbachol or bombesin and percent amylase secretion and cytosolic Ca2+ response was studied.
Results: There was significant increase in secretion in response to stimulation with different concentrations of carbachol (net maximal 11.8±2.0%) and bombesin (net maximal 11.0±1.3%). But no significant increase was observed in response to stimulation with different concentrations of caerulein. Increase in cytosolic Ca2+ was observed after stimulation with carbachol and bombesin but no significant increase was observed with caerulein.
Conclusions: Our data from acini prepared from nine healthy human donor pancreases suggest human acinar cells lack direct response to caerulein.
Pre-neoplastic Metaplastic Lesions Represent Epithelial Transdifferentiation to a Biliary-Like Phenotype
K.E. DelGiorno, E. Carpenter, K. Takeuchi, H.C. Crawford Department of Molecular and Cellular Pharmacology, SUNY Stony Brook, Stony Brook, NY
Pancreatic ductal adenocarcinoma (PDA) is consistently associated with a reactive epithelium known as metaplastic duct lesions (MDLs). MDLs are hypothesized to be a pre-neoplastic lesion in part because they have progenitor cell-like qualities, including expression of factors normally associated with pancreatic development, such as Pdx1. Here we set out to more thoroughly characterize the MDLs in PDA. Immunofluorescent and electron micrograph analysis of murine MDLs demonstrates transdifferentiation of acinar cells to tuft cell-containing ducts. Tuft cells, characterized by a thick tuft of microvilli at their apical surface and a well developed tubulovesicular system are not found in the normal murine pancreatic duct, but are commonly found in the developmentally related intestine and bile duct. In fact, expression of several biliary tract markers suggests that MDLs actually represent a transdifferentiation event resulting in a biliary phenotype. Interestingly, unlike the other cells in the MDL, metaplastic tuft cells are non-proliferative, but are highly biologically active, showing strong activation of several oncogenic signaling pathways and are a major source of prostaglandin synthesis, as revealed by their expression of COX1, COX2 and prostaglandin synthases; which has been shown to be pro-inflammatory and, thus, pro-tumorigeneic. This transdifferentiation event provides insight into the cellular programming leading to PDA progression and provides a potential source of early disease makers worthy of further investigation.
Human Ecdysoneless Promotes Aerobic Glycolysis in Pancreatic Cancer Cells
P. Dey, S. Chakraborty, S. Rachagani, S. Senapati, P. Singh, C.B. Gurumurthy, V. Band, M. Hollingsworth, S.K. Batra Department of Biochemistry and Molecular Biology, Eppley Cancer Institute, University of Nebraska Medical Center, Omaha, NE
Background and Hypothesis: Human Ecdysoneless (hEcd), a highly conserved orthologue of the D.melanogaster ecdysoneless protein, regulates synthesis of the steroid hormone ecdysone in insects. It is alternatively known as human suppressor of GCR2 (hSGT) since it can rescue growth defects in S.cerevisiae mutants with deletion of GCR2, a glycolysis regulatory gene. hEcd also interacts with and stabilizes p53, regulates E2F target gene expression and cell cycle progression. Due to its direct impact on cell cycle, which is often dysregulated in cancer, we hypothesize that hEcd plays a critical role in pancreatic cancer (PC) pathogenesis.
Results: Immunohistochemical analysis of hEcd expression in PanIN and PC tissues revealed that it is overexpressed in PC tissues compared to normal pancreas. Stable knockdown of hEcd in PC cell lines led to reduced proliferation in vitro and decreased tumor weight in vivo, indicating its involvement in PC progression. Microarray analysis revealed that insulin-regulated glucose transporter GLUT-4 is downregulated due to hEcd silencing, which was verified by qRT-PCR and immunoblotting. Membrane fractionation and confocal studies showed reduced GLUT-4 membrane localization upon hEcd knockdown. Further, hEcd silenced cells showed less uptake of radiolabeled glucose in vitro and upon orthotopic implantation in athymic mice, along with lower ATP and lactate production. hEcd depletion in PC cells also decreased the level of phosphorylated Akt, a major signaling molecule regulating glycolysis in cancer cells.
Conclusion: Overall, these results show that hEcd is aberrantly expressed in PC tissues and promotes tumorogenesis by modulating PC cell aerobic glycolysis.
Dual Inhibition of MEK and mTOR Signaling Pathways Decreases Proliferation of Human Pancreatic Neuroendocrine Tumor (PNET) Cells In Vitro
C. Djukom, K. Ives, S. O. Kim, X. Wang, M. Hellmich, C. Chao Department of Surgery, University of Texas Medical Branch; Galveston, TX, USA
Introduction: Patients with advanced PNET have limited therapeutic options. RAD001, an inhibitor of the mTOR pathway, has been shown to increase progression-free survival, but not overall survival, indicating a need to identify additional therapeutic targets. The BON cell line, derived from a metastatic PNET, has been used as a model to study PNET cell biology. Autocrine activation of Insulin Growth Factor 1 receptor on the BON cell stimulates proliferation through both the MEK/ERK and PI3K/mTOR pathways. Since both the MEK and mTOR pathways contribute to the regulation of proteins involved in apoptosis and cell growth, we hypothesized that dual inhibition of MEK and mTOR will overcome the limited activity of RAD001 alone.
Methods: Western blot analyses were performed to verify downregulation of downstream targets. Cell proliferation assays were performed with DMSO or the MEK (PD0325901, 50 nM) and mTOR (RAD001,50nM) inhibitors. Cell growth was measured over time in replicates; cells were quantified using a coulter counter.
Results: BON cells showed an 80% decrease in the activity of ppP70S6K and a 70% decrease in pERK levels when treated with RAD001 and PD, respectively, compared to DMSO control treatment. The number of BON cells 8 days after plating were 78%, 46%, and 15% of the DMSO-treated control cells in the presence of PD, RAD, and both PD and RAD, respectively. Cell proliferation significantly and synergistically decreased when the cells were treated with both PD and RAD.
Conclusion: The combination of MEK and mTOR inhibition significantly decreased cell proliferation compared to single drug inhibition alone in BON cells. Our data suggest that combination therapy is more effective in vitro than either inhibitor alone. Future pre-clinical studies will be necessary to examine the mechanism and effects of dual inhibition on tumor growth and metastasis in vivo.
Distinct Actions of Ethanol Metabolites in Pathologic Rat Pancreatic Acinar Exocytosis Underlying Alcoholic Pancreatitis
S. Dolai,* P. Lam,* T. Liang,* N. Fernandez, S. Chidambaram, H. Gaisano Depts of Medicine and Physiology, University of Toronto, Canada. *Equal contributors
Background & Aims: In alcoholic pancreatitis, it is not ethanol (EtOH) per se, but its oxidative (acetaldehyde) and non-oxidative metabolites (ethylpalmitate, ethyloleate) that mediate toxic injury. We reported that EtOH pre-treatment of pancreatic acini induced blockade of CCK-8-stimulated apical exocytosis and redirection of exocytosis to basolateral plasma membrane (PM) causing interstitial pancreatitis. We now examined how each EtOH metabolite contributes to pathologic exocytoses.
Methods: Dispersed rat pancreatic acini pre-treated with clinically-relevant doses of EtOH (20-50mM) or EtOH metabolites (1-3mM) were stimulated with CCK-8. We performed real-time FM1-43 epiflourescence, single zymogen granule (ZG) exocytosis by spinning-disk confocal microscopy and ultrastructural electron microscopy studies to explain the reduction in amylase secretion. Coimmunoprecipitation was performed to reveal distinct Munc18/SNARE complexes mediating apical, basolateral and ZG-ZG fusion.
Results: All 3 metabolites reduced CCK-8-stimulated apical exocytosis and apical exocytotic complexes (Munc18b/[Syn-2,SNAP23,VAMP2]), while acetaldehyde and ethyloleate redirected exocytosis to basolateral PM, promoting basolateral exocytotic complexes (Munc18c/[Syn-4,SNAP23,VAMP8]. Acetaldehye, like EtOH, promoted ZG-ZG fusion complexes (Munc18b/[Syn-3,SNAP23,VAMP8] while ethylpalmitate and ethyloleate reduced these complexes.
Conclusions: All 3 metabolites contribute to EtOH-induced perturbation in 3 different exocytotic events (apical blockade, basolateral exocytosis, ZG-ZG fusion); however, acetaldehyde, and to lesser degree ethyloleate, mimicked the pathologic effects of EtOH on CCK-8-stimulated pathologic exocytosis.
Inhibition of JAK-2 Sensitizes Pancreatic Cancer Cells to TRAIL Induced Cell Death
V. Dudeja, S. Skube, G. Beyer, S. Banerjee, V. Sangwan, N. Mujumdar, T.N. Mackenzie, R.K. Dawra, S.M. Vickers, A.K. Saluja Department of Surgery, University of Minnesota, Minneapolis, MN, USA
Introduction: Pancreatic cancer is resistant to conventional as well as novel anti-cancer therapies like TRAIL. JAK-2 pathway regulates cell survival and thus contributes to carcinogenesis. The aim of the current study was to evaluate whether inhibition of JAK-2 pathway sensitizes pancreatic cancer to TRAIL induced cell death.
Methods: Highly aggressive metastatic pancreatic cancer cell lines (S2013, S2VP10) were treated with JAK-2 inhibitors WP1066 (0-5μM) or FLLL-31 (0-5μM), TRAIL (0-40ng/ml) or a combination of JAK-2 inhibition and TRAIL. The effect on viability (MTT) and apoptosis (annexin V, caspase 3, 8 and 9 activation) was measured. The results were confirmed with specific inhibition of JAK-2 by siRNA.
Results: JAK-2 inhibition markedly increased TRAIL induced cell death in both pancreatic cancer cell lines. Viability, data expressed as % of Control (untreated cells), mean ±SEM. S2VP10 (48h): WP1066 (5μM) - 61.8±3.8%, TRAIL (2.5 ng/ml) - 100.6±2.5%, WP1066 (5μM) + TRAIL (2.5ng/ml) - 18.5±1.8%. Similar results were observed with the combination of TRAIL and the other JAK-2 inhibitor FLLL-31 as well as with JAK-2 downregulation by siRNA. JAK-2 inhibition noticeably augmented Caspase 8 and 3 activation and annexin V staining in response to TRAIL. Caspase 3, data expressed as % of Control, mean ±SEM. S2VP10 24h: WP1066 (5μM) - 410±7%, TRAIL (2.5 ng/ml) - 154±23%, WP1066 (5μM) + TRAIL (2.5ng/ml) - 2952±18%.
Conclusion: Inhibition of JAK-2 pathway sensitizes pancreatic cancer cells to TRAIL induced apoptosis and cell death. Combination of JAK-2 silencing and TRAIL has immense potential to emerge as a novel therapeutic strategy against pancreatic cancer.
Pancreatic Tumor Sensitivity to Plasma L-asparagine Starvation
E. Dufour,1 F. Gay,1 K. Aguera,1 J.Y. Scoazec,2 F. Horand,1 P. Lorenzi,3 Y. Godfrin,11R&D Department, Erytech Pharma, Lyon, France; 2Service d'Anatomie Pathologique, Hospices Civils de Lyon, Lyon, France; 3Department of Bioinformatics and Computational Biology, M. D. Anderson Cancer Center, Houston
Objectives: In this study, our aim was to test whether asparagine synthetase (ASNS) deficiency in pancreatic malignant cells can lead to sensitivity to asparagine starvation. We also investigated, in tumor-bearing mice, the efficacy of L-asparaginase entrapped in red blood cells (RBCs), a safe formulation, to induce asparagine depletion.
Methods: First, ASNS expression was evaluated by immunohistochemistry in sporadic pancreatic ductal adenocarcinoma. Then, four pancreatic carcinoma cell lines were examined by western blot, immunocytochemistry, and cytotoxicity assay to L-asparaginase and in asparagine-free or reduced-asparagine media. Finally, mice bearing the most in vitro sensitive cell line received RBC-entrapped L-asparaginase to investigate the anticancer efficacy of serum asparagine depletion in vivo.
Results: About 52% of pancreatic adenocarcinomas expressed no or low ASNS. The highest in vitro cytotoxicity to L-asparaginase or to reduced asparagine medium was observed with SW1990 line when ASNS expression was the lowest. In vivo sensitivity was confirmed for this cell line.
Conclusions: Plasma asparagine depletion by RBC-entrapped L-asparaginase in selected patients having no or low ASNS, may be a promising therapeutic approach for pancreatic cancer.
Transgastric Cooling Of the Pancreas; a Feasibility Study For Acute Pancreatitis (AP)
C. Durgampudi,1 J.A. Holmes,2 S. Narla,3 V.P. Singh,4Department of Medicine UPMC Passavant1, Swanson Center for Product Innovation2, Graduate School, Case Western Reserve University3, Division of Gastroenterology, University of Pittsburgh, Pittsburgh, PA4
Background: The pancreas is hypermetabolic on PET scans during AP. Multiple signaling pathways are rapidly activated simultaneously during AP. Hypothermia reduces metabolic demand, cell death and inflammation in other systems; however generalized hypothermia compromises vital systems. We attempted to cool the pancreas transgastrically to temperatures at which deleterious phenomena slow down in vitro.
Methods: A collapsible gastric balloon attached to a dual lumen tube was perfused with ice water in sedated rats. Rectal, pancreas body and tail temperatures were noted. Caerulein (100nM) induced trypsinogen activation (TGA), KC and MIP-2 mRNA levels were studied at 37°C and 23°C in acinar cells. Trypsin, Cathepsin B activities and cathepsin B mediated TGA to trypsin were studied at 37°C, 23 °C and 4°C.
Results: Pancreatic, but not rectal temperatures decreased from 37.8 +/-1.1 °C to 26.7+/-2.8 °C in 1 hour with similar body and tail temperatures (26.4+/-1.0 °C vs. 26.8+/-1.8 °C). Discontinuing cooling rewarmed the pancreas rapidly. In acini; TGA, KC and MIP-2 mRNA increase noted at 37 °C were reduced by >90% at 23 °C. While trypsin and cathepsin B activities at 23°C were 53% and 64% of 37°C, cathepsin B mediated TGA was reduced to 26% of that at 37°C.
Conclusions: Transgastric pancreatic cooling is rapid, reversible and inducible without causing generalized hypothermia. The temperatures achieved significantly slow mechanistically different phenomena and synergistically slow sequential steps in a signaling cascade. Therefore Transgastric hypothermia may be a therapeutic option to consider for AP.
Novel Model of Pancreatic Neoplastic lesions Induced by Smoking Compound NNK
M. Edderkaoui,1 C. Park,1 I. Lee,1 C. Nitsche,1 A. Gerloff,1 P.J. Grippo,2 S.J. Pandol,1 A.S. Gukovskaya,11University of California Los Angeles & VA-GLAHS, CA; 2Department of Surgery, Feinberg School of Medicine, Northwestern University, Chicago, IL
Introduction: Cigarette smoking is a major risk factor for pancreatic cancer. However, the molecular signaling mechanisms through which smoking promotes pancreatic cancer remain unknown. Experimental models of smoking-induced pancreatic cancer have not been developed.
Methods: Wild type and EL-Kras transgenic mice were injected with smoking compound NNK (100mg/Kg) once a week for 4 weeks, then sacrificed and pancreatic tissue analyzed. Immortalized human ductal cells HPDE6 were cultured for up to 1 week in the presence of NNK (1nM-1μM) or cigarette smoke extract (CSE) (0.04-4μg/ml). Apoptosis in cells was assessed by measuring DNA fragmentation. Autophagy was assessed by measuring autophagic marker LC3-II with western blot or with immunofluorescence. Pancreatic lesions and fibrosis in pancreatic tissue were analyzed by immunohistochemistry.
Results: NNK injections significantly increased the number of pancreatic neoplastic lesions in mice with activated Kras mutation. Number of neoplastic lesions was significantly increased by NNK (8 times). NNK injection stimulated fibrosis. NNK decreased apoptosis and autophagy both in vivo and in vitro. NNK and CSE increased Akt phosphorylation. Pharmacological and molecular inhibitions of NADPH oxidase and Akt kinase reversed the effects of smoking compounds on apoptosis and autophagy.
Conclusion: NNK-induced neoplasia progression in Kras mice represents a novel model to study the mechanisms underlying oncogenic effects of smoking on pancreas. Our in vivo and in vitro data indicate that one mechanism mediating smoking-induced pancreatic cancer progression is through activating NADPH oxidase and Akt kinase.
Three-dimensional (3D) Model of the Pancreas Using Routine CT Data and a 3D Printer: A Feasibility Study
K. Endo, N. Sata, Y. Kaneda, M. Koizumi, A. Lefor, Y. Yasuda Department of Surgery, Jichi Medical University
Background: Imaging technology has advanced significantly. The enormous amount of imaging data from modalities such as CT and MRI has not been adequately utilized. We propose a 3D model created from routine CT data for preoperative simulation and intraoperative real-time navigation in pancreatic surgery.
Material and Method: A healthy volunteer's dataset from a dynamic 4-phase CT study with a 64 detector-row CT (SOMATOM Definition AS Flush, Siemens Germany) was used. Pancreatic parenchyma, arteries and veins were segmented with an image processing workstation (SYNAPSE VINCENT Fujifilm Medical Japan). These data were sent to another workstation (FREEFORM, SenSable, USA) in 3D volume data saving format. After smoothing these surfaces, all elements were combined into one virtual 3D model. Finally a 3D real model was made on a 3D printer (ZPrinter, ZCOOPRATION, USA) using the same data.
Results: Using data from a routine CT study, 3D virtual and real models of the pancreas were created. The virtual model was created in 3 hours and a real model with a 3D printer was made in 5 hours.
Discussion: 3D visualization is an effective use of the enormous amount of imaging data available. There are two approaches for 3D visualization, virtual and real, each with its own merits. The virtual model is easy to make in a short time for simulating surgical procedures on the computer, but has limitations. 3D real models compensate for some of the limitations of virtual models and are useful to recognize size and stereoscopic relationship among surrounding organs. Both 3D virtual and real models may be valuable for planning and performing therapeutic procedures safely.
Conclusion: 3D visualization of the pancreas is a feasible and potent way to analyze valuable imaging data.
Pancreatic Star Alliance: After the First Meeting, Future Challenges
M. Erkan,1 J. Kleeff,1 H. Friess,1 M. Apte,2 M. Bachem,31Technische Universität München, Munich, Germany; 2The University of New South Wales, Sydney, Australia; 3Universitätsklinikum Ulm, Ulm, Germany
It is now well established that pancreatic stellate cells (PSC) are responsible for producing the stromal reaction (fibrosis) of two major diseases of the pancreas - chronic pancreatitis and pancreatic cancer. In contrast to research into hepatic stellate cells (the hepatic counterparts of PSC that were first described in 1870), the field of PSC biology is very young, since the essential in vitro tools to study these cells (i.e. methods to isolate and culture PSC) were only developed as recently as in 1998. Nonetheless, there has been an exponential increase in research output in this field over the past decade with numerous groups around the world focusing on elucidating the biology and function of these cells. In view of the rapidly accumulating information in the field, it was considered timely to convene a meeting of core PSC researchers to discuss and consolidate current knowledge, to outline and delineate areas of consensus (for example with regard to methodological approaches) and, more importantly to identify essential directions for future research. To this end, the Pancreatic Star Alliance (www.pancreaticstaralliance.com) convened its first meeting at the Technische Universitaet Muenchen in Munich, on the 4th of March 2011. The gaps in knowledge that warrant attention in order to increase our understanding of the role of PSC in health and disease are getting clearer. Here we summarize the aims, structure and functioning of the Pancreatic Star Alliance to tackle problems like access to primary human PSC and developing better animal models by further networking of research groups.
Vesicular Transport of ZG Proteins in the Early Secretory Pathway
J. Fang, P. Skallos, X. Chen Department of Physiology, School of Medicine, Wayne State University, Detroit, MI 48201
Introduction: Zymogen granule (ZG) proteins are cargoes of the early secretory pathway where they are exported from the endoplasmic reticulum (ER), modified in the Golgi then packaged into ZGs. Our goal is to understand the molecular mechanisms for ER to Golgi transport of ZG proteins.
Methods: A recombinant adenovirus was created expressing human chymotrypsin C (CTRC) fused with mCherry (a red fluorescent protein) at its C-terminal. The CTRC-mCherry fusion protein is a reporter to monitor ER to ZG transport. An adenovirus expressing mCherry alone was also created as a control. Rat pancreatic AR42J cells and isolated rat pancreatic acini were used to image CTRC-mCherry.
Results: Using a Leica laser scanning confocal microscope, pancreatic acinar cells or AR42J cells were imaged 16 hours after viral infection. While the mCherry adenovirus infected cells showed red fluorescence evenly distributed in the cytoplasmic region, those expressing CTRC-mCherry had punctuate red fluorescence in the ZG region consistent with its targeting to ZGs as a soluble content protein. To test the CTRC-mCherry fusion protein for studying ZG biogenesis, the acinar cells or AR42J cells expressing CTRC-mCherry were treated with brefeldin A (BFA) at 5 μg/ml which is known to inhibit protein transport from ER to Golgi. Dramatically different from the punctuate ZG localization in control acinar cells, CTRC-mCherry in BFA-treated cells showed a homogenous distribution in the basolateral region (excluding the nucleus) consistent with its being blocked in the ER. Currently, small GTPases such as Rab1 and Sar1 are being investigated for their roles in transport of ZG proteins in the early secretory pathway. In addition, CTRC-mCherry also showed punctuate secretory granule localization in MIN 6 cells.
Conclusions: Adenovirus mediated CTRC-mCherry expression can target to ZGs therefore is used to study ZG protein transport in the secretory pathway.
Down-Regulation of the CCK-B Receptor in Pancreatic Cancer Cells Promotes Apoptosis
K.K. Fino,1 G.L. Matters,2 C.O. McGovern,1 J.P. Smith,1Departments of Medicine1, Biochemistry and Molecular Biology2 Pennsylvania State University, Hershey, PA
Background: The gastrointestinal peptide gastrin has been identified as a growth factor in pancreatic cancer. Gastrin stimulates growth of pancreatic cancer through the cholecystokinin B (CCK-B) receptor.
Hypothesis: It is hypothesized that down-regulation of the CCK-BR, by small interfering RNAs (siRNAs) will inhibit proliferation of pancreatic cancer and promote apoptosis.
Methods: A series of human pancreatic cancer cells were screened by Real-Time RT-PCR for the presence and quantity of CCK-BR mRNA. Cells were transfected with siRNA specific to the CCK-BR or scrambled control. The effect of CCK-BR knock-down on proliferation was evaluated by cell counts, BrdU labeling, and cell cycle analysis by flow cytometry. Apoptosis was assessed by a caspase-3 activity assay and by XIAP protein expression by Western blot. Akt phosphorylation was also examined by Western blot.
Results: CCK-BR mRNA was detected in several human pancreatic cancer cell lines (PANC-1, MIA PaCa-2, AsPC-1, BxPC-3, and Capan-1) and expression was down-regulated by CCK-BR targeted siRNA. Cells with down-regulation of the CCK-BR displayed a significant decrease in proliferation by cell counting (75%) and BrdU (41%) labeling compared to scrambled control. G to S phase progression was inhibited and caspase-3 activity was increased by 80% in cells with CCK-BR down-regulation compared to scrambled control. Akt phosphorylation and XIAP expression was attenuated by CCK-BR down-regulation.
Conclusion: Down-regulation of the CCK-BR significantly inhibits growth, increases apoptosis, and decreases phosphorylation of Akt. These findings support the CCK-BR as a prime target for therapeutic intervention.
Assessment of Tissue Lysosomal Membrane Permeability in Acute Pancreatitis
F. Fortunato, H. Gu, M.W. Büchler, J. Werner Department of Surgery, University Clinic Heidelberg, Germany
Lysosomal membrane permeability (LMP) has been described as a condition by which the lysosomal membrane integrity becomes disrupted, leading to the release of lysosomal enzymes into the cytosol. LMP participates in the induction of cell death in numerous diseases, including hepatitis and pancreatitis. Cathepsin B activation appears to be involved in premature activation of trypsinogen, acinar cell death and pancreatitis.
Methods: Rats were fed a liquid diet containing either ethanol or control diet for 14 weeks, followed by one intravenous injection of LPS. The animals were sedated at 3 or 24 hours after LPS injection. The pancreata were extracted and FFPE tissue slides were prepared. Pancreatic tissue sections were stained for cathepsin B and immunofluorescence signals were evaluated by their whole expression profile and by their content of lysosomal vesicles, using TissueGnostics IF Microscope system.
Results: Cathepsin B expression profile did not significantly change among the treatment groups in an early phase of acute pancreatitis. However, puncta (vesicle) assessment, intensity of cathepsin B containing lysosomes, revealed a significant loss of the numbers of cathepsin B containing lysosomes, which suggested that cathepsin B is released into the cytosol due to the disrupted lysosomal membrane.
Conclusion: Our data suggest that premature trypsinogen activation is associated with LMP and cathepsin B release in the early onset of acute pancreatitis.
Pancreatic Stellate Cells Promote Migration of CD105+ Pancreatic Cancer Cells
K. Fujiwara,1 K. Ohuchida,1,2 S. Kozono,1 N. Ikenaga,1 L. Cui,1 M. Nakamura,1 K. Mizumoto,1,3 M. Tanaka,11Departments of Surgery and Oncology; 2Advanced Medical Initiatives, Graduate School of Medical Sciences, Kyushu University, Fukuoka, Japan; 3Kyushu University Hospital Cancer Center, Fukuoka, Japan
Introduction: CD105, endoglin, has been reported to be a component of TGF-β receptor. Its relation with the prognosis is pointed out in some kinds of cancer. However, the biological functions of CD105 in pancreatic cancer are unknown. Pancreatic cancer is characterized by abundant desmoplasia, and the interaction of cancer cells and pancreatic stellate cells (PSCs) plays an important role in the progress of pancreatic cancer. In the present study, we investigated the function of CD105 in pancreatic cancer cells and PSCs.
Methods: CD105 expression was assessed by quantitative real-time RT-PCR and flow cytometry, in pancreatic cancer cell lines and primary cultures of PSCs derived from pancreatic cancer tissues. Next, we analyzed the functional differences in proliferation, migration, and invasion between CD105+ and CD105- pancreatic cancer cells. In particular, we evaluated the relationship between cancer cells and PSCs.
Results: By flow cytometry, the frequency of CD105 in pancreatic cancer cells is various (0-99.2%), whereas most PSCs expressed CD105 (82.4-99.4%). We isolated CD105+ and CD105- subpopulations from pancreatic cancer cells. In vitro analyses revealed that the two subpopulations showed similar biological behaviors in their proliferation and invasion. By cocultures with PSCs, migration of CD105+ cancer cells was enhanced more than that of CD105- cells (P<0.05).
Conclusions These data suggest that CD105+ pancreatic cancer cells may be function-specific subpopulation, which is related with PSCs.
Human Pancreatic Stellate Cells as an in vitro Model for the Study of Bone Morphogenetic Proteins' Anti-Fibrogenic Role in the Pancreas
X. Gao,1 Y. Cao,1 C. Rostellini,2 C. Chao,2 M. Hellmich,2 T.C. Ko,1,21Dept. of Surgery, UTHSC-Houston; 2UTMB at Galveston, TX
Introduction: Pancreatic stellate cells (PSCs) activation is a key step in the development of pancreatic fibrosis. Transforming growth factor (TGF)-β is a key fibrogenic cytokine that activates PSCs. We have previously reported that bone morphogenetic protein (BMP) 2, a member of TGF-β superfamily, is expressed in the pancreas and inhibits TGF-β-induced activation of mouse PSCs, suggesting that BMP is an important anti-fibrotic pathway in the pancreas. However, whether BMP has similar effects on human PSCs are unknown.
Hypothesis: BMP antagonizes TGF-β activation of human PSCs.
Methods: Primary human PSCs were isolated from human pancreas specimen by outgrowth method. The cells were characterized by immunofluorescence with glial fibrillary acidic protein (GFAP, marker of PSCs), vimentin (marker of mesenchymal cells) and α-smooth muscle actin (α-SMA, indicator of activated PSCs). The cells were pretreated with BMP2 (250ng/ml) for 30 min followed by TGF-β (1ng/ml) for 48 h for the detection of α-SMA and fibronectin (an extracellular matrix protein) by immunofluorescence.
Results: Isolated human pancreatic cells were GFAP and vimentin positive, suggesting that these cells are PSCs. The majority of the cells (61.8%) were α-SMA positive and already activated. Compared to the vehicle control, TGF-β alone increased α-SMA by 5.0±0.8 folds and fibronectin by 1.7±0.2 folds; BMP2 pretreatment inhibited TGF-β-induced a-SMA by 34% and fibronectin by 65% (p<0.05).
Conclusions: BMP antagonizes TGF-β activation of human PSCs, which is similar to what we have previously shown in mouse PSCs. These results suggest that BMP is an important anti-fibrogenic pathway in the pancreas.
Combination of Alcohol and Smoking induce ER-Stress and Cell Death in Pancreatic Acinar Cells
A. Gerloff, A. Lugea, S. Pandol Pancreatic Research Group, Department of Veterans Affairs and University of California Los Angeles, Los Angeles, USA
Background: Recent epidemiologic studies demonstrate that smoking accelerates the development of pancreatitis in alcoholic patients. A recent study has shown that chronic alcohol feeding induces endoplasmatic reticulum (ER)-stress and activates an unfolded protein response (UPR) but the effect in combination with cigarette smoke is unkown.
Aim: To study the combined effect of ethanol and cigarette smoke on ER-stress and the unfolded protein response on pancreatic acinar cells.
Methods: Rat pancreatic AR4-2J cells were treated with ethanol (50 mM), cigarette smoke extract (CSE; up to 40 ug/ml; Murty Pharmaceuticals Lexington, KY) or the combination of both. After stimulation every 24h for up to 96h, cell viability was assessed by MTT assay and apoptosis determined by ELISA DNA fragmentation assay. Markers of ER-stress such as phosphorylation of PERK, eIF2α and expression of CHOP as well as caspase 3 and phosphorylation of AMPK were measured by Western blot analysis.
Results: With the conditions used, neither ethanol nor CSE alone had a significant effect on cell viability, apoptosis or parameters of ER-stress. However, combination of both treatments significantly decreased cell viability and markedly increased apoptosis. In addition, the combined treatment resulted in initiation of deleterious ER-stress signals including phosphorylation of eIF2α and expression of CHOP. The co-treatment also significantly increased the activation of the metabolic regulator AMPK, which has been shown to be related to ER-stress.
Conclusion: Our data demonstrate that the combination of ethanol and CSE have synergistic effects on pathologic ER-stress responses leading to cell death in this model of acinar cells. Studies into mechanisms of ER stress caused by the combination of smoking compounds and alcohol will provide insights into the pathology of this form of pancreatitis.
Role of MUC4 Glycans in Pancreatic Cancer Pathogenesis
V. S. Gnanapragassam,1 M. Jain,1 S. Kaur,1 S. Rachagani,1 S K. Batra,11Department of Biochemistry and Molecular Biology, University of Nebraska Medical Center, Omaha, NE, U.S.A
Aim: To investigate the expression of MUC4 associated glycan epitopes and corresponding glycogenes encoding glycosyl transferases in pancreatic cancer (PC).
Background: MUC4 is a heavily O-glycosylated and sparsely N-glycosylated mucin, differentially expressed in PC tissues, compared to the the normal pancreas. The role of MUC4 associated glycans in PC pathogenesis has not yet been investigated. We hypothesized that MUC4 associated glycans and corresponding glycosyl transferase enzymes are differentially expressed in PC cells.
Methods: Expression of specific glycans and glycogenes was analyzed in a panel of MUC4 (+) and (-) PC cell lines by a real time PCR array using HPNE cells as reference. Correlation of MUC4 with specific glycan epitopes was examined by reciprocal co-immunoprecipitation (IP).
Results: The expression of the glycosyl transferases GALNT3, GALNT7, GCNT3 and FUT8 was significantly increased in the MUC4 (+) cells. Further, GALNT3 and GCNT3 were undetectable in MUC4 (-) cells. Reciprocal co-IP revealed that MUC4 is associated with STn, SLeA, SLec and SLex epitopes in PC cell lines. These epitopes were absent in the MUC4 expressing normal colon tissue.
Discussion: GALNT3 and 7 are involved in the initiation of O-glycosylation, while GCNT3 is a Core2 glycan generating glycogene expressed abundantly and exclusively in MUC4 expressing cell lines. PC MUC4 is associated with the sialylated Lewis x glycan epitope while the normal colon is devoid of this glycotope. SLex is the major ligand for the E-selectins, a key receptor involved in the cancer metastasis. This indicates that altered pathological function of MUC4 may be driven by the aberrant expression of MUC4 glycans.
Conclusion: MUC4 associated glycans and respective glycogenes are aberrantly expressed in PC cells The MUC4 associated glycotopes STn, SLeA and SLe could be used as potential diagnostic markers for PC. MUC4 associated SLex may modulate MUC4 driven PC metastasis.
L-Asparaginase Loaded Red Blood Cells (RBC) in Pancreatic Cancer: A Phase I Clinical Study
Y. Godfrin,1 D. Liens,1 T. Andre,2 A. Adenis,3 F. Joly,41Erytech Pharma, Lyon, France; 2Hôpital Pitié-Salpêtrière, Paris, France; 3Centre Oscar Lambret, Lille, France; 4Centre François Baclesse, Caen, France
Background: L-asparaginase (L-asp) efficacy is based on sensitivity of tumoral cells to asparagine deprivation due to a lack of asparagine synthethase (ASNS). However its use is hampered by significant toxicities. Tentative to use L-asp in solid tumor, and especially in pancreatic cancer is reported in the literature but all attempts so far have failed due to toxicity. Deficiency in ASNS and therefore potential sensitivity to L-asp treatment has been reported in multiple tumor cell lines including pancreatic cancer.
Objectives: Thanks to the good safety profile of L-asparaginase loaded RBC (GRASPA®) in leukemic patients, the objective of this phase I clinical study (GRASPANC) was to confirm the safety profile of this cell-based formulation in patients with pancreatic carcinoma.
Methods: Primary objective was to find the Dose Limiting Toxicity (DLT) of GRASPA® in patients with non resectable, relapsed pancreatic cancer. Patients are treated successively by cohort of 3 with one injection of 25, 50, 100 or150 IU/kg of GRASPA®. An independent Safety Monitoring Board assessed toxicities at the end of follow-up of each cohort to decide for next dose. Twelve patients were included between Dec 2009 and Feb 2011.
Results/Conclusion: The mean age was 60.4 years (min: 42.6; max: 71.9) with 9 Male and 3 female. No limiting toxicity has been found up to the GRASPA® highest dose (150 IU/kg) planned in the study and the DLT was not reached. Preliminary conclusion is that the dose of 150 IU/kg appears suitable for further clinical development in patients with pancreatic carcinoma especially if they are screened for none or low expression of ASNS in the tumor cells.
TGF-β-Mediated Growth Inhibition of Pancreatic Cancer Cells Depends on RB Function
A.J. Gore, M. Korc Departments of Medicine, and Pharmacology and Toxicology, Dartmouth Medical School, Hanover, NH
Pancreatic ductal adenocarcinoma (PDAC) is a deadly malignancy. PDAC is thought to arise from precursor lesions, termed pancreatic intraepithelial neoplasia (PanIN), that progress from low to high grade. PDAC's aggressiveness derives from highly frequent KRAS, INK4A, TP53 and SMAD4 mutations, and mouse models with these alterations mimic the human disease. To study RB, which is functionally inactivated in PDAC, we generated a novel model with pancreatic RB deletion and oncogenic Kras expression. These mice rapidly developed highly proliferative low and high grade PanIN that progress to PDAC with high frequency, underscoring RB's tumor suppressive role in the pancreas. Mucinous cystic neoplasia were also common, as in another Kras-driven PDAC model lacking Smad4, a key TGF-β effector. Thus, our model's dramatic phenotype may be due to TGF-β dysfunction. To examine RB's role in TGF- β action, we established pancreatic cancer cell lines from our model. These cells were resistant to TGF-βı-mediated growth inhibition, whereas growth of murine cell lines that retained RB were markedly inhibited. RB loss did not alter TGF-βı signaling since TGF-βı - induced Smad2 phosphorylation, Smad3/4 nuclear translocation, and transactivation of TGF-βı-dependent reporters. Moreover, RB loss did not affect TGF-βı-induced epithelial-to-mesenchymal transition. These data suggest that RB loss specifically disrupts TGF- β's growth inhibitory functions while sparing its tumor promoting actions. In conjunction with our previous findings that Smad7 is overexpressed in PDAC and functionally inactivates RB, our data suggest that restoring RB function may represent a novel therapeutic approach in PDAC.
Pan-Negative Bone Marrow-Derived Cells Dramatically Worsen Pancreatitis via an ICAM-1 Dependent Mechanism
C. S. Graffeo,1 M. Hackman,1 A. Rehman,1 S. Rahim,1 A. Nguyen,1 M. Medina-Zea,1 H. Lebovics,1 M. Dorvil-Castro,1 M. Marr,1 J. Henning,2 G. Miller,1,2Departments of 2Surgery and 1Cell Biology, New York University School of Medicine, New York, New York
Background: Chronic pancreatitis (CP) is characterized by progressive fibro-inflammatory organ injury leading to exocrine and endocrine dysfunction. As bone marrow-derived cells (BMC) are known to be associated with inflammatory tissue injury, our goal was to investigate the role of BMC in CP. We postulated that BMC exacerbate pancreatic inflammation and fibrosis.
Methods: We FACS sorted BMC into three phenotypic subpopulations: hematopoetic progenitor cells (HPC: Lin-CD34+), hematopoetic stem cells (HSC: Lin-CD34-c-Kit+Sca+), and pan-negative cells (PN: Lin-CD34-c-Kit-Sca-). Each subpopulation was adoptively transferred (1×106 cells i.p. injection, thrice weekly) to mice undergoing caerulein pancreatitis (seven consecutive hourly i.p. injections [50μg/kg] thrice weekly for three weeks).
Results: BMC migration to the pancreas was increased in CP. HSC and HPC were noted to mildly exacerbate CP, while PN were associated with severe disease exacerbation as indicated by marked edema, advanced fibrosis, and pronounced inflammation. Further characterization of PN cells revealed that they acquired granulocyte-like characteristics in vitro and in vivo. PN cells required expression of ICAM-1 to affect pancreatic injury. Furthermore, effects were CD4+ and CD8+ T cell dependent.
Conclusions: PN cells dramatically worsen CP via an ICAM-1 dependant mechanism. As such, PN cells may make an attractive target for experimental therapeutics in CP.
Medium to Late Clinical and Social Results of Surgery for Chronic Pancreatitis (CP)
E.R. Grist, N. Kumar, V. Shah, M. Habib, N.W. Pearce, M. Abu-Hilal, C.D. Johnson University of Southampton, Southampton General Hospital, Southhampton, UK
CP is difficult to treat, many patients have long term pain and poor social function. Because success is not certain, some physicians are reluctant to offer surgery. Surgery can reduce pain in 60-80% in the short term, but longer term data are sparse.
We aimed to document pain, quality of life (QL) and social function in patients who had surgery for CP in a single centre over a 20 year period.
Methods: 75 patients who had surgery for CP were identified from a department register; 62 records were traced; 33 responded for telephone interview and completion of EORTC QLQ-C30 and PAN26. Aetiology was alcohol in 50%, unknown in 20% and due to stricture from acute pancreatitis or trauma in 15%.
Results: Indications for surgery were pain (48), pain and jaundice (9), suspicion of malignancy (12). Most operations were resections: pancreaticoduodenectomy 10, Beger/Frey 26, left 19 total 1. There were no postoperative deaths, median hospital stay was 10 days. 54 attended followup for at least 6 months and 27 for 2 years. 9 (145) died and 13 developed new diabetes. At final clinic followup, 38(66%) reported no or less pain; 17 (27%) were using opioids.
Of 33 patients interviewed, 16 (48%) were employed. 10 (30%) used opioids daily and 4 (12%) did so on 1-4 days per month. Overall QL was lower than a reference population (median 67%) but function scales (physical, role, emotion, cognitive and social) had median scores 80-100% indicating that many patients had good function. Satisfaction with health care was high (mean 88, median 100%).
Conclusion: Medium to longterm outcome after surgery for CP is good, with continued pain relief in 60-70% of patients. Half our sample were employed, and many patients had good QL. Resection, based on anatomic distribution of the disease, is appropriate treatment for painful CP.
In Severe Acute Pancreatitis (SAP) Without Chronic Kidney Disease (CKD), Increasing Cumulative 24h IV Fluids (IVF) Correlates with Falling BUN, Less Organ Failure and Complications (POF/LC) and Shorter Hospitalization (LOS)
S. Gupta, J. Spaete, N. Ahuja, R. Rizk, L. Napolitano, R. Hyzy, J. Desmond, E.J. Wamsteker, M.J. DiMagno Med, Surg, Emerg. U of Michigan, Ann Arbor
Background/aim: IVF resuscitation guidelines for AP recommend ≥6L in the 1st 24h, but supportive data are lacking. Our aims were to quantitate and determine if 24h IVF volumes correlate with normal or declining BUN (Ranson AJG 1982; Wu 2009), SAP (Atlanta), POF/LC, and LOS.
Methods: In an ongoing study we sequentially reviewed 254 records of persons who received an ICD-9 AP code (577.0) in the U of Michigan ER or Inpatient Unit between 5/30/06-12/31/06. 191 patients were excluded (transfers, chronic pancreatitis, pancreatic cancer, prior attack of AP, age≤18). We recorded diagnosis, comorbidities, predicted-SAP (SIRS, organ failure, APACHE II≥8), SAP, cumulative 24h IVF and measures of resuscitation.
Results: 63 patients had AP, SAP was predicted in 42 (66.7%) and 34 of these developed SAP, 5 had POF/LC and 1 (with SAP and POF/LC) died. 24h IVF (2.7+/-1.4L) was similar among presence or absence of predicted-SAP, SAP and POF/LC groups. In mild AP and SAP groups without CKD only, BUN declined significantly if ≥2.75L (P=0.039; R=0.49) and ≥3.25L (P=0.023; R=0.48), respectively were delivered within 24h. No one with a BUN decline of ≥4mg/dl developed POF/LC (P=0.044). Only in SAP without CKD did delivery of IVF within 24h (≥3.1L; P=0.04; R=0.38) and falling BUN (-2.25mg/dl; P=0.049, R=0.41) correlate with shorter LOS.
Conclusion: Patients with predicted SAP and mild AP received similar IVF volumes during the 1st 24h of hospitalization. If patients had SAP and CKD there was no correlation among 24h IVF, BUN and LOS. In SAP and no CKD, however, administering ≥3.25L in the 1st 24h results in falling BUN and shorter LOS.
The Intracellular Side of Pancreatic Cancer-Stellate Cell Interaction
H. Habisch, S. Zhou, M. Siech, T. Seufferlein, M. Bachem Department of Clinical Chemistry, University Hospital Ulm, Germany
One major hallmark of pancreatic cancer is the strong desmoplastic reaction resulting from the interaction of cancer (PCC) and stellate cells (PSC). This interaction promotes tumor growth, chemoresistance, extracellular matrix (ECM) turnover, and influences tumor-associated angiogenesis. The aim of our present study was to unravel the intracellular signal transduction pathways involved in PCC-PSC interaction.
Screening of multiple phosphoproteins followed by principal component analysis revealed the activation of the transcription factor CREB as well as Akt, Rsk, S6K, FAK and others in PSC upon stimulation with PCC-conditioned medium. Inhibition of focal adhesion kinase inhibits PSC cell motility (measured by single cell tracking) by 39±4%. Inhibition of the cAMP pathway by H-89 also revealed a concentration-dependent inhibition of cell motility by 32±2% (with serum) and 48±4% (w/o serum) and a change from the usual activated myofibroblast-like to a spider-shaped morphology with a small cell body and 3-5 filopodia. Cell death (measured by propidium iodide staining) increased by H-89 treatment to 41% after 24 h. Hence, continuous activation of CREB is necessary for the maintenance of the activated PSC phenotype and cell survival. Based on data from literature we are currently investigating the role of CREB in constitutive and PCC-induced expression of collagen I, fibronectin, and matrix metalloproteinase 2 (MMP2). In the context of PCC-PSC interaction, PSC are the main source of MMP2, but, as revealed by real-time PCR, zymography, and multipex immunoassays, various PCC lines (7 tested) also express variable amounts of MMP1, -2, -3, and -9. Importantly, mutual activation of various MMPs can aggravate local extracellular matrix degradation and facilitate cell spreading/metastasis.
Redo-Operations in Chronic Pancreatitis: Indications, Procedures and Outcome
Th. Hackert, Ch. Tjaden, S. Lutz, L. Schneider, W. Hartwig, O. Strobel, M.W. Büchler, J. Werner Department of Surgery, University of Heidelberg, Germany
Background: Surgery in chronic pancreatitis (CP) includes draining and resecting procedures. Besides chronic pain as the main indication, cholestasis and suspicion of malignancy are important indications for operations in CP. In a small number of CP patients a second operation is required due to recurrent CP symptoms. Aim of the study was to determine frequency, indications, surgical procedures and outcome of redo-operations in CP patients.
Methods: During an observation period of 8 years, data of all CP patients undergoing surgery were analyzed with regard to recurrent operations. Indications and surgical procedures for the first and second operation were analyzed as well as operative parameters, complications and outcome.
Results: Throughout the observation period 665 operations were performed in CP patients. In 34 patients (5.1%, 22m, 12f; median age 47 years) a second operation was performed. Indications for a redo-operation were recurrent pain (14/34 pat.), symptomatic pseudocysts (6/34 pat.) and obstruction of the pancreatic or bile duct (13/34 pat.) as well as suspected malignancy (1/34 pat.). After preceding duodenum-preserving pancreatic head resection (DPHHR, n=17) 13 patients underwent a Whipple operation, 3 patients received a hepatico-jejunostomy and one underwent a redo-operation of the pancreatic anastomosis. In 5 patients DPPHR or Whipple operations were performed after initial ampullectomies, in all other patients resections were performed after preceding drainage procedures or vice versa (n=12). Median time interval between both operations was 3.1 years. There were no perioperative deaths, overall morbidity was 26.5%.
Conclusion: Redo-operations in CP patients are rare with an overall frequency of app. 5%. Indications for a second surgical intervention mainly include recurrent episodes of pain and cholestasis. In most of the patients with an initially limited draining or resective procedure, a more extensive resection is required during the second operation. This can be performed with low morbidity and mortality and results in good long-term outcome.
Nestin Modulates EMT Molecules and Transcriptional Factors in Human Pancreatic Cancer Cells
M. Hagio, Y. Matsuda, T. Suzuki, Z. Naito, T. Ishiwata Departments of Pathology and Integrative Oncological Pathology, Nippon Medical School, Tokyo, Japan
Aim: Nestin, a class VI intermediate filament, expresses in pancreatic exocrine progenitor cells. We previously reported that nestin expression was detected in approximately 30% of pancreatic ductal adenocarcinoma (PDAC) cases, and that nestin promotes migration, invasion and metastasis in PDAC cells. In this study, we investigate the effect of nestin modulation on intracellular genetic change.
Methods: To clarify the effect of nestin, two PDAC cell lines that were transduced with overexpression or knockdown of nestin were used in these experiments. Gene expressions of cytoskeletal and epithelial-mesenchymal transition (EMT) factors were analyzed using real-time RT-PCR. Morphological changes were observed by confocal microscopy. For comprehensive analysis of specific cell signaling pathways, a DNA microarray was established.
Results: The expression of E-cadherin increased I the nestin-overexpressed PDAC cells, while nestin suppression by siRNA transfection induced a decrease of E-cadherin. Morphological change in agreement with mRNA alteration of cytoskeletal genes was confirmed. Among specific factors related to EMT, Snail gene expression was altered in parallel with changes in nestin expression. However, there was no association between nestin and other EMT markers, including Slug and Twist. By DNA microarray analysis, transcription factor 4 (TCF4) was selected as a candidate gene that responded to quantitative alteration of nestin.
Conclusion: The alteration of nestin expression affected EMT molecules and transcription factors. These observations suggest that nestin is not merely a structural protein, but that it has the capacity to participate in the control of various cellular processes.
Improved Effect of Chemoradiotherapy in Combination with Oncolytic Adenovirus Expressing Interferon-α for Pancreatic Cancer
J. Han,1 L. Armstrong,1 E. Brown,1 K. Aoki,2 S. Vickers,1 M. Yamamoto,1 J. Davydova,11Department of Surgery, University of Minnesota, Minneapolis, MN; 2National Cancer Center Research Institute, Tokyo, Japan
Pancreatic cancer remains as one of the deadliest diseases without a cure. A promising multimodal therapy, interferon-α (IFN) having one feature as a chemoradiotherapy sensitizer emerged in recent clinical studies, yet excess toxicity and insufficient level of IFN in tumor site remain as serious challenges. The application of oncolytic adenovirus as a vehicle system enables local production of IFN at therapeutic concentration. We hypothesize that replication competent adenovirus expressing IFN (Ad-IFN) in combination with either chemotherapy (5-FU) or radiotherapy would significantly enhance anti-cancer effect of existing IFN-based regimens while reducing toxicity. First, we analyzed the cytocidal effect of the combination therapy in vitro in human (MiaPaCa2) and hamster (HP1) pancreatic cancer cells. The cell viability assay revealed that Ad-IFN in combination with 5-FU killed 59% of MiaPaCa2 compared to the 21% and 10% of Ad-IFN and 5-FU therapy alone, respectively. In HP1, we observed greater oncolysis when Ad-IFN was combined with either 5-FU or radiotherapy. Furthermore, we established a fully syngeneic pancreatic cancer model in immunocompetent hamsters. Remarkably, Ad-IFN with radiotherapy showed significant decrease in tumor volume. At day 31, the tumor nearly disappeared. Thus, our data validate the feasibility of the combination effect of Ad-IFN with radiotherapy and 5-FU. These results reinforce the impact of Ad-IFN to stimulate immune response against pancreatic cancer and sensitize anti-tumor effect of chemoradiotherapy. The oncolytic adenovirus-based IFN expression combined with chemoradiotherapy may change a paradigm of pancreatic cancer treatment.
Role for apelin in Pancreatic Fibrosis
S. Han,1 E.W. Englander,1 C. Rastellini,1 T.C. Ko,3 Y. Cao,3 G. Gomez,1 M.R. Hellmich,1 C. Chao,1 J.F. Aronson,2 T. Quertermous,4 R. Kundu,4 G.H. Greeley Jr1Departments of 1Surgery and 2Pathology, University of Texas Medical Branch, Galveston, TX; 3Department of Surgery, The University of Texas Medical School at Houston, Houston, TX; 4Department of Medicine - Cardiovascular Medicine, Stanford University School of Medicine, Stanford, CA
Pancreatic apelin expression increases during cerulein-induced acute pancreatitis (AP). The aim of this study was to examine apelin's role in pancreatic recovery following AP induction.
Methods: Experiment (Exp) 1: pancreatic collagen production was compared during AP induction in wild type (WT) and apelin gene knockout mice (APKO), N=6/group. AP was induced by 7 hourly cerulein injections (50 ug/kg, IP). Collagen-4 mRNA and protein expression were measured. Pancreatic fibrosis was examined histologically. Exp2: the effect of apelin treatment (10-6 M) on collagen-4 protein production was examined in mouse pancreatic stellate cells exposed to cerulein (10-8 M) or vehicle.
Results: Exp1: 48 hours after AP induction, pancreatic collagen-4 expression levels were elevated in WT and APKO mice (P<0.05). Collagen-4 mRNA (WT: 0.8±0.1 vs. APKO: 1.1±0.1) and protein levels (WT: 0.65±0.03 vs. APKO: 1.03±0.02) were higher in APKO mice. Histologically, fibrosis was enhanced in APKO mice. Exp2: cerulein treatment increased collagen-4 production in stellate cells. Apelin treatment reduced cerulein-induced collagen-4 protein production.
Discussion: Results indicate that apelin influences pancreatic repair since collagen production and fibrosis are enhanced during the repair stage of AP in APKO mice. Furthermore, in vitro data show apelin reduces stellate cell collagen formation. Results imply that apelin plays a role in regulation of fibrosis during pancreatitis.
Pathobiological Role of MUC16 (CA125) in Pancreatic Cancer
D. Haridas,1 S. Chakraborty,1 M.P. Ponnusamy,1 I. Lakshmanan,1 S. Rachgani,1 E. Cruz,1 S. Kumar,1 S.M. Lele,4 J.M. Anderson,2 U.A. Wittel,3 M.A. Hollingsworth,1,2,4 S.K. Batra,1,2,41Department of Biochemistry and Molecular Biology, 2Eppley Institute for Research in Cancer and Allied Diseases, 4Department of Pathology, University of Nebraska Medical Center, Omaha, NE; 3Department of General and Visceral Surgery, Universitätsklinik Freiburg, Germany
Background: MUC16 (CA125) is a high-molecular weight mucin glycoprotein. Several mucins including MUC1 and MUC4 are reported to be aberrantly expressed during the initiation and progression of pancreatic cancer (PC). Although MUC16 is a well known biomarker in ovarian cancer, its expression and functional role in PC remains unknown. We hypothesized that MUC16 plays a role in the initiation and/or progression of PC.
Methods: MUC16 expression was examined in commercial tissue microarrays comprising normal and PC cases, in matched primary and metastatic PC tissues and cell lines. MUC16 was stably silenced in PC cells and its effect assessed in vitro and in vivo.
Results: MUC16 is not expressed in the normal pancreatic ducts or acini; however, a strong expression is observed in PC tissues with increasing grades (p-value of 0.0006 for moderate and 8.9E-06 for poorly differentiated cancer when compared to normal pancreas). MUC16 expression was observed in the high-grade precursors of PC called pancreatic intraepithelial neoplasia (PanIN grade II and III with a p-value of 0.0001 and 0.001, respectively when compared to the normal pancreas). Further, its expression was strongly upregulated in metastatic sites compared to the primary tumor (data obtained from 34 PC patients). PC cells with stably silenced MUC16 exhibited significantly decreased proliferation, motility (in vitro), tumorigenecity and metastasis (following orthotopic implantation in athymic mice) compared to scrambled RNA transfected cells. In addition, MUC16 knockdown cells accumulated in the G2/M phase of the cell cycle indicating that MUC16 might play a role in cell cycle regulation.
Conclusion: MUC16 is differentially expressed and regulates PC proliferation and metastasis suggesting its potential as a novel diagnostic and therapeutic target in PC.
Management of Relapses After Initial Therapy of Autoimmune Pancreatitis
P.A. Hart, S.T. Chari, the AIP Interest Group Mayo Clinic Rochester, MN
Background: Though autoimmune pancreatitis (AIP) responds dramatically to steroids, it frequently relapses on steroid withdrawal. Since AIP is rare there is a paucity of natural history data to guide long-term treatment decisions. We describe our experience with the management of relapses after initial therapy of AIP.
Methods: We reviewed our database of 103 AIP patients treated at our center who had pancreatic involvement, with or without biliary involvement. Relapse was defined as the recurrence of symptoms and/or abnormal liver biochemistries accompanied, when studied, by radiologic relapse. We compared the likelihood of developing a relapse and time to relapse among the initial treatment strategies.
Results: Initial treatment was with steroids (n=67), surgical resection (n=27) or no treatment (n=6); follow-up was incomplete in 3 patients. Mean followup was 42.2 months. Patients undergoing surgical resection were less likely to relapse than those treated with steroids (19% vs. 57%, p<0.01). Of those initially treated with steroids 38/67 (57%) had at least one relapse a mean 6.6 months from diagnosis. Initial relapses were managed with either steroids + an immunomodulator (n=20) or a second course of steroids (n=18). A second relapse was as common in those treated with immunomodulator as those treated with prednisone alone (35% vs. 39%, p=0.80), with similar relapse-free survival (11.3 vs. 8.7 months, p=0.36). Four patients relapsed while on immunomodulator treatment. Immunomodulators were discontinued in 6 patients with inactive disease for at least 12 months and they remain relapse free.
Conclusions: Relapses are common after initial treatment of AIP with steroids and after retreatment of initial relapse with steroids or steroids + immunomodulators. Role of immunomodulators in maintaining remission needs further study.
Reduction of Clinical Relevant Pancreatic Fistula Rate by Additional Coverage of the Pancreatic Remnant After Distal Pancreatectomy
M. Hassenpflug, U. Hinz, T. Hackert, W. Hartwig, O. Strobel, M.W. Büchler, J. Werner Department of General Surgery, University of Heidelberg, Germany
Introduction: Pancreatic fistula after distal pancreatectomy (DP) is a major source of morbidity and occurs in up to 60%. Several surgical techniques to decrease pancreatic fistula are used at present. However, the best management of the stump closure remains controversial.
Objectives: We prospectively evaluated whether the coverage of the resection margin after DP by autologeous serous tissue reduces the pancreatic fistula rate, morbidity, mortality, and hospital length of stay.
Patients and Methods: From 05/2009 to 09/2010, 117 consecutive Patients underwent a DP at the University hospital in Heidelberg, Germany. In 73 cases, a coverage was performed (falciforme patch. n=64; small bowel loop n=7; stomach n=2). In the remaining 44 patients the pancreatic remnant was closed by stapler or sutures only (standard control group). All patients were prospectively recorded and their clinical course was observed focusing on the occurrence of a pancreatic fistula that was graded with respect to the ISGPF-definition.
Results: Although overall fistula rate was not different between the groups, B and C fistula rate was decreased significantly in patients with coverage compared to the standard controls (Type B:7% vs. 9%; Type C: 7% vs 25%; p<0,002). The mortality in the coverage-group was 0% (vs. 4,5% in the controls; n.s.). In addition, the total hospital length of stay of patients with a pancreatic fistula was significant shorter when a coverage had been performed (p<0,02).
Conclusion: Coverage of the pancreatic remnant following DP decreases the rate of clinical relevant pancreatic fistulas and the hospital length of stay.
CA19-9 as a Predictor of Resectability in Patients with Pancreatic Cancer
Z. He, H. Lu, W. Hu, B. Tian, Z. Zhang Department of Hepato-bilio-pancreatic Surgery, West China Hospital, Sichuan University, Chengdu 610041, Sichuan Province, China
Objectives: CA19-9 is considered to be a useful marker in assisting with an initial diagnosis, and measuring the effectiveness of cancer treatment by studying the patient's CA19-9 level over time. The aim of the present study was to evaluate whether CA19-9 level related to the surgical resection.
Methods: Retrospectively, Logistic multivariate regression analysis was used to analyze data from 207 patients who underwent surgery at West China Hospital, during a 5-year period, and performed to identify CA19-9 levels contributing significantly to surgical resection. Patient Inoperable were excluded.
Results: Carbohydrate antigen 19-9(CA19-9)>150U/ml was proved to be of the essence risk factor for surgical resection. Patients with Carbohydrate antigen 19-9(CA19-9)>150U/ml had a frequency of surgical resection 35.8% vs. 11.9% in those patients with a lower level of CA19-9 (P<0.001). Using multivariate analysis adjusted the effects of other factors, high level of CA19-9 is considered to be an important risk factor for influencing surgical resection.
Conclusion: Obviously, CA19-9 should be a good predictor of surgical resection possibility in patients with pancreatic cancer. Furthermore, it is useful in diagnosis and measuring the effectiveness of cancer treatment.
Pancreaticocolonic Fistulae - Frequency, Presentation, and Management
Z. Heeter, E. Hauptmann, M. Gluck Virginia Mason Medical Center, Seattle, WA
Background: Pancreaticocolonic fistulae (PCF) are reported complications of severe acute pancreatitis (SAP) that are frequently managed surgically although radiological techniques have been described.
Objective: This IRB approved study describes the frequency, location, clinical presentation, and management of PCF's at a tertiary pancreatic referral center over 11 years.
Methods: Patients with SAP and PCF between January 2000 and March 2011 were identified by interrogating hospital databases for ICD-9 and CPT codes. Charts and imaging were queried for PCF course details.
Results: During the study period, 5,496 patients were admitted with acute pancreatitis. PCF was diagnosed in 18 (9 male, mean age 62). SAP etiology in 9 was choledocholitiasis, 4 idiopathic, 2 alcohol-induced, 2 hypertriglyceridemic, and 1 post-ERCP. Presenting symptoms were often nonspecific and included abdominal pain, diarrhea, and hematochezia. A diagnosis was made by fluoroscopic percutaneous drain study (13), ERCP (1), colonoscopy (1), contrast enema (1), or computed tomography (2). Percutaneous drains were present in 14 before PCF diagnosis. Median time from SAP diagnosis to PCF discovery was 79 days (range 13-395). Treatment included percutaneous catheter drainage and antibiotics in all, endoscopically-placed transpapillary pancreatic duct stents (10), endoscopically-placed internal stents through the stomach (3), and surgery (4).
Conclusions: PCF is an uncommon complication of severe acute pancreatitis often found incidentally. The majority of PCF's resolve with non-operative management including antibiotics, percutaneous drainage, and/or endoscopic internal stenting and heal without adverse consequence. Percutaneous drainage catheters placed in areas of organized pancreatic necrosis can resolve most PCF's but could also be an etiological agent in creating them.
Effects of Toxic Factors on the Expression of Aquaporins in CAPAN-1 Cells
P. Hegyi,1 L.V. Kemény,1 Z. Rakonczay Jr.,1 Á. Zvara,2 L. Puskás,2 V. Venglovecz,31First Dept. of Medicine, University of Szeged, 2Laboratory of Functional Genomics, Biological Research Centre, 3Dept of Pharmacology and Pharmacotherapy, University of Szeged, Szeged, Hungary
Background: Toxic agents inducing acute pancreatitis (AP) inhibit pancreatic ductal fluid secretion. Impaired fluid secretion by ductal cells may play an important role in the development of AP; however, the mechanism is not completely understood. Since aquaporins (AQPs) play critical roles in controlling water transport in epithelial cells, our aim in this study was to investigate the effects of bile acids, ethanol (EtOH) and tumor necrosis factor-α (TNF-α) on the expression of AQPs.
Methods: Human pancreatic cell line of ductal origin (CAPAN-1) was treated with EtOH (1, 10 and 100 mM), chenodeoxycholate (CDC; 0.1, 0.3 and 0.5 mM), glycochenodeoxycholate (GCDC; 0.1, 0.3 and 0.5 mM) or TNF-α (0.2, 20 ng/ml) for 6, 12, 24 and 48 hours and the expression of AQP isoforms (AQP1-12) was analyzed by real-time RT-PCR and immunocytochemistry (ICC).
Results: Messenger RNAs for AQP1-12 were detected in CAPAN-1 cells. AQP1, -3, -5, -6 and -11 were expressed at the highest levels while AQP2 and -4 were hardly detectable. ICC showed positive immunoreactivity against AQP1, -3 and -5. CDC and GCDC dose- and time-dependently decreased the expression of AQPs. 24 hours treatment with EtOH increased the expression of AQPs. Notably, the upregulatory effect of EtOH completely abolished or turned into downregulation after 48 hours. TNF-α did not significantly affect the expression of AQPs.
Conclusion: Conjugated and unconjugated bile acids downregulated the expression of AQPs in CAPAN-1 cells, which may play a key role in the impaired ductal fluid secretion and therefore in the pathogenesis of AP.
Supported by OTKA, NFÜ-TÁMOP and MTA.
Genetic Deletion of 5-Lipoxygenase Attenuates the Quantity of Pancreatic Neoplastic Lesions in EL-Kras mice
M.J. Heiferman, A. Samiei, S.B. Krantz, J.D. Phillips, J.R. Heiferman, D.J. Bentrem, P.J. Grippo Department of Surgery, Feinberg School of Medicine, Northwestern University, Chicago, IL; Robert H. Lurie Comprehensive Cancer Center, Northwestern University, Chicago, IL
Introduction: The role of inflammation in cancer is a critical area of research in the effort to elucidate the mechanisms of carcinogenesis. 5-lipoxygenase (5-Lox) is an enzyme involved in acrachidonic acid metabolism to leukotrienes. Overexpression of 5-Lox has been demonstrated in a variety of cancers, including pancreatic cancer. In this study, we evaluate the effect of 5-Lox gene knockout in a well-established model of pancreatic neoplasia: EL-Kras.
Methods: EL-Kras mice were crossed with 5-Lox knockout mice, and offspring were genotyped by polymerase chain reaction. EL-Kras mice were placed into the following groups based on 5-Lox genotype: homozygous (-/-), heterozygous (+/-), and wildtype (+/+). At least 6 mice in each group, age 6 to 9 months, were evaluated for number and relative size of pancreatic lesions.
Results: Homozygous null mice were found to have significantly fewer lesions compared to the heterozygous and wildtype mice; both P<0.01. Lesion size was not significantly different between groups. Bromodeoxyuridine analysis showed a lower proliferative index in neoplastic lesions of homozygous null mice compared to heterozygous and wildtype mice.
Conclusions: The global deletion of 5-Lox in EL-Kras model resulted in a marked reduction in the quantity of pancreatic neoplastic lesions.
Discussion: Since the deletion of 5-Lox was acquired in all tissues and cells, it is unclear what compartment is most susceptible to this effect. Further studies are needed to evaluate the loss of 5-Lox in epithelial, stromal, or hematopoetic compartments in order to determine which target sustains this effect.
Acinar-specific Xbp1 Ablation Triggers Apoptosis and Coordinate Acinar/Centroacinar Regeneration in the Adult Murine Pancreas
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
Background: Stress response pathways regulate cell survival and growth and are ideal candidates for therapeutic manipulation in treating pancreatic disease. The IRE1/Xbp1 axis of the unfolded protein response (UPR) is a key component of the regulatory machinery required for the heavy secretory activity characteristic of acinar cells.
Methods: Acinar-specific, Cre-inducible Xbp1 ablation was used to investigate the UPR in acinar cells over a 12 week period. Pancreas morphology, protein expression, and cell viability were investigated via protein blot, immunohistochemistry, and electron microscopy. Gene expression analyses were conducted using RT-qPCR. Additional experiments utilized shRNA knockdown of Xbp1 in culture.
Results: Xbp1 recombination was confirmed in ∼90% of acinar cells and resulted in ER stress accompanied by autophagy and apoptosis approximately four weeks after ablation. Unrecombined acini, as well as a subset of Sox9 and Hes-1 positive centroacinar cells, generated a robust proliferative response, regenerating the damaged exocrine tissue. Pancreatic protein and gene expression returned to near-wildtype levels following regeneration despite the appearance of enlarged acinar cells and Sox9-positive tubular complexes.
Conclusions: Ablation of Xbp1 results in uncontrollable ER stress leading to eventual apoptosis, and this damage triggers an acinar/centroacinar regenerative response that restores the organ. Current investigations are focused on suppressing Xbp1 in the context of activated Kras expression as a means of controlling the viability of transformed cells.
Can MAPK Signaling be Inhibited in vivo to Study Pancreatic Function?
B.J. Holtz,1 J.A. Williams,1,3Departments of 1Molecular and Integrative Physiology and 3Internal Medicine, University of Michigan Ann Arbor, MI
Introduction: During adaptive pancreatic growth the hormone cholecystokinin (CCK) has been shown to activate multiple MAPK signaling pathways and stimulate early-response genes necessary for cell-cycle processes and growth. Feeding a trypsin inhibitor (TI) elevates plasma CCK and serves as a model for adaptive growth.
Aim: To evaluate the role of pancreatic MAPK signaling following endogenous CCK release using the Mek1/2 inhibitors U0126 and PD0325901 and the JNK inhibitor SP600125.
Methods: Male ICR mice were administered U0126 and SP600125 IP; PD0325901 was given by oral gavage. Inhibitors were administered 2h prior to TI refeeding of fasted animals. Isolated acini were prepared using collagenase digestion and stimulated by CCK following pharmacological inhibition. Multiple signaling pathways were analyzed by Western blot.
Results: Administration of U0126 (15mg/kg) failed to inhibit MAPK signaling following TI refeeding. This may be due to solubility problems or a short in vivo half-life. SP600125 (15mg/kg) was effective in inhibiting JNK signaling in vivo however, specificity was compromised as other pathways including mTOR and Erk1/2 also showed inhibition. A similar non-specific effect was seen in vitro using CCK stimulation. PD0325901 (20mg/kg), known to have a longer in vivo half-life, was successful in specifically inhibiting Erk1/2 signaling in vivo following TI feeding while mTOR, JNK, and p-STAT pathways were unaffected.
Conclusion: PD0325901, a Mek1/2 inhibitor currently in trials for pancreatic cancer is a specific and potent inhibitor of Erk1/2 signaling in vivo following CCK stimulation. It should be a useful tool for evaluating in vivo participation of ERKs in pancreatic function.
Activation of Small G-Protein Rab27B Plays an Inhibitory Role in the Secretion in Mouse Pancreatic Acini
Y. Hou, X. Chen, J.A. Williams Molecular and Integrative Physiology, University Of Michigan, Ann Arbor, MI
Background: Previous Studies Have Shown That The Small G-Protein Rab27B Is Present On Pancreatic Zymogen Granules And Participate In Digestive Enzyme Secretion. However, Its Role And Regulation Of Activity In Mouse Pancreatic Acini Is Poorly Understood. We Identified EPI64B, A Tre-2/Bub2/Cdc16 (TBC) Domain Containing Protein, As A Potential Gtpase-Activating Protein (GAP) For Rab27B.
Aim: To Investigate The Effect Of EPI64B And Different Rab27B Mutants On Rab27B Activation And Amylase Release.
Methods: Isolated Pancreatic Acini Were Prepared From Mouse Pancreas By Collagenase Digestion And Amylase Secretion Was Measured. Effects Of EPI64B Over-Expression On GTP-Bound State Of Rab27B And Rab3D, And The Interaction Between Rab27B And Its Effectors Were Assessed By GST-Pulldown Assay. Over-Expression Of Rab27B Mutant Proteins Were Mediated By Adenoviruses.
Results: Over-Expression Of Epitope Tagged-EPI64B In Mouse Pancreatic Acini By Adenoviral Delivery Decreased The GTP Binding Of Endogenous Rab27B, While The GTP-Bound State Of Rab3D Was Not Affected. EPI64B Over-Expression Enhanced The Basal Level Of Amylase Release And Elevated The Entire Dose Response Curve Of CCK-Stimulated Amylase Release. The Over-Expression Of EPI64B Disrupted The Binding Of Slp1 To Rab27B. Over-Expression Of Wild-Type And Constitutively Active Form Of Rab27B Both Inhibited The Amylase Release Stimulated By CCK Or Carbachol.
Conclusions: EPI64B Is A Potential Gtpase-Activating Protein (GAP) For Rab27B And May Play An Important Role In Regulating The Activity Of Rab27B In Mouse Pancreatic Acinar Cells. Moreover, The Data Showed That Reducing The Amount Of Active Rab27B Enhanced Amylase Secretion.
Analysis of pRSS1 Transgenic Mice Prone to Pancreatitis Reveals that Apoptosis Is the Predominant Mode of Acinar Cell Death
W. Huang, R. Mukherjee, J.P. Neoptolemos, R. Sutton, N. Vlatković Institute for Translational Medicine, University of Liverpool, Liverpool, UK
Introduction: We previously reported generation of a transgenic mouse model for spontaneous hereditary pancreatitis with either wild type human PRSS1 gene or one of the two mutated forms R122H or N29I. To further elucidate the mechanisms leading to pancreatitis, we have investigated the response of all three strains to acinar cell injury, both in vitro and in vivo.
Methods: Isolated pancreatic acinar cells were exposed to 500 μM taurolithocholate sulphate (TLC-S) and assessed for apoptosis and necrosis using confocal fluorescence microscopy. Animals were injected with 20μg/kg cerulein and pancreatic tissue taken at 12 h for blinded histological scoring (oedema, inflammation and necrosis) and caspase-3 immunohistological staining.
Results: In vitro cell death assays demonstrated that whilst there was no difference in the number of necrotic cells, there was a statistically significant increase in the number of apoptotic cells in each of the transgenic strains, both with and without TLC-S treatment. In vivo, transgenic animals developed more severe pancreatitis with caerulein than the wild-type (wt) controls (statistically higher histopathological score). There was, however, no difference in necrosis. In addition, there were more apoptotic cells in each of the transgenic strains when compared to wt animals.
Conclusion: The expression of human PRSS1 in acinar cells, regardless whether it is wt or mutated, predisposes mice to develop pancreatitis. Our data suggest that the predominant mode of acinar cell death in this model is apoptosis rather than necrosis.
E-Cadherin Increases Human Equilibrative Nucleoside Transporter 1 Activity and Sensitizes Human Pancreatic Cancer Cells to Anti-Cancer Nucleoside Analogs
S.W. Hung,1 Y.D. Bhutia,1 B. Patel,2 D. Lovin,3 R. Govindarajan,1Departments of 1Pharmaceutical and Biomedical Sciences, 2Biology, and 3Biochemistry, University of Georgia, Athens, GA
E-cadherin (E-cad), a cell-cell adhesion molecule expressed in well-differentiated epithelial cells, was shown to increase the cytotoxic response of nucleoside drugs (e.g. gemcitabine; 2',2'-difluoro-2'-deoxycytidine). Since the precise mechanisms of this response are unclear, we hypothesized that E-cad acts on the gemcitabine activation pathway to improve chemosensitivity. To address this, we analyzed changes in expression of gemcitabine transporters, gemcitabine phosphorylating and metabolizing enzymes, and other DNA replication and repair enzymes that affect gemcitabine cytotoxicity in pancreatic cancer cells. Retroviral expression of human E-cad into MIA PaCa-2, a poorly-differentiated, E-cad-null pancreatic cancer cell line, showed maximal increase in human equilibrative nucleoside transporter 1 (hENT1) protein compared with the other players studied. Consistently, E-cad expression steeply augmented hENT1-mediated 3H-gemcitabine cellular transport and cytotoxicity, which were ablated by pharmacological inhibition of hENT1 activity. Further mechanistic studies in A431D, a cadherin-null skin carcinoma cell line, showed that the increase in hENT1 transport activity was due to increased hENT1 protein half-life and subsequent trafficking to the cell surface. These results suggest that E-cad enhancement of gemcitabine efficacy occurs predominantly through increased hENT1 expression and activity, leading to increased cellular transport and cytotoxicity of gemcitabine. These data demonstrate that cell adhesion molecules can induce and recruit drug transport proteins to the cell surface to modulate drug sensitivity in pancreatic cancer.
CD10 Positive Pancreatic Stellate Cells Enhance the Progression of Pancreatic Cancer
N. Ikenaga, K. Ohuchida, K. Mizumoto, K. Fujiwara, S. Kozono, T. Ohtsuka, M. Nakamura, M. Tanaka Departments of Surgery and Oncology, Kyushu University, Fukuoka, Japan
Background & Aims: Pancreatic stellate cells (PSCs) promote the progression of pancreatic cancer by producing extracellular matrix and soluble factors. However, the functional heterogeneity of PSCs has not been identified until now. Detailed characterization of the PSCs in human pancreatic cancer would provide a set of potential targets for stroma-directed therapy.
Methods: We isolated PSCs from fresh pancreatic ductal adenocarcinoma tissue and sorted them by flow cytometry according to cell surface expression of CD10, which is a stromal prognostic marker for various tumors. We analyzed the functional differences between CD10+ PSCs and CD10- PSCs.
Results: Immunohistochemical analysis showed that the frequency of CD10 expression by PSCs was markedly higher in tumor tissue than in normal tissue (33.7% vs. 0%, P = 0.028). In pancreatic ductal adenocarcinoma, CD10 expression by PSCs was associated with positive nodal metastases (P = 0.011) and a shorter survival time (P < 0.001). In vitro co-culture experiments showed that CD10+ PSCs promoted the invasiveness of SUIT-2 and Panc-1 cells more intensively than CD10- PSCs. CD10+ PSCs significantly increased the tumor growth and invasiveness of SUIT-2 cells in a murine co-transplantation model. CD10+ PSCs secreted higher levels of matrix metalloproteinase (MMP) 3 than CD10- PSCs, and knockdown of MMP3 in co-cultured PSCs reduced the invasion of SUIT-2 and Panc-1 cells.
Conclusions: CD10+ PSCs enhance the progression of pancreatic cancer cells. CD10+ PSCs may be a candidate for selective therapeutic targeting in the treatment of pancreatic cancer.
External Pancreatic Fistulas in Patients with Disconnected Pancreatic Gland Syndrome - Rendezvous Treatment to Avoid Surgery
S. Irani, M. Gluck, A. Ross, J. Brandabur, S.I. Gan, R. Crane, E. Hauptman, M. Fotoohi, R. Kozarek Digestive Disease Institute, Virginia Mason Medical Center, Seattle, WA
Background and Purpose: An external pancreatic fistula (EPF) generally results from an iatrogenic manipulation of a pancreatic fluid collection. Severe necrotizing pancreatitis can lead to complete duct disruption causing a disconnected pancreatic gland syndrome. Distal pancreatectomy, often the only permanent solution, carries a high morbidity and mortality. We describe a combined endoscopic & percutaneous rendezvous treatment as an alternative to surgery.
Methods: Retrospectively (3pts) & prospectively (10 pts) collected data.
Results: Thirteen pts, (10 M, 3 F) with a median age of 52 yrs (24 - 65 yrs) were diagnosed with EPF & disconnected pancreatic gland syndrome (cut off/blow out of pancreatic duct, with inability to demonstrate upstream body/tail of pancreas on pancreatogram) resulting from severe necrotizing pancreatitis (mean CTSI 8.3). Prior to undertaking rendezvous treatment of the EPF, all fluid collections had resolved with percutaneous drain treatment. Pts were either non-surgical candidates or had refused a distal pancreatectomy. The EPF had failed medical management. 2 pts had failed attempts at fistula drainage via EUS alone. The median duration of EPF prior to treatment was 5 months (1-48 months) & median output was 200 ml/day (50-700 ml/day). Approach and puncture of the gastric/duodenal wall was percutaneous through the existing drain tract/fistula in 10 of 13 pts, using a combination of stiff wires, and a transjugular intrahepatic portosystemic shunt (TIPSS) needle. In 3 pts, EUS guided fistula puncture was performed to avoid large varices, and the wire was grasped by the interventional radiologist. With a wire secured at both ends (percutaneously by radiologist & endoscopically through the scope), 2 stents were usually deployed into the fistula. Two pts were treated by transpapillary rendezvous with eventual reconnection of the entire pancreatic duct. The external drain was capped & removed when the EPF output stopped, or after capping for 2-4 weeks, no recurrent fluid collection was identified on CT scan. The median duration to EPF closure was 10 days (1-73 days). There was no EPF recurrence in any of the 13 pts. (median f/u 25 months). One pt had recurrence of a pancreatic fluid collection at 30 months due to spontaneous migration of both transduodenal stents, which was successfully drained via EUS alone. Another pt developed an abscess due to stent fragmentation within the fistula. This was retrieved percutaneously, and was restented, with a final successful outcome.
Conclusions: 1) Management of EPF, especially in the setting of a disconnected pancreatic gland syndrome is challenging. 2) Combined endoscopic & percutaneous rendezvous treatment is a safe, possibly durable alternative to surgery, in the management of this difficult problem.
RUNX3 is a Tumor Suppressor in Human Pancreatic Cancer
M. Jain,1 M.J. Baine,1 S. Senapati,1 S.K. Batra,1,21Department of Biochemistry and Molecular Biology, 2Eppley Institute for Research in Cancer and Allied Diseases, University of Nebraska Medical Center, Omaha, NE, USA
Introduction: Human RUNX proteins (RUNX1, RUNX2, and RUNX3) coordinate members of the TGF-β superfamily to regulate cell growth and differentiation while acting as context-dependent transcription activators or repressors. Similar to TGF-β, RUNX proteins have a paradoxical role in cancer development as they have been shown to act as both dominant oncogenes (RUNX1) and tumor suppressors (RUNX3). Interestingly, pancreatic cancer (PC) cell lines have been demonstrated to downregulate RUNX3 by both hemizygous deletions and methylation, eliciting the potential for loss of RUNX3 to be of significant importance in the development and pathophysiology of pancreatic cancer.
Methods: We studied the effects of ectopic RUNX3 expression restoration in CD18/HPAF pancreatic cancer cells using both in vitro and in vivo models. Particularly, RUNX3 transfected cells (CD18/HPAF-R3) were compared to their empty vector-transfected counterparts (CD18/HPAF-NEO) for growth pattern and growth kinetics in vitro and tumorigenicity and metastasis in vivo using an orthotopic transplant model. Comparative global transcriptome analysis using whole-genome cDNA microarray was performed on control and RUNC3 restored cells.
Results: CD18/HPAF-R3 cells were found to exhibit a reduced growth rate and formed adherent clumps as compared to the more random and disperse growth pattern seen in CD18/HPAF-NEO controls. These growth-pattern changes in RUNX3 transfected cells were associated with increased E-cadherin expression. Orthotopic implantation of CD18/HPAF-R3 cells resulted in a significant decrease in tumorigenicity (p=0.01) and metastasis (p<0.01) as compared to their control counterparts. Upon global transcriptome analysis, restoration of RUNX3 expression was found to result in the differential expression of 332 genes by 2-fold or greater, with the majority (283/332) being downregulated. Ingenuity pathway analysis of these 332 genes found that the most significantly implicated molecular and cellular functions associated with the loss of RUNX3 expression in PC are Cellular Movement (P<0.005, 7 associated proteins), Cellular Development (P<0.005, 9 associated proteins), and Cellular Growth and Proliferation (P<0.005, 15 associated proteins). The Disease States found to be most closely associated with the loss of RUNX3 were Cancer (p<0.005), 19 associated proteins) and Gastrointestinal Diseases (p<0.005, 16 associated proteins).
Conclusion: These studies provide substantial evidence that RUNX3 acts as a tumor suppressor in pancreatic cancer by inhibiting cellular growth and metastasis. The highly aggressive and metastatic nature of PC makes these observations of considerable importance as they provide a novel potential target for preventing PC progression. With these results, further studies are required to understand the mechanisms underlying tumor suppressive function of RUNX3 in pancreatic cancer as well as the potential diagnostic and therapeutic implications of our findinngs.
Contemporary Outcomes from Synchronous Portal/Superior Mesenteric Vein (PV/SMV) Resection at Pancreaticoduodenectomy for Cancer
S. Jegatheeswaran, A.K. Siriwardena HPB Unit, Manchester Royal Infirmary, UK
Introduction: Pancreaticoduodenectomy (PD) is the standard of care for localised cancer of the pancreas. Resectability can be improved by synchronous resection of the portal/SM vein. The procedure may add morbidity without benefit. We previously reported outcome in PV/SMV resection from 1966 - 2005 but included patients having arterial and venous resection and predated the era of high-volume centres. This study examines outcome from PV/SMV resection at PD in the 21st century.
Methods: Medline and Embase searches identified 44 manuscripts giving data on 1905 patients undergoing PV/SMV resection reported during 2000 -20010. Data were only included from original reports providing resection details and outcome data. Data were extracted to Cochrane criteria to populate a pre-determined database. Reporter variables included numbers of vein resections (total and proportion of all PD), outcome and survival. Data are presented as medians (range) of pooled data.
Results: Forty four centres report outcome after PD with vein resection to 2010. Of 1905 vein resections, 1638 were in centres which provided denominator data on PD without vein resection. PD with vein resection comprised 24% of 6,923 PD. Two centres used artificial grafts; all others used either partial circumference resections or autologous vein reconstruction. Median 30-day mortality ranged from 0 - 11%. The modal 30-day mortality was 0%. Median survival was 15 (9-33) months.
Conclusion: This is thought to be the largest pooled report of portal/SMV resection at PD. The data show that vein resection is undertaken in up to a quarter undergoing PD with acceptable operative mortality. Median survival is 15 months. Vein resection has become an established option in the modern operation of pancreaticoduodenectomy.
Returning Genetic Research Results to Individuals: The International Cancer Genome Consortium Initiative Experience
A.L. Johns,1 N. Zeps,2 L. Chantrill,1 A.J. Gill,4 D.K. Chang,1 C.J. Scarlett,1 S.M. Grimmond,4 A.V. Biankin,1,51Cancer Research Program, Garvan Institute of Medical Research, Sydney Australia, 2St John of God Pathology, Perth Australia, 3Department of Anatomical Pathology, Royal North Shore Hospital, Sydney Australia, 4QLD Centre for Medical Genomics, Institute for Molecular Bioscience, University of Queensland, Brisbane Australia, 5Department of Upper GI Surgery, Bankstown Hospital, Sydney Australia. On behalf of the Australian Pancreatic Cancer Genome Initiative (APGI)
Disclosure of individual research results to participants has emerged as a complex and contentious issue. Approaches and practices of research investigators, funding bodies and human research ethics committees are diverse. The development and approval of ethically justified policies regarding the disclosure of results is important as genetic research is increasingly prevalent, and the focus has shifted from studying rare diseases to determining the role of genetics in common disorders such as cancer. This is the main aim of the International Cancer Genome Consortium (www.icgc.org); to elucidate comprehensively the genomic, transcriptomic and epigenomic changes present in many forms of cancers that are of clinical and societal importance across the globe.
Ethical principals of beneficence, respect and reciprocity provide justification for routinely offering certain results to research participants. The Australian Pancreatic Cancer Genome Initiative (APGI), the Australian arm of the ICGC, has employed a result-evaluation approach to returning results, which assesses the information and the context of the study in order to decide if results should be offered. Using this approach the analytical validity and clinical utility of results are considered and help determine what information is disclosed, at what time point and to whom. We present 2 cases where results were returned to participants based on this approach, and discuss the process, issues and outcomes.
The Effect of Timing of Cholecystectomy for Biliary Pancreatitis on Patient Outcomes
M. Johnstone, P. Marriott, C.E. Richardson, E. Hepburn, T. J. Royle, A. Torrance, A. Patel, T. D. Pinkney on behalf of the West Midlands Research Collaborative, Birmingham, UK
Introduction: There is no consensus regarding optimum timing of cholecystectomy for biliary pancreatitis. The aim of this study was to assess if timing of operation affects the subsequent morbidity of patients diagnosed with biliary pancreatitis.
Methods: Multi-centre, retrospective study of patients with an index admission of biliary pancreatitis across the West Midlands, UK from 2006 to 2008.
Results: 524 patients were identified from 7 NHS hospitals (36% male; median age 63 (12-99) years). 365 (70%) underwent cholecystectomy of which 96% were laparoscopic (6.5% converted). The median time from presentation to cholecystectomy was 11.5 weeks. 58 (16%) were performed during the index admission; operative complications occurred in 8 (14%) of these patients versus 21/307 (7%) after discharge (p=0.07). Complications included 6 bile leaks, 3 wound infections, 2 common bile duct injuries and 1 enterotomy.
The rate of pre-interventional adverse events, such as recurrent pancreatitis or unplanned readmission, increased with time from index admission: 2.4% (1/42) in those treated in week 1, to 14% (23/164) within 10 weeks, and 15.7% (26/350) by a year. Perioperative complications were 16.7% (7/42) in patients operated on in week 1 dropping to 6.7% (11/164) at 10 weeks, and remaining at this level.
Conclusion: Within the first 10 weeks of presentation, early intervention reduces the risk of further adverse events but is associated with a higher risk of post-intervention complications. Overall there is no clear benefit of a specific time limit to perform cholecystectomy by, although the lowest rate of operative complications is seen when cholecystectomy is performed more than 3 months after index admission.
Pancreatic Duct Cell Function is Impaired in Cigarette Smokers
V. Kadiyala,1 L.C. Lee,1 J. Rosenblum,1 J.A. Paulo,1 N.I. Sainani,2 K. Mortele,2 P.A. Banks,1 D.L. Conwell,11Division of Gastroenterology, Endoscopy and Hepatology; 2Department of Radiology; Brigham and Women's Hospital, Harvard Medical School, Boston MA
Background: There is evidence to suggest that smoking impacts pancreas function. As such smoking cessation may be an important component in the management of chronic pancreatitis (CP).
Aim: Compare pancreatic acinar cell exocrine and duct cell secretory function in subjects with and without cigarette smoking exposure (current and past).
Methods: Cross sectional retrospective chart review: demographics, smoking status (former, current, never), alcohol intake, clinical data and laboratory studies (pancreas fluid (PF) peak [HCO3] and stool pancreatic elastase (PE-1). Statistical analysis (SPSS v. 16.0) to assess association between cigarette smoking exposure and pancreas function.
Results:Duct Cell Secretory Function: 131 subjects underwent ePFT PF bicarbonate analysis. 74/131 (56.5%) subjects smoked and 57/131 (43.5%) never smoked. Controlling for age, gender and alcohol intake, smoking exposure was found to be an independent predictor of abnormal peak PF [HCO3] (OR 4.05 [1.79, 9.16]; p= 0.001). There was no significant difference in [HCO3] between past and current smokers (p=0.572). The risk of duct cell dysfunction in smokers was 56.78% [45.41, 67.44], and 26.32% [16.55, 39.07] in non-smokers. Acinar Cell Exocrine Function: 61 subjects underwent stool PE-1 testing. No significant association was found between smoking exposure and abnormal PE-1 (p=0.39).
- Smoking exposure is independently associated with pancreatic duct cell secretory dysfunction.
- Smoking was not associated with pancreatic acinar cell exocrine dysfunction.
- Smoking cessation is strongly advised as part of long term CP management.
Cigarette Smoking Impacts Chronic Pancreatitis Clinical Feature Score, Severity Index and Pancreas Imaging Severity
V. Kadiyala,1 L.C. Lee,1 J. Rosenblum,1 J.A. Paulo,1 N.I. Sainani,2 K. Mortele,2 P.A. Banks,1 D.L. Conwell,11Division of Gastroenterology, Endoscopy and Hepatology; 2Department of Radiology; Brigham and Women's Hospital, Harvard Medical School, Boston MA
Background: Cigarette smoking is a known risk factor for chronic pancreatitis. However, the effect of smoking on clinical severity and morphology is unclear.
Aim: Compare chronic pancreatitis (CP) clinical severity in cigarette smokers and non-smokers, using the M-ANNHEIM classification system.
Methods: Cross sectional, retrospective chart review: demographics, smoking exposure (past, current, none), alcohol intake, clinical data, imaging and laboratory data. M-ANNHEIM CP clinical feature score (0-24), severity index (grade A-E) and morphologic (imaging) changes (normal-marked changes) determined. Univariate analysis (SPSS v. 16.0) to assess association between smoking and CP clinical severity.
Results: 131 subjects were evaluated for CP. 74/131 (56.5%) subjects smoked (former or current) and 57/131 (43.5%) never smoked. 31/131 (23.7%) of subjects consumed increased/excessive alcohol. Clinical Feature Score: Increased clinical feature scores were associated with smoking (p<0.001), but not with increased alcohol intake (p=0.361). Severity Index: Increased severity index was associated with smoking (p<0.001), but not increased alcohol intake (p=0.407). Morphology (Imaging): Smoking (p=0.0156) and increased alcohol intake (p=0.0137) were associated with increased risk of moderate/marked imaging changes.
- Smoking, not alcohol, was associated with increased M-ANNHEIM CP clinical feature score and severity index grade.
- Smoking and alcohol were associated with marked/moderate radiologic imaging changes.
- Smoking cessation is strongly advised in acute and long term CP management.
A Novel Mouse Model to Study the Importance of Mist1-Dependent Transcriptional Regulation in Pancreatic Acinar Cells
A. Karki, S. Humphrey, D. DiRenzo, Y. Sun, S. Konieczny Department of Biological Sciences and the Purdue Center for Cancer Research, Purdue University, West Lafayette, IN
Introduction: Mist1 is a bHLH transcription factor expressed in adult acinar cells. Gene array and ChIP analyses have identified target genes in which Mist1 binding can activate or repress gene expression. The ability of Mist1 to serve as both an activator and repressor was surprising since in vitro mutagenesis studies revealed that the central bHLH domain was sufficient for full transcriptional activity in transgenic mice. We hypothesize that Mist1 utilizes various nuclear cofactors to regulate its transcriptional activity in the normal pancreas and in disease states.
Aim: To generate a Mist1BT/+ targeted mouse line that can be used to identify the cofactors of Mist1 by LC-MS/MS via BirA ligase-dependent biotinylation.
Methods: Mist1BT/+ knock-in mice were generated by homologous recombination in which the Mist1 coding region was replaced with a BT-Mist1Myc coding region. BT-Mist1Myc consists of a biotin tag (BT) at the N-terminus and a C-terminal Myc-epitope tag. R26HA-BirA mice expressing an HA-tagged BirA ligase were crossed to Mist1BT/+ mice, generating Mist1BT/BT; R26HA-BirA animals. Biotinylated protein complexes from Mist1BT/BT; R26HA-BirA pancreata were subjected to tandem immunoprecipitation using anti-Myc magnetic beads followed by streptavidin-coated magnetic beads. The pull-down material was processed through LC-MS/MS to identify all Mist1-interacting proteins.
Results and Conclusion: Preliminary results show that Mist1BT/BT acinar cells are normal and biotinylated Mist1 can be immunopurified from mouse pancreas. Current studies are focused on characterizing Mist1-interacting proteins to understand how Mist1 functions as a dual transcriptional activator and repressor.
Connecting the Cords: Mucin MUC4 and Acute Phase Protein NGAL
S. Kaur,1 S. Chakroborty,1 I. Lakshmanan,1 S. Guha,2 S.K. Batra,11Department of Biochemistry and Molecular Biology, UNMC, Omaha, NE, USA, 68198; 2Department of Gastroenterology, University of Texas, M. D. Anderson Cancer Center, Texas, USA
Background and hypothesis: MUC4, a large-sized membrane-anchored mucin is overexpressed in pancreatic cancer (PC). It promotes pancreatic tumor growth and metastasis. Neutrophil gelatinase associated lipocalin (NGAL) is multifaceted acute phase protein aberrantly up-regulated during PC and is potential diagnostic marker for PC. Simultaneous overexpression of NGAL and MUC4 transcripts were observed in the ileal mucosa in response to enteric infection. Similarly, we observed simultaneous expression of NGAL and MUC4 in pancreatic intraepithelial neoplastic lesions as early as PanIN2. Based on these observations, we hypothesized that MUC4 and NGAL co-regulates each other's expression during PC pathogenesis.
Methods: Quantitative RT-PCR, immunoblotting and immunocytochemistry was performed to analyze the effect of MUC4 knockdown and over-expression on NGAL and vice-versa. Cells were treated with specific chemical inhibitors of AKT and NF-Κβ followed by immunoblotting to study the molecular mechanism responsible for MUC4-mediated regulation of NGAL. Immunohistochemistry was performed to analyze the correlation between the expression of NGAL and MUC4.
Results and Discussion: The stable knockdown of MUC4 caused significant decrease in NGAL expression in PC cells (CD18/HPAF, CaPan1), while NGAL overexpression and/or knockdown did not alter the MUC4 expression. Analysis of downstream signaling pathway showed that MUC4, by stabilizing epidermal growth factor receptor 2 (ErbB2/HER2), activates PI3K/AKT signaling cascade. Furthermore, AKT through NF-ΚB found to regulate the transcription of NGAL. Interestingly, in rapid autopsy cases, MUC4 positive subjects showed 100% positivity for NGAL in the area of primary tumor while only 69% cases were positive for NGAL if MUC4 was negative.
Conclusion: The present study shows for the first time a novel mechanism of mucin MUC4-mediated regulation NGAL via HER2/PI3K/AKT/NF-ΚB pathway in PC.
Chronic Pancreatitis: New Direction of Surgery Treatment (Alternative Way versus Frey and Beger Procedures)
A.V. Klymenko, V.N. Klymenko, A.A. Steshenko Zaporozhye State Medical University, Zaporozhye, Ukraine
Introduction: Resected operations (Frey and Beger procedures) significantly make worse had existed before the operation exocrine and endocrine pancreatic insufficiency.
Aim: To study long term outcomes developed parenchyma-preserving surgical procedure in patients with CP as there had no disadvantages of Frey and Beger procedures.
Methods: We have analyzed long-term (5 years) outcomes of surgery in 84 patients (men - 76, women - 8) with different forms of CP in main (n=39) and control (45) groups. The main group patients underwent a new procedure: longitudinal total pancreaticowirsungoduodenopapillotomy with pancreaticojejunoduodenostomy by Roux-en-Y. The control group patients underwent resected procedures: Frey - 29, Beger - 16. Alcohol CP was in 76 (90,5%). Patient examined with: US, CT, ERCP, intraoperative US, C-peptide, endogen insulin, parathormone, CA 19-9, Ig G, fecal ellastase-1. Quality of life in both groups was assessed by MOS SF-36.
Results: In 32 patients (32/39, 82.1%) main group the size of enlarged head of pancreas turn to normal. All patients reduced abdominal pain. Manifestations of pancreatic diabetes remained only in 4 (4/39, 10.3%), before the operation were 16 (16/39, 41.1%) (P<0,05); exocrine insufficiency were 7 (7/39, 17.9%) against to 25 (25/39, 64.1%) (P<0,05) before surgery. In the control group after Beger procedure there was exocrine insufficiency - 13 (13/16, 81.25%), Frey - 14 (14/29, 48.3%); endocrine - 9 (9/16, 56.3% - Beger), 11(11/29, 37.9% - Frey). Quality of life of patients in the main group was better comparing control (p<0,05).
Conclusion: Parenchyma-preserving procedure reduce abdominal pain, pancreatic ductal hypertension, restore the physiological passage of pancreatic juice, leads to normalization of pancreatic functions and is an alternative to Frey and Beger procedures in patients with CP.
TGF-β/NFAT-dependent Regulation of the Fas Ligand Expression on Pancreatic Cancer Cells Influences T-cell Microenvironment
A. Koenig,1,2 J-S. Zhang,1 J. Gorman,1 K. Reutlinger,2 T. Gomez,1 V. Ellenrieder,2 D. Billadeau,11Dep. of Oncology Research, Mayo Clinic, Rochester, MN. 2Dep. of Gastroenterology, University of Marburg, Marburg, Germany
NFAT proteins are calcium sensitive transcription factors playing an important role in T cell activation where they regulate the expression of cytokines and immunomodulatory proteins. However, recent studies have demonstrated that NFATc1 and NFATc2 are expressed in pancreatic cancer while normal exocrine pancreatic tissue shows no expression of either protein. In pancreatic cancer cells, we have shown that NFAT regulates the expression of the oncogene c-Myc leading to increased cellular proliferation.
In addition to its pro-proliverative function, we have found that NFAT expression in pancreatic cancer cells results in an upregulation of multiple immunomodulators. In the present work we demonstrate that Fas ligand (FasL) is overexpressed in pancreatic cancer cells in a TGF-β and NFAT dependent manner. Genetic depletion of NFATc1 or c2 as well as inhibition of NFAT activity by cyclosporine A or GSK-3β reduces the expression of FasL at mRNA and protein level. We show that NFAT directly binds to DNA and cooperates with Ets to induce FasL promoter activity.
In fact, syngeneic cocultivation of activated murine CD8 T-cells with murine pancreatic cancer cells revealed a Fas/FasL dependent induction of apoptosis in these CD8 T-cells when cancer cells were pretreated with TGF-β resulting in an increased FasL expression. Decreased expression of FasL following genetic depletion of NFAT in these tumor cells led to increased numbers of activated CD8 T-cells without any signs of apoptosis induction in cocultivation experiments.
Taken together, our results identified NFAT proteins as important modulators of the pancreatic cancer microenvironment.
Semaphorin 4D, Acting through Plexin-B1 and CD72, Exerts Autocrine Promalignant Effects in Pancreatic Cancer
D. Koliogiannis,1 M. Müller,2 N. Giese,1 A. Heller,1 T. Giese,1 J. Werner,11Department of Surgery, University Hospital of Heidelberg, Germany; 2Department of Surgery, Hospital of Bad Canstatt, Stuttgart, Germany
Introduction: As a constituent of a large family of secreted and transmembrane molecules, semaphorin 4D (Sema4D) has been implicated in tumor growth and metastasis by acting through its receptors, plexin-B1 and CD72.
Aim: We investigated, whether Sema4D exerts autocrine pro-malignant effects in pancreatic ductal adenocarcinoma.
Material & Methods: Pancreatic tissues of healthy donors (n=34) and of patients with pancreatic cancer (n=69), as well as pancreatic cancer cell lines were processed under identical conditions by QRT-PCR-analysis. The probability of autocrine effects was assessed by analysing growth and motility of pancreatic tumor cells upon siRNA-based modification of Sema4D expression. Furthermore we performed immunohistochemical analysis to reveal concentration in tumor cells.
Results: QRT-PCR revealed triple overexpression of Sema4D in pancreatic cancer tissue compared to donor tissue (mean 43.28; SEM 3.34 vs. mean 12.65; SEM 1.65; p < 0.01). It originated from up-regulation of synthesis in nerves and immune cells but not in tumor cells, which however showed high-level receptor expression. Higher levels of Sema4D were associated with reduced survival. In 6 out of 7 pancreatic cell lines we could detect a co-expression of Sema4D (low) and its receptors (high).
Conclusion: Inflammatory reactive changes in pancreatic parenchyma raise local level of Sema4D, potentially able to impact several direct (tumor cells) and indirect (angiogenesis, immune response) mechanisms influencing tumor progression. Although a paracrine effect on tumor cells may prevail, an autocrine regulation in a small population remains a possibility.
HNF1A-mediated MIA2 Expression Regulates Metabolism of Pancreatic Cancer and Affects Response to Chemotherapy
B. Kong, C. W. Michalski, W. Wu, C. Tosolini, N. Valkovska, M. Erkan, H. Friess, J. Kleeff Department of Surgery, Technische Universität München, Munich, Germany
Hepatocyte nuclear factor 1A, HNF1A, a recently discovered pancreatic cancer susceptibility gene, participates in a transcription factor network that regulates development of the pancreas and metabolic processes including carbohydrate and protein metabolism in the adult organ. Here, we show that melanoma inhibitory activity 2, MIA2, is a target of the HNF1A network in pancreatic cancer sustaining the activity of the AKT/mTOR axis to control protein translation; loss of MIA2 in pancreatic cancer cell lines reduced the activity of AKT/mTOR. In line, a common genetic variant of MIA2 (the 617GG and 1833CC haplotype), which potentially affects its cellular localization, was associated with a decreased activity of AKT/mTOR. Pancreatic cancer cells expressing this haplotype endo- or exogenously were highly sensitive to gemcitabine treatment. Because pancreatic cancers that share a similar molecular signature with gemcitabine-sensitive cell lines have recently been shown to be clinically aggressive, we tested pancreatic cancer patients for hetero- or homozygosity of the MIA2 haplotype; here, patients hetero- or homozygous for the haplotype survived significantly shorter than those without it. This implies that pancreatic cancer cells, which are sensitive to non-targeted chemotherapies are actually more aggressive than their chemoresistant counterparts. Thus, our data suggest that HNF1A-mediated MIA2 expression is an important regulatory component of AKT/mTOR signaling in pancreatic cancer. A common genetic variant of MIA2, which significantly influences the metabolism of pancreatic cancer cells, may affect the clinical response to gemcitabine therapy and would thus be a relevant diagnostic target.
Targeting the Metastasis Suppressor NDRG1: A New Strategy for the Treatment of Pancreatic Cancer
Z. Kovacevic, S. Chikhani, S. Sivagurunathan, D.B. Lovejoy, D.R. Richardson Iron Metabolism and Chelation Program, Department of Pathology and Bosch Institute, University of Sydney, Sydney, New South Wales, 2006, Australia
The metastasis suppressor gene, N-myc downstream regulated gene-1 (NDRG1), is negatively correlated with tumor progression in multiple neoplasms, including pancreatic cancer. However, the exact molecular function of NDRG1 remains to be established and is important to elucidate. In the current study, we have used gene array analysis to identify potential molecular targets of NDRG1 in MIAPaCa-2 pancreatic cancer cells. For the first time, we have identified two novel molecular targets of NDRG1 in pancreatic cancer, NEDD4L and GLIS3, both of which were up-regulated by NDRG1 hyper-expression. Further studies examining the down-stream effects of NEDD4L led to the discovery that NDRG1 was able to affect the transforming growth factor β (TGF-β) pathway, leading to up-regulation of two key tumor suppressor proteins, namely phosphatase and tensin homologue on chromosome 10 (PTEN) and SMAD4. Moreover, NDRG1 inhibited the phosphatidylinositol 3-kinase (PI3K) and Ras oncogenic pathways in these cells. The identified target genes of NDRG1 and their effect on the TGF-β pathway reveal its molecular function in pancreatic cancer and a novel therapeutic avenue.
We further demonstrate that novel anti-cancer agents that target NDRG1 expression in pancreatic cancer are a potential new strategy for the treatment of this disease. These compounds belong to a new class of thiosemicarbazones that have shown to be potent and selective against a range of different neoplasms in vivo due to their ability to target iron. In the current study, we show that these agents are more effective than the currently used gemcitabine at inhibiting the growth of pancreatic cancer in vivo.
Pirfenidone Inhibits Proliferation, Invasiveness, the Chemokine and Stromal Component Production of Pancreatic Satellate Cells
S. Kozono, K. Ohuchida, H. Takanami, T. Eghuchi, K. Fujiwara, M. Zhao, N. Ikenaga, L. Cui, 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 chemoradiotherapy. Desmoplasia produced by pancreatic satellate cells(PSCs) is known to enhance the chemo-radioresistancy in pancreatic cancer. In this study, we investigated whether the antifibrotic angent pirfeniodne decreases the biological activity of PSCs and chemokine and stromal component production in PSCs.
Methods: Human primary cultures of PSCs (hPSCs) were established by outgrowth method from pancreatic cancer tissue obtained during surgery. Then, hPSCs were treated with pirfeniodne and examined proliferation and invasion activity by PI assay and invasion assay. We analyzed the change of mRNA and protein expression of chemokine and stromal component in pirfenidone-treated hPSCs using quantitative RT-PCR and western blotting.
Results: Pirfenidone inhibited proliferation and invasiveness of hPSCs in the concentration-dependent manner. PDGF and periostin mRNA expression were reduced by pirfenidone. Pirfenidone decreased the protein expression of PDGF, collagen-1 and periostin in PSCs. And the supernatant of pirfenidone-treated PSCs decreased the protein expression of PDGF, TGF-β1, CTGF, collagen-1 and periostin compared to non-treated PSCs.
Conclusions: This study shows that pirfenidone can attenuate the bilogical activity of PSCs and inhibit the chemokine and stromal component production related to desmoplasia augmentation. These findings suggest that Pirfenidone may be promising agents for pancreatic cancer.
NCOA3 Regulates MUC4 Expression in Pancreatic Cancer
S. Kumar,* S. Das,* M. Torres-Gonzalez, S. Rachagani, S.K. Batra Dept of Biochemistry and Molecular Biology, UNMC, Omaha, NE
Background and Hypothesis: Deregulated mucin production is the hallmark of neoplastic disorders of the pancreas. MUC4, a transmembrane mucin is aberrantly over expressed in pancreatic cancer (PC) and appears very early in the progression of PC and has strongly been implicated in the initiation, progression and metastasis of PC. Studies have demonstrated the synergistic up regulation of MUC4 by retinoic acid (RA) and interferon gamma (IFN-γ). MUC4 is differentially overexpressed very early in pancreatic cancer, suggesting the involvement of chromatin remodeling events at MUC4 locus. To understand the role of chromatin remodeling enzymes in regulating MUC4 expression, we performed a chromatin modifying enzyme PCR array using MUC4 expressing (CAPAN-1) and non-expressing (PANC-1) cells, which resulted in the identification of a nuclear receptor co-activator NCOA3. NCOA3 was found to be overexpressed in all the MUC4 expressing PC cells and was nuclearly localized as confirmed by both biochemical fractionation and immunofluorescence studies. Based on our preliminary studies we hypothesized that NCOA3 is a critical regulator of MUC4 in PC.
Results: We stably knocked down the expression of NCOA3 in HPAF-CD18 and T3M4 PC cells, which resulted in approximately 70-80% downregulation of MUC4 in both the cell lines at transcriptional level. Immunohistochemical analysis on PC tissue microarray for NCOA3 and MUC4 established a significant positive correlation between NCOA3 and MUC4 expression (Normal ducts were negative for both). Further, we were able to demonstrate in Ptf1a-cre-ER/K-rasG12D tamoxifen inducible PC mouse model that the NCOA3 positive PanIN lesions (three months after tamoxifen induction) start expressing MUC4. Immunohistochemical analysis of both human and mouse tissues demonstrate nuclear localization of NCOA3 and the corresponding ducts showed membranous MUC4 expression. NCOA3 binds to steroid receptors (including RAR) in a ligand dependent manner and acetylates the histones. Here we demonstrate that RA in NCOA3 knock down cells fails to upregulate the MUC4 expression, underscoring the importance of NCOA3 in regulating MUC4 expression. Interestingly, the normal immortalized colonic epithelial cells (NCM460), which normally express MUC4; knock down of NCOA3 fails to downregulate MUC4 expression highlighting the pathological significance of NCOA3 in MUC4 regulation in PC.
Conclusion: Taken together our results demonstrate for the first time the role of nuclear receptor co-activator NCOA3 in regulating MUC4 expression in pancreatic cancer.
Comparison of CT and EUS to Differentiate Pseudocyst from Walled-Off Pancreatic Necrosis
N. Kumar,1 V. Sahni,2 C. Thompson,11Division of Gastroenterology, Brigham & Women's Hospital, Boston, MA; 2Department of Radiology, Brigham & Women's Hospital, Boston, MA
Background: Distinction between walled-off pancreatic necrosis (WOPN) and pseudocysts (PC) is critical as management differs.
Aim: To determine sensitivity and specificity of computed tomography (CT) and endoscopic ultrasound (EUS) for WOPN, accuracy for differentiation between WOPN and PC in patients referred for endoscopic cystgastrostomy (ECG), and interobserver agreement.
Methods: Patients undergoing ECG with direct endoscopic visualization for suspected WOPN were selected randomly. 7 gastroenterologists and 1 radiologist interpreted 15 deidentified CT taken within 15 days before ECG and EUS videos taken at cyst entry from a different set of 15 patients (10 in common). 120 CT and 120 EUS interpretations were done. Positive (PPV) and negative predictive value (NPV) and kappa were determined.
Results: Using CT, WOPN were correctly categorized in 36/64 interpretations (sensitivity 56.3%). PC were correctly categorized in 26/56 (specificity 46.4%). CT PPV was 54.5% and NPV 48.1%. Interobserver agreement was low (kappa=-0.15).
Using EUS, WOPN were correctly categorized in 44/56 interpretations (sensitivity 78.6%). PC were correctly categorized in 39/64 (specificity 60.9%). EUS PPV was 63.8% and NPV 76.5%. Interobserver agreement was low (kappa=-0.14).
EUS was more accurate than CT: 69.2% vs 51.7% (p=0.008). 10 patients had CT and EUS: when imaging the same cyst, accuracy for WOPN was 28.2% for CT and 87.5% for EUS; accuracy for PC was 50.0% for CT and 71.9% for EUS.
Conclusions: EUS was more accurate than CT, but neither reached satisfactory sensitivity or specificity. Other imaging modalities should be investigated. Endoscopic visualization via ECG is the gold standard for diagnosis of WOPN.
Safety of Endoscopic Cystgastrostomy in Patients with Gastric Varices
N. Kumar, C. Thompson Division of Gastroenterology, Brigham & Women's Hospital, Boston, MA
Background: Pseudocyst (PC) drainage via endoscopic cystgastrostomy (ECG) is increasingly performed. The most worrisome complication is hemorrhage. Patients with PC have a high rate of gastric varices (GV); some literature reports that GV are a contraindication to ECG.
Aim: To determine safety of ECG in patients with GV.
Methods: We retrospectively studied patients referred for ECG for drainage of PC or necrosectomy of walled-off pancreatic necrosis (WOPN). All patients having ECG with GV confirmed on EGD or EUS were included.
Results: 6 patients with GV, median age 53.5 (27-86), 3M+3F, had ECG. 19-g ultrasound needle was used to enter the cyst via transgastric approach; needle-knife was used with Endocut settings in one. Doppler confirmed lack of vascular structures in the needle path. 5 patients had balloon dilation of the cystgastrostomy site to mean 18.6 ±1.0mm; one to 6mm. A median 2.5 (1-3) 10 Fr stents were placed. 4/6 cysts were entered with a gastroscope and 3/4 of these had endoscopic necrosectomy. All patients had hematocrit within 10 days; no bleeding or other complications were noted.
On endoscopic cyst evaluation, 2 patients had bridging GV through the cyst and 2 had GV along the cyst wall. On follow-up cyst endoscopy, 2 patients had ulceration overlying GV in the cyst.
Conclusions: Our experience in patients with GV suggests that ECG can be performed safely with some modifications. EUS with Doppler should be used to guide needle insertion. GV should be kept under visualization during stent insertion and guidewire used during stent placement and subsequent removal to prevent vascular injury. Care should be taken to avoid hooking pigtail stents around bridging varices. Given finding of ulceration overlying GV in the cyst cavity, we recommend PPI in patients with GV who undergo ECG.
Response to Pancreatic Duct Stenting Predicts Outcomes after Modified Lateral Pancreaticojejunostomy
R. Kwon,1 B. Young,1 W. Marsteller,2 C. Lawrence,2 B. Wu,3 D. Mullady,4 D. Klibansky,5 T. Gardner,5 D. Simeone,61Div of Gastroenterology, Univ of Michigan, Ann Arbor, MI; 2Div of Gastroenterology, Medical Univ of South Carolina, Charleston, SC; 3Div of Gastroenterology, Brigham & Womens Hospital, Boston, MA; 4Div of Gastroenterology, Washington Univ, St. Louis, MO; 5Div of Gastroenterology, Dartmouth Hitchock Medical Center, Lebanon, NH; 6Dept of Surgery, Univ of Michigan, Ann Arbor, MI
Background: For chronic pancreatitis, current options for pancreatic duct decompression are ERCP and endoscopic PD stent (EPD) placement or surgical drainage by modified lateral pancreaticojejunostomy (LPJ).
Aim: To determine if the pain response to EPD predicts clinical outcome of LPJ.
Methods: A retrospective review of pts with successful EPD bypassing an obstruction prior to LPJ at 5 academic centers between 2001 - 2010 was performed. Primary outcome was narcotic independence (NI) within 2 mo after EPD or LPJ.
Results: 34 narcotic dependent pts had successful EPD prior to LPJ. Ten (29%) achieved post-LPJ NI (mean f/u 15.3 mo (SD 19)). Mean dilation of PD was higher in NI group (12.8 v 7.6mm, p=0.04). There was no difference in age, disease duration, diabetes, current EtOH or tobacco use, PD strictures or stones. 8 of 10 pts with NI post-EPD achieved NI post-LPJ. Two of 24 without NI post-EPD achieved NI post-LPJ. NI-post EPD was strongly associated with NI post-LPJ with OR=44 (p=0.001) and predicted post-LPJ NI with a sensitivity (SN), specificity (SP), PPV, NPV of 80%, 91%, 80%, 91% respectively.
Conclusion: NI after EPD is associated with NI after LPJ. Failure to achieve NI post-EPD predicts failure to achieve NI post-LPJ. Larger prospective studies are needed confirm the predictive value of PD stenting for selection of pts for surgical intervention.
In Human Pancreas Exposed to Surgical Trauma and Hypoxia, Early Activation of Inflammation Markers Is Different in Acinar-cell Rich Compared to Fibrotic Pancreas
M. Laaninen, M. Bläuer, J. Sand, I. Nordback, J. Laukkarinen Dept. of Gastroenterology and Alimentary Tract Surgery, and Tampere Pancreas Laboratory, and *Dept. of Pathology, Tampere University Hospital, Tampere, Finland
Introduction: Postoperative pancreatitis often precedes other complications after pancreato-duodenectomy operation. We have shown that the risk for postoperative complications increases significantly, when there are more than 40% of acinar cells in the transsection line of pancreas. We hypothesized that the intra-operative pancreatic injury leads to immediate activation of inflammation cascade in the transsection line of the remnant of pancreas, and that this activation might be different in acinar cell-rich compared to fibrotic pancreas.
Objectives: The aim of this study was to analyse the expression of inflammation markers in the transsection line of human pancreas after pancreato-duodenectomy both in acinar cell-rich and fibrotic pancreata.
Materials and Methods: In pancreato-duodenectomy operation, several pancreatic samples from six patients, 3 with acinar cell-rich and 3 with fibrotic pancreas, were exposed to surgical trauma, and thereafter to hypoxemia for 15 min, 1h, 2h, 4h or 6h, to mimic the conditions at the transsection line of the pancreas remnant in the patient. Immunohistochemical analysis of inflammation markers was performed on formalin-fixed, paraffin-embedded samples.
Results: In the acinar cell-rich pancreata, intra-acinar cell NFkB -activation, and intra-acinar and intra-ductal MCP-1 expression increased from mild/none at 15 min to high during the first 4 hours. Also in the fibrotic pancreata, intra-acinar cell activation of NFkB and intra-acinar and intra-ductal cell expression of MCP-1 were detected during the 6h monitoring, but due to the fewer overall number of acinar cells, the tissue expression of these markers remained lower.
Conclusions: In human pancreas which is rich in acinar cells the inflammation cascade begins almost immediately after induction of injury by surgical trauma and hypoxemia. Fibrosis may protect the pancreas from developing clinically relevant inflammation.
HER2 Mediated MUC4 Expression and Role of MUC4-HER3 Interaction in Pancreatic Cancer Proliferation
I. Lakshmanan,1 M.P. Ponnusamy,1 V.S. Gnanapragassam,1 P. Seshacharyulu,1 S. K. Batra,1,2,31Department of Biochemistry and Molecular Biology, 2Eppley Institute for Research in Cancer and Allied Diseases, 3Department of Pathology, University of Nebraska Medical Center, Omaha, Nebraska, U.S.A
Background: MUC4 is a type I transmembrane glycoprotein shown to be involved in pancreatic cancer (PC) progression. Previously, we have reported that MUC4 interacts with HER2 and potentiates aggressiveness of PC cells. However, the synergistic role of MUC4 and HER2 in PC has not yet been elucidated.
Method: HER2 expression was stably silenced in PC cells by specific shRNA followed by in vitro and in vivo studies (control and HER2 knockdown cells were subcutaneously injected into immunodeficient female nude mice (1 × 106 cells/mouse). Interaction of MUC4 with other EGFR family members (EGFR, HER3 and HER4) was analyzed by Reciprocal co-immunoprecipitation method.
Results: Stable silencing of HER-2 in HPAF/CD18 and Capan-1 cells resulted in significant upregulation of MUC4 expression (2.5 Fold changes) associated with increased proliferation of PC cells in vitro (p<0.05) and increased tumorigenicity in vivo (p<0.05). Reciprocal co-immunoprecipitation analysis revealed that MUC4 predominantly interacts with HER3 in PC cells. Additionally, the expression of proliferation marker c-Myc was significantly upregulated (1.5 fold changes) in HER2 knockdown cells.
Conclusion: Loss of HER2 leads to increased expression of MUC4 and promotes aggressiveness of PC cells through activation of c-Myc. Further, HER3 may participate in PC progression through interactions with MUC4.
Next Generation Sequencing Identifies Multiple, Complex Etiologies In An Idiopathic Hereditary Pancreatitis Kindred
J. LaRusch, M.M. Barmada, S. Solomon, D.C. Whitcomb University of Pittsburgh, Department of Medicine
Pancreatitis is a complex inflammatory syndrome variably associated with heavy alcohol consumption, smoking, mutations in PRSS1, CFTR, SPINK1, CTRC, and CASR, and copy number variants in PRSS1.
We evaluated a large, multigenerational family with pancreatitis that was typical for hereditary pancreatitis. Direct sequencing of all exons of PRSS1 and exons 2-3 of SPINK1 resulted in no apparent familial mutation. To determine whether this family had undetected variants in known pancreatitis susceptibility genes, we applied whole exome sequencing (WES) as a screening method, focusing on PRSS1, SPINK1, CFTR, CTRC, and CASR.
We identified multiple, complex etiologies in affected cases with epistasis between common and rare mutations in CFTR and SPINK1, copy number variants in PRSS1, and smoking but not alcohol. These data illustrate the utility of WES to screen for rare mutations in known genes in epistasis and to provide guidance on the practical application of next generation sequencing for personalized medicine.
An important application of genomic sequencing is in the guidance of clinical decision-making, particularly in managing complex chronic inflammatory disorders. In this study, we used WES to screen for underlying genetic risk for pancreatitis by focusing on known pancreatitis susceptibility genes. WES saved significant time at about the same cost compared with direct sequencing and copy number analysis of individual candidate genes. Even within a single pedigree, there were multiple genetic etiologies of pancreatic disease, underlining the need for complete individual genetic workup for effective prognosis and therapy.
The Second Most Common PRSS1 Haplotype Lowers Trypsin Expression and Reduces Pancreatitis Risk
J. LaRusch, L. Orlichenko, V. Singh, D.C. Whitcomb University of Pittsburgh, Department of Medicine
Trypsin is the master enzyme of the pancreas encoded by the trypsinogen gene (PRSS1). Mutations that result in uncontrolled trypsin activity lead to hereditary pancreatitis (HP). We hypothesize that mild variations in PRSS1 that result in variable exogenous trypsin expression levels may play a role in other pancreatic etiologies as well.
We genotyped all cases and controls in the NAPS study for variations in PRSS1, with complete data in 952 cases and 646 controls. We compared the frequencies of common SNPs between controls and cases in subsets of patients and controls with and without genetic or environmental risk factors. We then performed quantitative rtPCR of PRSS1, PRSS2 and CTRC from cDNA obtained from 11 different pancreatic surgical samples and compared relative gene expression between PRSS1 haplotypes.
We identified 4 common SNPs that did not change the protein sequence, but formed two frequent haplotypes that account for the majority of alleles (Hap1 60.5%, Hap2 35.4%). Germline HP mutations in PRSS1 were not significantly associated with either haplotype. The Hap1 allele was identified more frequently in cases than controls (OR 1.31 p=0.001), and the Hap2 allele less frequently (0.71 p=0.002). Hap2 carrier status was a significant protective factor among the entire cohort, especially against chronic disease and those with genetic lesions in CFTR or SPINK1, but not heavy smokers or drinkers.
Pancreatic tissue samples with at least one Hap2 allele showed lower relative PRSS1 cDNA levels than hap1 homozygotes, indicating that these haplotypes may result in different trypsin expression levels.
We have identified a common PRSS1 haplotype that is a significant protective factor against genetic pancreatitis by reducing trypsin expression levels.
Large Pancreatic Cystic Neoplasms are Not Associated with Current Biomarkers of Malignancy
L.S. Lee, V. Kadiyala, S. Mehta, P.A. Banks, D. Conwell Center for Pancreatic Diseases, Brigham and Women's Hospital, Boston, MA
Background: Predictors of malignancy in pancreatic cystic neoplasms include symptoms, pancreatic main duct dilation, mural nodularity, and cyst size. Cysts ≥3 cm are believed to correlate with malignancy, and this impacts current management and practice guidelines.
Aim: To identify clinical, endosonographic, laboratory, and pathologic factors that are associated with pancreatic cyst size ≥3 cm or <3 cm.
Methods: Retrospective study performed at a Pancreas Referral Center. All subjects underwent endoscopic ultrasound-guided fine needle aspiration (EUS-FNA) of pancreatic cystic neoplasms from 6/2006 to 5/2011. Data collected include: demographics (age, gender, race), symptoms, EUS findings (size, septation, nodularity), laboratory data (cyst fluid CEA, k-ras, LOH, cytology), and surgical pathology (malignancy defined as high-grade dysplasia, carcinoma in situ, and invasive carcinoma). Univariate and multivariate logistic regression analyses were performed to identify predictive variables.
Results: 239 patients with pancreatic cysts had EUS-FNA; 68/239 (28.5%) were ≥3 cm. Mean age was 63.7 yr with 66% (157/ 239) female and median follow-up 10 months. Evaluation of Pancreas Cyst Size: symptoms (p= 0.001) and absence of k-ras mutation (p=0.043) were associated with cysts ≥3 cm while malignant pathology was not (p=0.315). Only symptoms (p=0.008) remained associated with cysts ≥3 cm in multivariate logistic regression analysis when controlling for age, gender, and race.
Conclusion: Pancreatic cystic neoplasms ≥3 cm are not associated with malignancy and k-ras mutation compared to cysts <3 cm. Larger pancreatic cysts are more likely to be symptomatic. Improved biomarkers are needed to guide management of pancreatic cystic neoplasms regardless of cyst size.
Function and Regulation of Polo-Like Kinase 3 in Human Pancreatic Cancer
Z. Li, Z. Chang, P.J. Chiao Department of Molecular and Cellular Oncology, The University of Texas M. D. Anderson Cancer Center, Houston, Texas, USA
Pancreatic cancer is currently the fourth leading cause of cancer death in the United States. Despite the recent advances in pancreatic cancer research, patients with this devastating disease still have a poor prognosis. Therefore, deciphering pancreatic cancer-related gene expression and defining the individual gene's specific functions will make a significant impact on the diagnosis and treatment of pancreatic cancer. Recently, our lab has demonstrated the tumorigenic transformation in an immortalized pancreatic cell line, HPNE/hTERT by sequential introduction of multiple genetic alterations. We found that Polo-like kinase 3 (Plk3) is significantly down-regulated in transformed tumor cell lines. Plk3 belongs to the Polo-like kinase family and is a multi-functional kinase. We previously reported that Plk3 played a pro-apoptotic role in pancreatic cancer lines when it was overexpressed. Plk3 down-regulation in transformed HPNE cells suggests that Plk3 may have tumor suppression function during pancreatic tumor development. In addition, we found Plk3 expression is regulated by Pten. In Pten-knockout MEF cells, Plk3 expression was significantly down-regulated. So Plk3 may be downstream of Pten in the tumor suppression pathway. These data suggest that Plk3 play an important role in pancreatic cancer development.
Estrogens May Increase the Probability of Pancreatitis and Reduce Pancreatic Function in Women with Chronic Abdominal Pain, Possibly Independent of Triglycerides
J. Lieb II,1 C. Curington,2 P. Toskes,2Divisions of Gastroenterology, Universities of 1Pennsylvania, Philadelphia, PA and 2Florida, Gainesville, FL
Introduction: The relationship between estrogens and acute pancreatitis (AP)/acute relapsing pancreatitis (ARP)/chronic pancreatitis (CP)/pancreatic function outside of triglycerides is not well characterized.
Aims: To determine if exogenous estrogen use is related to (1)pancreatitis, (2) peak bicarbonate (PB) and (3)triglycerides.
Methods: Clinical information on 224 patients who had undergone Secretin Stimulation Testing (SST) for chronic abdominal pain was recorded into a retrospective database. The database was queried for females with available medication histories, who were then divided into 2 groups: Those taking estrogens (E) and those not on estrogens (N). Mann Whitney U, t-, and Fisher's exact testing were used.
Results: 70 of the patients in the database were females with available medication histories. 35 were taking estrogens. 19 in the E group and 15 in the N group had CP by clinical criteria, p = 0.23. Relative Risk 1.27 95% CI (0.8-2.1). 29 of the E's experienced any type of pancreatitis (AP, ARP, CP) while only 19 of the N's did, p = 0.0194. RR 1.52 (1.08-2.14).
17 pts in the E group and 16 in the N group had available lipid profiles with mean triglycerides of 280±250mg/dL and 286±198mg/dL respectively, p =0.631. Only 2 of the E and 3 of the N were believed-as documented in the pancreatic clinic notes-to have had pancreatitis from hypertriglyceridemia.
During SST, the PB levels for E and N patients respectively were 80 ±18 and 90±23 mEq/L, p =0.058. Total volume was not significantly different (212±91vs 213±107ml, respectively p=0.96). When patients with any type of pancreatitis were excluded, E's still displayed decreased PB levels in response to secretin: 90±18 vs. 104±19mEq/L, p=0.0213. Total volumes were still similar at 208.6±95 vs 208.3±72ml, p =0.945.
Among the 2 groups (E and N respectively), weight (kg) (65.9+/-15.5 and 64.8 +/- 16.7) and age (yrs) (52.2+/-10.9 and 47.6 +/-15.6) did not differ significantly.
Conclusions: These data suggest exogenous estrogens may be related to the development of AP and ARP, and probably to a lesser degree CP, perhaps through a triglyceride independent mechanism such as by proinflammatory pathways. During SST, PB-but not volume-production may be diminished in women on estrogens-even in those who have never had pancreatitis. Further study is necessary to better define the relationship between estrogen use, pancreatitis, and pancreatic function.
Bi-shRNAPDX-1 Targeting PDX-1 Therapy Suppresses Tumor Growth and Prolongs Survival in a Human Pancreatic Cancer Mouse Model
S. Liu,1 Z. Wang,2 K.M. Stehling,1 D.D. Rao,2 G. Zhou,1 P.B. Maples,2 J. Nemunaitis,2,3 W. Fisher,1 F.C. Brunicardi,1* 1Michael E. DeBakey Department of Surgery, Baylor College of Medicine, Houston TX. 2Gradalis, Inc, Dallas, TX; 3Mary Crowley Cancer Research Center, Dallas, TX
Introduction: This study aims to explore a new strategy for PDX-1 targeted silencing therapy for metastatic pancreatic cancer by using a unique RNAi bi-functional shRNA (bi-shRNA) platform.
Methods: Bi-shRNAPDX-1 vector was cloned and screened using Western blot. Cell proliferation was determined by MTS after bi-shRNAPDX-1 and pSUPER shRNAPDX-1 transfection of PANC-1 cells. Following i.p. injection of 106 PANC-1 cells in SCID mice, mice received 3 biweekly iv cycles of either a) liposomal bi-shRNAPDX-1 or b) empty vector (n=30 mice/group). Glucose and insulin levels were monitored biweekly. Immunostaining and TUNEL assay were performed on tumor and islets. Survival analysis was performed using Kaplan-Meier in SPSS statistical software (p<0.05).
Results: Bi-shRNAPDX-1 resulted in more efficient knock down of PDX-1 expression than pSUPER shRNAPDX-1 with greater inhibition of cell proliferation (p<0.05). 3 cycles of iv bi-shRNAiPDX-1 resulted in remarkable suppression of tumor growth (51 vs 1200mm3 controls (p<0.05), greater survival (115d vs 85d controls with no differences in glucose and insulin levels. Immunostaining and TUNEL assay revealed decreased PDX-1 expression, disruption of cell cycle proteins, increased cell apoptosis in residual tumor cells.
Conclusion: Multiple cycles of L-bi-shRNAPDX-1 therapy effectively ablate tumor cells from SCID mice bearing PANC-1 tumor and prolong mice survival. This study demonstrates feasibility to use L-bi-shRNAPDX-1 in a phase 1 trial for metastatic pancreatic cancer.
Risk Factors of Resectability for Patients with Pancreatic Cancer: A Multivariate Analysis
H. Lu, Z. He, W. Hu, B. Tian, Z. Zhang Department of Hepato-bilio-pancreatic Surgery, West China Hospital, Sichuan University, Chengdu 610041, Sichuan Province, China
Objectives: Surgical resection at present offers the only chance of cure for the patients with pancreatic cancer. Unfortunately, most patients lost the opportunity for curative resection when diagnosed. The aim of the study was to investigate whether there are some related factors associated with the unresectability.
Methods: Retrospectively, the medical records of all the patients diagnosed with pancreatic cancer in a single institute during a 5-year period were reviewed. A total of 207 patients were included in this study. Univariate and multivariate analysis were performed to identify the potential factors contributing significantly to unresectability.
Results: Only 44 patients in 207 (21.3%) underwent curative resection. Our results show that the following factors were related to unresectability: history of diabetes [odds ratio (OR) 5.44, 95% confidence interval (CI) 1.23-24.16]; carbohydrate antigen 19-9 (CA19-9) (≥150 U/ml) (OR 1.69, 95% CI 1.04-2.72); American society of anesthesiologists (ASA) class (OR 5.91, 95% CI 3.10-11.27); TNM stage (OR 3.03, 95% CI 1.72-5.34).
Conclusion: Resectability of patients with pancreatic cancer is affected adversely by the presence of high level of CA19-9, history of diabetes, advanced ASA class and TNM stage.
Integrated Traditional Chinese and Western Medicine for Severe Acute Pancreatitis
H.M. Lu,1 Q. Guo,1 W.M. Hu,1 B.L. Tian,1 X.B. Liu,1 G. Mai,1 Z.W. Huang,2 G.Y. Chen,2 W.F. Tang,2 X.D. Jin,3 Q. Xia,2 Z.D. Zhang,11Hepato-bilio-pancreatic Surgery, West China Hospital, Sichuan University, Chengdu 610041, Sichuan Province, China, 2Department of Integrated Traditional Chinese and Western Medicine, West China Hospital, Sichuan University, Chengdu 610041, Sichuan Province, China, 3Intensive Care Unit, West China Hospital, Sichuan University, Chengdu 610041, Sichuan Province, China
Objectives: Traditional Chinese medicine (TCM) is considered to be an alternative therapy for acute pancreatitis, and the outcome of the disease may be improved by integrating TCM and Western medicine approaches, based on the results of certain trials. The aim of this study was to investigate the efficacy of integrated TCM and western medicine for treating severe acute pancreatitis (SAP).
Methods: In this study, we reviewed the medical records of 9421 patients who were admitted with a diagnosis of acute pancreatitis from 2002 through 2009. Basic information and therapeutic data were collected for analysis, and 4417 of the 9421 patients were determined to have presented with SAP. The primary end point was death or surgery.
Results: Of the 4417 SAP patients who were identified, 3390 received integrated TCM and Western medicine therapy, and the mortality and surgery rates were 4.0% (135/3390) and 8.2% (278/3390), respectively. In contrast, in the remaining 1027 SAP patients who received only Western medicine, the mortality and surgery rates were 13.0% (135/1027) and 13.3% (134/1027), respectively. Thus, the integrated TCM and Western medicine approach yielded significantly lower surgery and mortality rates (P <0.001).
Conclusion: An integrated TCM and Western medicine approach reduced the surgery and mortality rates among patients with SAP when compared with Western medicine only.
PLGA/Poloxamer Nanoparticle-Encapsulated MBD1-siRNA Effectively Inhibits Pancreatic Cancer Growth in vivo
G. Luo, X. Yu,* J. Long Department of Pancreatic & Hepatobiliary Surgery, Shanghai Cancer Center, Fudan University, Shanghai, China
Objectives: Methyl-CpG binding domain protein 1 (MBD1) is a transcriptional regulator that binds the methylated CpG islands of tumor suppressor genes and represses their transcription. In our previous study, we found that MBD1-siRNA plasmid loading PLGA/poloxamer nanoparticles (M-NPs) exert therapeutic effect on human pancreatic cancer cell growth in vitro. Our current study will focus on testing the inhibitory effect of M-NPs on pancreatic cancer growth in vivo.
Methods: M-NPs were prepared by a modified solvent diffusion technique, and the distribution of nanoparticles in tumors was examined by transmission electron micrography and fluorescence microscopy. Tumor animal models were established by subcutaneous injection of pancreatic cancer cells at subaxillary. Tumor volume was measured weekly. The transfection efficiency, gene expression level and anti-tumor effect of M-NPs in vivo were further evaluated.
Results: The mean size of M-NPs was 210.1±24.3 nm and the distribution of nanoparticles in tumors was confirmed. MBD1 protein expression was decreased after day 2. With immunostaining, the presence of DNA fragments resulted from apoptosis can be observed. An apoptotic rate of 21.53% in the M-NPs treated group was found to be higher than that of the control group. Moreover, the tumor volume in the M-NPs treated group was found to be much smaller than that of the controlled group (P=0.0059).
Conclusions: Our data suggest that MBD1 is a novel therapeutic target in pancreatic cancer with PLGA/poloxamer nanoparticle being a promising delivery vehicle in cancer treatment.
Urinary 1H-NMR Metabolomics Discriminates Patients with Acute and Chronic Pancreatitis from Healthy Controls
E.R. Lusczek,1 J.A. Paulo,2 J.R. Saltzman,2 V. Kadiyala,2 J. Rosenblum,2 P.A. Banks,2 D.L. Conwell,2 G. Beilman,11Department of Surgery, University of Minnesota, Minneapolis, MN; 2Center for Pancreatic Disease, Brigham and Women's Hospital, Harvard Medical School, Boston, MA
Background: The characterization of the metabolic response in pancreatitis is a potentially useful strategy to gain insights into the cellular and subcellular effects of pancreatic injury in acute and chronic pancreatitis.
Aim: To profile and compare urinary metabolites in patients with acute and chronic pancreatitis.
Methods: Urine was obtained from healthy controls (n=5), inpatients with mild acute pancreatitis (AP, n=5), and outpatients with chronic pancreatitis (CP, n=5). Proton nuclear magnetic resonance (1H-NMR) spectra were obtained for each sample. Urinary metabolites were characterized in each spectrum with Chenomx software (Chenomx, Edmonton AB). Resulting metabolite concentrations were normalized to account for differences in dilution between samples. Principal Components Analysis and Mann-Whitney U tests were performed with R software (http://cran.r-project.org/) and SPSS (Armonk, NY) respectively.
Results: A total of 60 metabolites were identified and quantified. Three urinary metabolites (citrate, adenosine and acetone) differentiated between controls and patients with AP and CP. Citrate is decreased and adenosine is increased in patients with AP and CP when compared with controls (p<0.05). Acetone is increased in the urine of CP as compared to AP (p=0.03). Principal components analysis of the entire urine metabolic profile separates controls from both AP and CP.
Conclusions: This exploratory metabolomics investigation demonstrates that there are differences in the urinary metabolome between healthy controls and patients with acute pancreatitis and chronic pancreatitis.
Relationship Between Exocrine Pancreas and Small Intestine L-Glutamine and L-Glutamate Transport
C. Lutz,1 L. Mariotta,1 K. Huggel,1 B. Herzog,1 F. Verrey,1 T. R. Graf,2 R. Graf,2 T. Lahoutte,3 S.M.R. Camargo,11Institute of Physiology and ZIHP, University of Zurich, Zurich, Switzerland; 2Department of Surgery, University Hospital Zurich, Zurich, Switzerland; 3ICMI, Vrije Universiteit Brussel, Brussels, Belgium
The amino acids L-glutamine (Gln) and L-glutamate (Glu) are an important source of energy for intestine and pancreas. The pancreas efficiently absorbs amino acids for the synthesis of enzymes, but also secrets free amino acids in the pancreatic juice which reach the small intestine and are (re-)absorbed. Glu concentration in pancreatic juice was shown to be increased after intravenous administration of Gln and to be more concentrated in the pancreatic juice than in plasma. We hypothesize that the dietary protein content could modulate the expression of Gln and Glu transporters in pancreas and small intestine. The mRNA expression of Gln transporters (snat2,3,5 (slc38a2,3 and 5), Lat1 (slc7a5), Lat2 (slc7a8)), and Glu transporters (EAAT1-3 (slc1a3,2,1), xCT (slc7a11)) was analyzed in the pancreas and small intestine of mice fed with free (PFD), high (HPD), or normal-protein diet (NPD). The plasma levels of Gln increased after PFD and HPD when compared to NPD, whereas the Glu plasma concentration tended to be lower in animals receiving PFD. The pancreatic expression of Na+-dependent (snat5) and -independent Gln transporter (Lat1) mRNAs was higher in PFD, while the Glu transporter mRNAs were unchanged. In intestine, the level of luminal enterocyte Glu transporter (EAAT3) mRNA was increased. These preliminary results suggest a recycling of Gln and Glu between the pancreas and the intestine. The on-going analysis of the intestinal protein expression and of the pancreatic juice composition will help us to identify the underlying mechanism of this collaboration.
Dietary Regulation of Amino Acid Transporter Expression in Exocrine Pancreas: Effect of Low Protein Diet
C. Lutz,1 L. Mariotta,1 K. Huggel,1 B. Herzog,1 F. Verrey,1 T. R. Graf,2 R. Graf,2 T. Lahoutte,3 S.M.R. Camargo,11Institute of Physiology and ZIHP, University of Zurich, Zurich, Switzerland; 2Department of Surgery, University Hospital Zurich, Zurich, Switzerland; 3ICMI, Vrije Universiteit Brussel, Brussels, Belgium
Dietary protein intake can modulate the secretion of exocrine pancreas, but the effect of dietary protein levels on the transport of amino acids into acinar cells is not known. The aim of this study is to analyze the effects of a chronic and acute low protein diet on the expression levels of L-glutamine (SNAT2, 3 and 5), branched chain (BCAA) and aromatic amino acid (TAT1, LAT3 and LAT4) transporters in mouse exocrine pancreas. In mice fed for 4 days a low protein diet (7% casein) we observed a decreased level of BCAA and aromatic amino acid transporter mRNAs (TAT1, LAT3 and LAT4) as well as a non-significant reduction in the expression of mRNAs encoding glutamine transporters (SNAT2 and 3). In contrast, at the end of a chronic low protein treatment (5 weeks), the level of BCAA transporters (LAT3 and LAT4) mRNAs was clearly increased and that of glutamine transporters (SNAT2, 3 and 5) non-significantly. A short treatment of the animals with a protein-free diet had diverging effects on the expression of different transporter mRNAs. The variable impact of the diets on amino acid transporter mRNA expression might be secondary to differences in plasma amino acid concentrations. This new insight might lead towards an improved understanding of the mechanism that regulates the changes in metabolism during low protein diet or even to the identification of amino acids to be used as supplements in pathological states.
Mast Cells Promote Fibrotic Tumor Microenvironment of Pancreatic Ductal Adenocarcinoma
Y. Ma,1 R. Hwang,2 C. Logsdon,3 S.E. Ullrich,1Department of 1Immunology, 2Surgical Oncology, 3Cancer Biology, University of Texas M.D. Anderson Cancer Center, Houston, TX
Aim & Background: Pancreatic ductal adenocarcinoma (PDAC) is notoriously resistant to most therapies. In the patient, cancer cells exist in a complex microenvironment containing pancreatic stellate cells and immune cells. These components of the microenvironment provide a fibrotic niche that is an impediment to successful cancer therapy. We and others have found that mast cells are essential to PDAC tumorigenesis. Whether mast cells contribute to fibrosis is unknown. Here we test the hypothesis that mast cells contribute to the fibrotic stromal response that promotes PDAC development.
Methods: In an in vitro co-culture system, we quantified the crosstalk of human mast cells, PDAC cells, and pancreatic stellate cells (HPSCs). Effects of mast cells on PDAC cells and HPSC were measured by proliferation, migration, and flow cytometry. To determine whether targeting mast cell migration has a therapeutic effect in vivo, we treated tumor-bearing mice with AMD3100, which inhibits mast cell migration.
Results: Tumor cells promoted mast cell migration to the tumor site (P<0.05). Treatment with AMD3100 depressed PDAC growth (P=0.0045), and promoted increased survival (P=0.014). PDAC cells and HPSCs stimulated mast cell activation, as measured by degranulation and tryptase release (P<0.05). Conversely, conditioned media from mast cells stimulated PDAC proliferation (P<0.001). Mast cells promoted HPSC proliferation by direct cell-cell interaction.
Conclusions: Tumor cells promote mast cells activation and migration to the tumor site, where they activate HPSC proliferation and the fibrotic tumor microenvironment found in PDAC. Therefore, therapy targeting mast cells may overcome stromal formation and improve PDAC therapy.
The Novel Sterol Guggulsterone Modulates Novel microRNAs Targeting MUC4 Expression in Pancreatic Cancer Cells
M. A. Macha, S. Rachagani, S. Chakraborty, M. P. Torres, M. P. Ponusamy, S. K. Batra Department of Biochemistry and Molecular Biology, University of Nebraska Medical Center, Omaha, NE, U.S.A
Background and Objective: Resistance of pancreatic cancer (PC) cells to chemo-therapeutic agents has led to the search for novel anti-cancer agents. The glycoprotein MUC4 is aberrantly expressed in and contributes to PC progression, making it a novel target for PC therapy. microRNAs (miRNA), short non-coding RNAs have also emerged as novel targets in cancer therapy. In this study, we investigated the effect of Guggulsterone (GS), a farnesoid X receptor antagonist on PC cells behavior and its ability to modulate expression of MUC4 and select MUC4 targeting miRNAs.
Methods: MUC4 expressing PC cells (HPAF/CD18 and Capan-1) were incubated with GS for upto 72 hrs to determine the IC-50. The effect of the dose corresponding to IC-50 was then investigated on PC cell behavior through various functional assays. Effect of GS treatment on expression of MUC4 and select MUC4 targeting miRNAs was assessed by western blotting and real time PCR respectively.
Results: At a dose of 50 μM (i.e. the IC-50), GS significantly downregulated MUC4 expression in PC cells in a time-dependent manner (maximum effect at 48 hrs post-treatment). It also induced apoptosis, and significantly inhibited motility and invasion in PC cells (p<0.001). In-silico analysis with web-based miRNA target prediction software (http://www.mirz.unibas.ch/ElMMo/ and Human miRNA Targets (http://www.microrna.org) identified three novel miRNAs, Let-7b, miR-345 and miR-96 with potential binding sites to the 3' UTR of MUC4. GS treatment upregulated the expression of Let-7b but had no effect on either miRNA-345 or miRNA-96.
Conclusion: GS downregulates MUC4 possibly through upregulation of miRNA Let7b. Its anti-cancer effects suggest potential utility as a novel therapeutic agent in PC.
Histone Deacetylase Inhibitor Valproic Acid Inhibits MiaPaCa2 Cells Growth in vitro and in vivo: a New Promising Class of Anti-neoplasic Agents in Pancreatic Cancer?
M.C.C. Machado, M.S. Kubrusly, N.T. Molan, L.A.C. D′Albuquerque Department of Gastroenterology (LIM-37), School of Medicine, University of São Paulo, São Paulo, Brazil
Introduction: Pancreatic adenocarcinoma is a dismal disease without effective treatment therefore new therapeutic strategies are needed. Valproic acid (VPA) has been used as a therapeutic drug for epilepsy and other neuropsychiatric disorders. VPA is a histone deacetylase inhibitor being currently used in clinical trials for various cancers.
Aim: To investigate the effect of VPA in vitro and in vivo on pancreatic cancer cells.
Methods: Human pancreatic cancer cell lines MiaPaCa2 were cultured. Viability of MiaPaCa2 cells (20mM of VPA) was determined using MTT assay. Cell cycling was examined by FACS analysis. The animals were divided into two groups of 11 animals each (treated and non-treated groups). Cells were injected subcutaneously into the lateral flank of 11 athymic male BALB/c mice. The treated group received orally 16mg daily of VPA for 30 days. Tumor size was measured every 3 days, and tumor volumes calculated. Mice were sacrificed and the tumors were again measured.
Results: VPA treatment results in statistically significant reduction of tumor xenograft growth in treated group (V= 28.4mm3) when compared with non-treated group (V= 503mm3) (p=0,0030). Treatment with VPA resulted in a significant reduction (45%) of viability in cultivated MiaPaCa2 cells compared with non-treated cells. Cell cycle evaluation demonstrated a significant increasing in DNA degradation in treated cells.
Conclusion: These results demonstrated that VPA inhibits the growth of MiaPaCa2 cells in vitro and in a mice xenograft model, suggesting that VPA is of value for further exploration as potential anti cancer agent targeting pancreatic adenocarcinoma.
Triptolide-Induced Expression of MicroRNA-142-3p Mediates Apoptosis of Pancreatic Cancer Cells by Inhibition of Heat Shock Protein 70
T.N. MacKenzie,1 S. Vickers,2,3 A.K. Saluja,1,2,31Department of Pharmacology; 2Department of Surgery; 3Masonic Cancer Center, University of Minnesota, Minneapolis, MN
Triptolide inhibits pancreatic cancer cell growth in vitro and blocks growth and metastatic spread in vivo. Our lab has shown that triptolide triggers apoptosis via inhibition of heat shock protein 70 (HSP70) expression. Triptolide is hypothesized to decrease HSP70 expression via inhibiting the transactivation of heat shock factor 1. As miRNAs are now recognized as powerful gatekeepers of apoptosis, we are testing whether triptolide may be inhibiting HSP70 via a novel mechanism of inducing the expression of HSP70-targeting microRNAs.
In this study we show that triptolide induces the expression of miR-142-3p and that this miRNA may play a pro-apoptotic role as its overexpression induces apoptosis via decreasing HSP70 expression. MicroRNA-142-3p was found by miRNA microarray and qRT-PCR to increase after triptolide treatment in MiaPaCa-2 and S2-013 cells. Overexpression of miR-142-3p decreases cell viability, triggers caspase-3 activation, decreases HSP70 expression and sensitizes. MiaPaCa-2 cells to triptolide treatment. To demonstrate direct binding between miR-142-3p and HSP70, we performed a luciferase reporter assay in HEK-293 cells co-transfected with a HSP70 3'UTR reporter vector (renilla) and a control vector (firefly). Overexpression of miR-142-3p decreases the renilla-to-firefly luciferase ratio significantly as compared to control.
These results show for the first time a miRNA mechanism of regulating HSP70 expression in cancer. Furthermore, we show a proof-of-principle for restoring miR-142-3p expression as a novel therapeutic strategy for triggering apoptosis and inducing chemosensitization of pancreatic cancer cells.
The Chemical Chaperon 4-Phenylbutyric Acid Reduces Hallmarks of Acute Pancreatitis as well as Endoplasmic Reticulum Stress in Rat
A. Malo, B. Goke, CH. Kubisch Department of Medicine II, University of Munich, Germany
Background: The accumulation of unfolded protein in the Endoplasmic reticulum (ER) early during acute pancreatitis (AP) initiates the unfolded protein response (UPR), is important in pancreatic development and function but also in the induction of pro-apoptodic signaling. Incubation with the fatty acid and ER-chaperon 4-phenylbutyric acid (4-PBA) showed a reduction in UPR and trypsin activation in pancreatic acini. Our study now aimed to examine the effects of 4-PBA on AP and the UPR.
Materials and Methods: Rats received an i.p. injection of 50μg/kg caerulein (Cer) for AP induction. Controls received saline. One half of the rats received 250mg/kg 4-PBA two hours prior i.p. UPR components (BiP expression, PERK phosphorylation, XBP-1 splicing, caspase 3 and JNK activation) were analyzed, as were effects on amylase secretion, trypsin activation, edema formation and myeloperoxydase (MPO) activation in lung tissue 0.5 to 4h after Cer injection.
Results: Cer caused an amylase increase, trypsin activation and edema starting 30min after injection. MPO started to rise at 1h. Cer injection after 4-PBA was followed by a significant smaller increase in trypsin activity (1122.12 vs. 574.6 fmol/mg), edema formation (89.1 vs. 82.6 %) and MPO activity (6.7 vs. 4.6 mU/mg). Amylase did not change. Cer increased BiP, PERK phosphorylation, XBP1 splicing, caspase 3 activation, and JNK phosphorylation. All of this was significantly less after 4-PBA treatment.
Conclusions: Preincubation with an ER-chaperone reduced hallmarks, as well as systemic inflammatory consequences of the AP. Several UPR elements and their pro-apoptotic signal partners were reduced by 4-PBA. Future efforts should be directed at understanding these mechanisms in the pancreas and the use in patients.
Inhibition of GSK3 Activity Promotes Autophagy in Pancreatic Epithelial Cells
B. Marchand, M.J. Boucher Gastro Unit, Dept. of Medicine, FMSS, University of Sherbrooke, Sherbrooke, Canada
Background: We have previously demonstrated that prolonged inhibition of GSK3 activity induces human pancreatic cancer cell death. Recently, in prostate cancer cells, it was suggested that inhibition of GSK3 promotes both apoptosis and autophagy, the latter playing a protective role against cell death. The AIM of the study was to evaluate whether GSK3 also controls autophagy in pancreatic epithelial cells and to determine whether inhibition of autophagy sensitizes these cells to GSK3 inhibition-induced apoptosis.
Methods: Experiments were performed using pancreatic cancer (PC) cell lines (PANC1 and MIAPaCa2) and the non-tumorigenic HPDE cells. GSK3 activity was inhibited by treatment with specific inhibitors: SB216763(20μM) or CHIR99021(5μM. Apoptosis was measured by assessment of PARP cleavage and caspase 3 and 7 activities. Autophagy was evaluated by the detection of the membrane-bound LC3B-II isoform. Inhibition of autophagy was achieved by treatment with the autophagy inhibitor 3-methyladenine 3-MA (10mM).
Results: 1-In PC cells, prolonged inhibition of GSK3 (48-72h) provoked autophagic and apoptotic responses. 2-Treatment of PC cells with the autophagy inhibitor elicited an apoptotic response and strongly promoted GSK3 inhibition-induced apoptosis. 3-Conversely, prolonged inhibition of GSK3 activity in HPDE promoted autophagy without inducing apoptosis. 4-3-MA treatment did not significantly affected HPDE cell viability but rendered HPDE cells responsive to GSK3 inhibition i.e. combined treatment with GSK3 and autophagy inhibitors induced apoptosis of HPDE cells.
Conclusion: Our data demonstrate that inhibition of GSK3 activity promotes autophagy in pancreatic epithelial cells and suggest that this autophagic response protects cells against GSK3 inhibition-induced apoptosis.
Lysosomal Dysfunction in Pancreatitis
O.A. Mareninova, I. Yakubov, I. Gukovsky, A.S. Gukovskaya VA Greater Los Angeles Healthcare System & University of California Los Angeles; and Southern California Research Center for ALPD and Cirrhosis, Los Angeles, CA
Background & Aims: We recently showed that lysosomal proteolytic function and autophagy is impaired in models of acute pancreatitis, mediating key pathologic responses of this disease. Here we further investigate the mechanisms underlying lysosomal dysfunction in pancreatitis.
Methods: We measured processing and activities of cathepsins and other lysosomal hydrolases; levels of lysosomal membrane proteins LAMP-1 and -2; and markers of early (EEA1) and late (Rab7) endosomes in rat and mouse pancreatitis induced by cerulein or L-arginine.
Results: Conversion of cathepsin (Cat)B and CatL proforms to their active mature (double-chain) forms was greatly inhibited in both cerulein and L-arginine pancreatitis, indicating impaired cathepsin processing. Pancreatitis also altered cathepsins' subcellular distribution. In control pancreas, activities of CatB and L, as well as the amounts of their active forms, were maximal in subcellular fractions enriched in late endosomes and lysosomes. Pancreatitis caused a decrease in both activities and the levels of CatB and L active forms in these fractions, with a concomitant increase in other fractions. These effects are not restricted to cathepsins, as we observed similar changes for alpha-mannosidase and alpha-galactosidase. With immunofluorescence, cathepsins in control pancreas localized to small juxtanuclear puncta, representing endo/lysosomes. In pancreatitis, CatL was localized to much larger structures distributed throughout the cell. We found a marked decrease in LAMP-1 and -2, as well as the markers of early and late endosomes, in cerulein pancreatitis.
Conclusion: The results demonstrate profound disturbances in key proteins regulating the function and structure of endo/lysosomes in experimental acute pancreatitis, indicating a possible causative role for these abnormalities in the pathogenesis of pancreatitis.
PUFAs Employ EP Receptors to Alter pAkt in HPDE/HPDE-Kras Cells
W. Mascariñas, M. Heiferman, M. Barron, M. Wojtanek, B. Mullapudi, T. Stratton, M. Tsao, D. Bentrem, P. Grippo Department of Surgery, Northwestern University
The fatty acids (FAs) Ω-3 and Ω-6 are known to have differential roles in cancer progression. Previously we have shown that Human Pancreatic Ductal Epithelial (HPDE) cells and HPDE cells with a Kras mutation (HPDE-Kras) treated with DHA, an Ω-3 FA, show a marked decrease in pAKT, a pro-survival factor in tumorigenesis. HPDE and HPDE-Kras cells treated with LA, an Ω-6 FA, showed only a marginal increase in pAKT. The mechanism modulating these events is poorly understood. The goal of this study is to identify a pathway that is crucial in establishing a link between these PUFAs and cell proliferation/apoptosis. Experiments with HPDE and HPDE-Kras cells and PGE3 showed a modest trend in suppression of pAkt. Treatment of HPDE and HPDE-Kras cells with DHA and an EP 1/2/3 inhibitor generated an increase in pAkt compared to cells without the inhibitor. Administration of DHA and an EP1 or EP4 inhibitor did not provide a significant difference compared to cells treated with DHA only. This suggests that there may be a mechanistic relationship between the EP2/3 receptors and DHA. Treatment of HPDE and HPDE-Kras cells with LA and an EP1 or EP4 inhibitor suggests that there is a possible mechanistic relationship between LA and these receptors. Oddly, suppression with an EP 1/2/3 inhibitor did not yield a significant change suggesting an antagonistic relationship between EP 1 and EP 2/3. Based on these EP inhibition studies, it is likely that FA metabolism yields increased levels of prostaglandins which could bind EP receptors. Ongoing work seeks to determine if fat metabolism is involved in the mechanism for pAKT attenuation/amplification, and future work will define the exact interaction between EP receptors and Akt in a high FA environment.
Human Pancreatic Cancer Cell Lines Derived from Metastatic Tumor in NOG Mice Possesses High-stemness Ability
Y. Matsuda,1 K. Yamahatsu,1,2 J. Ueda,1,2 K. Kawahara,1 M. Akiyama,1 M. Hagio,1 Z. Naito,1 T. Ishiwata,1Departments of Pathology and Integrative Oncological Pathology, Nippon Medical School, 2Surgery for Organ and Biological Regulation, Graduate School of Medicine, Nippon Medical School
Background: Pancreatic cancer has extremely high mortality rates due to rapid progression and a high incidence of metastases. Injection of human pancreatic cancer cells (PANC-1) in the spleens of NOD/Shi-scid/IL-2Rγnull (NOG) mice can highly metastasize to other organs. In this study, we established cancer cell lines from the tumors of metastasized organs, and analyzed cell behaviors including their stemness ability.
Methods: PANC-1 (1×105 cells) was injected into the spleen of NOG mice. The tumor cells of these mice at 8-weeks after injection were collected from metastatic livers and lungs. We repeatedly performed xenotransplantation using these cells three times in total, and biological and morphological characteristics of the established metastatic cell lines were compared with those of parental cells.
Results: In the metastatic cells, cell growth tended to be lower and cell migration was higher than those in parental cells. Metastatic cells exhibited polygonal and anaplastic appearances and showed high sphere-forming ability, one aspect of stemness ability. Nestin, one of the stem cell markers, showed significantly highly expression in liver and lung metastatic cells than in parental cells, and metastatic cells of the third xenotransplantation showed a higher nestin expression level than those of first xenotransplantation.
Conclusion: These findings suggest that these liver and lung metastatic cell lines formed by highly metastatic variants have different characteristics from parental cells. Stemness ability may play important roles in the metastasis of pancreatic cancer.
Imaging and Pathological Characteristics of Small (T2) Acinar Cell Carcinomas of the Pancreas: A Report of 3 Cases
S. Matsumoto, N. Sata, M. Koizumi, A. Lefor, Y. Yasuda Department of Surgery, Jichi Medical University
Introduction: Acinar cell carcinoma of the pancreas (ACC) is a rare malignant tumor of the exocrine pancreas accounting for less than 1% of primary pancreatic neoplasms. Most cases of ACC are detected at a late stage with large tumors and ultimately more than 50% of ACCs are unresectable. We present three cases of small (T2, <4cm) ACCs and discuss the clinical and pathological features.
Cases:[Case 1] A 34 year-old woman suffered from acute pancreatitis during her first pregnancy at age 29 and was found to have a small oblique hypodense lesion in the head of the pancreas. Since CT and MRI failed to demonstrate a distinct tumor, the lesion was observed and showed no change over four years. In her next pregnancy five years later, she had recurrent abdominal pain and CT revealed the lesion had become a 2.5cm iso- or hyperdense tumor. Enucleation was performed because the intra-operative pathological diagnosis was SPN. Final diagnosis showed an ACC with a positive surgical margin, and PpPD was then performed. [Case 2] A 61 year-old man suffered from acute pancreatitis and was found to have an encapsulated 30mm hypervascular tumor in the head of pancreas. PpPD was performed with an intra-operative diagnosis of an islet cell tumor but the final pathological diagnosis was ACC. The patient is alive without recurrence. [Case 3] A 66 year-old woman was incidentally found to have a 2cm hypervascular tumor in the tail of the pancreas. Distal pancreatectomy was performed and histology showed an ACC. Despite adjuvant chemotherapy and additional resection, peritoneal dissemination developed 16 months later and she died 30 months after resection.
Discussion: The endocrine counterpart of ACC is the pNET and the latest WHO classification ranges from benign to malignant pathological categories. However, the classification system for pancreatic exocrine tumors is poor and diagnostic options are limited to SPN and ACC. These three small ACCs were found to have common clinical features and two presented with benign-like behavior. ACCs should be classified more precisely according to their size and clinical behavior.
Neuropilin-1 Is an Important Therapeutic Target in Pancreatic Cancer
A. Matsushita,1 T. Götze,2 M. Kawamoto,1 Y. Nakamura,1 T. Aimoto,1 T. Ishiwata,3 Z. Naito,3 E. Uchida,1 M. Korc,21Department of Surgery, Nippon Medical School, Tokyo, Japan; and 2Departments of Medicine, and Pharmacology and Toxicology, Dartmouth Medical School, Hanover, NH, 03755
Introduction: Pancreatic ductal adenocarcinoma (PDAC) is a leading cause of cancer death and characterized by multiple transmembrane tyrosine kinase receptors and their ligands. Neuropilin-1 (Np-1) is a co-receptor for VEGF-A, that is also overexpressed in PDAC. The role of Np-1 in PDAC is less clearly defined.
Methods and results: PANC-1 pancreatic cancer cells were transfected with the Np-1 antisense cDNA. By comparison with sham transfected cells, Np-1 antisense expressing clones (Np-1AS) exhibited decreased adhesion and invasiveness. Immunoprecipitation followed by immunoblotting revealed that Np-1 associated with integrin β1, and integrin β1 blockade attenuated adhesion, mimicking the effects observed in Np-1AS clones. Furthermore, not VEGF-A, but HGF promotes cell invasion in PANC-1 dependent on Np-1.
Next, COLO-357 pancreatic cancer cells were stably transfected with the Np-1 cDNA. Overexpression of Np-1 was associated with enhanced invasiveness of the cells across a Matrigel membrane in response to HGF, and these effects were abolished by siRNA-mediated down-regulation of c-Met. Immunoprecipitation studies revealed that Np-1 associated with c-Met. By confocal microscopy, this association occurred on the plasma membrane, and HGF promoted the internalization of Np-1-c-Met complex. Third, function blocking Np-1 antibody study was performed. The antibody inhibited HGF-induced invasiveness.
Conclusions: Np-1 is a co-receptor of integrin β1 and c-Met that promotes pancreatic cancer cell adhesion and invasion. Targeting Np-1 may lead to novel therapeutic strategies in this deadly malignancy.
A Five-Marker Panel of Glycoforms for the Accurate Differentiation of Malignant from Benign Pancreatic Disease
K.A. Maupin,1 B. Fallon,1 K. Partyka,1 W. Tembe,2 Karen Kaul,3 Z. Feng,4 A.J. Moser,5 H. Zeh,5 M.A. Anderson,6 D. Simeone,6 R.E. Brand,5 B.B. Haab,11Van Andel Research Institute, Grand Rapids, MI; 2Translational Genomics Institute (TGen), Phoenix, AZ; 3Northshore University Health System, Evanston, IL; 4Biostatistics Program, Fred Hutchinson Cancer Research Center, Seattle, WA; 5Department of Gastroenterology, University of Pittsburgh, Pittsburgh, PA; 6Department of Surgery, University of Michigan Medical School, Ann Arbor, MI
Many patients have incidentally detected pancreatic abnormalities discovered by non-invasive imaging that have no clinical significance but are referred for further procedures. We sought to reduce this high rate of unnecessary referral through the development of an accurate, blood-based diagnostic biomarker. We used lectin and glycan-binding antibody detection of antibody arrays to systematically measure the suite of glycan structures that are related to the CA19-9 antigen in plasma samples from 121 pancreatic cancer patients (early and late stage) and 76 patients with pancreatitis and other benign conditions. Using newly-developed software that scans through all logical combinations of markers, a five-marker panel was discovered with 88% sensitivity 94% specificity for identifying cancer, superior to existing biomarkers for pancreatic cancer. The panel is based on simple combination rules between two CA19-9-related assays and three glycoforms of MUC5AC, a mucin that is elevated early in pancreatic cancer development. The performance of the panel is being validated in blinded sample sets from three different institutions. If validated, future prospective studies will focus on defining its role in a diagnostic setting such as those patients presenting with pancreatic abnormalities incidentally detected on abdominal imaging studies.
Blockage of CTLA-4 Indicates That Autoimmune Pancreatitis is a T-Cell Mediated Disease Responsive to Ciclosporin A and Rapamycin Treatment
J. Mayerle,1 T. Schwaiger,1 S. Zaatreh,2 J. Emmrich,2 B. Fitzner,2 A. Dummer,2 C. van den Brandt,2 H. Nizze,3 M. Evert,4 Tsalem,3 M.M. Lerch,1 R. Jaster,21Department of Medicine A, Ernst-Moritz-Arndt Universität Greifswald, Germany; 2Department of Medicine II, Division of Gastroenterology, University of Rostock, Rostock; 3Institute of Pathology, University of Rostock, Rostock, Germany 4Institute of Pathology, Ernst-Moritz-Arndt Universität Greifswald, Germany
Background: Autoimmune pancreatitis (AIP) responds to steroid treatment. However, the pathogenesis and alternative treatment options are poorly investigated.
Aims: We evaluated the pathogenesis and efficacy of alternative immunosuppressants in the MRL/Mp mouse model of AIP.
Methods: MRL/Mp-mice were pretreated for 4 weeks with polyinosinic:polycytidylic acid to trigger AIP. In a pilot study, the mice received daily injections of dexamethasone for two weeks, while poly I:C application continued. Subsequent therapeutic studies were performed for a period of four weeks, using the immunosuppressants ciclosporin A or rapamycin. Blockage of CTLA-4 was achieved by i.p. antibody treatment with 2 μg/g anti-mouse-CD152 and purified NA/LE Hamster-IgG1 as control. Severity of AIP was assessed by histopathology, FACS analysis, characterisation of the pancreatic inflammatory infiltrate, measurement of serum cytokine levels, and quantitative assessment of fibrosis.
Results: Blockage of CTLA-4 suppressed Treg function and raised the Teff response with subsequent histomorphological organ destruction. This indicates that autoimmune pancreatitis is a T-cell driven disease. Using a histopathological score, we found that dexamethasone (at 1 μg/g), ciclosporin A (40μ g/g) and rapamycin (1 μg/g; all drugs applied daily) significantly reduced pancreatic lesions. However, the beneficial effect of ciclosporin A and rapamycin was achieved by different mechanisms: ciclosporin A inhibits Teff activation and proliferation while rapamycin leads to selective expansion of Tregs subsequently suppressing the Teff response.
Conclusions: The calcineurin inhibitor ciclosporin A and the mTOR inhibitor rapamycin improve the course of AIP in MRL/Mp mice by different mechanisms. Both drugs specifically inhibit overall Teff-cell activation. While ciclosporin A has a direct effect on Teff, rapamycin leads to expansion of Tregs. This finding further supports the concept of autoreactive T cells as key players in the pathogenesis of AIP. We suggest that ciclosporin A and rapamycin should be considered for treatment of AIP in humans.
Myocardial Alteration in Experimental Acute Pancreatitis
A. Meyer,1 J. Jukemura,1 M. S. Kubrusly,1 A. M. Coelho,1 R. A. Patzina,2 V. Salemi,3 M. C. C. Machado,1 J. E. M. Cunha,1 C. Mady,3 L. A. C. D′Albuquerque,11Department of Gastroenterology (LIM-37), 2Department of Pathology, 3Heart Institute (InCor), University of São Paulo, São Paulo, Brazil
Introduction: Evidences suggest that proinflammatory cytokines (IL-1, TNF-α, IL-6 and IL-8) act as mediators of local and systemic manifestations in acute pancreatitis (AP) and correlate with the severity of the disease. The mechanisms of myocardial injury in AP are not completely understood. The production in situ into the myocardium of pro-inflammatory cytokines may lead to acute myocardial damage with functional changes and, eventually, chronic sequelae. TGF-β cytokine is responsible for the modulation of collagen synthesis (type I and III) by triggering the pancreas fibrogenesis and collagen deposition, playing an important role in regulating mechanism of cellular repair.
Aim: To evaluate the histological and functional changes of the heart in AP and to correlate with the production of cytokines in situ into the myocardium.
Methods: Adult Wistar rats were subjected to experimental AP induced by pancreatic duct infusion of Na-taurocholate. Myocardial function was evaluated by using echocardiography. Rats were sacrificed for biochemical and histological analysis and TGF- β, IL-6 and TNF-α gene expression study at 30 min; 2, 12 and 24h and 15 days.
Results: We observed an increased myocardial production of cytokines (TNF-α, IL-6, TGF-β), myocardial histological lesions associated with decreasing in diastolic and systolic function and ventricular compliance.
Conclusion: We concluded that in acute pancreatitis there is myocardial damage probably related to in situ production of myocardial pro-inflammatory cytokines followed by the increasing TGF-β gene expression related to the occurrence of fibrosis and cellular repair.
Contemporary Retrospective Review of Open Pancreatic Debridements - How Are We Doing?
M. Michailidou, G. Chiou, A. Warshaw, C. Fernández-del Castillo, P. Fagenholz Department of Surgery, Massachusetts General Hospital, Harvard Medical School, Boston, MA
Introduction: Pancreatic necrosis remains a serious complication of acute pancreatitis. As minimally invasive techniques for managing pancreatic necrosis evolve, it is valuable to assess the most contemporary results for open surgical debridement. We retrospectively reviewed all patients who underwent pancreatic debridement at our institution from 2005-2010.
Methodology: We identified 67 patients who underwent pancreatic debridement for necrotizing acute pancreatitis. Indications for surgery, evidence of infected necrosis, presence of organ failure, post-operative complications, and mortality were the main endpoints.
Results: Infected necrosis was the most common indication for pancreatic debridement (63.2 %). Overall mortality was 13%. Twelve (18%) patients required re-operation, and 27 (39.7%) required percutaneous drainage following debridement. New post-operative organ failure occurred in 23.5% (n=16). Infection was documented preoperatively in 63.2% of patients, but final cultures revealed infection in 75% of cases. Advanced age, etiology of pancreatitis, high APACHE score, presence of organ failure, intensive care unit admission, and early reoperation were all significantly linked to mortality in univariate analysis (p<0.05). Timing of debridement and presence of infected necrosis were not linked to mortality (p=0.907 and p= 0.302 respectively).
Conclusion: Mortality in this contemporary series of pancreatic debridements for acute pancreatitis compares favorably with published results for minimally invasive methods of treating pancreatic necrosis. Post-operatively, new organ failure, percutaneous drainage, and reoperation remain common. This series provides a reference point against which newer interventions can be compared.
Resection of Pancreatic Cancer invading the Portal Vein - a Case-Control Study
C. W. Michalski, S. Kloe, M. Erkan, H. Friess, J. Kleeff Department of Surgery, Technische Universität München, Munich, Germany
Introduction: Resection of pancreatic cancer invading the portal (splenic/superior mesenteric) vein has been controversially discussed. It is perceived that such resections are justified to achieve (macroscopic) complete removal of the tumor.
Methods: We conducted a prospective case-match study to investigate the outcome following resections of pancreatic cancer with venous infiltrations. 87 patients operated on between July 2007 and July 2009 were included into the study. In 47 patients, venous resections were performed; these were matched with 40 patients - in whom no venous infiltration was present - according to age, gender and operative technique as well as to grade, nodal and resection status of the tumor. Morbidity was registered according to the international study group of pancreatic surgery definitions on a daily basis until discharge. Patients were followed-up at three-month intervals until June 2011. Adjuvant chemotherapy was administered in most of the patients.
Results: Morbidity and mortality were comparable between the groups as was the length of hospital stay. Kaplan-Meier analysis demonstrated a median survival of 17.9 months in the group of patients in whom venous resections were performed versus 20.9 months in the conventionally resected patients (no statistically significant difference, log rank test, p=0.97).
Conclusion: Pancreatic cancers infiltrating the portal vein can be safely resected with a median survival comparable to a matched group of patients without venous invasion.
Prospective Application of the Four Category (Mild, Moderate, Severe, Critical) Classification of Acute Pancreatitis
C.J. Miranda, B.I. Babu, A.K. Siriwardena HPB Unit, Manchester Royal Infirmary, UK.
Introduction: Episode severity in acute pancreatitis (AP) follows a skewed distribution with the majority following a mild course. Accruing knowledge suggests that binomial (mild/severe) categorisation of the 1992 Atlanta consensus lacks accuracy as it does not allow for transient organ failure or distinguish critical from less acute forms of severe. The four division (mild, moderate, severe, critical) categorisation of AP has been proposed. This study carries out a prospective application of the system.
Methods: Patient-level data with critical care occupancy and outcome were collected in a consecutive series of 128 patients admitted with a clinical diagnosis of AP. The 4-category system was modified for practical use by adding the following descriptors: Pancreatic infection was indicated by radiological confirmation of gas in necrosis and/or positive cultures from fine needle aspiration of necrosis and a Marshall organ dysfunction score of ≥ 2 was defined as organ failure. The allocation system is intended for use early in the episode and the study examined application at day 2. Data were compared to1992 Atlanta (end-of episode). Data are presented as medians (range).
Results: 101 had mild AP: APACHE II 3 (0-8), inpt stay 4 days (2-96), 0 critical care and 0 mortality. 12 had moderate AP: APACHE II 10 (9-13), inpt stay 12 (3-17), (92-5) critical care and 0 mortality. 15 had severe AP: APACHE II 14 (10-23), 17 (0-53) critical care and 0 mortality. No patients fulfilled criteria for the critical category. If the 1992 Atlanta criteria are applied to the same data, 95 patients are mild and 33 severe.
Conclusion: The 4-category system can be used in clinical practice and it may provide better categorisation than the Atlanta composite "severe".
Pancreatic Cancer-Selective Adenovirus with Redesigned AB-loop Isolated via Adenovirus Library Shows Specificity to Mesothelin Expressing Cells
Y. Miura, J. Davydova, M. Yamamoto Department of Surgery, University of Minnesota, Minneapolis, MN
Pancreatic cancer is resistant to adenovirus (Ad) infection. Genetically coded viral capsid modification has achieved increased infectivity but not much target specificity. Direct selection of targeted vector from Ad library is a promising approach to overcome this issue, whereas it has not come to reality due to low diversity of library. We have established a novel Ad vector generating system enabling a huge-size Ad library. We hypothesize that Ad with redesigned AB-loop isolated by the screening of the new library system would result in a new class fiber structure for the gene therapy of pancreatic cancer. Ad AB-loop library was screened with Panc-1 based on the replication, and 2 dominant clones were selected. We analyzed the binding and the replication of the obtained clones with 3 pancreatic cancer cells and 293 cells. Panc-1 was the only cell line that the clones showed significant binding and replication. Thus, the clones shows strong binding and replication in the cells used for screening. After the re-cloning of fiber region into the new vector, the each clone was produced. One of the selected clone showed particular binding and replication capability which corresponded with MSLN expression. An intratumoral injection of this clone significantly suppressed the growth of Panc-1 subcutaneous tumors, whereas didn't suppress that of MiaPaca-2. In this study, we applied our novel library system to AB-loop region. Interestingly, the clone screened with Panc-1 exhibited selectivity to mesothelin expressing cells in vitro and in vivo. This new genetically modified Ad clone transductionally re-targeted to pancreatic cancer may embody a next generation targeting for this devastating disease in clinical settings.
Nicotine Enhances the Metastasis of Pancreatic Cancer via MUC4 Mucin Up-Regulation
N. Momi,1 M.P. Ponnusamy,1 S.K. Batra,1Department of Biochemistry and Molecular Biology1, University of Nebraska Medical Center, Omaha, NE, U.S.A
Background and Hypothesis: Studies have determined that smokers have a 2.5 times greater risk of developing pancreatic cancer (PC) compared to non-smokers. Interestingly, in our previous studies, nicotine and its metabolic derivative cotinine were detected in the blood and pancreata of smoke-exposed animals. Our previous investigations have revealed an aberrant expression of MUC4 mucin in pancreatic tumors compared to normal pancreas. Moreover, studies have demonstrated the direct association of colonic and gastric mucin secretion with nicotine uptake. Based on all the previous studies we hypothesized that nicotine-mediated pancreatic cancer pathogenesis is MUC4 dependent.
Results: Our results showed that nicotine significantly upregulates MUC4 mucin in PC cells. Nicotine treatment resulted in increased expression levels of α7nAChR and exhibited activation of JAK2/STAT3 downstream signaling cascade. On the other hand, treatment with α7nAChR subunit antagonists led to a reduction of MUC4 expression. Furthermore, MUC4 expression was abrogated by short interfering RNA (siRNA)-mediated inhibition of STAT3 expression, hence strengthening the relevance of STAT3 in MUC4 regulation. Interestingly, nicotine treatment imparted an increased migration potential to PC cells which was decreased by siRNA-mediated inhibition of MUC4. Increased migration of PC cells was accompanied by enhanced expression levels of pHER2, tHER2, c-Scr and pFAK. Additionally, our in-vivo studies showed significantly higher tumor metastasis from pancreas to various organs such as liver and lung, in the smoke-exposed mice orthotropically implanted with PC cells. Furthermore, pancreatic and liver tissues from smoke-exposed mice showed high expression of MUC4 as compared to the sham control. In the sham control, no tumor metastasis was observed at all, hence confirming our in-vitro observations.
Conclusion: Our study shows for the first time that nicotine upregulates MUC4 via α7nAChR subunit-mediated downstream signaling, thereby leading to an increased metastasis of PC.
Utility of EUS-FNAB in the Diagnosis of Cystic Pancreatic Endocrine Neoplasms
V. Morales-Oyarvide,1 T. Ingkakul,1 V. Deshpande,2 D.G. Forcione,3 W.R. Brugge,3 C. Fernández-del Castillo,1 M.B. Pitman,2Departments of 1Surgery; 2Pathology; and 3Gastroenterology Unit, Massachusetts General Hospital, Harvard Medical School, Boston, MA
Aim: To evaluate the utility of endoscopic ultrasound-guided fine-needle aspiration biopsy (EUS-FNAB) and cyst fluid analysis (CFA) in the diagnosis of cystic pancreatic endocrine neoplasms (CPEN).
Background: 17% of all pancreatic endocrine neoplasms (PEN) are cystic, and currently, 1 of every 12 resected pancreatic cysts is a CPEN. Their preoperative diagnosis and distinction from primary pancreatic cysts that may not require resection remains a challenge.
Methods: Cytopathology records between 1992-2010 were searched for all reports of cysts interpreted as PEN and crossed referenced with our surgical database. Patient demographics, clinical and radiological information, CFA and pathology findings were recorded. Performance characteristics of EUS-FNAB and cytology for accurate diagnosis of PEN were calculated.
Results: 53 CPEN were identified. EUS-FNAB was performed in 24 cases; of these, 16 (67%) were true positives. There were 8 false negatives (4 non-diagnostic, 3 benign and 1 adenocarcinoma); no false positives were identified. Fluid analysis was performed in 12 cases; all revealed low CEA (mean 0.93 ng/mL, range 0.3 - 2.0 ng/mL) and 11 of 12 had a low amylase (mean 59 U/L, range 16-90 U/L) with a single outlier (1,493 U/L). Of the 28 cases not evaluated by EUS-FNAB, only in 13 (46%) was CPEN considered in the differential diagnosis.
Conclusion: EUS-FNAB has a sensitivity of 67% and a positive predictive value of 100% for the preoperative diagnosis of CPEN. CPEN, like serous cystadenomas, are characterized by low CEA and amylase levels. EUS-guided aspiration is a very useful tool to distinguish CPEN from other pancreatic cysts.
IPMNs Presenting with Acute Pancreatitis are Associated with an Intestinal Phenotype
V. Morales-Oyarvide,1 M. Mino-Kenudson,2 S.P. Thayer,1 J.A. Wargo,1 C.R. Ferrone,1 K.D. Lillemoe,1 A.L. Warshaw,1 C. Fernández-del Castillo,1Departments of 1Surgery; and 2Pathology, Massachusetts General Hospital, Harvard Medical School, Boston, MA
Aim: To evaluate the histological features of IPMNs presenting with acute pancreatitis (AP).
Background: Acute pancreatitis has been shown to be a sentinel symptom in up to 15% of IPMNs. Its association with their epithelial morphology remains unknown. We hypothesized that a highly viscous mucin containing MUC2 glycoprotein, which is found in the intestinal phenotype, predisposes to development of acute pancreatitis.
Methods: Demographic and clinical data of 325 cases with resected IPMNs were evaluated. Pathologic characteristics of patients with and without a history of AP were analyzed.
Results: A history of AP was found in 74 patients (23%). Of those, 33 (45%) experienced a single episode, and some patients had as many as 10 distinct attacks; 2 patients presented with necrotizing pancreatitis requiring surgical debridement. The elapsed time between the first attack of pancreatitis and surgery ranged from 1 month to 24 years (median 2 years). IPMNs with a history of acute pancreatitis had a higher proportion of intestinal phenotype (OR 4.51, 95% CI 2.61 to 7.80, p < 0.001) and a higher degree of histologic main duct involvement (OR 1.82, 95% CI 1.06 to 3.14, p = 0.039). No difference was observed between the group with a history of AP and the group without AP regarding alcohol use, cholelithiasis, grade of epithelial atypia or presence of invasive carcinoma (16% vs. 21%).
Conclusion: Acute pancreatitis is a frequent presentation of IPMN. These patients have a higher likelihood of harboring an intestinal phenotype that involves the main pancreatic duct.
PDAC with Tumor-Related Retention Cysts, a Mimicker of Invasive IPMN, May Be Associated with Poor Prognosis
V. Morales-Oyarvide,1 C. Fernández-del Castillo,1 N.P. Valsangkar,1 D.V. Sahani,2 M.B. Pitman,3 S.P. Thayer,1 M. Mino-Kenudson,3Departments of 1Surgery, 2Radiology and 3Pathology, Massachusetts General Hospital, Boston, MA
Aim: To evaluate the clinicopathological features and survival of pancreatic ductal adenocarcinoma containing tumor-related retention cysts (PDAC-cyst).
Background: Tumor-related retention cysts are not infrequent in PDAC. PDAC-cyst may exhibit mucinous metaplasia and/or cancerization, and is often difficult to differentiate from invasive carcinoma arising in IPMN, particularly of the gastric type (invasive IPMN-G) on pathologic examination. While we and others have described clinicopathological features and biologic behavior of invasive IPMN, those of PDAC- cyst remain unknown.
Methods: The cohort comprised 555 patients with PDAC without a cystic component, 21 with PDAC-cyst, and 25 with invasive IPMN-G resected at a single institution. We compared clinicopathologic characteristics and survival between PDAC-cyst and the other groups.
Results: PDAC-cyst was associated with a higher incidence of nodal metastasis and lymphatic and vascular invasion compared to invasive IPMN-G, and compared to conventional PDAC, had an older age at presentation (73 vs. 66, P = 0.0009), a higher incidence of vascular invasion, and a trend toward a higher incidence of nodal metastasis (81% vs. 64%, P = 0.16). As for prognosis, the 5-year overall survival rate of PDAC-cyst was 8%, which was lower than invasive IPMN-G (27%, log-rank P = 0.12) and conventional PDAC (16%, P = 0.13).
Conclusions: PDAC with tumor-related retention cysts that histologically mimics invasive IPMN is often associated with adverse pathologic features and may have a worse prognosis than conventional PDAC. Special attention should be paid to differentiating PDAC-cyst from invasive IPMN.
Preliminary Study for Detection of Pancreatic Cancer Using the Duodenal Juice
Y. Mori, T. Ohtsuka, H. Kono, N. Ideno, T. Aso, Y. Nagayoshi, K. Ohuchida, S. Takahata, M. Nakamura, K. Mizumoto, M. Tanaka Department of Surgery and Oncology, Graduate School of Medical Sciences, Kyushu University, Fukuoka, Japan
Objective: Assessment of several markers in the pancreatic juice (PJ) has been reported to be useful to diagnose pancreatic cancer. We have been collecting the PJ for cytology as well as molecular analyses during ERCP; however, the sensitivity of these assessments to detect pancreatic cancer is not high, and ERCP has a risk of pancreatitis. The aim of this study was to establish a minimally invasive screening test for detection of pancreatic cancer using the duodenal juice (DJ) without cannulation into the pancreatic duct.
Methods: DJ was collected prospectively during ERCP in 48 patients who were suspected to have a pancreatic disease between April 2010 and April 2011. There were 29 patients with pancreatic cancer, 9 with intraductal papillary mucinous neoplasm, 3 with serous cyst neoplasm, 3 with neuroendocrine tumor, 2 with mucinous cyst neoplasm, one with chronic pancreatitis, and one with retention cyst. A protease inhibitor was not added to the samples collected during initial 2.5 minutes, while it was added to the samples of the latter 2.5 minutes. Thereafter, secretin was administered intravenously, and the DJ was subsequently collected for additional 10 minutes. Then the pancreatic tube was inserted into the pancreatic duct for pancreatography, and after that, the pure PJ was collected for 15 minutes. The sensitivities of carcinoembryonic antigen (CEA), S100 calcium binding protein P (S100P), and Interleukin-8 (IL-8) in PJ and DJ were calculated as possible markers to detect pancreatic cancer. Cut-off values were determined as the maximum value of each marker in the benign diseases. The sensitivities of cytological diagnosis using PJ, and the serum concentrations of CEA and carbohydrate antigen 19-9 (CA19-9) were also measured.
Results: The average volume of DJ during every 5 minutes was 2 to 4mL. The sensitivity of CEA in the DJ for predicting pancreatic cancer was 90% in any section even without the protease inhibitor. S100P also showed 70% sensitivity when using the protease inhibitor. However, IL-8 in the DJ showed undetectable level in about half of the patients. Cytological diagnosis of PJ to diagnose pancreatic cancer was 58%, and the sensitivities of serum CEA and CA19-9 concentrations were 29% and 46%, respectively.
Conclusions: CEA and S100P in the DJ might be good markers to diagnose pancreatic cancer.
Sensitivity to mTOR Inhibition in a Subset of Pancreatic Ductal Adenocarcinomas
J.P. Morton,1 D.C. Morran,1,2 N. Jamieson,2 C. McKay,2 R. Carter,2 T.R.J. Evans,1 O.J. Sansom,11Beatson Institute for Cancer Research, Glasgow, UK; 2Pancreatic Unit, Glasgow Royal Infirmary, Glasgow, UK
Pancreatic cancer is a leading cause of cancer-related death, and in most patients, current chemotherapies have negligible survival benefit. Evaluation of targeted therapies however, is a relatively recent development. In view of this, we have attempted to model genetic 'subsets' of the human disease in the mouse. Mutations in KRAS and in genes involved in one if its major effector pathways, the PI3K/Akt/mTOR pathway, often occur concurrently in human tumors. We have now shown that activated PI3K signaling impairs Ras-induced senescence in PanINs, the precursor lesions of pancreatic ductal adenocarcinoma (PDAC). Loss of Pten and thus activation of PI3K signaling led to acceleration of KrasG12D-driven PDAC progression in the mouse, while in human tumors, activation of the pathway was independently prognostic of decreased survival. Importantly, these patients represent a discrete cluster in which mTOR inhibitors might be effective, in contrast with the majority of patients. In a mouse model driven by KrasG12D and Pten deficiency, mTOR inhibition significantly enhanced survival of animals presenting with advanced PDAC. Ultrasound imaging showed regression of tumors in some cases. In contrast, in tumors driven by KrasG12D and mutant p53, with normal PI3K signaling, mTOR inhibition had little survival benefit, suggesting that its chemotherapeutic effect is specifically due to the reliance on mTOR signaling in this subset of mice. Our results suggest that genetic signatures of tumors could be used to direct therapy in the future. Clearly, promising treatments may work only in certain patient groups, and thus, careful consideration should be taken before selecting models to test new agents.
The Ca2+ Target Calcineurin Mediates Early Pancreatitis Events
K.A. Muili, A.I. Orabi, M.U. Ahmad, Y. Luo, D. Wang, S.Z. Husain Department of Pediatrics, Yale University, New Haven, CT
Acute pancreatitis is a major health burden for which there are currently no targeted therapies. The premature activation of digestive proenzymes, specifically proteases, within the pancreatic acinar cell is an early and critical event. Our previous studies demonstrate that pathologic protease activation requires a high amplitude, sustained rise in cytosolic Ca2+. In this study, using both a pharmacologic and genetic approach, we tested the hypothesis that a target of aberrant Ca2+ in acinar cells is the Ca2+/calmodulin-dependent phosphatase calcineurin (CN). Acinar cells were freshly isolated from mice and pretreated for 30 min with the CN inhibitors FK506 (10 uM), CN inhibitory peptide (10 uM; CiP), or cyclosporine (1 uM; CsA). Supra-physiologic concentrations of the Ca2+ -activating agonists carbachol (1 mM; Ach analogue), or bombesin (100 nM; gastrin releasing peptide analogue) were administered for 30 min. The CN inhibitors reduced pathologic protease activation by 93, 67, and 80% down to control levels, respectively (n=3; P<0.05). Cell injury was reduced by 20, 15, and 25%, respectively (n=3; P<0.05). We found that the predominant isoform of the catalytic CN-A subunit was Aβ. For this reason, we obtained acinar cells from Aβ deficient mice and demonstrated that compared to wild type cells, protease activation with carbachol was reduced by 93% (n=3; P<0.05). There were no significant differences in physiologic secretion from wild type acinar cells treated with the CN inhibitors or the CNA-β deficient cells. These data suggest early pancreatitis events that occur within the acinar cell are dependent on Ca2+, CN activation. Further, because CN modulation does not affect key physiological function in the acinar cell, CN inhibitors may serve as a potential therapy for pancreatitis.
Triptolide Activates Unfolded Protein Response in Pancreatic Cancer Cells
N. Mujumdar,1 S. Banerjee,1 T. MacKenzie,2 V. Sangwan,1 S. Vickers,1 A.K. Saluja,1,21Divison of Basic and Translational Research, Department of Surgery, 2Department of Pharmacology, University of Minnesota, Minneapolis, Minnesota
Background: The endoplasmic reticulum (ER) is an organelle with crucial biosynthetic and signaling functions in eukaryotic cells. Apart from folding and modification of proteins, ER is a major intracellular calcium storage organelle which is also involved in calcium homeostasis and calcium mediated signaling pathways. Many pathological conditions cause an imbalance between ER protein folding load and capacity, leading to the accumulation of unfolded proteins in the ER lumen, a condition known as "ER stress."Adaptation to ER stress is mediated by induction of the unfolded protein response (UPR), which is a signal transduction pathway that transmits information about protein folding status in the ER lumen to the nucleus in order to increase the folding capacity which aids in cell survival. Pancreatic epithelial cells have a highly developed ER due to heavy engagement in insulin and digestive enzyme secretion and are sensitive to ER stress-induced apoptosis. Our previous work has shown that treatment of pancreatic cancer cells with triptolide, a diterpene triepoxide, leads to an increase in intracellular calcium levels. Also, triptolide induces apoptosis and autophagy in pancreatic cancer cells. Both these pathways are associated with an increase in the levels of intracellular calcium. Hence, triptolide could be a potential ER stress inducer in pancreatic cancer cells.
Methods: Pancreatic cancer cell lines, MiaPaCa-2 and S2-VP10, were exposed to triptolide for different periods of time and the expression of various players involved in UPR was assayed by either qRT-PCR or by western blotting.
Results: In the current study we demonstrate that treatment of both MiaPaCa-2 and S2-VP10 cells with triptolide shows a sustained increase in the phosphorylation of eIF2α, a downstream effector of the ER stress sensor PERK. S2-VP10 cells show a robust increase in the levels of the ER stress sensor, Ire1α, following triptolide treatment. The signal is transduced from the sensor to its downstream target Xbp-1 as evidenced by the formation of a spliced variant Xbp-1s following triptolide treatment. There is also a transient increase in the expression of the downstream effectors of ER stress, CHOP and GRP78, following triptolide treatment in both the cell lines.
Conclusions: Our data indicates that triptolide activates two different arms of the unfolded protein response in both MiaPaCa-2 and S2-VP10 cells. Not only are the sensors activated but the signal is also transduced downstream as evidenced by the increase in the expression of the downstream targets. Hence, triptolide could be a potential ER stress inducer, which activates UPR in pancreatic cancer cells thereby shedding light on the mechanism of the action of this potential chemotherapeutic agent.
Inhibition of GRP78, ER Stress Master Regulator, Triggers Cell Death in Pancreatic Cancer Cells
N. Mujumdar, S. Banerjee, V. Sangwan, S. Vickers, A.K. Saluja Division of Basic and Translational Research, Department of Surgery, University of Minnesota, Minneapolis, Minnesota
Background: Heat shock proteins (HSPs), molecular chaperones which aid in proper protein folding, help cells to survive under unfavorable conditions. One of the members of the HSP family of proteins which resides in the endoplasmic reticulum is the Glucose-Regulated Protein 78 (Grp78). The primary function of GRP78 is to bind to the hydrophobic patches of nascent misfolded polypeptides and aid in protein folding. If polypeptide production exceeds a certain threshold, unfolded protein response (UPR) is triggered. The result is a decrease in the biosynthetic burden of the ER, and an increase in the folding capacity by induction of GRP78 expression. Several studies report that, relative to normal tissue, GRP78 levels are increased in various cancer types including prostate, stomach, breast, lung, gastric cancers and hepatocellular carcinomas and GRP78, like HSP70, is known to protect cells against apoptotic cell death.
Methods: Pancreatic cancer cell lines, MiaPaCa-2 and S2-VP10, were exposed to different doses of triptolide and GRP78 expression was monitored by qRT-PCR and western blotting. GRP78 expression was inhibited either using a specific siRNA or by exposure to (-)-Epigallocatechin gallate (EGCG) and cell viability was assayed using CCK8 reagent. After an inhibition of GRP78, apoptosis was measured by monitoring the number of Annexin V positive cells and caspase-3 activation, and autophagy was measured by immunofluoresence by monitoring the conversion of LC3-I (cytosolic) to LC3-II (membrane bound). The effect of inhibition of GRP78 on the expression of other chaperones: HSP70 and GRP94 were monitored by Western Blotting.
Results: In the present study we show that there is a dose dependent decrease in the expression of GRP78 after treatment with triptolide. In accordance with this inhibition of GRP78 expression with siRNA or by EGCG cell viability decreases. Inhibition of GRP78 expression in MiaPaCa-2 but not in S2-VP10 cells results in an increase in Annexin V positive cells and caspase-3 activation, whereas an inhibition of GRP78 expression in S2-VP10 but not in MiaPaCa-2 cells results in an increase in the conversion of cytosolic LC3-I to membrane-bound LC3-II. Inhibition of GRP78 does not alter the levels of HSP70 expression but increases the levels of GRP94.
Conclusions: Our results indicate that GRP78 is essential to the survival of pancreatic cancer cells. Inhibition of GRP78 results in cell death of pancreatic cancer cells by two different mechanisms: apoptosis in MiaPaCa-2 and autophagic in S2-VP10 cells. Although the effect of GRP78 downregulation is independent of HSP70 levels, there appears to be a compensational effect by another ER chaperone, GRP94. Targeting GRP78 can be considered for clinical utilization to block tumor progression.
STAT3 Mediates Regulation of the Tumor Microenvironment in Pancreatic Cancer
N.S. Nagathihalli, Y. Beesetty, M. Reyzer, C. Shi, R. Caprioli, N.B. Merchant Departments of Surgery, Cancer Biology, Pathology and Biochemistry, Vanderbilt-Ingram Cancer Center, Vanderbilt University Medical Center, Nashville, TN
Aim: To investigate the mechanism of regulation of STAT3 on the tumor microenvironment (TME).
Background: We have previously identified constitutively activated STAT3 as a mediator of treatment resistance in pancreatic ductal adenocarcinoma (PDAC). STAT3 signaling is a central regulator of tumor development, progression and also mediates communication within the TME. Agents that improve drug delivery by targeting the TME represent an important future direction for PDAC therapy.
Methods: STAT3 protein expression was determined in human pancreatic tissues (n=106) and human and genetic mouse pancreatic cell lines generated from PanIn, primary PDAC and liver metastasis. In vivo effects of Src, EGFR and STAT3 inhibition on SPARC, fibronectin, VEGF and CD31 was performed and drug delivery was analyzed by MALDI-MS. PDAC cells treated with STAT3 inhibitors or siRNA were evaluated for the mRNA and protein expression of HIF-1α, MMP9, MMP7, VEGF, SPARC, cyclin-D1, c-Myc, c-Fos and survivin, and VEGF release by ELISA. ChIP assay was performed to study STAT3 binding to the c-Myc, cyclin-D1 and iNOS promoters.
Results: STAT3 activation is necessary for the malignant phenotype and affects survival in PDAC. STAT3 inhibition or siRNA depletes the tumor stroma and inhibits angiogenesis, hypoxia and proliferation markers. Targeting STAT3 increases drug delivery to the tumor. Nuclear STAT3 forms a transcriptional complex with c-Myc promoter.
Conclusion: Targeting STAT3 overcomes drug resistance and affects the TME by regulating the tumor stroma, tumor angiogenesis and hypoxia-suggesting a novel mechanism to enhance drug delivery and improve therapeutic response.
Association of c-Src and TACE/ADAM-17 Induce Amphiregulin-Dependent EGFR Signaling
N.S. Nagathihalli, M.K. Washington, R. Coffey, N.B. Merchant Departments of Surgery, Cancer Biology, Cell and Developmental Biology and Pathology, Vanderbilt-Ingram Cancer Center, Vanderbilt Medical Center, Nashville, TN
Aim: To determine the mechanism of bile acid induced EGFR signaling.
Background: Ectodomain shedding of EGFR ligands amphiregulin (AR) and TGF-α is dependent on tumor necrosis factor-α converting enzyme/a disintegrin and metalloprotease (TACE/ADAM-17). Interactions between c-Src and TACE in sheddase activity can be stimulated by secondary bile acids, principally deoxycholic acid (DCA), which is implicated in promoting cancer growth, progression and invasion of gastrointestinal malignancies.
Methods: TACE, TGF- α and AR protein expression was determined in human pancreatic tissues (n=106). Pancreatic ductal adenocarcinoma (PDAC) cells were treated with DCA and analyzed for Src, TACE, AR and TGF-α expression and activity. Cells treated with inhibitors or siRNA of Src, TACE and AR with/without DCA were evaluated for the mRNA and protein expression of AR, TGF-α, total and activated TACE, Src, EGFR, AKT, c-Jun, MAPK. Effects of AR inhibition on tumorigenicity and cyclin-D1 promoter activity were determined.
Results: TACE and AR are overexpressed in PDAC compared with normal tissues. Exposure of PDAC cells to DCA results in co-localization of Src and TACE at the cell membrane and is associated with AR-dependent EGFR activation. Inhibition of Src or TACE attenuates DCA-induced AR shedding. Moreover, AR siRNA cells significantly reduce DCA-induced phosphorylation of both EGFR and MAPK and inhibit tumorigenicity.
Conclusion: These findings provide a novel mechanism of Src- and TACE-mediated shedding of AR dependent EGFR activation underlying the oncogenic effects of secondary bile acids. Targeting the Src-TACE-AR-EGFR axis may enhance the therapeutic response of PDAC treatment.
Glucagon-like Peptide 1 Induces Cell Proliferation of Rat Pancreatic Stellate Cells Through ERK and PKCζ Pathway
T. Nakamura, T. Ito, M. Uchida, R. Takayanagi Department of Medicine and Bioregulatory Science, Kyushu University, Fukuoka, Japan
Background: The cause of irreversible pancreatic atrophy is still unclear and prevention of this cycle is one therapeutic target against chronic pancreatitis (CP). Glucagon-like peptide 1 (GLP-1) not only stimulates insulin secretion but also induces proliferation of beta cells. However, until now, it is not well known of GLP-1 effect on exocrine pancreas. The GLP-1 serum level is decreased in patients with CP and pancreatic enzyme substitution therapy recovers the level, indicating the possible deficiency of GLP-1 in CP patients.
Aims: To clarify the possible role of GLP-1 as a regenerative factor for damaged exocrine pancreas.
Methods: Male WBN/KOB rats were used as chronic pancreatitis model. Pancreatic stellate cells (PSCs) were isolated from the Wistar rats. The expression of GLP-1 receptor (GLP-1R) was evaluated using reverse transcription (RT)-PCR. Localization of GLP-1R was analyzed using a specific antibody. PSCs were incubated with GLP-1 and its analogues (0-1000 nM, 0-24H) and then proliferation was quantified by BrdU assay.
Result: Increased expression of GLP-1R was confirmed by RT-PCR, indicating the possible deficiency of GLP-1 in pancreas. In normal pancreas, GLP-1R was expressed in not only islets but also PSCs. In chronic pancreatitis, almost all cells were positive for GLP-1R. We performed BrdU assay using activated PSCs, which expressed GLP-1R more than quiescent PSCs. GLP-1 and its analogues induced proliferation of PSCs dose-dependently. Using specific inhibitors for ERK and PKCζ, we confirmed strong inhibition of GLP-1 induced proliferation.
Conclusion: GLP-1 induces cell proliferation of PSCs. GLP-1 might be a therapeutic agent for progressive and irreversible pancreatic atrophy in CP.
The Mechanisms of ETHANOL Induced FRACTALKINE Release in Chronic Pancreatitis
T. Nakamura, T. Ito, M. Uchida, M. Hijioka, Y. Niina, N. Fujimori, T. Oono, H. Igarashi, R. Takayanagi Department of Medicine and Bioregulatory Science, Kyushu University, Fukuoka, Japan
Background: Fractalkine (CX3CL1) is involved in pathogenesis of chronic pancreatitis. We have previously reported the increased serum level of soluble CX3CL1 in patients with chronic pancreatitis, especially in alcoholics. However, there are many unclear points about the mechanisms of CX3CL1 release.
Aims: The aim of our study was to clarify the mechanisms of CX3CL1 release induced by ethanol.
Methods: Male WBN/KOB rats were used as chronic pancreatitis model. Pancreatic stellate cells (PSCs) were isolated from male Wistar rats. The expression of CX3CL1 was evaluated using reverse transcription (RT)-PCR and immunofluorescent histo/cytochemistry. PSCs were incubated with LPS, Poly (I:C) (10 μg/ml) with lipofectamine2000, TNF-α and IL-1β(10 ng/ml). Then, the CX3CL1 mRNA and soluble CX3CL1 protein were assessed using a realtime PCR method and ELISA.
Results: CX3CL1 mRNA expression was increased in the pancreas with chronic pancreatitis. We confirmed the strong expression of the CX3CL1 in rat PSCs in vivo and in vitro using confocal microscopy. In response to infectious pathogens and cytokines, PSCs released CX3CL1 dose-dependently in ELISA. Using inhibitors of ERK, double-stranded RNA dependent kinase (PKR) and sheddases (MMPs and ADAMs), the CX3CL1 release was attenuated dose-dependently, indicating the possible role of these pathways in CX3CL1 release. Interestingly, ethanol could not increase, but synergistically increased the CX3CL1 release with other stimuli. This synergistic effect was also seen at transcriptional level which was through ERK and PKR pathway.
Conclusion: It is suggested that ERK and PKR play an important role in ethanol-induced CX3CL1 release.
Portal Vein Resection is Beneficial for Pancreatic Cancer Patients in the Era of Gemcitabine
M. Nakamura,1,2 T. Kayashima,1 K. Tsutsumi,1,2 H. Nakashima,1,2 T. Ohtsuka,1 J. Urakami,2 S Takahata,1 K. Mizumoto,1 M. Tanaka,11Department of Surgery and Oncology, Graduate School of Medical Sciences, Kyushu University, Fukuoka, Japan; 2Digestive Surgery, Kawasaki Medical School, Kurashiki, Japan
Aim: To clarify the benefit of portal vein resection (PVR) on advanced pancreatic cancer in the era of adjuvant chemotherapy using gemcitabine (GEM).
Backgrounds: PVR is controversial as a treatment of pancreatic cancer because of the poor prognosis. GEM improved the prognosis of resectable pancreatic ductal adenocarcinoma (PDAC) as well as nonresectable PDAC. However, the effect of GEM on the prognosis of patients with borderline resectable pancreatic cancer is not clear. Here we show that the combination therapy with PVR and GEM has improved the prognosis of borderline resectable PDAC.
Patients and Methods: We retrospectively analyzed the prognosis of 177 pancreatic cancer patients who underwent curative pancreatectomy. Of them, 42 patients underwent PVR (PVR(+) group), and 135 patients did not undergo PVR (PVR(-) group).
Results: Mean survival time (MST) was 25.6 months in PVR(-) group, and 13 months in PVR(+) group (p=0.021). When we focused on the patients who underwent adjuvant chemotherapy with GEM, there was no significant difference in MST between PVR(+) group (42 months) and PVR(-) group (31 months). In contrast, MST of the patients without GEM adjuvant therapy in PVR(+) group (9 months) was significantly shorter than that in PVR(-) group (22 months) (p=0.005). Furthermore, we specifically analyzed the patients who underwent PVR and PV invasion was histologically proven. MST was 42 months in the group with adjuvant GEM chemotherapy, and 7 months in the group without GEM (p=0.0002).
Conclusion: Pancreatic cancer involving the portal vein is thought to be borderline resectable because of the poor prognosis of the old data. However, the prognosis of PVR(+) group can be greatly improved by adjuvant chemotherapy with GEM. Combination therapy of PVR and GEM may be a new option to improve the prognosis of borderline resectable PDAC with portal vein involvement.
Periampullary Cancer ESPAC-3(v2) trial: a Randomised Controlled Phase III Trial of Adjuvant Chemotherapy Versus Observation in Patients With Periampullary Adenocarcinomas of the Head of the Pancreas
J. Neoptolemos,1 M. Moore,2 T. Cox,1 J. Valle,3 D. Palmer,1 A. Mcdonald,4 R. Carter,5 N. Tebbutt,6 C. Dervenis,7 D. Smith,1 B. Glimelius,8 F. Coxon,9 F. Lacaine,10 M. Middleton,11 P. Ghaneh,1 C. Bassi,12 C. Halloran,1 A. Olah,13 C. Rawcliffe,1 M. Büchler,141Liverpool University, UK; 2Princess Margaret, Toronto, Canada; 3Christie, Manchester, UK; 4Birmingham University, UK; 5Glasgow Royal Infirmary, UK; 6Austin Hospital, Heidelberg, Australia; 7Aiga Olga, Athens, Greece; 8University Hospital, Uppsala, Sweden; 9Northern Cancer Centre, Newcastle upon Tyne, UK; 10Hospital Tenon, Paris, France; 11Radcliffe Hospitals, Oxford, UK; 12Verona University, Italy; 13Petz Aladar Hospital, Györ, Hungary; 14Heidelberg University, Germany
Background: The effect of adjuvant treatment on periampullary adenocarcinomas is not known. We compared the overall survival (OS) of chemotherapy (CTX) vs observation (OBS) after resection and within CTX also 5-fluorouracil/folinic acid (5-FU/FA) vs gemcitabine (GEM).
Methods: Patients were stratified by R0/R1 margins then randomised into 3 arms.
Results: Of 434 patients (ampullary=310, bile duct=89, other=35) 289 were randomised to CTX (143 5FU, 146 GEM) and 145 to OBS. Median (range) age = 62 (35-81) yrs; men = 187 (61.5%). Median (IQR) max tumor diam. = 20 (15-30) mm, mod. well diff. = 258 (62%), involved LN = 256(59%) R0 resection = 367(85%). Independent prognostic factors were tumor diam. and grade, LN status and R0/R1 status. The median (95% CI) OS was 38.9 (32.5-45.7) mths; overall HR (95% CI) of CTX vs OBS = 0.88 (0.68-1.14), p=0.33; for R0 the HR=0.83 (0.61-1.11), p=0.21. Adjusting for risk factors for R0 patients HR=0.73, p=0.048.
Conclusion: There was a benefit for adjuvant chemotherapy in patients with clear resection margins.
Analysis of Hereditary Pancreatitis Families in Europe (EUROPAC): Incidence of PRSS1 Associated Pancreatic Adenocarcinoma Does Not Correlate To Symptoms of Pancreatitis
J.A. Nicholson,1 S. Harrison,1 R. Sutton,1 A. Rousseau,2 R. Mountford,2 P. Hammel,3 V. Rebours,3 J. Mayerle,4 R. Charnley,5 J.P. Neoptolemos,1 M. Lerch,4 W. Greenhalf,11NIHR Pancreas Biomedical Research Unit, Liverpool, UK; 2Liverpool Regional Genetics Laboratory, Liverpool, UK 3Hôpital Beaujon, Clichy, France; 4Klinikum der Ernst-Moritz-Arndt-Universität, Greifswald, Germany 5Freeman Hospital, Newcastle-upon-Tyne, UK
Background: Chronic pancreatitis is associated with an increased risk of pancreatic ductal adenocarcinoma (PDAC) but the risk is much greater with hereditary pancreatitis (HP). The reason is generally thought to be earlier onset.
Aim: To determine whether disease severity is associated with a predisposition to pancreatitis or just with the symptoms of pancreatitis.
Methods: Kaplan Meier analysis was used with different ages of onset of pancreatitis: early (lower quartile) to late (upper quartile). Mantel-Cox logrank testing was used to establish significance. Unaffected obligate mutation carriers were excluded if younger than upper quartile onset.
Results: 1002 individuals were identified from the database as having symptomatic hereditary pancreatitis of whom 50 developed pancreatic cancer and 148 individuals were asymptomatic of whom 10 developed pancreatic cancer. Although diabetes mellitus and smoking were significantly associated with PDAC (p<0.01) there was no correlation between age of onset (early or late) of pancreatitis and PDAC (p=0.18), furthermore exocrine failure and other measures of disease severity were not shown to confer increased cancer risk.
Conclusions: Symptomatic attacks of pancreatitis do not serve as a surrogate for risk of PDAC in patients with HP. This could be the result of chronic inflammation in patients who report themselves to be well. Screening should be considered for all patients with HP, regardless of their symptoms of pancreatitis.
The Role of Innate Immunity in the Pathogenesis of Alcoholic Pancreatitis in Mice
A. Nishio, S. Nakayama, M. Yamashina, K. Uchida, T. Fukui, K. Okazaki Third Department of Internal Medicine, Kansai Medical University, Moriguchi, Japan
Background: Chronic alcohol ingestion has been considered as one of the major causes of chronic pancreatitis. Recent study reported that endotoxemia induced by alcohol abuse may lead to pancreatic damage. The aim of the study was to determine the role of innate immunity in the pathogenesis of alcoholic pancreatitis using a murine model.
Methods: Eight-week-old C57BL6 mice were fed with 20% ethanol (AL) in drinking water and injected intraperitoneally with peptidoglycan (5mg/kg body weight), polyinosinic polycytidylic acid (poly I:C; 5mg/kg) or lipopolysaccharide (LPS, 4mg/kg) twice weekly for 4 weeks. Eight-week-old SCID mice with or without reconstitution with CD4 or CD8 T-cells from wild type mice were treated with AL + LPS. The mice were killed and the severity of pancreatitis was graded using a histological scoring system. Serum cytokine levels were measured by a cytokine bead assay kit. Inflammatory cell infiltration into the pancreas was immunohistochemically examined.
Results: Feeding of 20% AL for 4 weeks did not cause pancreatitis. Administration of peptidoglycan or poly I:C did not cause pancreatitis in AL-fed mice. However, pancreatitis was induced by treatment with both AL + LPS and LPS alone. The severity of pancreatitis was greater in mice treated with AL + LPS than in those treated with LPS alone. Intense infiltration of neutrophils, accompanied by CD4 T-cells, CD8 T-cells, and B-cells, were observed in the pancreas. Serum levels of IL-1β, IFN-γ and TNF-α levels were elevated in AL+ LPS-treated mice. SCID mice reconstituted with CD4 T-cells, developed pancreatitis.
Conclusion: Specific stimulation of innate immunity augments alcohol-induced pancreatic damage via activation of CD4 T-cells.
Tumor-Supressor Role of PHLPP1 in Pancreatic Ductal Adenocarcinoma is Mediated Through Inhibiting of Akt2
C. Nitsche,1,2 R. Moore,2 C.D. Heidecke,1 M. Edderkaoui,2 G. Eibl,2 N. Kasahara,2 J. Mayerle,1 M. M. Lerch,1 A. S. Gukovskaya,2University Greifswald, Germany1 UCLA, Los Angeles, USA2
Background & Aims: Several isoforms of Akt kinase are present in pancreatic ductal adenocarcinoma (PDAC), however, their differential roles in tumorigenesis have not been established. We recently reported that phosphatases PHLPP1 and PHLPP2 dephosphorylate and inactivate Akt in pancreatic cancer (PaCa) cells. The specificity of PHLPPs towards individual Akt isoforms remain unknown. Here we investigate the role of phosphatases PHLPP1 and 2 in regulating Akt1 and Akt2, cell death and tumor development in mouse and human PDAC.
Methods: The effects of PHLPPs on Akt isoforms, cell death and tumor development were measured in PaCa cells, human PDAC and orthotopic model using MiaPaCa-2 cells overexpressing PHLPP1 or 2.
Results: Akt isoforms 1 and 2 and phosphatases PHLPP1 and 2 are present in PaCa cells and PDAC tissue samples. PHLPP1 selectively dephosphorylated Akt2, while PHLPP2 dephosphorylated Akt1. The significance of the Akt isoforms for pancreatic tumorigenesis is different: Akt2, but not Akt1, was upregulated in nearly all PDAC. Consistent with these results, high level of PHLPP1, which dephosphorylates Akt2 (but not PHLPP2, which dephosphorylates Akt1), correlated with better survival of PDAC patients. Correspondingly, overexpressing PHLPP1 or silencing Akt2 both stimulate PaCa cell death, the effects of PHLPP2/Akt1 on PaCa death responses were less pronounced. Furthermore, in the orthotopic mouse model PHLPP1 overexpression inactivated Akt, stimulated cancer cell death, and decreased tumor growth.
Conclusion: The results demonstrate that PHLPP1 supresses pancreatic tumor through Akt1 inactivation and suggest PHLPP1 as a potential therapeutic and diagnostic tool in PDAC.
An In-silico Study of the Heparin Interactome in Pancreatic Disease
Q. Nunes,1 B. Lane,1 R. Sutton,1 D. Fernig,2 O. Vasieva,21Liverpool NIHR Pancreas Biomedical Research Unit, Liverpool, United Kingdom; 2Institute of Integrative Biology, University of Liverpool, Liverpool, United Kingdom
Background: Heparin binding proteins (HBPs) abound in the extracellular space and influence fundamental biological processes via the heparin interactome network of HBPs. We have used an in-silico approach to investigate the role of HBPs in acute pancreatitis (AP), chronic pancreatitis (CP) and pancreatic ductal adenocarcinoma (PDAC).
Methods: HBPs associated with AP, CP and PDAC were identified using online databases and published data. HBP networks were compared by topological features, gene ontology terms and canonical pathway enrichment using Ingenuity Pathway Analysis and Cytoscape.
Results: HBPs uniquely associated with AP (n=17), CP (n=11) and PDAC (n=21) were identified. Hepatic fibrosis (HF)/Hepatic Stellate Cell Activation (HSCA) was the top canonical pathway for the AP (p=1.07E-30) and PDAC HBP datasets (p=1.09E-18) and was significant for the CP set (p=1.5E-13). 'Cellular Growth and Proliferation' was the top bio-function in the CP set (5.83E-30 to 3.60E-07). The clustering coefficient was 0.632, 0.431 and 0.378 and number of connected components 1, 1 and 2 for the AP, CP and PDAC heparin interactomes respectively.
Conclusion: HBPs in AP, CP and PDAC form highly connected networks, with subtle differences that may be responsible for significant differences in biological outcomes. 'HF/ HSCA' is a significant canonical pathway, likely in part due to the role of pancreatic stellate cells (PSCs). HBPs unique to these pancreatic diseases may be explored in wet lab investigation as biomarkers.
Novel Simple Method for the Isolation of Pancreatic Stellate Cells
Q. Nunes,1 D. Latawiec,1 M. Awais,1 D. Fernig,2 R. Sutton,11Liverpool NIHR Pancreas Biomedical Research Unit, Liverpool, UK; 2Institute of Integrative Biology, University of Liverpool, Liverpool, UK
Background: Pancreatic stellate cells (PSCs) are important players in the pancreatic microenvironment. Various techniques to isolate PSCs from normal and diseased pancreas have been described, the majority of which are complex. We have developed a simplified technique to isolate and purify activated PSCs.
Methods: Cerulein hyperstimulation-, bile acid injection- or alcohol-induced experimental acute pancreatitis was induced in CD1 mice then pancreata removed and digested with Collagenase NB 8 Broad Range (Serva Electrophoresis, Heidelberg, Germany). Digested pancreata were plated in tissue culture flasks containing DMEM: F12 medium (Dulbeco's modified Eagle medium: Ham's F12 nutrient mixture) containing 10% fetal calf serum, glutamine and antibiotics. Media were replaced every 48 h. After attaining significant confluence, the isolated cells were purified by density gradient centrifugation using Histodenz (Sigma Aldrich). Isolated PSCs were characterized by fluorescence (FM) and immunoelectron microscopy (IEM).
Results: Activated PSCs were successfully isolated (> 95% purity) using the same technique from all experimental models (hyperstimulation 3, bile acid 1, alcohol 1) as well as normal CD1 pancreas (n=3). PSCs stained positively for glial fibrillary acidic protein (GFAP), desmin and alpha-Smooth Muscle Actin on FM. GFAP staining was demonstrated on IEM using the post-embedding technique.
Conclusions: The isolation and purification techniques that we describe are easy to perform. IEM is useful for characterizing PSCs.
Follow-up Study after Resection of Intraductal Papillary Mucinous Neoplasm of the Pancreas; Special References to the Multifocal Lesions and Development of Ductal Carcinoma in the Remnant Pancreas
T. Ohtsuka, H. Kono, Y. Nagayoshi, Y. Mori, N. Ideno, T. Aso, S. Takahata, M. Nakamura, K. Mizumoto, M. Tanaka Department of Surgery and Oncology, Graduate School of Medical Sciences, Kyushu University, Fukuoka, Japan
Background: Frequency and characteristics of metachronous occurrence of multifocal intraductal papillary mucinous neoplasms (IPMNs) of the pancreas or distinct pancreatic ductal adenocarcinomas (PDACs) in the remnant pancreas during follow-up after pancreatectomy for IPMNs have not been well known. The aim of this study was to investigate the outcomes after resection of IPMNs, especially focusing on the metachronous occurrence of multifocal IPMNs and distinct PDACs.
Methods: Medical records of 172 patients who underwent resection of IPMNs were retrospectively reviewed, and the data regarding the occurrence of metachronous IPMNs or PDACs in the remnant pancreas were collected during a mean postoperative follow-up period of 64 months were collected.
Results: The incidence including synchronous and metachronous multifocal occurrence of IPMNs was 20%, and that of distinct PDACs was 9.9%. Ten metachronous IPMNs developed in the remnant pancreas after mean time of 23 postoperative months, and 2 with main duct IPMNs required remnant pancreatectomy. Six distinct PDACs developed in the remnant pancreas after mean time of 84 postoperative months. Four of them were found to have a tumor with the size of less than 2cm, while the remaining 2 PDACs were found to be unresectable over 10 years after resection of IPMNs.
Conclusions: Intense long-term follow-up is necessary for the early detection of metachronous occurrence of distinct PDACs as well as malignant IPMNs after resection of IPMNs.
A Murine Model of Acute Pancreatitis Demonstrating Age-dependent Increases in Mortality, Systemic Inflammation, and Activation of ERK and JNK
D. Okamura, J. Ienaga, M.E. Starr, B.M. Evers, H. Saito Markey Cancer Center and Department of Surgery, University of Kentucky, Lexington, KY
Patient age is a major risk factor for the severity of acute pancreatitis (AP); however, the underlying mechanisms for this remain largely unknown partly due to a lack of appropriate animal models. The purpose of this study was to establish a murine model of AP, which displays age-associated severity, and to use this model to examine the mechanisms for increased morbidity and mortality in the aged with AP.
Methods: AP was induced in young (4-5 months) and aged (23-25 months) C57BL/6 mice by caerulein injection (50 μg/kg, i.p., total of 9 injections at hourly intervals); mice were sacrificed at multiple time points for histological analysis, myeloperoxidase (MPO) and plasma protein analysis. To investigate inflammatory signaling, activation of ERK and JNK was assessed in the pancreas of the mice with AP and in caerulein-stimulated pancreatic acinar cells derived from young and aged mice.
Results: While no mortality was noted in young mice, approximately 10% of the aged mice died during AP. While edema and inflammation were observed in the pancreas, lung and kidney of both young and aged mice during AP, only aged mice showed sustained severe tissue damage and cell death. Significant age-associated increases in pancreatic MPO activity, and plasma levels of amylase, IL-6, and creatinine were also observed (p<0.05). Both in vivo and in vitro experiments showed significant age-dependent activation of ERK and JNK at early time-points.
Conclusions: This caerulein-induced murine AP model exhibits age-dependent increases in mortality, multiple tissue injury and inflammation. Augmented activation of ERK and JNK in pancreatic acini may be partly responsible for age-associated vulnerability to AP.
Effects of Volumetric Fat Parameters on Severity of Acute Pancreatitis
D. O'Leary Cork University Hospital, Wilton, Cork, Ireland
Introduction: Obesity is a well-established risk factor for acute pancreatitis. Increased visceral fat adiposity has been shown to exacerbate the pro-inflammatory milieu experienced by patients.
Aims: This study aimed to investigate the relationship between severity of acute pancreatitis and individual fat volumes measured on Computed Tomography (CT) scan.
Methods: Consecutive patients admitted to Cork University Hospital with acute pancreatitis between 2005 and 2010 were evaluated for inclusion in the study. Web-based image analysis software, namely Osirix v3.9, was used to calculate individual fat volume parameters from CT scans by segmentation of abdominal tissues.
Results: A total of 214 patients were admitted with pancreatitis between January 2005 and December 2010. 62 patients underwent a CT scan and were thus eligible for inclusion. Visceral fat volume was the volumetric fat parameter that had the most significant association with severe acute pancreatitis (p=0.003). There was a significant association between visceral fat volume and subsequent development of systemic complications of severe acute pancreatitis (p=0.003). There was a strong association between mortality and visceral fat volume (p=0.019). Multivariate regression analysis, adjusted for sex, did not identify any individual fat area as an independent risk factor for severe acute pancreatitis.
Conclusion: Overall, CT volumetry indicates a strong association between visceral fat, severe acute pancreatitis and the subsequent development of systemic complications. This data suggests that visceral fat volume analysis should be incorporated into future predictive scoring systems.
Inositol 1,4,5-Trisphosphate Receptor Type 2 Deficiency Leads to Accumulation of Zymogen Granules in Pancreatic Acinar Cells
A.I. Orabi, Y. Luo, K.A. Muili, Z. Mannan, D. Wang, S.Z. Husain Department of Pediatrics, Yale University, New Haven, CT
Ca2+ signals from within pancreatic acinar cells are a critical component of the initiation of acute pancreatitis. Ca2+ signals are generated by intracellular Ca2+ channels, notably the inositol 1,4,5-trisphosphate receptor (IP3R). IP3R generally mediates physiologic exocytosis of zymogen granule contents from the apical region. We hypothesized that a chronic deficiency in IP3R would lead to either reduced exocytosis and/or accumulation of zymogen granules. In this study, we examined the genetic loss of IP3R type 2 (IP3R2), which comprises 50% of the acinar cell IP3R. 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.
ARF-1 Regulates Trypsinogen Activation via Procathepsin B Trafficking and Autophagic Maturation During Caerulein Pancreatitis
L. Orlichenko,1 D. Stolz,2 J. Behari,1 S. Liu,1 V. Singh,1Departments of 1Medicine, 2Cell Biology and Physiology, University of Pittsburgh, PA
Background and Aims: Several recent studies suggest that autophagy may play a deleterious role in acute pancreatitis via intracinar activation of digestive proteases. The prototype for this phenomenon is cathepsin B mediated trypsin generation. Our aim was to determine the organellar basis of this process.
Methods: We initially studied the subcellular distribution of the cathepsin B precursor procathepsin B. Based on procathepsin B enrichment in Golgi-containing microsomes, we studied the effect of the ADP ribosylation factor (ARF) inhibitor Brefeldin A (BFA) on caerulein-induced changes in procathepsin B trafficking, active trypsin generation and autophagic outcomes.
Results: BFA inhibited ARF-1, caused dismantling of the Golgi, prevented caerulein-induced depletion of procathepsin B resulting in reduced cathepsin B activity (280±37.4% vs. 150±29% control, p=.035), and consequently reduced trypsinogen activation (2.3±0.1 vs. 1.0±0.1 fold control, p=0.0001). Simulating the effect of retained procathepsin B, its propeptide inhibited cathepsin B activity in acinar lysates in a dose-dependent fashion. BFA similarly inhibited caerulein-induced ARF-1 activation in vivo during the progression of caerulein-induced pancreatitis. This process was accompanied by a reduction in trypsin generation. However, formation of LC3-II suggested that BFA did not prevent autophagic initiation by caerulein. Instead, sucrose density gradient centrifugation and electron microscopy showed that BFA arrested caerulein-induced autophagosomal maturation.
Conclusions: ARF-1 dependent trafficking of procathepsin B and autophagic maturation results in cathepsin B-mediated trypsinogen activation during caerulein-induced pancreatitis.
Two-phase Indirect Pancreatic Function Tests for Individual Enzyme Replacement Therapy
A. Pap National Institute of Oncology, Budapest, Hungary
Introduction: Indirect function tests use substrates of pancreatic enzymes and appearence of end-products demonstrates activity of the measured enzyme.Specificity depends on function of organs involved in elimination of the substrate.Fals-positive results of the indirect pancreatic function tests can be corrected by repeating the test with pancreatin administration and individual pancreatic enzyme insufficiency can be estimated by the dose of pancreatin normalizing the test,thus tailored substitution therapy may be applied.The repeated starch loading can be useful for amylase replacement therapy according to this scenario.
Methods: Glucose loading with 75g of dextrose on Day1 and starch loading with the same amount of starch on Day2 was performed in 8 patients with chronic pancreatitis and in 3 cases of functional dyspepsia.Althausen formula was used to calculate ATT value (percent differences between levels of blood glucose).On Day3,4,5 starch loading was repeated with administration of 1-2-3 doses of pancreatin preparations.
Results: In chronic pancreatitis patients starch loading demonstrated levels of pancreatic insufficiency and optimal doses of pancreatin corrected ATT values almost totally while larger doses somewhat deteriorated starch digestion.In dyspepsia cases pancreatin administration did not corrected the fals-positive results but frequently further deteriorated it.
Conclusion: 2-phase starch tolerance test can evaluate amylase insufficiency and estimate optimal dose of pancreatin for enzyme substitution therapy.Supraoptimal doses of pancreatin progressively deteriorate results of replacement therapy possibly by increasing proteolytic inactivation of amylase.The 2-phase starch loading can differenciate between pancreatic insufficiency and functional dyspepsia or other motility disorders without pancreatic damage.
Garcinol Sensitizes Human Pancreatic Adenocarcinoma Cells to Gemcitabine by Modulating MicroRNA Signatures
M. A. Parasramka,1 S. V. Gupta,21Linus Pauling Institute, Oregon State University, Corvallis, OR; 2Department of Nutrition and Food Science, Wayne State University, Detroit, MI
Background: Alterations in microRNA (miRNA) genes are of prominent biological importance in the pathophysiology of all human cancers, including pancreatic cancer (PaCa). Although there is exponentially accumulating evidence supporting the role of miRNA in cancers, little is known regarding their response to dietary phytochemicals. We previously showed that garcinol, a bioactive ingredient found in the rind of the fruit Garcinia indica, induces PaCa cell growth arrest and apoptosis in vitro. In addition, we observed a potent chemo-sensitization effect of garcinol in synergism with first-line PaCa chemotherapy drug, gemcitabine. In the present study, we evaluated by microarray analysis the alterations in miRNA expression profile of gemcitabine-resistant Panc-1 cells treated with garcinol and/or gemcitabine. This is the first study of miRNA modulation by garcinol and gemcitabine in PaCa cells.
Methods and Results: Scatter plot, hierarchical cluster analysis, volcano plot, quantitative PCR data, and in silico analyses using TargetScan5, PicTar, and DNA Intelligent Analysis, microT-V.B4 database showed that garcinol and/or gemcitabine modulated a number of miRNAs (miR-21, miR-196a, miR-495, miR-605, miR-638, and miR-453) linked to various canonical oncogenic signaling pathways such as cell proliferation, apoptosis, angiogenesis and metastasis.
Conclusion: We identified garcinol-specific miRNA biomarkers that sensitize PaCa cells to gemcitabine treatment, thus attenuating the drug-resistance phenotype. These results prompt further interest in garcinol and gemcitabine combination strategy as a drug modality to improve treatment outcome in patients diagnosed with PaCa.
Correlation of Early Recurrence with In Vitro Adenosine Triphosphate Based Chemotherapy Response Assay in Pancreas Cancer with Postoperative Gemcitabine Chemotherapy
J. Seong Park, J. Keun Kim, H. Wook Shin, D. Sup Yoon Pancreatobiliary Cancer Clinic, Department of Surgery, Gangnam Severance Hospital, Yonsei University Health System, Seoul, Korea
Background: Pancreas cancer remains the fourth leading cause of cancer related mortality in Korea. Overall survival remains poor despite advances in therapeutics. Gemcitabine-based regimens represent the standard systemic first line treatment in patients after pancreatic resection. The clinical impact of gemcitabine remains modest owing to the high degree of chemoresistance, so, identification of nonresponders before initiation of treatment may help to avoid first line chemotherapeutics. An in vitro adenosine triphosphate-based chemotherapy response assay (ATP-CRA) was designed to require only a limited number of cells and shorten test turnaround time with a high success rate. This study investigated the correlation between in vitro gemcitabine sensitivity of tumor cells and early recurrence after curative resection.
Methods: From January 2007 to December 2009, the ATP-CRA for gemcitabine was tested in 39 patients among pancreas cancer treated at Gangnam Severance Hospital, Seoul, Korea. All patients received gemcitabine chemotherapy. Selected patients were divided into a chemosensitive group and a non-chemosensitive group, according to a 20% cell death as a cut-off value. We analyzed the relationship between chemosensitivity and early systemic recurrence in patients with pancreas cancer.
Results: The mean age of the patients was 65.2 years old, the mean follow up period was 24.5 months. Twenty seven recurrence were observed during follow-up. The patients were divided into two groups: early recurrence (≤9 months) and late recurrence (>9 months). In the early recurrence group, non-chemosensitive tumors was more common in comparison with the late recurrence group.
Conclusions: Gemcitabine sensitivity measured by ATP-CRA was well correlation with in vivo drug responsibility to predict early recurrence against gemcitabine based adjuvant chemotherapy in patients with pancreas cancer.
Pattern and Clinical Predictors of Lymph Node Involvement in Neuroendocrine Neoplasms of the Pancreas
S. Partelli,1 R. Cherif,3 L. Boninsegna,1 S. Gaujoux,3 S. Crippa,1 A. Couvelard,4 A. Scarpa,2 A. Sauvanet,3 P. Ruszniewski,5 M. Falconi,11Departments of Surgery and 2Pathology - University of Verona, Verona, Italy; 3Department of HPB Surgery - PMAD, 4Departments of Pathology and 5Gastroenterology - Hopital Beaujon - Clichy - AP-HP, France
Background: Pancreatic neuroendocrine neoplasms (PNENs) are often indolent without pathological lymph node metastasis (pN1). Therefore, in patients with low risk of pN1, a lymphadenectomy could be avoided.
Aim: To construct a model for predicting the risk of pN1 prior to surgical resection.
Methods: Databases from the University of Verona and the Beaujon Hospital were queried. Data of all patients with resected (R0 or R1) non-functioning PNEN between 1993 and 2009 were analyzed.
Results: Data were analyzed for 194 patients. Metastases were present in the dissected lymph nodes of 58 patients (30%). The 5-year disease free survival for pN1 patients was significantly lower than for pN0 (66% vs 93%, P<0.0001). Multivariable analysis suggested the independent predictors of pN1 were radiological nodal status (rN) (OR 3.4, P=0.008), localization in the pancreatic head (OR 3.4, P=0.002) and the degree of differentiation (G2 vs G1 OR 3.5, P=0.001). When the degree of differentiation was excluded, on multivariable analysis rN1 (odds ratio 4.1, P=0.001), localization in the pancreatic head (odds ratio 3.2, P=0.002), and radiological size > 4 cm (odds ratio 2.5, P=0.012) were independent predictors of pN1.
Conclusions: Patients with PNEN-G1 of the pancreatic body, in the absence of radiological node involvement, have a low risk of pN1. If a preoperative cytological diagnosis is not achieved, radiological size of the lesion is a powerful alternative predictor of pN1.
CEA & Long-Term Follow-Up of Mucinous Cystic Neoplasms (MCN)
M. Patel, M. Othman, T. Woodward, M. Wallace, J. Stauffer, H. Asbun, M. Raimondo Divisions of Gastroenterology & Surgery, Mayo Clinic, Florida
Introduction: Long-term follow-up data is lacking in patients with MCN and IPMN. The utility of intracystic CEA to differentiate malignant from benign mucinous cysts is controversial. We sought to examine the natural history and the utility of cyst CEA in differentiating benign from malignant MCN and IPMN.
Methods: Retrospectively, we reviewed patients' medical records diagnosed with MCN who underwent EUS between 1998 and 2010. The demographics, cyst characteristics, cyst CEA level, surgical outcome, and follow-up data were collected. We compared the median cyst CEA level in benign and malignant mucinous cysts by Mann-Whitney test and determined the relation between cyst CEA level and progression in cyst size by R2 coefficient of determination.
Results: 143 patients (88 F/55 M) were included with mean age of 68.9 years. 63 patients received surgery and 80 patients were followed conservatively. In the surgery group; the median CEA for benign mucinous cyst (n=27) was 796 ng/ml, while for malignant mucinous cyst (n=17) was 438 ng/ml. There was no significant difference among the groups' median CEA level (p=0.79). In the follow-up group (n=80), the median follow-up duration was 21 months. Cyst malignant transformation was observed in 4 (4.7%) patients (1 MD-IPMN, 3 BD-IPMN). Cyst CEA was 116 ng/ml in MD-IPMN patient and 160 ng/ml in one Br-IPMN patient. There was no correlation between cystic CEA level and progression in cyst size (>5 mm) in patients with >6 months of follow-up (R2=0.0002).
Conclusion: Intracystic CEA level was not predictive of malignancy or the cyst size progression of the pancreatic mucinous cysts during follow-up. Based on this data, cyst CEA level in MCN should not be used in medical decision with regard to operative or conservative management of MCN.
Proteomic Analysis of a Mouse Pancreatic Stellate Cell Line in the Activated and a Pseudo-Quiescent Cell State Identifies Differentially-Expressed Proteins
J.A. Paulo,1,2,3 P.A. Banks,3 D.L. Conwell,3 H. Steen,1,21Department of Pathology, Children's Hospital Boston, Boston, MA; 2Proteomics Center at Children's Hospital Boston, Boston, MA; 3Center for Pancreatic Disease, Division of Gastroenterology, Hepatology and Endoscopy, Brigham and Women's Hospital and Department of Medicine, Harvard Medical School, Boston, MA
Background: Pancreatic stellate cells (PaSC) have emerged as mediators in both chronic pancreatitis and pancreatic cancer pathogenesis. Proteins regulating the biomolecular pathways involved in the conversion of activated to quiescent PaSC may have a significant influence in the development of chronic pancreatitis.
Aim: We aim to compare differentially expressed proteins from a cell line of mouse PaSC in the activated and pseudo-quiescent cell states.
Methods: PaSC in the activated and serum-starved cell states using a mass spectrometry-based proteomics strategy. An immortalized mouse PaSC cell line was cultured in DMEM media supplemented with 10% fetal bovine serum (FBS). In parallel, we serum starved PaSC to promote the cellular transition to a "pseudo-quiescent" state. Using the activated and pseudo-quiescent mouse PaSC, we performed a comparative gel-based mass spectrometry (GeLC-MS/MS) proteomic analysis. PaSC grown in FBS maintained their activated fibroblast morphology, however the serum-starved PaSC developed a rounded morphology within 24 hrs.
Results: We identified over 2000 non-redundant proteins in activated and/or pseudo-quiescent PaSC. Qualitative and label-free quantitative (spectral counting) analysis revealed several hundred proteins that were differentially abundant between the two cellular states. Proteins that were more abundant in activated PaSC included cytoskeletal proteins and ribosomal proteins. Conversely, proteins that were more abundant in pseudo-quiescent PaSC included those in protein degradation-related pathways (lysosome, ubiquitin-mediated proteolysis, and the proteasome).
Conclusion: We have identified differentially expressed proteins in activated and pseudo-quiescent PaSC. Investigation of the role of PaSC in the pathogenesis of chronic pancreatitis using the mass spectrometry-based proteomics strategy described herein will lead to further insights into the mechanism of the fibrosis associated with the disease.
Use of "BrU-Seq" for Global Analysis of Enhancers, Transcription Start Sites and Synthesis, Stability and Splicing of RNAs in Pancreatic Cancer Cells
M.T. Paulsen,1 J. Prasad,1 A. Veloso,1 C. Kumar-Shina,2 D. Simeone,3 T.E. Wilson,2 M. Ljungman,1Departments of 1Radiation Oncology, 2Pathology and 3Surgery, University of Michigan Medical School, Ann Arbor, MI
Transcriptional and post-transcriptional regulation of gene expression, such as the regulation of mRNA stability and alternative splicing, is often perturbed in pancreatic cancer. Many RNAs are aberrantly regulated at the level of stability by RNA-binding proteins and microRNAs and the pattern of alternatively spliced transcripts is affected by attenuated expression of specific splice factors. Some of these alternatively spliced transcripts produce oncogenic proteins and thus may play important roles in pancreatic carcinogenesis. To obtain a comprehensive global picture of gene expression in normal and cancer cells we developed the BrU-Seq technique, which is based on bromouridine pulse-chase labeling of nascent RNA followed by isolation of the BrU-containing RNA and deep sequencing. We are using the BrU-Seq to obtain global signatures of enhancers, transcription start sites and synthesis, stability and splicing of all transcripts in normal and cancerous pancreatic cells. In preliminary experiments using the BrU pulse-chase labeling technique with real-time PCR analysis, we have found that the intrinsic stabilities of KRAS and C-MYC mRNAs vary dramatically in three pancreatic cell lines tested and that the newly discovered pancreatic oncogene ATDC can mediate effects on both the synthesis and stability of a subset of mRNAs involved in promoting tumor metastasis. The generation of comprehensive, whole-genome profiles with BrU-Seq should provide us with important new information that would help us better understand the unique etiology and physiology of pancreatic cancer and may open up new possibilities for therapeutic intervention.
Cachexia but not Obesity Worsens the Postoperative Outcome after Pancreaticoduodenectomy in Pancreatic Cancer
T. Pausch,1 W. Hartwig,1 T. Swolana,1 U. Hinz,1 B.D. Bundy,2 T. Hackert,1 L. Grenacher,2 M.W. Büchler,1 J. Werner,11Department of Surgery, University of Heidelberg, Germany; 2Department of Radiology, University of Heidelberg, Germany
Introduction: Prognosis after pancreaticoduodenectomy for adenocarcinoma of the pancreas is determined by tumor, surgeon and by the patient's physical condition.
Objectives: Our study aimed to assess the impact of body mass and fat distribution on the postoperative course.
Methods: 408 patients who underwent Whipple operation at our department between 2001 and 2010 were followed-up in a prospective database. Preoperative CT-scans were analysed for following parameters: abdominal wall (AW), hip girdle (HG) and visceral (VF) fat thickness and "depth" of the surgical site. These and preoperative weight and height were stratified to equally sized subgroups. Postoperative parameters including mortality, non-surgical and surgical complications (e.g. delayed gastric emptying, pancreatic fistula) were evaluated and statistically correlated with body mass and fat distribution.
Results:Overall mortality was 3%. Patients with low BMI had a significantly higher 90-day mortality (p<0.05) and higher rates of complications and relaparotomies. Accordingly patients with high AW fat thickness showed a significant reduction of in-hospital mortality (p=0,019) and after 90 days (p=0,007). High AW fat thickness patients had less surgical complications, relaparotomies and significantly less abscesses (p<0.05). Thin patients had a worse outcome though having less preoperative comorbidities. HG and VF fat thickness and "depth" had no impact on the outcome.
Conclusions: Our study shows a worse outcome for patients with low body mass and little fat and emphasizes the need for therapeutic improvements in the field of pancreatic cancer-associated cachexia.
Circulating Lymphocyte Subsets in Pancreatic Adenocarcinoma
R. Pezzilli, D. Fabbri, A. Imbrogno, A.M. Morselli Labate Department of Digestive Diseases and Internal Medicine, St. Orsola-M. Malpighi Hospital, University of Bologna, Bologna, Italy
Objective: To evaluate the immune response in patients with pancreatic adenocarcinoma (PA) as compared to chronic pancreatitis (CP), other malignant digestive cancer (MDC) and healthy subjects (HS) by determining circulating lymphocyte subsets.
Patients: Ninety-four subjects were studied: 25 consecutive patients with histologically proven PA, 17 patients with proven CP, 14 patients with histologically confirmed MDC and 38 HS. None of the study cancer patients were treated medically or surgically at the time of the study. One PA patient was in Stage I, 5 in Stage II, 4 Stage III, and 15 in Stage 4.
Methods: Total and subset lymphocyte counts (total T lymphocytes, T helper and T suppressor lymphocytes, activated T lymphocytes, B lymphocytes, NK cells) were evaluated. Statistical analysis was carried out using the one-way ANOVA design and simple contrast. Total lymphocytes and lymphocyte subset counts are reported as mean±SD.
Results: Only T helper, T suppressor and B lymphocytes were significant different among the four groups of patients studied (P<0.05); these differences were due to the fact that PA patients had T suppressor lymphocytes (560.4±399.8 mm 3) and B lymphocytes (227.3±106.3 mm 3) significantly higher (P<0.05) than MDC patients (T suppressor lymphocytes 438.9±412.0 mm 3; B lymphocytes 154.6±94.5 mm 3). AP patients with Stage I and II had B lymphocytes subset (147.0 ± 75.0 mm 3) significantly lower (P<0.05) than those with Stage III and IV (252.6 ± 103.4 mm 3).
Conclusions: Circulating total and lymphocytes subjects of PA patients are similar to that of both patients with CP and HS. The increase of circulating B lymphocytes according to the AP stages seems to play an important role in pancreatic cancer immunology.
No Effect of Drugs that Reduce Gastric Acid Production on the Efficacy of CREON® in Randomized Trials of Patients With Exocrine Pancreatic Insufficiency
P. Pollack,1 S. Sander-Struckmeier,2 K. Beckmann,2 G. Janssen-van Solingen,3Abbott, 1Chicago, IL, USA; 2Hannover, Germany; 3Allschwil, Switzerland
Objective: To determine whether concomitant use of proton pump inhibitors (PPIs) or histamine-2 receptor antagonists (H2RAs) may affect CREON® efficacy.
Methods: This was a subanalysis of data from three double-blind, randomized, multicenter, placebo-controlled trials of CREON® (pancrelipase delayed-release capsules) in patients with confirmed exocrine pancreatic insufficiency (EPI). Trials 3.124 (NCT00414908) and 4.009 (NCT00705978) were parallel-group trials enrolling patients ≥18 years old with EPI due to chronic pancreatitis (CP) or pancreatic surgery (PS; 3.124 only) and 3.126 (NCT00510484) was a cross-over trial in patients ≥12 years old with EPI due to cystic fibrosis (CF). In all studies: patients received a diet providing ≥100 g fat/day; those already taking PPIs/H2RAs could continue them at a stable dose; and the primary endpoint was the coefficient of fat absorption (CFA). Prospectively planned exploratory analysis of CFA outcomes according to PPI/H2RA use vs no use was carried out in each trial (statistical comparisons not performed). No subjects took H2RAs in 4.009.
Results: There were no meaningful differences in CFA values on CREON® at the end of the double-blind phase according to PPI/H2RA use. In 3.124, 4.009, and 3.126, mean CFAs on CREON® for PPI/H2RA use vs no PPI/H2RA use were 87.0% (n=9) vs 85.7% (n=13), 86.2% (n=12) vs 86.0% (n=20), and 88.5% (n=18) vs 88.8% (n=13), respectively.
Conclusion: In three double-blind, randomized, placebo-controlled trials enrolling patients with EPI due to CP, PS, or CF, there were no differences in CREON® efficacy related to PPI/H2RA use, as determined by CFA values on CREON®.
PD2/hPaf1 a Novel Marker for the Maintenance of Self-Renewal and Drug Resistance of Pancreatic Cancer Stem Cells
MP Ponnusamy,1* AP Vaz,1* SK Batra,1,2,31Dept of Biochemistry and Molecular Biology, 2Eppley Institute for Research in Cancer and Allied Diseases, 3Dept of Pathology, University of Nebraska Medical Center, Omaha, Nebraska
Background: Over the last several years, it has been evident that a small population (less than 5%) of cancer cells, referred to as "cancer stem cells (CSCs)" or "side population (SP)", is responsible for the disease aggressiveness, metastasis and resistance to therapy. Cancer stem cells have been demonstrated to have roles in many cancers including pancreatic cancer. To date, only a few CSC maintenance markers like CD44, CD24 and CD133 have been identified. These markers, however, do not play a functional role in cancer stem cell maintenance and their expression varies in cancer types. In search of a new marker and its role associated with self-renewal and drug resistance of pancreatic cancer stem cells, we have focused on the human polymerase association factor 1 (hPaf1).
Hypothesis: Recently, we identified the novel role of PD2 protein in the self-renewal of mouse embryonic stem cells. Several studies have shown that self-renewal is a common feature for all types of stem cells including CSCs. Based on the aforementioned seminal work; we hypothesize that PD2/hPaf1 is involved in the maintenance of self-renewal and drug resistance of pancreatic CSCs.
Results: In the present study, we have isolated CSCs from SW1990 pancreatic cancer cells and maintained in stem cell specific condition. Isolated CSCs maintained their cancer stem cell characteristics as shown by their phenotype, spherical colony formation, tumorspheres and in vivo tumor formation. Further, CSCs overexpress hPaf1/PD2 along with other known CSC markers (ALDH1, CD44 and CD133) and self-renewal markers (Shh and Oct3/4). Finally, the knockdown of PD2/hPaf1 in CSCs with Gemcitabine treatment decreased the viability, CD133 and Multi drug resistant gene 2 gene expressions.
Conclusion: Overall, our results suggest a novel role of PD2/hPaf1 in the maintenance of cancer stem cells and also involved in the drug resistance by either directly or indirectly controlling MDR2 gene.
Serum Osteopontin and TIMP-1 as Diagnostic and Prognostic Biomarkers for Pancreatic Adenocarcinoma
K.E. Poruk,1 M.A. Firpo,1,5 C.L. Scaife,1,5 D.G. Adler,2,5 L.L. Emerson,3 K.M. Boucher,4,5 S.J. Mulvihill,1,5Departments of 1Surgery; 2Internal Medicine; 3Pathology; and 4Oncological Sciences, University of Utah School of Medicine, and Huntsman Cancer Institute5, Salt Lake City, UT, 84132, USA
Background: Pancreatic adenocarcinoma (PA) is the fourth most common cause of cancer mortality in the United States and has a dismal 5-year-survival rate. There is an urgent need to identify patients while surgically resectable. Osteopontin (OPN) and tissue inhibitor of metalloproteinase 1 (TIMP-1) have been identified as potential diagnostic serum markers. We assessed serum OPN and TIMP-1 as diagnostic and prognostic biomarkers in a novel cohort of pancreatic cancer patients.
Methods: Serum OPN and TIMP-1 levels were determined by ELISA in a cohort of 86 PA patients, 86 healthy control subjects, and 48 chronic pancreatitis patients. Linear regression models were used to relate OPN and TIMP-1 to gender, age, stage, class, treatment, and survival.
Results: Serum levels of both OPN and TIMP1 distinguished PA from chronic pancreatitis (P ≤ 0.0001) and healthy control subjects (P <0.0001). Neither OPN (ROC AUC = 0.721) nor TIMP1 (ROC AUC = 0.769) had improved diagnostic capability compared to CA 19-9 (ROC AUC = 0.918) in this patient cohort. However, a diagnostic algorithm utilizing all three biomarkers could be devised that improved sensitivity (0.87) and specificity (0.91) over the sensitivity (0.84) and specificity (0.88) of CA 19-9 alone. Serum levels of both OPN and TIMP1 distinguished early stage, resectable PA from chronic pancreatitis and healthy controls (P < 0.04 and P < 0.0001 respectively) whereas CA 19-9 did not (P >0.16). Serum TIMP1 was an independent prognostic factor for survival in PA (P = 0.046), while serum OPN was not (P = 0.055).
Conclusions: Serum OPN and TIMP-1 are potentially useful early detection biomarkers for PA. TIMP-1 is a significant prognostic factor for PA. A panel utilizing CA19-9, OPN, and TIMP-1 increases the sensitivity and specificity for differentiating pancreatic cancer from chronic pancreatitis and normal subjects.
The Effect of Initial Antibiotic Therapy with Moxifloxacin in Infected Necrosis in 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: Antibiotic therapy in acute necrotizing pancreatitis does not have an impact on the development of infected necrosis and the overall lethality. To further examine the failure of antibiotics in acute necrotizing pancreatitis we experimentally evaluated early antibiotic therapy in infected necrosis in necrotizing pancreatitis in mice with respect to the local pancreatic pathology as well as systemic pancreatitis induced adverse events.
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. Antibiotic therapy was performed with 10 mg/kg bwt. moxifloxacin prior to pancreatitis induction (AN). After 24 hours animals were sacrificed and serum as well as organs related to SIRS were examined.
Results: Early antibiotic therapy significantly reduced bacterial count in pancreatic lysates of animals with infected pancreatic necrosis (IN 4.1 107±2.4 107 vs. AN 4.9 104±2.6 104 CFU/g; p<0.001). However, antibiotic therapy did not have an impact on the pancreatic histology (Histology score: IN 23.8±2.7 vs. AN 22.6±1.7). While pulmonary damage was unaltered, systemic immunoactivation was reduced by the administration of moxifloxacin (Serum IL-6: IN 330.5±336.6 vs. 38.7±25.5 pg/ml; p<0.001). Additionally, early moxifloxacin treatment reduced hypoglycaemia, which is a parameter of impaired liver function (serum glucose: IN 105.8±12.7 vs. AN 155.7±39.5 mg/dl; p<0.001).
Conclusion: The initial antibiotic therapy with mocifloxacin in experimental infected necrosis in acute pancreatitis in mice does not have an impact on pancreatic pathology or pulmonary damage. However, other systemic complications induced by infected necrosis in acute pancreatitis are reduced by the administration of moxifloxacin.
Pigment Epithelium-Derived Factor (PEDF) Deficiency Accentuates Pancreatic Fibrotic Responses
P. Protiva, J. Schmitz, R. Hoque, T. Utsumi, Y. Iwakiri, A.G. Neto, A. Gattu, S.E. Crawford, C. Chung Section of Digestive Diseases, Departments of Medicine and Surgery, VA CT, Yale University School of Medicine; NorthShore Research Institute, University of Chicago
Background and Aims: Pigment epithelium-derived factor (PEDF) regulates tissue homeostasis by maintaining vascular and stromal quiescence. We reported that absence of PEDF results in stromal and metabolic disturbances in the liver. Our aim was to determine whether loss of PEDF promoted fibrosis in the pancreas. To examine potential mechanisms by which PEDF modulates the fibrotic response, levels of thrombospondin-1 (TSP-1), a TGF-β activator, were evaluated after PEDF overexpression and inhibition in vitro.
Methods: Repetitive episodes of cerulein-induced pancreatitis were induced in adult PEDF null and wild type mice (n=6/group). Saline-injected controls assessed baseline characteristics. The pancreas was harvested following recovery periods of one and four weeks after the last episode of pancreatitis. Quantitative real-time PCR, immunoblots and immunohistochemical stains were performed for fibrogenic and inflammatory markers. Trichrome and Sirius Red stains assessed the extent of fibrosis.
Results: At baseline, PEDF null animals revealed an increase in activated stellate cells denoted by alpha smooth muscle actin positivity, higher vascular density, and a four-fold increase in TGF-β mRNA in the pancreas versus controls, p<0.001. Inflammatory cytokines MCP-1, TNF-alpha and VEGF were all significantly higher (p<0.05) in PEDF null compared to wildtype mice. After the induction of pancreatitis, increased stromal expansion occurred in PEDF null compared to wildtype mice as seen on trichrome stains and this was associated with strong TSP-1 immunolocalization within the fibrotic regions. A 43% increase in the area stained with Sirius Red was observed in PEDF null compared to control mice in the one-week recovery group (p<0.001) but not in animals with a four-week recovery period. PEDF overexpression in vitro suppressed TSP-1 levels more than four-fold.
Conclusions: PEDF modulates the pancreatic stroma by maintaining stellate cell quiescence and negatively regulating matrix proteins such as TSP-1, an activator of fibrogenic cytokines. PEDF maintains normal pancreatic stromal homeostasis.
Anti-Inflammatory Macrophages Activate Invasion in Pancreatic Adenocarcinoma by Increasing the MMP-9 and ADAM-8 Expression
P. Puolakkainen,1 H. Seppänen,1 H. Mustonen,1 S. Vainionpää,1 Z.H. Shen,1 H. Repo,2 E. Kemppainen,11Department of Surgery, Helsinki University Central Hospital, 2Department of Bacteriology and Immunology, University of Helsinki, The Hartmann Institute, Helsinki, Finland
Introduction: Patients with chronic pancreatitis have high risk for pancreatic cancer. The aim of this study was to examine the role of the inflammatory cells in the invasion and metastasis in pancreatic cancer.
Methods: Monocytes from healthy donors were differentiated into macrophages by GM-CSF incubation. Cancer cells (primary cancer: PANC-1, MiaPaCa-1; metastatic: HPAFII and ASPC-1) were cultured either alone or with differentiated macrophages in matrigel. The cancer cell migration rate in matrigel was measured by imaging fluorescently stained cells for 24h. After invasion the cells were sorted in CD14 positive / negative macrophages and the cancer cells with magnetic separation. The expression of ADAM-8, MMP (2, 9) and TIMP (1, 3) were measured by the real-time PCR.
Results: The macrophage co-culture increased migration and MMP-9 expression in all cancer cell lines. MMP-9 inhibitor TIMP-3 expression was increased in all but the metastatic HPAF cell line. There was a positive correlation between macrophages induced increase in MMP-9 expression in cancer cells and increase in migration rate due to macrophages (R2=0.94, p=0.03). MiaPaca had the largest macrophage induced increase in ADAM-8 expression and migration rate. Pancreatic cancer cells upregulated MMP-2 and TIMP-1 in CD14+ macrophages.
Conclusion: MMP-9 is one of the main gene affecting the macrophage induced increase in cancer cell migration, although also ADAM-8 has a role. The cancer cell induced upregulation of MMP-2 in CD14+ macrophages might increase their migration.
Identifying Molecular Signatures for the Earliest Kras-Induced Transformation Events in Pancreatic Adenocarcinoma Development
C. Qu, D. DiRenzo, S. F. Konieczny Department of Biological Sciences and the Purdue Center for Cancer Research, Purdue University, West Lafayette, IN
Background: Over 90% of pancreatic ductal adenocarcinoma (PDAC) patients are diagnosed at a late metastatic stage necessitating early detection and intervention. In mouse models, PDAC can be derived from KrasG12D-expressing acinar cells which undergo a process of acinar-ductal metaplasia (ADM), pancreatic intraepithelial neoplasia (PanIN) and then on to PDAC with a rapid accumulation of stromal cells surrounding the lesion. What remains unknown are the signaling pathways that initiate the earliest changes in Kras-dependent ADM and stromal cell activation.
Methods: Mist1CreER/+; LSL-KrasG12D mice were used to induce KrasG12D exclusively in adult acinar cells and individual pancreata were examined for early molecular alterations by IHC.
Results: Despite the fact that ∼90% of acinar cells express KrasG12D, virtually all cells appear normal during the first 2-4 weeks. The very earliest events observed in isolated morphologically normal acini involved expression of the duct cell transcription factor Sox9 and activation of pErk and pStat3. At later stages additional duct markers (K19) and proliferation markers (Ki67) were detected. Vimentin+ PSCs were observed surrounding relatively normal pErk+ acini followed by expression of α-smooth muscle actin and collagen I.
Conclusions: Our study supports a model in which the MAPK and Stat3/JAK pathways are activated shortly after KrasG12D expression in a subset of acinar cells. Sox9 expression in the acini promotes cells to go toward an ADM transition. Stromal cells are then recruited to the lesion area which in turn supports progression toward PanIN/PDAC.
MUC4 induces Epithelial Mesenchymal Transition Through N-Cadherin Upregulation in Pancreatic Cancer
S. Rachagani, M. A. Macha, M. P. Ponnusamy, S. Chakraborty, S. K. Batra Department of Biochemistry and Molecular Biology, Eppley Cancer Institute, University of Nebraska Medical Center, Omaha, NE
Background: MUC4, a transmembrane mucin is has previously been shown to enhance progression of pancreatic cancer (PC). Recent studies have demonstrated an important role of epithelial to mesenchymal transition (EMT) in tumor progression, making tumor cells more invasive and metastatic. N-cadherin is a key mediator of the EMT process in PC cells. In this study, we investigated the mechanism underlying MUC4 mediated upregulation of N-cadherin and its interacting partner fibroblast growth factor receptor 1 (FGFR1) in PC cells as well as signaling pathways involved in EMT process.
Method: MUC4 expression was silenced by specific shRNA in Capan1 and BXPC3 PC cells. The effect of MUC4 knockdown on EMT was assessed by various in vitro and in vivo assays. Effect MUC4 knockdown on cell signaling was assessed by western blotting and Real time PCR.
Results: MUC4 silencing led to down-regulation of N-cadherin and its interaction partner FGFR1 through destabilization of pHer2, followed by decreased activation of pScr, FAK and its downstream molecules, such as MKK7, JNK1/2 and c-Jun. This was associated with a more compact growth of the cells, increase in epithelial markers (E-cadherin, Occludin and cytokeratin-18) and decrease in mesenchymal markers (vimentin, N-cadherin and Twist) in the Capan1/BXPC3-shMUC4 cells. The MUC4 knock down cells also showed a significant decrease in motility (p<0.00001) and invasion (p<0.0001) and increased apoptosis (p<0.05) in vitro and decreased tumorogenicity (p<0.05) and incidence of metastasis upon orthotopic implantation in athymic mice associated with down-regulation of pro-invasive proteins like Her2, FAK, AKT, ERK. uPA and MMP9.
Conclusion: Taken together, our results suggest that MUC4 promotes EMT in PC cells through upregulation of N-cadherin and its interacting partner FGFR1.
Results of Pancreaticogastrostomy Versus Pancreaticojejunostomy in Reconstruction After Cephalic Duodenopancreatectomy in Patients With Soft Pancreas and Small Pancreatic Duct-PANAM Study
D. Radenkovic,1 Dj. Bajec,1 N. Ivancevic,2 P. Gregoric,2 V. Jeremic,2 V. Djukic,2 D. B. Kardzic,2 A. Antic,1 I. Pejovic,11Clinic for Digestive Surgery and 2Clinic for Emergency Surgery, Clinical Center of Serbia, Belgrade, Serbia
Introduction: Pancreatic fistula (PF) remains a most common complication after pancreaticoduodenectomy (PD) and the main cause of other morbidities and mortality. Pancreaticojejuno (PJ) anastomosis is the most often used method of reconstruction after PD. Several technique modifications such as placement of the stents, reinforcement of anasomosis with fibrin glue, pancreatic duct occlusion and pancreaticogastrostomy (PG) type of anastomosis was used in order to decrease PF rate. It was shown that the higher risk of PF was noticed in patients with soft residual pancreas and small diameter of pancreatic duct. The only one randomized study did not reveal any significant differences between PG and PJ in patients with soft pancreas and small duct. In order to investigate once more this important issue, randomized multicenter controlled trial was conducted.
Methods: One hundred patients with patients with soft pancreas and small duct will be randomly allocated to two groups: I) reconstruction by PJ anastomosis or II) reconstruction by PG anastomosis. Patients will be recruited from 5 hospitals during 3 years period. The primary endpoint is development of abdominal complications comprising: PF, acute fluid collection, acute pancreatitis, billiay fistula, gastric fistula, enteral fistula, hemorrhage and delayed gastric emptying. Secondary endpoints are mortality, duration of hospital and ICU stay, and cost of hospitalization. A total sample size of 100 patients was calculated to demonstrate that PG anatomosis can reduce development of abdominal complications rate from 40% to 20% with 80% power at 5% alpha.
Conclusion: PANAM study is designed to reveal a reduction in development of abdominal complications by using PG type of reconstruction after PD in patients with soft pancreas and small pancreatic duct in comparison with PJ anastomosis.
MicroRNA-200c Modulates the Expression of MUC4 and MUC16 in Human Pancreatic Cancer
P. Radhakrishnan, A.M. Mohr, P.M. Grandgenet, M.A. Hollingsworth Eppley Institute for Research in Cancer and Allied Diseases, University of Nebraska Medical Center, Omaha, NE
Background: Transmembrane mucins, MUC4 and MUC16 are aberrantly overexpressed and associated with tumor progression and metastatic potential in human pancreatic adenocarcinoma. MicroRNAs (miRNAs) bind to specific mRNA sequences and induce degradation or inhibit translation of the targeted mRNA. Studies of miR-200c in multiple cancers indicate this miRNA has a significant role in cancer biology by modulating a set of proteins that enact epithelial-to-mesenchymal transition (EMT). The study presented here demonstrates that miR-200c also regulates MUC4 and MUC16 expression in pancreatic cancer.
Methods: miR-200c was overexpressed in pancreatic cancer cells and its effect on MUC4 and MUC16 expression was assessed by quantitative real time PCR (RT-PCR), western blot and luciferase reporter assays.
Results: We discovered that miR-200c interacts with specific sequences within the MUC4 and MUC16 mRNAs coding sequences. S2.028 and T3M-4/miR200c pancreatic cancer cells showed a 4.18 and 8.50 fold down regulation of MUC4 mRNA, and 4.68 and 4.82 fold down regulation of MUC16 mRNA compared with mock cells, respectively. A significant reduction of glycoprotein expression was also observed.
Conclusions: Our findings indicate that miR-200c overexpression regulates MUC4 and MUC16 mucins in pancreatic cancer cells by directly targeting the mRNA coding sequence of each, resulting in reduced levels of MUC4 and MUC16 mRNA and protein. These data suggest that, in addition to EMT, miR-200c influences expression of cell surface mucins in pancreatic cancer.
Pancreatic Ductal HCO3- and Fluid Secretion Are Reduced in SLC26a6 Knock-Out Mice
Z. Rakonczay Jr.,1 P. Pallagi,1 V. Venglovecz,2 T. Takács,1 T. Wittmann,1 A.K. Singh,3 R. Engelhardt,3 B. Riederer,3 U. Seidler,3 P. Hegyi,11First Dept. of Medicine, 2Dept. of Pharmacology and Pharmacotherapy, University of Szeged, Hungary; 3Dept. of Gastroenterology, Hepatology, and Endocrinology, Medical School of Hannover, Germany
Introduction: Pancreatic duct cells secrete a HCO3-rich isotonic fluid. The mechanism of this HCO3- secretion is poorly understood, especially concerning transport occurring at the apical cell membrane. However, cystic fibrosis transmembrane conductance regulator and SLC26 anion exchangers are required for these functions.
Aim: To evaluate the role of SLC26a6 (PAT1) in pancreatic anion and fluid secretion.
Methods: Interlobular pancreatic ducts were isolated from wild-type (WT) and PAT1 knock-out (KO) mice. Anion exchange activity was determined by measuring the intracellular pH (pHi) by microfluorimetry. We used the inhibitory stop and alkali load methods to determine the HCO3- efflux across the apical membrane. Fluid secretion into the closed luminal space of the cultured ducts was analysed by using a video technique.
Results: Exposing the ducts to 0.2 mM H2DIDS and 0.2 mM amiloride caused an acidification of pHi due to inhibition of the basolateral Na+/HCO3- cotransporters and Na+/H+ exchangers. HCO3- secretion was significantly lower in PAT1 KO vs. WT mice. We also analysed the recovery of pHi from an alkali load induced by exposure to 20 mM NH4Cl in a HCO3-/CO2-containing solution. The recovery from alkalosis was significantly lower in PAT1 KO vs. WT mice. The forskolin (5μM)-stimulated fluid secretory rate was significantly lower in KO vs. WT mice.
Conclusion: Our results suggest that SLC26a6 is necessary for pancreatic ductal secretion.
This study was supported by OTKA, MTA/DFG and NFÜ (TÁMOP).
AGR2/C4.4A Pathway - A Novel Target for PDAC
V. Ramachandran,1 T. Arumugam,1 R.F. Hwang,2 C.D. Logsdon,1,3Depts. of 1Cancer Biology, 2Surgical Oncology, 3Medical Oncology, UT MD Anderson Cancer Center, Houston, TX
Background: We previously reported that anterior gradient 2 (AGR2) is an autocrine factor in PDAC that facilitates tumor aggressiveness and resistance to gemcitabine. Very recently we identified C4.4A, a GPI-linked receptor as the first functional receptor of AGR2. Current study, we further characterized its signaling complex and mechanisms and developed blocking antibodies against the ligand and the receptor.
Methods: Gene silencing (C4.4A/panel of laminins/integrins) was obtained using siRNA. Recombinant AGR2 was used for analysis of proliferation, migration and invasion of PDAC cell lines (MPanc96/BxPC-3/MiaPaCa-2). Apoptosis after siRNA silencing +/- gemcitabine and AGR2 was conducted by FACS analysis. Erk, Akt, Src, Fak activation and k-Ras activity was measured in AGR2 treated cells. Purified mouse monoclonal blocking antibodies were developed against AGR2 and C4.4A. PDAC patient RNA was analyzed for C4.4A expression by Affymetrix and outcome was analyzed.
Results: Silencing of laminins 1(1.5 fold) & 5 (2 fold) and integrin β1 (1.3 fold) increased the level of Gem-mediated apoptosis and blocked AGR2 mediated survival effects (p<0.05). Exogenous AGR2 stimulated Erk, Akt, Src and Fak phosphorylation within 5mins. K-Ras activity was stimulated at 5 mins on wild type k-ras bearing NIH 3T3 cells, while in mutated k-ras bearing MPanc96 cells the activity was sustained on treatment with AGR2. Signaling effects of AGR2 were abolished on silencing C4.4A. Blocking antibody against AGR2 and C4.4A showed a clean binding on western blotting and blocked the effects of basal and AGR2 mediated migration, invasion and survival (p<0.05). IHC of pancreatic TMA using the blocking antibodies showed 85% positivity for AGR2 and 91% positivity for C4.4A. C4.4A mRNA levels were associated with poor outcome in PDAC.
Conclusion: The AGR2/C4.4A autocrine loop may be a reasonable target for new therapeutics against PDAC.
Comparison of BISAP, Ranson's, and CTSI Scores, and C-reactive Protein in Predicting Organ Failure, Complications and Mortality in Acute Pancreatitis
L. Ricardo, F. Cardoso, A. Oliveira, C. Rodrigues, D. Horta, A. Figueiredo, J. Deus Department of Gastroenterology, Hospital Fernando Fonseca, Amadora, Universidade Nova, Lisboa, Portugal
Background: Risk stratification early in the course of acute pancreatitis (AP) is important to improve outcome. The aim of this study was to compare the new bed side index for severity in AP (BISAP) with Ranson's and computed tomography severity index (CTSI) scores, and c-reactive protein (CRP) in predicting organ failure, pancreatic necrosis and in-hospital mortality.
Methods: Demographic, laboratory and radiographic data from consecutive patients with AP admitted to our institution was collected between January 2009 and December 2010. Predictive accuracy of the scores and CRP was measured by the area under the receiver-operating curve (AUC).
Results: There were 299 patients with AP, with a median age of 61 years and 52.8% men. Sixteen patients (9%) developed organ failure and were classified as severe AP. Thirty-five (20.7%) developed pancreatic necrosis and 10 (3.3%) died. The number of patients with BISAP ≥3 was 42 (14%), Ranson's ≥3 was 95 (31.8%), CTSI ≥3 was 49 (29%) and CRP ≥15mg/dl was 100 (51.5%). AUCs for BISAP, Ranson's, CTSI and CRP at 48 hours in predicting severity were 0.73 (95% CI 0.61-0.86), 0.66 (95% CI 0.53-0.78), 0.84 (95% CI 0.72-0.95) and 0.83 (95% CI 0.73-0.93), respectively, and in predicting in-hospital mortality were 0.84 (95% CI 0.72-0.96), 0.85 (95% CI 0.77-0.93), 0.93 (95% CI 0.87-0.98) and 0.86 (95% CI 0.78-0.95), respectively.
Conclusions: BISAP is a simple and accurate method for the identification of patients at increased risk for in-hospital mortality within the first 24 hours of AP course. The BISAP value in predicting organ failure in AP is acceptable, but inferior to that of the CTSI and CRP.
Activation of NFkB Pathway is Independent of Trypsinogen Activation in Chronic Pancreatitis
R.P. Sah, R. Dawra, A. Bekolay, V. Dudeja, A.K Saluja Division of Basic and Translational Research, Department of Surgery, University of Minnesota, Minneapolis, MN, USA
Background and Aims: Trypsinogen activation, observed early during pancreatitis, has been considered the central event in pancreatitis for several decades. Recently, NFkB activation has been shown to occur temporally parallel but independently of trypsinogen activation during pancreatitis. In this study we aimed to evaluate the role of these crucial events observed early during pancreatic injury in the pathogenesis of chronic pancreatitis (CP).
Methods: CP was induced by caerulein (50μg/kg i.p. every hour × 6) twice a week for 10 weeks. Novel knock-out mice lacking cationic trypsinogen gene (T-/-) were used. These mice lack pathologic trypsinogen activation. Groups with (WT) and without (T-/-) pathologic trypsinogen activation were compared for histopathological severity of CP, and for activation of NFkB and its downstream events that are known to be associated with chronic inflammation (N=20 each group, saline injected mice as controls). Clinical specimens were obtained from CP patients who underwent surgical intervention for chronic pain. Normal pancreases collected for islet transplants were used for comparison.
Results: WT and T-/- mice developed comparable CP as indicated by pancreas atrophy, histological changes, fibrosis, stellate cell activation and ductular metaplasia. Similar changes were noted in clinical CP samples. Staining for p65 subunit of NF-kB in clinical CP samples revealed nuclear translocation of p65 in significantly higher number of acinar cells compared to normal pancreas, suggesting chronic NFkB activation in CP. A very similar pattern of p65 staining was seen in mice with CP, with comparable increase in p65 nuclear translocation in WT and T-/- groups. COX-2 and TGFβ are downstream of NFkB activation and are known to be key players in chronic inflammation. Comparable increase (by Western Blots) in protein levels of COX-2 and TGFβ were seen in WT and T-/- mice with CP compared to controls. The increase in COX-2 and TGFβ were confirmed and localized to acinar cells by immunohistochemistry. CD3 staining revealed similar extent of T-cell infiltration in WT and T-/- mice with CP compared to controls.
Conclusion: Intra-acinar trypsinogen activation is not required for the pathogenesis of chronic pancreatitis. Chronic NFkB activation is seen in chronic pancreatitis independent of trypsinogen activation. Persistent COX-2 activation and overexpression of TGFβ as a result of NFkB activation are drivers of chronic ongoing inflammation independent of trypsinogen activation and this may be responsible for the pathogenesis of chronic pancreatitis.
Pathogenesis of Pain in Chronic Pancreatitis is Independent of Intra-Acinar Trypsinogen Activation
R.P. Sah, R. Dawra, S. Banerjee, A. Bekolay, V. Dudeja, A.K Saluja Division of Basic and Translational Research, Department of Surgery, University of Minnesota, Minneapolis, MN, USA
Background and Aims: Chronic pain is a major problem in Chronic Pancreatitis (CP). Unfortunately, very little is known about its pathogenesis. Involvement of intra-acinar trypsinogen activation, an event widely considered to be central in pancreatic injury, has been hypothesized recently. Here we aim to examine the role of intra-acinar trypsinogen activation in pathogenesis of pain in CP.
Methods: CP was induced by caerulein (50μg/kg i.p. every hour × 6) twice a week for 10 weeks. A three-fold approach incorporating behavioral, neuronal, and histopathological responses was used for quantitative pain assessment. Cationic trypsinogen gene knock-out mice (T-/-), which do not demonstrate pathologic trypsinogen activation (Dawra RK et al, Pancreas 2009;38:992-93), were used in the study. We compared pain response in CP in wild type (WT) and T-/- mice (n=15-20, each group).
Results: The severity of CP was similar in WT and T-/- mice as previously reported by us. Mice with CP demonstrated a consistent increase in MGS score (range 0-2) which has recently been validated as a quantitative behavioral marker of pain in mice (Langford et al, Nature Methods 2010; 7(6): 447). MGS score was similar in WT and T-/- mice with CP (WT: 0.44±0.09, T-/-: 0.42±0.06; p=0.98) as compared to 0.09±0.04 in controls (p<0.001 for each pair). The activation state of 2nd order sensory neurons conducting pain from the pancreas (neurons in lamina 1 of posterior horn at T9 spinal level) was assessed by c-fos positivity. WT and T-/- mice with CP had 29±3 and 33±3 positive neurons (p=0.43) Vs 9±1 in controls (p<0.001 for each pair). Nerve Growth Factor (NGF) immune-positivity in the pancreas was markedly increased compared to controls to comparable extent in WT and T-/- mice with CP. Significant but comparable increase in mRNA expression (quantified by qPCR) of NGF, Substance P (NK1), NK2 and CGRP was noted in WT and T-/- mice with CP compared to controls.
Conclusion: Pain response was quantified and found to be similar in CP in the presence (WT) and absence (T-/-) of trypsinogen activation. Therefore, pathogenesis of pain in CP is independent of intra-acinar trypsinogen activation. Given the growing recognition of the role of protease activated receptors (PAR-2) (trypsin being one of its natural ligands) in pain pathogenesis, this study suggests that the involvement of PAR-2 in the pathogenesis of pain in CP is independent of pancreatic trypsin. Another novel aspect of this study is characterization of pain response in a standard mouse model of CP. This model can be used to study pathogenesis as well as therapeutics of pain in CP.
Minnelide, a Potent Chemotherapeutic Agent Against Pancreatic Cancer
V. Sangwan, R. K. Chugh, D.M. Johnson, S. Banerjee, V. Dudeja, N. Mujumdar, T. N. Mackenzie, S. M. Vickers, A. K. Saluja Division of Basic and Translational Research, Department of Surgery, University of Minnesota, Minneapolis, MN 55455, U.S.A
Background: Pancreatic cancer carries one of the most dismal prognoses, with a median survival of less than six months. Previous data from our group has shown that triptolide, a diterpenoid, and its water soluble variant, Minnelide, inhibit HSP70 expression leading to cell death in pancreatic cancer cells, as well as prevent progression of pancreatic tumors in vivo.
Aim: To evaluate the efficacy of Minnelide as a potential therapeutic agent for pancreatic cancer in vivo.
Methods: In order to assess the ability of Minnelide to prevent tumor progression, and, more importantly, to cause tumor regression, we used the following pancreatic tumor models: 1) Pancreatic cancer cells derived from ascites (Aspc-1) were orthotopically implanted into the pancreas of nude mice, and, 2) tumor from a human patient was implanted subcutaneously into SCID mice. Both models were treated with either vehicle or Minnelide (0.21 or 0.42 mg/kg QD) at different stages of tumor formation. Animal survival and tumor weight/volume were used as a measure of disease progression. HSP70 expression was measured in RNA from tumors from Minnelide or saline-treated mice by real time PCR, and immunohistochemistry.
Results: In the Aspc-1 model, Minnelide dramatically extended survival from 36 days in the saline group to more than 384 days in the Minnelide group. The average tumor volume in the saline group was 1200 mm3, whereas that in the Minnelide group was 100 mm3. In the human xenograft subcutaneous model, Minnelide treatment was started either when tumor size was 330 mm3 to study tumor progression, or 1000 mm3 to study tumor regression. In the tumor progression model, tumor size in the saline group increased to 1000 mm3 by Day 40 post-treatment, but in the Minnelide treated group, tumor size decreased to nearly zero at the same point. In support of these data, average tumor weight decreased from 1.2g in the control group to less than 0.1g in the treatment group. In the tumor regression model, Minnelide was given at either 0.42 or 0.21 mg/kg QD. In both groups, tumor volume decreased from 1000 mm3 to nearly zero forty days post-treatment. Histological assessment of tumors from these animals show ablation of pancreatic dysplasia, accompanied by a decrease in HSP70 levels.
Conclusion: We have exciting data using orthotopic pancreatic cancer cell lines and human xenograft transplants, which demonstrate that Minnelide is not only able to prevent tumor progression, but also dramatically increase survival and cause tumor regression. We are currently in the process of initiating Phase I clinical trials for this drug.
Is the Lack of Insulin Signaling on Pancreatic Acinar Cells the Cause of Pancreatic Insufficiency in Diabetes?
M.D. Sans, R.K. Amin, N.L. Vogel, L.G. D'Alecy, J.A. Williams Department of Molecular and Integrative Physiology, University of Michigan, Ann Arbor, MI
Background: Type-1 diabetes is characterized by pancreatic insufficiency and experimental diabetes inhibits digestive enzyme synthesis and secretion. However, it is unclear whether effects are due to insulin deficiency or secondary to diabetes.
Aim: To demonstrate that direct insulin action on acinar cells is required for the synthesis of pancreatic digestive enzymes without systemic diabetes.
Methods: Pancreatic acinar cell insulin receptor knock out (PACIRKO) mice were generated by crossing IRlox/lox mice with tamoxifen regulated Ela-Cre mice. Control and PACIRKO mice were either fasted for 12 h or refed for 2 h. Pancreas samples were analyzed for the activation of translation initiation factors, polysomal profiles and the mRNA for different digestive enzymes. Pancreatic juice was collected by canulation of the biliopancreatic duct.
Results: Plasma glucose levels (mg/dL) were: Fasted-C 104; Refed-C 189; Fasted-PACIRKO 114; and Refed-PACIRKO 182; and insulin levels (μU/mL) were <2.5 in both fasted groups and >150 in both re-fed groups. Pancreas/body weight was reduced about 28% in PACIRKO mice. No major histological changes in acini or islets were observed. Akt, S6 and 4E-BP1 phosphorylation was reduced by 30%, 41% and 69% respectively. Polysomal profiling revealed an increase in the free ribosomal subunit peaks for the Refed-PACIRKO mice; indicating a decrease in the number of mRNAs engaged in translation. Amylase content was reduced to 50% of control. Total pancreatic fluid secretion was not changed, but protein concentration and content of amylase and chymotrypsin were reduced in the PACIRKO mice.
Conclusions: Insulin action is required for both short- and long-term pancreatic function.
COX-2 Depending CXC-Chemokines Production in Human Pancreatic Cancer Cells
M. Satake,1,3 G. Eibl,2 N. Kuroda,1 T. Hirano,1 H Ishikawa,3 M Nishii,3 A. Babaya,3 V. L. Go,2 H. Reber,2 Y Fukuda,3 O. J. Hines,2 J Fujimoto,11Departments of Surgery, Hyogo College of Medicine: Nishinomiya, Hyogo, JP. 2David Geffen School of Medicine at U.C.L.A: Los Angeles, CA, USA. 3Sasayama Medical Center, Hyogo College of Medicine, Sasayama, Hyogo, JP
Background: Pancreatic cancer (PaCa) is a lethal disease. Prostaglandin E2 (PGE2), generated by cyclooxygenase-2 (COX2) from arachidonic acid (AA), stimulates PaCa growth through EP receptors. This effect is suppressed by COX2-inhibitor (Coxibs) and selective EP2 and EP4 receptor antagonists (EP2-Ant. and EP4-Ant). Recent studies suggest a correlation between COX2 and ELR-positive CXC chemokines (ELR+CXCLs) production. ELR+CXCLs show growth stimulation through CXC-receptor-2 (CXCR2). In PaCa, the correlation of COX and CXCL production has not been identified. The aim of this study was to establish the role of COX2 in CXCLs production in PaCa. We used Coxibs, EP2-Ant and EP4-Ant to evaluate their inhibitory effects.
Methods: The COX-2 positive (COX+) PaCa cell line BxPc-3 (B) and COX-2 negative (COX-) cell line Mia-PaCa-2 (MP) were analyzed for ELR+CXCL by ELISA and Immunofluorescence (IMF). After stimulated with AA or PGE2, ELR+CXCL were measured, and cell numbers were compared. CXCLs levels were also measured after stimulation by AA in the presence of Coxibs, EP2-Ant and EP4-Ant.
Results: CXCR2 was highly expressed in B. AA and PGE2 enhanced CXCLs production in B. CXCLs production was significantly suppressed by Coxibs, EP2-Ant. and EP4-Ant.
Conclusion: In PaCa, ELR+CXCLs production was stimulated by a COX2/PGE2/EP receptor pathway. Inhibition of this pathway decreased ELR+CXCLs and cell growth, suggesting a possible therapeutic role of these agents in PaCa.
Proposal of a Step-Down Approach for the Antibiotic Therapy in Preoperatively Stented Patients with Distal Bile Duct Obstruction
D. Sauliunaite,1 M. Erkan,1 A. Herner,2 C. Reiser-Erkan,1 T. Miethke,3 C.W. Michalski,1 H. Friess,1 J. Kleeff,11Department of Surgery; 22nd Department of Internal Medicine; 3Institute of Medical Microbiology, Immunology and Hygiene, TUM, Munich, Germany
Background: Preoperative stenting of the bile duct in the patients with distal bile duct obstruction increases infectious postoperative complications. A step-down antibiotic therapy may reduce infectious complications, costs and hospital stay.
Methods: 198 patients who received pancreatic head resection due to distal bile duct obstruction between Juli 2007 and February 2010 were analysed retrospectively. Complications were assessed according to Clavien-Dindo classification.
Results: Ninety patients underwent preoperative ERCP and stenting due to obstructive jaundice. Intraoperative bile cultures were significantly more often contamined in the stented patiens (100% vs 34.8%, p=0.03) with a broader microbiological spectrum (40 vs. 19 species). Resistance to prophylactic antibiotics were more often in stented patients (34.2% vs. 26.2%, p=0.2). Antibiotic therapy with Imipenem would have covered >95% of the patients. The average hospital stay of the patients with the postoperative complications was significantly longer then patients without complications (15.6 vs 28.4, p<0.0001). Duration of the hospital stay also increased significantly in the patients with complications if they were preoperatively stented (24.1 vs 32.7, p=0.03).
Conclusions: Preoperative biliary stenting increases postoperative complications, hospital stay and costs significantly in patients undergoing pancreatic head resections. Due to the high prevalance of resistant bacteria, a step down approach of therapeutic antibiotics instead of a standard culture oriented escalation of the antibiotic therapy may reduce morbidity.
Effects of Ethanol on Pancreas Regeneration
M.A. Scheer, K.J. Mahan Schneider, D.L. Clemens Nebraska and Western Iowa Veterans Administration Medical Center and Department of Internal Medicine, University of Nebraska Medical Center Omaha, NE 68105
Introduction: Alcohol abuse is one of the most common factors associated with acute and chronic pancreatitis. Although it is evident that alcohol abuse can have an important role in the development of pancreatitis, it does not appear that alcohol abuse alone is responsible for this disease. Rather, it appears that ethanol sensitizes the pancreas to damage and that other factors are required to develop alcoholic pancreatitis. Many studies have identified mechanisms by which ethanol sensitizes the pancreas to injury, but few have investigated its effects on recovery from damage.
Methods: A biologically relevant model of alcoholic pancreatitis combining chronic ethanol consumption and coxsackievirus infection was used to investigate the effects of ethanol on pancreatic regeneration. Mice were provided 20% ethanol in their drinking water for 6-8 weeks prior to infection with coxsackievirus (CVB3/CO). Tissues were harvested and analyzed by RT-PCR and immunoblot.
Results: Data from these studies demonstrate that chronic ethanol consumption delays the structural repair of the exocrine pancreas. This is accompanied by a delay in the restitution of amylase and lipase expression. Additionally, alterations in the expression of the critical pancreatic transcription factors, PDX1 and PTF1, and the mediator of Notch signaling, HES1, were observed.
Conclusions: Chronic ethanol consumption impairs the structural repair and functional restitution of the pancreas after severe injury. These impairments may, in part, be explained by alterations in the expression of factors important in the development and regeneration of the pancreas. Impaired pancreatic regeneration may have a role in the pathogenesis of alcoholic pancreatitis.
Rare PRSS1 Mutations Found in Patients With Chronic Pancreatitis are Harmless Variants
A. Schnúr, P. Hegyi, M. Sahin-Tóth Department of Molecular and Cell Biology, Boston University, Boston, MA and First Department of Medicine, University of Szeged, Szeged Hungary
Background and Aims: Mutations in the human cationic trypsinogen gene (PRSS1) are causative agents for hereditary pancreatitis (p.R122H, p.R122C, p.N29I) or risk factors for chronic pancreatitis (p.A16V). These mutations result in a gain of function, manifested by increased autoactivation and resistance to degradation by chymotrypsin C. As genetic screening of pancreatitis patients has become increasingly routine, a large number of rare or private PRSS1 mutations have been found and reported as pancreatitis-associated. Our aim was to establish possible clinical relevance of published rare PRSS1 mutations on the basis of their phenotypic characteristics.
Methods: Wild-type cationic trypsinogen and 8 mutants were produced recombinantly and purified to homogeneity. Trypsinogen activation, degradation and trypsin activity were investigated by enzyme activity assays and gel electrophoresis.
Results: Five of 8 mutations studied (p.P36R, p.Q98K, p.D100H, p.T137M and p.G208A) had no demonstrable functional effect. Mutation p.K92N decreased autoactivation of cationic trypsinogen. Unexpectedly, two mutants (p.G83E and p.V123M) exhibited markedly increased degradation by chymotrypsin C. This loss of function is particularly surprising for mutant p.V123M, which is located next to the autolytic site Arg122, and was previously postulated to increase trypsin stability.
Conclusion: Rare mutations of the PRSS1 gene observed in patients with chronic pancreatitis are innocuous variants with no functional consequence or loss-of-function character. The study reinforces our contention that assignment of clinical relevance to rare PRSS1 variants should not be based on a perceived analogy with genuine disease-causing PRSS1 mutations.
Using In Vivo Phage Display to Identify Non-VEGF Mediated Tumor Angiogenic Proteins
M.E. Seaman, K.A. Kelly Department of Biomedical Engineering, University of Virginia, Charlottesville, VA
Angiogenesis is vital to the growth, progression and ultimately metastasis of tumors. As such, many therapies have attempted to eradicate tumors through antiangiogenic strategies. Most current and past therapies focus on the vascular endothelial growth factor (VEGF) pathway. Unfortunately, these VEGF therapies have not shown much success. Therapies against novel or non-VEGF proteins could hold the key to effective antiangiogenic cancer therapies. To identify novel non-VEGF mediated angiogenesis factors, we used an in vivo peptide phage display screen.
After selection, 30 clones were isolated and validated in vivo and in vitro. In vivo validation showed phage binding to 75% of tumor vessels and binding to less than 15% of normal pancreas vessels. Interestingly, one selected clone resembles previously published VCAM-1 binding clones, a known angiogenic protein. The clone bound recombinant VCAM-1 2.2-fold better than controls. To identify clones that bind tumor vessels, but not in the VEGF context, in vitro validation on HUVECs grown in different media was completed using phage ELISA. Four clones specifically bound HUVECs grown in tumor-conditioned media. These clones were selected for binding partner identification. The binding partner of clone 9 has been identified and preliminarily validated using mRNA expression, ICC studies, and IHC on mouse and human PDAC tissue. In fact this protein is ONLY expressed in HUVECs treated with tumor-conditioned media but not vehicle or VEGF conditioned media.
Our phage display approach has identified a novel, non-VEGF mediated potential therapeutic target that could provide success for antiangiogenic therapies. Future work includes identifying remaining phage binders and testing therapeutic target feasibility with knockout studies.
Inhibition of Tumour Growth in a Pancreatic Cancer Xenograft Model by AT13387, an HSP90 Inhibitor
E. Shaw,1 A. Thomas,1 P. Ghaneh,1 W. Greenhalf,1 M. Davies,1 E. Costello,1 F. Gibbs,1 J. Lyons,2 J.P. Neoptolemos,1 D.R. Sibson,11Department of Molecular and Clinical Cancer Medicine, Institute of Translational Medicine, University of Liverpool, Liverpool, UK;2 Astex Therapeutics, Cambridge, UK
Background: Pancreatic cancer continues to have a poor prognosis with over 7700 deaths in the UK per year. Gemcitabine the main standard treatment for pancreatic cancer has a poor response rate, only improving average survival by weeks. Targeting specific pathways in tumourgenesis may provide new more effective therapies. Heat shock protein 90 (Hsp90) is a molecular chaperone affecting multiple key cellular signalling pathways of particular importance in pancreatic cancer. Astex Therapeutics' AT13387, a novel HSP90 inhibitor, has shown efficacy in a number of cancer Xenograft models and is here investigated for the treatment of pancreatic cancer.
Methods: Anti-tumoral activity of AT13387 and Gemcitabine was assessed in nude mice using a human pancreatic cancer cell line (MiaPaCa2) xenograft model. Cell proliferation in 7 pancreatic cancer cell-lines was measured using EZ4U assay (Biomedica). Cell cycle analysis was carried out with propidium iodide staining and flow cytometry.
Results: Administered as single agents, AT13387 and gemcitabine were tolerated in xenograft experiments with no mortality or significant morbidity. Tumour growth was significantly reduced over 16 days treatment with AT13387 (p=0.0387) or gemcitabine (p=0.0038) compared to vehicle treated controls. Treatment with AT13387 inhibited proliferation in multiple pancreatic cancer cells (IC50 values 29-100nM) comparable to other cancer types previously reported (10-400nM). Similar sensitivity to AT13387 was also observed in a gemcitabine resistant cell-line, SUIT-2GR, (gemcitabine IC50 60-fold higher than parental cell-line). Cells treated with AT13387 alone accumulated in the G1 and G2/M phases of the cell cycle. Gemcitabine caused an accumulation of cells in S-phase of the cell cycle which was intensified by combination with AT13387. Combination studies in the xenograft model are underway and will also be reported on.
Conclusion: The ability of AT13387 to inhibit tumour growth in vivo, while demonstrating good tolerability, suggests potential for this compound as an additional treatment for this aggressive disease.
Frondoside A Enhances the Antiproliferative Effects of Gemcitabine in Pancreatic Cancer Cells
J. Al Shemaili,1 K. Parekh,1 S.A. Thomas,1 S.S. Halas,1 M. Al Sultan,1 P. Collin,2 T.E. Adrian,11Department of Physiology, Faculty of Medicine, United Arab Emirates University, Al Ain, UAE and 2Coastside Research, Stonington, Maine, USA
Pancreatic cancer has a very poor prognosis. Gemcitabine is the mainstay of therapy, but although it improves the quality of life it has little impact on survival. More effective treatment options are desperately needed for this disease. Frondoside A is a triterpenoid isolated from the Atlantic sea cucumber, Cucumaria frondosa. We have previously shown that Frondoside A potently inhibits pancreatic cancer cell growth and induces apoptosis in vitro and in vivo. The aim of the present study was to investigate whether Frondoside A could enhance the anti-cancer effects of gemcitabine.
Effects of frondoside A and gemcitabine alone and in combination on viable cell number were investigated in two human pancreatic cancer cell lines, AsPC-1 and S2013. Both drugs alone had marked inhibitory effects on proliferation. To investigate possible synergistic effects, combinations of low concentrations of the two drugs were used for a 72 hr treatment period. Growth inhibition was greater with the combination of both drugs than with the calculated additive effects of the drugs in vitro (P<0.0001). The effect of combinations of frondoside A and gemcitabine were tested in vivo using the athymic mouse model. Xenografts of AsPC-1 and S2013 cells were allowed to form tumors for several days prior to treatment with the drugs alone or in combination for 30 days. Tumors grew rapidly in placebo-treated animals. Tumor growth was reduced in frondoside A-treated animals and was completely inhibited in animals receiving the combination of frondoside A and gemcitabine. Combination therapy with frondoside A and gemcitabine may be valuable in the treatment of pancreatic cancer.
Cerulein Hyperstimulation Decreases Amp-Dependent Protein Kinase Levels At the Site of Maximal Zymogen Activation
C. Shugrue,1 M. Alexandre,1 A. Diaz de Villalvilla,1 E. Thrower,1 F. Gorelick,1,21Department of Internal Medicine, Section of Digestive Diseases, 2Department of Cell Biology, Veterans Administration Connecticut Healthcare, West Haven and Yale University School of Medicine, New Haven, Connecticut
Early events leading to acute pancreatitis include premature activation and retention of digestive zymogens within the acinar cell. AMP-dependent protein kinase (AMPK) is a heterotrimer consisting of a catalytic α subunit and regulatory β and γ subunits. Preliminary studies from our laboratory indicate that active AMPK may play a protective role in protease activation. To examine the mechanism of this AMPK effect, we examined the cellular distribution and levels of AMPK using the cerulein model of acute pancreatitis. Using immunofluorescence, the γ subunit was found primarily at the apical membrane in the acinar cell, with some intracellular puncta also visible. Pancreatic homogenates from unstimulated and ceruelin hyperstimulated (i.p. injection, 60 min) rats were fractionated by differential centrifugation. AMPK α and β subunits were identified in all isolated organelle fractions by immunoblot. In cerulein stimulated rats, overall AMPK protein levels (α and β subunits) were reduced compared to unstimulated; the greatest reduction was in the zymogen granule-enriched fractions (1750g, 3000g). We have shown that zymogen activation was maximal in the zymogen granule-enriched fractions. Similar results were obtained in isolated acinar cells. We speculate that AMPK activity reduces zymogen activation in the basal state; the selective loss of AMPK in distinct subcellular compartments may enhance zymogen activation during acute pancreatitis.
Systematic Detection of Transcriptomic Aberrations in Pancreatic Cancer by High-Throughput Sequencing
C. K.-Sinha,1,2 S. Shankar,1,2 L. Ma,1 D. R. Robinson,1,2 S. K.-Sundaram,1,2 C. S. Grasso,1,2 M. Quist,1 R. J. Lonigro,1,2 I. A. Asangani,1,2 X. Cao,1,6 Y.-M. Wu,1, 2 S. Laurinec,3 J. Siddiqui,1, 2 T. J. Giordano,2 D. Simeone,3,5 A. M. Chinnaiyan,1,2,4,5,61Michigan Center for Translational Pathology, 2Department of Pathology, 3Surgery, 4Urology, 5Comprehensive Cancer Center, 6Howard Hughes Medical Institute, University of Michigan Medical School, Ann Arbor, Michigan, USA
Aim: To identify therapeutically targetable aberrations in pancreatic cancer using RNA-Seq.
Background: Pancreatic Cancer is the fourth leading cause of cancer deaths in the US due largely to late detection and absence of druggable therapeutic targets. We sought to apply the recent technology of high throughput transcriptome sequencing to detect chimeric transcripts, missense mutations and outlier gene expression patterns, to nominate therapeutic targets in individual pancreatic cancer cell lines and primary patient tumors to serve as a model system for personalized medicine.
Methods: A panel of 22 pancreatic cancer and 2 benign cell lines, 4 pancreatic adenocarcinoma, 4 pancreatic endocrine neoplasia, 2 normal pancreas samples and 4 low passage primary human pancreatic cancer xenografts were subjected to RNA-seq, followed by bioinformatic analysis of the sequence reads to nominate chimeric transcripts, mutations in cancer genes, and genes showing outlier expression. Novel observations were validated using real time PCR or Pyrosequencing, as well as through siRNA based knockdown of candidate genes.
Results: Pancreatic cancer cell lines and primary tumor tissues were found to harbor multiple oncogenic aberrations that could represent potential vulnerabilities of individual tumors. We identified genes known to be commonly mutated in pancreatic cancer such as KRAS, SMAD4, CDKN2A, and P53 etc. Several novel gene fusions (e.g. MIG7-GCLM, MAT2B-HMMR, WDR62-HCST) as well as sample specific outlier kinases (e.g. AXL, MET, MST1R, PLK2) with therapeutic implications in pancreatic cancer samples were also identified and their functional significance is currently being tested in in vitro (cell lines) and in vivo (pancreatic cancer xenograft) model systems.
Conclusion: High throughput sequencing of tumor samples can help identify personalized therapeutic avenues. Functional validation of novel targets using appropriate pre-clinical model systems may ultimately lead to new therapeutic approaches in the clinical arena.
Germline Brca2 Heterozygosity Promotes Krasg12d-Driven Carcinogenesis in a Murine Model of Familial Pancreatic Cancer
F. Skoulidis,1 L.D. Cassidy,1 V. Pisupati,1 J.G. Jonasson,2 H. Bjarnasson,3 J.E. Eyfjord,3 F.A. Karreth,4 M. Lim,1 L.M. Barber,1 S.A. Clatworthy,1 S.E. Davies,5 K.P. Olive,6 D.A. Tuveson,4 A.R. Venkitaraman,11Department of Oncology and the Medical Research Council Cancer Cell Unit, University of Cambridge, Hills Road, Cambridge CB2 0XZ, UK; 2Department of Pathology, Faculty of Medicine, Landspitalinn-University Hospital, University of Iceland and the Icelandic Cancer Registry, Reykjavik, Iceland; 3Cancer Research Laboratory, Faculty of Medicine, University of Iceland, Reykjavik, Iceland; 4Li Ka Shing Centre, Cambridge Research Institute, Cancer Research UK, Robinson Way, Cambridge, UK; 5Addenbrooke's Hospital, Cambridge, UK 6Herbert Irving Comprehensive Cancer Center and Department of Medicine and Pathology, Columbia University, New York, USA.
Inherited heterozygous BRCA2 mutations predispose to tissue-specific cancers, but somatic deletion of the wild-type allele is considered essential for carcinogenesis. We find in a murine model of familial pancreatic cancer that germline heterozygosity for a pathogenic C-terminal Brca2 truncation suffices to promote pancreatic ductal adenocarcinomas (PDACs) driven by KrasG12D, irrespective of Trp53 status. Unexpectedly, tumor cells retain a functional Brca2 allele. Correspondingly, three out of four PDACs from patients inheriting BRCA2999del5 did not exhibit loss-of-heterozygosity (LOH). Three tumors from these patients displaying LOH were acinar carcinomas, which also developed only in mice with bi-allelic Brca2 inactivation. We suggest a revised model for tumor suppression by BRCA2 with implications for the therapeutic strategy targeting BRCA2 mutant cancer cells.
Survival of Pancreatic Cancer is Associated with a Single Nucleotide Polymorphism of the Cholecystokinin-B Receptor
J.P. Smith,1 J.F. Harms,2 G.L. Matters,3 C. McGovern,1 F. Ruggiero,4 J. Liao,5 K. Fino,1 E. Ortega,1 E. Gilius,1 J. Phillips III6Depts. of Medicine1, Biochemistry and Molecular Biology3, Pathology4, and Public Health Sciences5, Pennsylvania State University, Hershey, PA Dept. of Biological Sciences2, Messiah College, Grantham, PA, Division of Medical Genetics & Genomic Medicine6, Vanderbilt University School of Medicine, Nashville, TN
Background and Aims: Gastrin stimulates growth of human pancreatic cancer through the cholecystokinin B receptor (CCKBR) and an alternatively spliced isoform called CCKCR. The aim of this study was to determine the etiology of the splice variant and compare CCKBR and CCKCR expression in individuals with pancreatic cancer.
Methods: Surgically resected normal, benign and malignant pancreatic tissues were prospectively evaluated for CCKBR genotype and CCKCR protein expression by immunohistochemistry. Characteristics predicting survival were assessed in relationship to genotype.
Results: CCKCR expression was associated with a single nucleotide polymorphism (SNP) (C>A) at position 32 of the intron 4 (IVS 4) of the CCKBR gene. Only patients with the A/A or A/C genotypes, but not the C/C genotype, exhibited immunoreactivity to a selective CCKCR antibody. Survival among pancreatic cancer patients with C/C genotype was significantly longer (p=0.0006) compared to individuals with an A-allele. The SNP was the only factor that predicted survival (p=0.0022) compared to surgical margins, histologic grade, perineural invasion, adjuvant chemotherapy, or lymph node status. Age at diagnosis occurred earlier (p = 0.006) in those with the A allele. Cancer cells transfected to over-express the CCKCR demonstrated increased proliferation over controls.
Conclusions: A novel SNP in the CCKBR gene predicts survival in pancreatic cancer patients. Genetic screening for this SNP may aid in identifying high risk patients for increased surveillance.
Endoscopic Versus Surgical Necrosectomy for Patients with Symptomatic Pancreatic Necrotic Collections: A Retrospective Cohort Study
I. Spofford,1,2 D. Conwell,2 B. Wu,2 K. Mortele,3 R. Khorasani,3 S. Yu,2 P. Banks,2 C. Thompson,21Division of Pediatric Gastroenterology, Massachusetts General Hospital, Boston, MA; 2Division of Gastroenterology, Brigham & Women's Hospital, Boston, MA; 3Department of Radiology, Brigham & Women's Hospital, Boston, MA
Background: Endoscopic necrosectomy has evolved as a less invasive alternative to surgical necrosectomy for patients with symptomatic pancreatic necrotic collections.
Aim: Compare outcomes of patients undergoing endoscopic and open surgical necrosectomy.
Methods: A retrospective medical record review of patients undergoing either surgical or endoscopic necrosectomy from 2003-2010 was conducted. Outcomes included clinical success, length of hospital stay, recurrence of pancreatitis, radiologic findings, complications and mortality. Outcomes measures were compared using Fisher's exact test and Wilcoxon Rank Sum test.
Results: 19 endoscopic and 19 open surgical necrosectomy patients met inclusion criteria. Groups did not differ in severity of pancreatitis; however, the surgical group had less well defined necrotic collections. Length of hospital stay was significantly shorter in the endoscopic group (6 [2, 8]) as compared to the surgical group (13 [7, 27]; p=0.003). There was no difference in clinical success (79% vs 63.2%; p=0.476) or complication rate (21.1% vs 47.4%; p=0.1071) between the endoscopic and surgical group, respectively. There were no differences in radiologic resolution or recurrence of acute pancreatitis and no mortalities in either group.
Conclusion: Endoscopic necrosectomy should be considered in the management of symptomatic pancreatic necrotic collections given shorter hospital length of stay and similar radiologic resolution, complication rate and clinical success as compared to open surgical necrosectomy.
Investigating CXCR2 Signalling in Pancreatic Cancer
C.W. Steele,1,2 J.P. Morton,1 K.O. Oien,1 C.R. Carter,2 C.J. McKay,2 T.R.J. Evans,1 O.J. Sansom,11Beatson Institute for Cancer Research, University of Glasgow, Glasgow, Scotland; 2Department of Pancreaticobiliary Surgery, Glasgow Royal Infirmary, Glasgow, Scotland
Background: Current therapies for pancreatic cancer (PDAC) are hindered by aggressive progression. Local inflammation, chronic pancreatitis, increases the risk of PDAC development. Molecules driving tumour-associated inflammation have potential as therapeutic targets. CXCR2 directs homing of pro-tumourigenic leukocytes including neutrophils, while CXCR2 expression by tumour cells can mediate growth, survival, and angiogenesis.
We have shown CXCR2 inhibition suppresses inflammation-mediated tumourigenesis in intestinal cancer models. Microarray data shows marked upregulation of CXCR2 and its ligands in PDAC, suggesting CXCR2 may be a novel therapeutic target.
Aims: Assess if CXCR2 deletion suppresses pancreatic inflammation.
Assess if CXCR2 inhibition slows pancreatic tumourigenesis
Elucidate the mechanisms by which CXCR2 loss modifies pancreatitis and tumourigenesis.
Methods: To investigate the impact of CXCR2 on pancreatitis, we used mice where CXCR2 has been genetically inactivated. We chemically induced chronic pancreatitis and examined the immune response in wild type and CXCR2 knockout mice. To examine the influence of CXCR2 inhibition on murine models of PDAC we used a validated CXCR2 blocking pepducin with expression of constitutively active KRasG12D and p53R172H within the pancreas.
Results: Murine pancreata were protected from chronic pancreatic inflammation by CXCR2 knockout in a neutrophil dependent fashion.
Chemotherapy with CXCR2 pepducin in murine PDAC shows improved survival compared to controls. Reduced rates of angiogenesis and metastases have been observed in CXCR2 pepducin treated mice. Neutrophil infiltration is currently under study.
Genomic Alterations of Single Disseminated Tumor Cells (DTC) Isolated From Patients With Operable Pancreatic Ductal Adenocarcinoma (PDAC)
N.H. Stoecklein, A.C. Vaerst, W.T. Knoefel Department of General, Visceral and Pediatric Surgery, University Hospital Duesseldorf, Duesseldorf, Germany
Background: Clinically occult DTC that are left behind after an intentionally curative surgery must be the origin for metatstic relapse in organ confined PDAC. In order to elucidate the genetics of these potential metastatic precursor cells, we perfomed a genome-wide screen of single DTC isolated from patients with operable PDAC.
Methods: We screened bone marrow (BM) and lymph node (LN) preparations from 76 patients with operable PDAC (UICC0-IV) for DTC with an immuno-fluorescence assay. 41 marker-positive DTC were isolated from BM and/or LN of 24 patients via micromanipulation. Matched primary tumors were laser-microdissected. Global amplification of single cell DNA and dissected tumor DNA was performed with an adapter-linker PCR. Subsequently, comparative genomic hybridization (CGH) was done for the genome-wide screening of DNA-gains and -losses.
Results: DTC were detected in 38% of the PDAC cases. CGH revealed in almost all DTC chromosomal alterations, which were typical for PDAC (deletions at 3p, 4pq, 9p, 18 and amplifications at 3q, 8q, 20q). interestingly, amplifications at 11q (harbouring the gene CCDN1) were noted more frequently in DTC than in corresponding primary tumors. CGH of cases with more than one DTC from one site (BM/LN) displayed signs of clonal expansion.
Conclusion: Our study provides a description of genomic alterations in minimal residual tumor cells detected in BM and LN of patients with operable PDAC. The altered chromosomal regions of DTC may help to identify genes that regulate the growth of metastases. Moreover, genomic aberrations detected in disseminated cancer cells might lead to the identification more effective anti-metastatic therapies in PDAC.
Epithelial to Stromal Redistribution of Primary Cilia During Pancreatic Carcinogenesis
O. Strobel, S. Kneller, N. Dadabaeva, J. Werner Department of General Surgery, University Hospital of Heidelberg, Heidelberg, Germany
Introduction: The Hedgehog (Hh) pathway is upregulated as an important mediator in pancreatic ductal adenocarcinoma (PDAC). Surprisingly, previous studies suggested that Primary Cilia (PC), the essential organelles for Hh signal transduction, are lost in PDAC. In contrast, others identified PC in several cancer cell lines. The Hh pathway is known to mediate signals from cancer cells to the associated stroma, but the presence of PC in the stroma has not yet been analyzed.
Aim: To reassess the presence of PC in human PDAC with focus on both epithelium and stroma.
Methods: PC were identified in paraffin sections by double immunofluorescence for acetylated tubulin and gamma tubulin. Co-staining for the Hh receptor Ptch was performed. Analysis included both the fraction of cells with PC and the length of PC.
Results: In normal pancreata PC increase in both number and length towards smaller ducts. PC are gradually lost during pancreatic carcinogenesis: The fraction of cells with PC gradually and significantly decreased from 32% in ducts in normal pancreata, to 21% in ducts in chronic pancreatitis, 18% in PanIN1a, 6% in PanIN2, 3% in PanIN3 and 1.2% in PDAC (G2). The length of persisting PC decreased from 1.5μm in normal ducts to 0.8μm in PanIN2. This loss of PC in the neoplastic epithelium was accompanied by a gain of PC in the associated stroma: The fraction of stromal cells with PC significantly increased from 13% in normal ducts to about 30% in PanIN and G2 cancers. The length of PC increased from 0.9μm to 1.5 μm.
Conclusion: PC are not lost during pancreatic carcinogenesis but their formation is redistributed from the epithelium to the stroma. This redistribution may be the explanation for a redirection of Hh signaling towards the stroma in PDAC.
siRNA Mediated Blockade of DCAMKL-1 Results in Pancreatic Tumor Xenograft Growth Arrest by let-7a and miR-200a MicroRNAs-Dependent Mechanisms
S.M. Sureban,1,2 R. May,1,2 Dongfeng Qu,1 S. Anant,3 C.W. Houchen,1,21Department of Medicine, University of Oklahoma Health Sciences Center, Oklahoma City, OK; 2Veterans Affairs Medical Center, Oklahoma City, OK; 3Kanasas University Medical Center, Kansas City, KS
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, and there is a gain of stem cell properties during EMT. Here, we sought to determine the potential role of the stem cell/CSC marker DCAMKL-1 in pancreatic cancer. We have utilized Nanoparticle (NP) technology to deliver DCAMKL-1 specific siRNA.
Methods: Total RNA isolated from human pancreatic tumor xenografts (AsPC-1 cells), following NP-based siRNA-mediated knockdown of DCAMKL-1, was subjected to real-time RT-PCR for mRNA (DCAMKL-1, c-Myc, KRAS, ZEB1, ZEB2, Snail, Slug and Twist) and microRNA (pri-miR-200a and pri-let-7a) analysis. Immunohistochemistry was performed (DCAMKL-1, c-Myc, Snail and Slug) on AsPC-1 tumor xenografts following siRNA treatments.
Results: Administration of NP-siDCAMKL-1 into AsPC-1 xenografts resulted in tumor growth arrest, downregulation of proto-oncogenes c-Myc and KRAS via let-7a miRNA-dependent mechanisms. Furthermore, siRNA-mediated knockdown of DCAMKL-1 in AsPC-1 tumor xenografts resulted in down-regulation of ZEB1, ZEB2, Snail, Slug and Twist following induction of miR-200a (EMT inhibitor).
Conclusion: These findings demonstrate a positive regulatory role of DCAMKL-1 in pancreatic tumorigenesis. Furthermore, these data also suggests that NP-based delivery of siRNAs directed against critical target such as DCAMKL-1 may provide a novel approach to treat cancer through the regulation of endogenous miRNAs.
A Multi-Center Study of a MicroRNA-Based Assay for the Diagnosis of Pancreatic Ductal Adenocarcinoma in Fine Needle Aspirates
A.E. Szafranska-Schwarzbach,1 D.C. Whitcomb,2 M.B. Lloyd,1 A.T. Adai,1 M.S. Sanders,2 R.E. Brand,2 L.S. Lee,3 D.L. Conwell,3 G. Rateb,4 C. Menard,4 J.A. Morisset,4 S. Vignesh,5 B. Centeno,5 M. Hartleb,6 A. Wiechowska-Kozłowska,7 L. Stefanczyk,8 C.L. Lefferts,9 G.J. Tsongalis,9 S. Hahn,10 B.F. Andruss,11Asuragen Inc., Austin, TX; 2University of Pittsburgh Medical Center, Pittsburgh, PA; 3Brigham and Women's Hospital, Boston, MA; 4University of Sherbrooke, Fleurimont, QC; 5Moffitt Cancer Center & Research Institute, Tampa, FL; 6Silesian Medical University, Katowice, Poland; 7Hospital of the Ministry of Internal Affairs and Administration, Szczecin, Poland; 8Medical University of Łódź, Poland; 9Dartmouth Hitchcock Medical Center, Lebanon, NH; 10Ruhr University of Bochum, Germany
Introduction: Pancreatic ductal adenocarcinoma (PDAC) and chronic pancreatitis (CP) often present with similar symptoms and mass imaging features and coexist, resulting in a misdiagnosis rate as high as 25%. We previously reported a miRNA-based laboratory developed test (LDT) for PDAC with a Se and Sp of 95% in FFPE specimens. In this multicenter investigation, we developed and validated a miRNA LDT for pancreatic fine needle aspirates (FNAs).
Methods: RNA was extracted from whole serial sections of 95 FFPE specimens (52 PDAC, 43 CP; assay development) and 71 endoscopic ultrasonography (EUS) FNA specimens stored in RNARetain® Stabilization Solution (59 PDAC and 12 CP, with diagnoses confirmed by final surgical pathology; assay validation). Expression levels of up to 12 selected miRNAs were interrogated using TaqMan® qRT-PCR assays.
Results: The diagnostic performance of the miRNA assay alone in FNAs was Se: 79.7%, Sp: 91.7%, PPV: 97.9%, NPV: 47.8%. When initial FNA cytology was combined with the miRNA assay, the accuracy improved from 87.3% for FNA cytology alone (Se: 86.4%, NPV: 57.1%) to 97.2% (Se: 98.3%, NPV: 91.7%). Of note, five histologically confirmed PDAC samples were called CP by FNA cytology, while the miRNA assay identified 5/5 as PDAC. Furthermore, within the atypical and suspicious FNA cytology specimens (n=10), the miRNA assay correctly identified 7/7 as PDAC and 2/3 as CP for a corresponding 100% and 66.7% agreement with final pathology, respectively.
Conclusions: This newly developed and validated miRNA-based assay is complementary to conventional FNA cytology by identifying false negative aspirations and resolving atypical and suspicious cytopathology results.
MicroRNA Biomarkers in Cyst Fluid May Improve the Management of Pancreatic Cysts
A.E. Szafranska-Schwarzbach,1 H. Matthaei,2 D. Wylie,1 M.B. Lloyd,1 J. Kemppainen,1 L. Langfield,1 R. Schulick,2 B.F. Andruss,1 R.H. Hruban,2 A.T. Adai,1 A. Maitra,21Asuragen Inc., Austin, TX; 2The Sol Goldman Pancreatic Cancer Research Center, Johns Hopkins University School of Medicine, Baltimore, MD
Introduction: There has been a dramatic increase in the diagnoses of pancreatic cysts. While most lesions are benign, some represent precursors of pancreatic cancer (IPMNs). Therapeutic stratification in IPMNs is challenging without precise information on grade of dysplasia and presence of invasion. We assessed the diagnostic benefit of miRNAs in pancreatic cyst fluid focusing on IPMNs due to their frequency and malignant potential.
Methods: RNA from 61 microdissected FFPE IPMNs and 65 cyst fluids was extracted using Asuragen protocols. miRNA expression profiling of 754 miRNAs was performed with TaqMan® MicroRNA Arrays using FFPE (n=23) and cyst fluid (n=15) specimens. Verification of differential expression was carried out in FFPE (n=38) and cyst fluid (n=50) specimens using TaqMan® MicroRNA Assays.
Results: Bioinformatics analysis yielded 26 and 37 differentially expressed miRNAs between low-grade (LG) and high-grade (HG) IPMNs, respectively, with 4 overlapping miRNAs. Nineteen miRNAs selected using both FFPE and cyst fluid data were shown to separate HG from LG IPMNs/SCAs as well as from uncommon cysts such as solid pseudopapillary neoplasms (SPNs) and cystic neuroendocrine tumors (NETs). Logistic regression model allowed prediction of cyst pathology that could be used to select resection (NETs, SPNs) vs. conservative patient management (LG IPMNs, SCAs), with an accuracy of 90% and 100%, respectively.
Conclusions: miRNA classifiers can aid in identification of patients with HG IPMN and rule out non-mucinous cysts. Validation of these classifiers in a prospective study is needed to demonstrate the clinical utility of miRNAs for stratification of cystic lesions.
Proteomic Forecast of Postoperative Prognosis of Pancreatic Cancer using Formalin-Fixed Paraffin-Embedded Tissue
T. Takadate, T. Onogawa, F. Motoi, T. Morikawa, S. Maeda, K. Nakagawa, T. Rikiyama, Y. Katayose, S. Egawa, M. Unno Division of Hepato-Biliary-Pancreatic Surgery, Department of Surgery, Graduate School of Medicine, Tohoku University, Sendai, Japan
Background and Aims: This study aimed to identify the novel prognostic biomarker using mass spectrometry (MS)-based proteomic analysis with formalin-fixed paraffin-embedded (FFPE) tissue.
Patients and methods: The two groups with poor prognosis (n=4) and with better prognosis (n=4) had been carefully chosen among 96 resected cases of pancreatic cancer during 1998 to 2007 in Tohoku University Hospital. Although those 2 groups had adjusted background (UICC-Stage IIB, Grade2, R0, gemcitabine adjuvant), there was a significant difference in postoperative mean survival time (poor 21.0M, better 67.0M, P<0.05). Cancerous cells collected from FFPE tissue by laser microdissection were processed for liquid chromatography/MS. Furthermore, identified proteins were validated by the selective reaction monitoring (SRM) quantitative analysis and immunohistochemical analysis.
Results: Totally 1,229 proteins were identified and 170 candidate proteins selected by semi-quantitative comparison were verified by SRM. In result, we identified 18 proteins overexpressed in poor prognostic group. These proteins were validated in 96 cases by immunohistochemical analysis.
Discussion: Adjusting background, we could clarify the target molecules related with biological aggressiveness. These candidate proteins include the molecules that play a role in adhesion and migration of cells and progression of cell cycle. Validation in the larger cohort can elucidate the practical prognostic markers.
Conclusion: We identified the 18 candidate postoperative prognostic biomarkers for pancreatic cancer.
Results of Spleen-preserving Distal Pancreatectomy
Y. Takeyama, H. Ishikawa, T. Yasuda, M. Yamasaki, T. Nakai Department of Surgery, Kinki University Faculty of Medicine, Osaka-sayama, Osaka Japan
Introduction: The number of the patients with the diagnosis of intraductal papillary mucinous neoplasm (IPMN) has increased due to the progress in image diagnostic technology. Moreover, elucidation of the pathophysiology of the disease makes it possible to apply less-invasive operation, such as spleen-preserving distal pancreatectomy (SPDP) in borderline lesion, such as IPMN or endocrine tumors. We aimed to investigate the safety and usefulness of SPDP.
Method: SPDP was performed on 15 patients from 2004 to 2011. We compared their background, indications and results with those of the 70 patients which underwent on pancreatectomy with splenectomy (DP) during the same period.
Results: The patients with SPDP consist of 8 with IPMN, 4 with endocrine tumors, and 3 with other diseases. In contrast, the 70 patients with DP consist of 34 with invasive ductal cancer, 17 with cystic tumors, one with endocrine tumors, and 18 with other diseases. Although there was no significant difference between these two groups in operative time, and in blood loss, post-operative hospital stay in SPDP was significantly shorter than that in DP (11 days vs. 15 days). Among 15 patients with SPDP, the splenic vessels were preserved in 13 patients, and were resected in 2 cases. Neither mortality nor splenic abscess was experienced in both groups. Frequency of pancreatic fistula did not differ between both groups. Splenic vein obstruction was observed in only one case with SPDP 6 years after operation.
Conclusion: SPDP is safe and reasonable procedure for borderline lesions. Long-term prognosis including immunological function should be evaluated.
Evaluation of Long-Term Condition after Total Pancreatectomy
Y. Takeyama, H. Ishikawa, T. Yasuda, M. Yamasaki, T. Nakai Department of Surgery, Kinki University Faculty of Medicine, Osaka-sayama, Osaka Japan
Introduction: Quality of life after total pancreatectomy (TP) is mainly defined by complete loss of pancreatic function, and the patients are forced to receive insulin injections and oral supplementation of digestive enzymes. Sometimes, they suffer from hypoglycemic attack and malnutrition. Recently, however, the cases of TP increased due to increase of patients with main duct type intraductal papillary mucinous neoplasm. We explored the long-term condition after total pancreatectomy.
Method: TP was performed on 16 patients from 2005 to 2010. Changes in nutritional status and HbA1c 1 year after operation were able to be analyzed on 10 patients among them. The patients were managed with digestive enzyme supplementation and intensive insulinotherapy.
Results: Preoperative body mass index was 21.5 in average, and it decreased to 20.1 one year after operation. Serum albumin at preoperative period was 3.8 g/dl in average, and it increased to 4.0 g/dl. Serum total cholesterol at preoperative period was 166 mg/dl in average, and it increased to 175 mg/dl. HbA1c at preoperative period was 6.6% in average, and it increased to 7.5%.
Conclusion: The nutritional status and diabetic control after TP were maintained at an acceptable level at 1 year after operation as indicated. We should reappraise TP as an acceptable operation and may expand its indication.
"Binding" Pancreato-Yeyuno Anastomosis Versus a Classical Ducto-Mucosa Anastomosis After Pancreatoduodenectomy for Periampullary and Pancreatic Neoplasm
J. Targarona, L.A. Barreda Department of Surgery Pancreatic Unit, Edgardo Rebagliati Martins Hospital, Lima Peru
Abstract: Pancreatic fistula is the most feared complication after a duodenopancreatectomy and is the most common related factor for postoperative mortality. Recently a new technique of telescope pancreatico-jejunal anastomosis has been published by Peng et al, called "Binding anastomosis" reporting 0% of post operative pancreatic fistula (POPF) in a ramdomized study of 217 patients. The aim of this study was to evaluate and validate this technique compared with a classical termino-lateral pancreatico-jejunal anastomosis (TLDM).
Methods: We conducted a prospective study of a consecutive series of patients between January 2007 and February 2010, who underwent a pancreaticoduodenectomy for pancreatic or ampullary neoplasm, operated by the same surgeon.
Results: Were performed 63 duodenopancreatectomies, in 30 patients we realized a Peng anastomosis (Group A), and in 33 patients a TLDM was conducted (Group B). The postoperative morbidity in the Group A was 36.7% against 24.2% in Group B (p = 0.29). The POPF was 6.7% (2/30) in the Group A, against 12.1% (4/33) in Group B (p = 0.67). There were no significant differences regarding postoperative hospital stay and mortality (0% Group A vs. 3% in the Group B, p = 1.00).
Conclusions: In our study, the Binding Anastomosis described by Peng is a safe procedure, but it's not associated with lower rates of pancreatic fistula, overall morbidity or mortality, when we compared this procedure with a classical pancreatico-jejunal anastomosis, although more studies are needed to validate it.
S100A2 and S100A4 as Prognostic and Predictive Biomarkers in Patients Receiving Adjuvant Therapy for Pancreatic Cancer (PC): a Secondary Analysis of RTOG 9704
M. Tempero,1 J. Moughan,2 G.E. Kim,1 S. Kakar,1 T.S. Hyun,1 W.F. Regine,3 R. A. Mowat,4 K.P. Charpentier,5 W. Small Jr.6 C. Guha,7 D. K. Chang,8 A.V. Biankin,81University of California San Francisco, San Francisco, CA; 2RTOG Statistical Center, Philadelphia, PA; 3University of Maryland Medical Systems, Baltimore, MD; 4Toledo Community Hospital Oncology Program CCOP, Toledo, OH; 5Alpert Medical School, Brown University, Providence, RI; 6The Robert H. Lurie Comprehensive Cancer Center of Northwestern University Hospital, Chicago, IL; 7Montefiore Medical Center, Bronx, NY; 8Garvan Institute of Medical Research, Darlinghurst, Sydney, NSW, Australia
Background: S100A2 expression is a poor prognostic marker in patients with PC who did not receive adjuvant therapy. Nuclear expression of S100A4 is associated with gemcitabine (gem) resistance.
Methods: S100A2 and S100A4 expression was analyzed on archival tissue from patients on a prospective adjuvant trial, RTOG 9704, comparing 5FU to gem. IHC was performed on triplicate samples. DSS was estimated univariately with the Kaplan-Meier method and subgroups were compared using the log-rank test. Cox proportional hazards models were utilized to identify the impact of S100A2 expression and S100A4 on DSS.
Results: The analysis was based on 150 head of pancreas (HOP) specimens. For S100A2 related DSS, the HR for no, low, mod, or high expression for each subgroup was as follows: 1.0, 1.22 (95% C.I. = [0.70, 2.12]), 1.36 (95% C.I. = [0.77, 2.41]), and 1.35 (95% C.I. = [0.86, 2.12]), respectively. For high vs. low/no expression, the curves separated at 1 year but were not significantly different (p=0.09). For S100A4 related DSS, the HR for negative and positive expression was 1.00 and 1.83 (95% C.I. = [0.88, 3.79]), and 1.00 and 1.11 (95% C.I. = [0.59, 2.09]) for gem and 5FU, respectively.
Conclusions: This study was limited by the small number of unique samples available, especially in subgroups of interest. Analysis of S100A2 expression shows a trend supporting S100A2 as a prognostic biomarker in patients with HOP cancers. Likewise, the improvement in survival for S100A4 negative patients treated with gem compared to 5FU supports the hypothesis that S100A4 expression is a predictor for gem resistance.
Investigation of AT7519, a Novel CDK Inhibitor, in a Xenograft Model of Pancreatic Cancer
A. Thomas,1 E. Shaw,1 P. Ghaneh,1 W. Greenhalf,1 E. Costello,1 K. Dajani,1 M. Davies,1 J. Lyon,2 R. Sibson,1 J.P. Neoptolemos,11Department of Clinical and Cancer Medicine, University of Liverpool, Liverpool, UK; 2Astexs Therapeutics, Cambrigde, UK
Background: Pancreatic cancer continues to have a poor prognosis with over 34, 000 deaths across the US in 2008 and ranks fourth among cancer related deaths. Chemotherapy is the treatment of choice in advanced disease and has a proven role in the adjuvant setting though current clinical agents, such as gemcitabine, only prolong survival by a matter of weeks; highlighting the need for more effective novel therapies.
The Cyclin Dependent Kinase (CDK) family of protein kinases are pivotal in regulating the cell cycle. AT7519, a general CDK inhibitor providies potent inhibition of CDK 1, 2, 4, 5 and 9 (Astex Therapeutics) and is here investigated in vitro and in vivo.
Methods: Cell proliferation assays to determine IC50 values and isobolar analysis were performed in triplicate using EZ4U assay (BioMedica), measured 48 h or 72 h after drug treatment. Cell cycle analysis was performed 4 h,7 h,24 h and 48h after treatment of cells with 10mM AT7519, using flow cytometry to determine propidium iodide incorporation. Western blot analysis was performed using standard methods with ECL detection to assess downstream markers.
In vivo studies were conducted using a murine xenograft model, BALB/c (CAnN.Cg-Foxn1nu/Cr) nude mice with MiaPaCa2 pancreatic cancer cells. Tumour volumes were measured with external callipers and a subgroup of mice underwent CT imaging.
Results: AT7519 and gemcitabine were tolerated in our xenograft model with no mortaility or significant morbidilty. Mice treated for 17 days with AT7519 showed significant reduction in tumour growth (p0.0446) compared with vehicle control. Reduction in tumour growth was also apparent with gemcitaibine (p0.0038) and drug combination studies are now in progress.
AT7519 inhibited proliferation in a range of pancreatic cancer cell with IC50 values ranging from 5-2000nM. In cell cycle analysis AT7519 and gemcitabine in combination causes an accumulation of cells in S phase. Isobolar analysis of AT7519 combined with gemcitabine suggested an additive effect. Downstream effects were observed. CDK1 levels were unchanged but phosphorylation of pp1-α was inhibited, shown to result in mitotic collapse after nuclear envelope breakdown. Phosphorylation of Rb and NPM was also inhibited.
Conclusions: Pancreatic cancer cell lines develop resistance to gemcitabine (standard chemotherapy) after relatively brief exposure to the drug. AT7519 inhibited growth in a range of pancreatic cancer cell lines including one that has acquired gemcitabine resistance and was effective in combination with gemcitabine.
Downstream effects of AT7519 were observed, including decreased phosphorylation of pp1-a and Rb, and may be used as biomarkers for drug activity.
In vivo AT7519 reduces tumour growth confirming activity: Combination of AT7519 and gemcitabine is now being investigated.
Eps8 Modulation of Integrin-Dependent Pancreatic Cancer Invasion
J. Tod, M. Chrzan, V. Jenei, D. Fine, G. Thomas Experimental Pathology Group, University of Southampton School of Medicine
Background: Tumor cell motility requires reorganization of the actin cytoskeleton. EGFR pathway substrate 8 (Eps8) regulates actin remodeling, through interactions with numerous intracellular proteins, including palladin, F-actin and integrins. The integrin αvβ6 is over-expressed in many carcinomas, and promotes invasion and TGF-β1 activation. The aim was to investigate the role of Eps8 in integrin dependent invasion in pancreatic cancer (PC).
Methods: We used immunochemistry to examine the expression of Eps8 and αvβ6 in normal pancreas and PC in vivo. Western Blotting was used to examine Eps8 and αvβ6 expression in a panel of PC cell lines. Cell migration and invasion was investigated using Transwell assays and organotypic cultures. The ability of PC cells to promote transdifferentiation of fibroblasts was examined using TGF-β activation assays and co-culture assays with fibroblasts.
Results: Eps8 and αvβ6 were markedly upregulated in >60% of PC in vivo, but were not generally detectable in normal pancreas. Eps8 was expressed in all 5 PC cell lines and αvβ6 in 4 of 5 lines. Eps8 promoted cell motility through a mechanism dependent on αvβ6. PC cell lines expressing high levels of αvβ6 showed integrin-dependent activation of TGF-β1, and promoted myofibroblastic transdifferentiation when co-cultured with fibroblasts.
Conclusions: We have shown that Eps8 promotes PC cell motility, modulated through αvβ6 integrin. Moreover, αvβ6 regulates invasion indirectly, through modulating a pro-invasive stroma. This may be particularly important in PC, which is characterized by a desmoplastic stroma.
Combined LC/MS-MS and iTRAQ Analysis of Serum and Pancreatic Juice for the Detection of Biomarkers for Pancreatic Cancer: Influence of Biliary Obstruction
S. Tonack,1 C. Jenkinson,1 V. Elliot,1,2 K. Dajani,1 R.E. Jenkins,3 N.R. Kitteringham,2 B. Lane, J.P. Neoptolemos,1 E. Costello,11Liverpool Cancer Research-UK Centre; 2Liverpool National Institute for Health Research Pancreatic Biomedical Research Unit; 3MRC Centre for Drug Safety Science, Department of Pharmacology and Therapeutics, University of Liverpool, UK
Advanced pancreatic cancer patients often experience biliary obstruction, a feature that has received little attention in biomarker discovery or validation protocols. We used iTRAQ to compare serum from 60 patients, pooled into groups of PDAC, chronic pancreatitis (CP), benign biliary obstruction (BBO), and healthy controls (HC). Pancreatic juice samples were analysed by LC-MS/MS +/- iTRAQ labeling. In total, 159 serum and 108 juice proteins were identified and quantified. Ten proteins (7 serum, 3 juice) were selected for initial validation and 5 qualified for further validation in up to 150 serum samples, comprising 54 PDAC, 45 CP, 30 BBO and 29 HC. All five candidates and two reference markers (CA19-9 and Reg3A) were significantly elevated in patients with BBO. When the patients were divided into obstructed (bilirubin >20μmol/L) versus unobstructed (bilirubin <20μmol/L), all five candidate markers and reference markers distinguished the obstructed PDAC patients from HCs (p<0.001). However, in unobstructed patients, only CA19-9, Reg3A and two candidate markers remained significantly elevated in PDAC versus the HC group. Only CA19-9 could distinguish CP from PDAC and then, only in patients without obstruction. Markers will be presented along with their combined performance. In conclusion, when translating pancreatic cancer biomarkers to clinical use, it is essential to understand how the markers behave in the context of obstruction as well as chronic pancreatitis.
Cytotoxic and Antitumor Effects of Annona Muricata in Pancreatic Cancer Cells
M.P. Torres,1,2 P. Pandey,3 S. Joshi,2 V. Purohit,1 P. Singh,1 S.K. Batra,1,21Eppley Cancer Institute, 2Department of Biochemistry and Molecular Biology, 3Department of Environmental, Agricultural & Occupational Health, University of Nebraska Medical Center, Omaha, NE
Aim: Currently, the chemotherapeutic drug gemcitabine remains the standard line of pancreatic cancer treatment and, unfortunately, no other drug, or combination of drugs, has significantly improved the benefits conferred by gemcitabine alone. We aim to find alternative, natural-derived chemotherapeutic drugs to improve the poor prognosis of pancreatic cancer patients.
Background: Traditionally, the leaves from the tropical tree Annona Muricata, also known as Graviola or Soursop, have been used for a wide range of human diseases including inflammation, rheumatism, neuralgy, diabetes, hypertension, insomnia, cystitis, parasitic infections, and cancer. Although few in vitro studies have shown the cytotoxic characteristics of Graviola against a wide range of cancer cell lines, including pancreatic cancer cells, scientific evidence that confirms the antitumor mechanism of the plant is still missing.
Methods: The main objective of this study was to evaluate the effect of a commercially available Graviola supplement on pancreatic cancer cells and determine the role of the product on cytotoxicity, protein and gene expression, tumorigenicity and metastatic properties on the pancreatic cancer cell lines FG/COLO357 and CD18/HPAF. Experimental techniques used included western blot, PCR, confocal, flow cytometry, immunohistochemistry analyses among other functional assays.
Results: The results indicated a decrease in cell viability at increasing concentrations of Graviola which correlated with a deactivation of Akt and Erk pathways. In agreement with previous studies, Graviola inhibited ATP production in pancreatic cancer cells which correlated with a decrease in glucose uptake. Additionally, the components present in the supplement induced the production of reactive oxygen species, which ultimately lead to the necrosis of pancreatic cancer cells. The expression of mucin glycoproteins, which have been related to pancreatic cancer tumorigenicity and metastasis (i.e. MUC1, MUC4, MUC16), was significantly downregulated in pancreatic cancer cells after being incubated with Graviola.
Conclusion: The compounds that are naturally present in a supplement of Annona Muricata targeted multiple signaling pathways in pancreatic cancer cells including cell cycle, survival, and metastatic pathways. Collectively these pathways can lead to a decrease in tumorigenicity and metastasis of pancreatic tumors. The antitumor effect of the Graviola supplement on pancreatic tumor xenografts and spontaneous pancreatic cancer animal models is currently being tested.
Isolated Peri-Pancreatic Necrosis is Associated With Better Outcomes Compared to Pancreatic Necrosis With or Without Peri-Pancreatic Necrosis
Y. Tsuji,1 N. Takahashi,2 R. Talukdar,1 S.T. Chari,1 S.S. Vege,11Pancreatic Interest Group, Department of Gastroenterology and Hepatology, Mayo Clinic, Rochester, MN; 2Department of Radiology, Mayo Clinic, Rochester, MN
Aim: There is scant data that isolated peri-pancreatic necrosis (PPN) might be less serious than pancreatic necrosis (PN). We investigated the relationship between PN with/without PPN and isolated PPN with regards to important clinical outcomes in patients with acute pancreatitis (AP).
Method: Between 2005-2008, 356 patients with a diagnosis of AP were admitted to our institution and had CT scan. These patients were categorized into three groups based of presence or absence of PN and PPN; Group A (PN-PPN-,#232), Group B (PN-PPN+,#49), Group C (PN+PPN-/+,#75). The persistence organ failure (POF), length of hospital stay (LOS), need for ICU, length of ICU stay (LOICU), need for intervention and mortality were recorded. The clinical outcome were compared between Group A, B and C.
Results: For the three groups A, B and C, POF rates were 39(16.8%), 12(24.5%) and 41(54.7%), LOS 8.9 ± 14.2, 22.8 ± 50.3 and 40.1 ± 50.2 days, need for ICU 45(19.3%), 15(30.6%) and 41(54.6%), LOICU 1.5 ± 6.1, 5.0 ± 16.4 and 6.6 ± 16.6 days, need for intervention 3(1.3%), 16(32.7%) and 50(66.7%), and the mortality 2.6%, 12.2% and 12.0%, respectively.
LOS, need for ICU, and LOICU need for intervention and mortality were significantly different between A and B (P<0.05). All outcomes showed significant differences between A and C (p<0.05). POF, LOS, need for ICU, and intervention showed significant differences between B and C (P<0.05).
Discussion: Patients with PN had higher morbidity compared to those with isolated PPN. This finding is very important and may help the recent efforts at revision of Atlanta classification beyond the mild and severe types.
EUS-FNA Can Obviate a Large Number of Unnecessary Surgeries While Missing Very Few Potentially Resectable Pancreatic Cancers in Jaundiced Patients with Suspected Pancreatic Cancer
P. Tummala, S. Munigala, B. Agarwal Department of Internal Medicine, Division of Gastroenterology and Hepatology, Saint Louis University School of Medicine, St. Louis, MO
Background: Patients with obstructive jaundice (ObJ) and biliary stricture on ERCP or an identifiable mass lesion on CT/MRI scan are believed to have a very high probability of pancreatobiliary malignancy (PBM) even though there is limited supporting data. Unless an unresectable mass lesion is noted on CT/MRI, surgery is recommended. The role of EUS-FNA is debated for fear of missing potentially resectable PBM. In above-defined patient subset, we evaluated 1) the final diagnosis and prevalence of PBM, 2) prevalence of lesions that do not require surgery and, 3) prevalence of potentially resectable PBMs in patients with false negative diagnosis by EUS-FNA.
Patients and Methods: This is a retrospective analysis of patients from our database of patients who underwent EUS/EUS-FNA in our university based practice. From 2002-2009, there were 342 patients who presented with ObJ and a biliary stricture on ERCP (n=342) and 172 of these had an identifiable mass lesion on CT/MRI scans. Patients in whom there was evidence of unresectability on imaging (locally advanced tumor or suspected metastasis) were not included. Final diagnosis was based on definitive cytology, surgical pathology or clinical follow-up of ≥1 year. Atypical, suspicious or highly suspicious cytology was considered negative for malignancy.
Results: The mean age of 342 patients (176 male) was 68.0 ± 12.5 years. A final diagnosis of malignancy was made in 248 patients (72.5%, 95%CI 67.7, 77.2) and included pancreatic adenocarcinoma (n=210), cholangiocarcinoma (n=28), malignant lymphoma (n=3), gallbladder carcinoma (n=3), neuroendocrine tumor (n=3) and pleomorphic carcinoma (n=1). A malignant neoplasm was identified in 248 of 342 patients with ObJ and biliary stricture (72.5%, CI 67.7, 77.2) and 148 of 172 patients with identifiable mass lesion (86.0%, CI 80.8, 91.2) on CT/MRI. The overall accuracy of EUS-FNA for diagnosing malignancy in this cohort was 92.4% (89.5, 95.2), with 91.5% sensitivity (87.1, 94.5) and 80.9% NPV (72.0, 87.5). Amongst 21 patients with false negative diagnosis of malignancy; 8 had cholangioCa and 13 had pancreatic adenoCa. R0 surgical resection could be achieved only in 7 of these 21patients. EUS-FNA identified malignant celiac and/or mediastinal lymph nodes in 24 patients with PBM.EUS-FNA obviated the need for surgery in 116 patients: in 89 patients diagnosed as true negatives, in 24 patients with distant malignant lymphadenopathy and in 3 patients with true positive diagnosis with lesions that did not require surgery(Malignant Lymphoma n=3).
Conclusions: In patients who present with ObJ and biliary stricture on ERCP ± a mass lesion on CT/MRI, the prevalence of PBM is about 72.5%. The risk of missing resectable PBM in these patients by EUS-FNA is rather small. Information from EUS-FNA can obviate surgery in many more patients.
Survival Benefit After R0 Resection of Pancreatic Adenocarcinoma is Largely Seen in Patients with Tumor Size ≤35 mm and ≤1 Involved Lymph Node
P. Tummala,1 T. Howard,2 B. Agarwal,11Department of Internal Medicine, Division of Gastroenterology and Hepatology, Saint Louis University School of Medicine; 2Missouri Baptist Medical Center, St. Louis, MO
Background: Surgical resection is the standard of care for pancreatic adenocarcinoma (PaCa) that seems resectable on imaging. Patients with R0 resection have significantly better survival than those with R1/R2 resection. Whether R0 resection confers survival benefit in all patients or if there are factors that predict survival benefit following R0 resection is however not known.
Patients and Methods: This is a retrospective analysis and included 921patients who were treated for pancreatic adenocarcinoma at Saint Louis University Hospital or Missouri Baptist Medical Center from 2001-10. Patients with radiologic evidence of unresectability (n=451) and those lost to follow up (n=177) were excluded. Patients with metastatic disease noted during surgery (n=64), those received pre-operative chemoradiation (n=15), those with cystadenocarcinoma (n=26), those who had multiple cancers (n= 15) and those who died of unrelated causes (n=16) were excluded. Post-operatively, patients were managed as clinically indicated as per the NCCN guidelines. Survival was calculated from the date of surgery. Patients were classified as having R0 and R1/R2 resection based on operative findings and surgical pathology. Tumor size was based on surgical pathology.
Results: The median age of 157 patients finally included for analysis was 65.4±10.7 years. The mean and median tumor size was 33.6±13.8 mm and 31 mm. Surgical pathology revealed R0 resection in 105 patients and regional lymph node (LN) metastasis in 104 patients. The median and mean survival after R0 resection was 26.6 months (95%CI 17.3-35.9) and 40.8±4.6 months and after R1/R2 resection was 17.4 (95%CI 8.7, 26.0) and 22.7 ± 2.4 months respectively (p=0.004). Tumor size and tumor infiltration of peripancreatic LN(s) were significant determinants of survival benefit after R0 resection in univariate analysis and multivariate analysis. Amongst patients with R0 resection, those with tumor ≤35 mm in size and ≤1 involved peripancreatic LN had median and mean survival of 70.9 and 60.5 months. The remaining patients with R0 resection had median survival of 19.7 months that was identical to the survival in patients with R1/R2 resection (17.4months).
Conclusions: In conclusion, the survival benefit from upfront surgery with R0 resection (vs. R1/R2 resection) of pancreatic adenocarcinoma is not uniform in all patients. Patients with tumor >35 mm in size and/or ≥2 involved peripancreatic LN(s) seem not to have a significant survival benefit with R0 resection. If confirmed in future studies, the above mentioned criteria may potentially be used to identify patients who would most benefit from upfront surgical resection.
NF-κB and AP-1 Crosstalk in Exocrine Pancreatic Cells Involves ELK-1
E. Twait, D. Williard, Z. Yuan, I. Samuel VAMC and UI CCOM Dept of Surgery, Iowa City, IA
We previously showed that dominant negative ERK (DN.ERK) expression inhibits NF-κB and AP-1 in vitro and improves mortality in murine ligation-induced acute pancreatitis. ELK-1 is the prototypical transcription factor activated by ERK, while we showed that NF-κB and AP-1 protein cJun also regulate each other. We hypothesize that ELK-1 is involved in the crosstalk between NF-κB and AP-1. We first confirmed that DN.ERK inhibits CCK-stimulated ELK-1 activation in isolated acinar cells using immunoblots. To investigate transcription factor activation, we used AR42J cells co-infected with DN mutant protein adenovirus (or empty virus control) and activated transcription factor-dependent luciferase reporter adenovirus. CCK-stimulated increases in ELK-1-dependent luminescence were subdued by DN.ERK, DN.IκBα or DN.cJun, indicating that ELK-1 is activated by ERK, NF-κB and AP-1 protein cJun. Interestingly, CCK-stimulated increases in AP-1-dependent luminescence were inhibited by DN.IκBα or DN.ELK-1, suggesting that modulation of AP-1 by NF-κB involves ELK-1. Conversely, TNF-α-stimulated increases in NF-κB-dependent luminescence were inhibited by DN.cJun or DN.ELK-1, suggesting that modulation of NF-κB by AP-1 protein cJun also involves ELK-1.
Our findings indicate that NF-κB-dependent gene transcription is regulated by AP-1, that AP-1-dependent gene transcription is modulated by NF-κB, and that these cooperative interactions between the NF-κB and AP-1 transcription factor pathways involve ELK-1. As DN.ERK improves mortality in ligation-induced acute pancreatitis in mice, it is possible that its beneficial effects are related at least in part to inhibition of ELK-1-mediated crosstalk between NF-κB and AP-1, in addition to separate inhibition of the NF-κB and AP-1 nuclear transcription factor pathways due to ERK inhibition.
Possible Role of ICOS and IL-10 Positive Regulatory T Cells in the Development of IgG4-related Autoimmune Pancreatitis
K. Uchida, T. Kusuda, Y. Sakaguchi, K. Yoshida, T. Fukui, A. Nishio, K. Okazaki The Third Department of Internal Medicine, Division of Gastroenterology and Hepatology, Kansai Medical University, Moriguchi, Japan
Objectives: IgG4-related autoimmune pancreatitis (AIP) is a new clinical entity of pancreatic disorder. There are immunologic and histological abnormalities, including increased serum IgG4 levels and the infiltration of lymphocytes and IgG4-positive plasmacytes. However, the role of IgG4 is unclear. Recently, regulatory T cells (Tregs) were reported to contribute to the development of various autoimmune diseases as well as in B cell shifting to IgG4-producing plasmacytes. For this report, we studied regulatory T cells in the pancreas by immunohistochemistory and ICOS+ Tregs and IL-10 producing cells in the peripheral blood lymphocytes by flowcytometry.
Methods: We recruited 44 patients with IgG4-related AIP. For comparison, we recruited 37 patients with other pancreatic diseases and 27 healthy subjects as controls. We studied infiltrating cells in the pancreas by immunohistochemistry, and analyzed ICOS+ Tregs and IL-10+ Tregs in the peripheral blood by flow cytometry.
Results: The ratio of Foxp3-positive cells to infiltrated mononuclear cells (Foxp3/Mono) in AIP patients (0.091 ± 0.023) was significantly higher than in patients with alcoholic chronic pancreatitis (0.012 ± 0.003; p<0.05). In AIP, Foxp3/Mono and IgG4/Mono were positively correlated (p<0.05; R =0.91). ICOS+ Tregs (%ICOS-positive Tregs of total Tregs) were significantly higher in AIP patients (3.45% ± 1.58%) than in the patients with other pancreatic diseases (alcoholic chronic pancreatitis; 1.71% ± 0.98%, idiopathic chronic pancreatitis; 1.80% ± 0.86%) and the healthy control group (1.57% ± 0.69%, p<0.05). IL-10+ Tregs were significantly higher in AIP patients than in the healthy control group.
Conclusions: Increased quantities of ICOS+ Tregs may influence IgG4 production in IgG4-related AIP.
Hamster Pancreatic Cancer Model for Research on Metastasis and its in vivo Imaging
E. Uchida,1 A. Matsushita,1 Y. Nakamura,1 K. Yamahatsu,1 T. Aimoto,1 J. Ueda,1 Y. Matsuda,2 T. Ishiwata,2 Z. Naito,21Department of Surgery; 2Department of Pathology, Nippon Medical School, Tokyo, Japan
We developed short-term pancreatic cancer models in hamsters using PGHAM-1 cells and estimated the utility of the models for research on metastasis and therapeutic trials. With three PGHAM-1 models; 1) primary pancreatic cancer and simultaneous liver metastasis by intrapancreatic transplantation, 2) liver metastasis alone by intrasplenic transplantation, 3) peritoneal dissemination by intraperitoneal transplantation, within twenty-one days after inoculation, we studied the specific characteristics of metastases and the effect of several anti-angiogenic substances on primary and metastatic pancreatic tumors.
In addition, PGHAM-1-Luc, which is luciferase-positive PGHAM-1 cells, was newly developed. Administration of luciferin and usage of IVIS Imaging System (Xenogen, USA) could provide us with PGHAM-1 tumor imaging (primary tumor and metastasis) without sacrifice. This system might be useful not only for the research on tumor invasion and metastasis but also for the application to therapeutic trial with new substances through vital observation.
Expression and Role of Epithelial Splicing Regulatory Protein 1 in Human Pancreatic Cancer
J. Ueda,1,2 Y. Matsuda,1 K. Yamahatsu,1,2 E. Uchida,2 Z. Naito,1 T. Ishiwata,11Departments of Pathology and Integrative Oncological Pathology, Nippon Medical School, Tokyo, Japan; 2Surgery for Organ and Biological Regulation, Graduate School of Medicine, Nippon Medical School, Tokyo Japan
Background: Epithelial splicing regulatory protein 1 (ESRP1) binds the FGFR2 auxiliary cis-element ISE/ISS-3, located in the intron between exon IIIb and IIIc, and primarily promotes the expression of FGFR2IIIb. In this study, we examined the expression and role of ESRP1 in pancreatic ductal adenocarcinoma (PDAC).
Methods: Immunohistochemical analysis was performed using anti-ESRP1, FGFR2IIIb and FGFR2IIIc antibodies in PDAC tissue. ESRP1-expression vector and small interference RNA (siRNA) targeting ESRP1 were transfected into human PDAC cells, and cell growth, morphology, migration and invasion were analyzed.
Results: ESRP1 was strongly expressed in the nuclei of cancer cells in well and moderately differentiated adenocarcinoma, while weakly expressed in poorly differentiated adenocarcinoma and normal pancreatic ducts. The expression pattern was high-FGFR2IIIb/low-FGFR2IIIc for well and moderately differentiated adenocarcinomas, but the opposite for poorly differentiated adenocarcinoma. Clinicopathologically, the overall survival of the high-ESRP1 group was statistically longer than that of the low-ESRP1 group. In vitro, ESRP1-gene-transfected PDAC cells showed an increase in FGFR2IIIb expression and a decrease in migration and invasion. In contrast, ESRP1 siRNA-transfected PDAC cells induced FGFR2IIIc expression and increased cell growth, migration and invasion.
Conclusion: ESRP1 regulates the expression pattern of FGFR2 isoforms and modulates cell growth, migration and invasion in PDAC cells. These findings suggest that ESRP1 is one of the prognostic factors in PDAC.
The G12D Mutation of KRAS Predicts Early Recurrence and Poor Overall Survival in Ampullary Adenocarcinoma
N.P. Valsangkar,* T. Ingkakul,* C. Correa-Gallego,* A.L. Warshaw, C. Fernández-del Castillo, A.S. Liss, S.P. Thayer General and GI Surgery, Warshaw Institute, Massachusetts General Hospital, Harvard Medical School, Boston, MA
Background: Ampullary Adenocarcinoma (AA) is usually diagnosed in its early stages; however, a subset of patients present with advanced disease or progress to early local recurrence. This study evaluates the role of KRAS status in predicting poor outcome.
Methods: The tumor registry of an academic referral center was reviewed for data on surgically managed patients with AA. KRAS genotypes were determined for these patients using clinical specimens. ANOVA and χ2 tests were used to compare continuous and categorical variables between patients. Survival analyses were performed using the Kaplan-Meier (KM) and Cox methods.
Results: Sixty-three patients who underwent a resection for AA between 1982 and 2008 had available tissue specimens and their genotyping revealed 60.3% as wild type (WT) and 39.7% as KRAS mutant. There were no significant survival differences between WT and the total group of mutant KRAS patients. However, among the KRAS mutants, 36% were aspartate mutations at codon 12 (G12D) and KM analysis demonstrated significantly poorer three-year survival for G12D patients (40%) compared to non-G12D mutants (68%) and WT patients (73%), p < 0.05. More patients with G12D also developed early (≤ 24 Mo) recurrence: G12D = 66%, non-G12D = 33%, WT = 22% (p < 0.01). The Cox model showed higher risk of death for the G12D population regardless of nodal status (hazard ratio [HR], 2.80; 95% CI, 1.09-7.16; p < 0.05).
Conclusions: The detrimental effects of mutant KRAS in AA are seen only with G12D genotype. Stratification based on G12D identifies patients at risk of early recurrence and poorer survival who may benefit from adjuvant therapy.
Probiotic Prophylaxis in Predicted Severe Pancreatitis: a Monocenter Retrospective Cohort
M.C. van Baal,1 P. Kohout,2 M.G. Besselink,3 H.C. van Santvoort,3 G.T. Rijkers,1 H.G. Gooszen,11Dept. Operating Room/Evidence Based Surgery, Radboud University Nijmegen Medical Centre, Nijmegen, the Netherlands, 2Dept. of Internal Medicine, Faculty Thomayer's Hospital, Prague, Czech Republic, 3Dept. of Surgery, University Medical Center, Utrecht, the Netherlands
Background: The use of probiotics in acute pancreatitis has become highly controversial. Although initial studies suggested that probiotics could prevent infectious complications, a large randomized multicenter trial (PROPATRIA) found that probiotics may increase mortality in patients with predicted severe pancreatitis. As part of an ongoing research project to elucidate the latter unexpected findings we analyzed the use of probiotic prophylaxis in patients with predicted severe pancreatitis in Prague.
Objectives: To analyse the outcome of probiotic prophylaxis in patients with predicted severe pancreatitis in terms of infectious complications and mortality.
Methods: In this monocenter retrospective cohort study, patients with predicted severe pancreatitis (Ranson ≥3 or Imrie ≥3 or C-reactive protein ≥150 mg/L, all within 48h after hospital admission) were admitted to an internal medicine medium care facility of a teaching hospital in Prague from January 2003 to December 2010. After admittance, a nasojejunal feeding tube was inserted and a mixture of seven probiotic strains (Probioflora®) was administered twice daily during the whole hospital admission. Total parenteral nutrition was started immediately for 10 days with a gradual shift towards total enteral nutrition. Primary endpoint was the composite of infectious complications (i.e. infected pancreatic necrosis, bacteraemia, pneumonia) and mortality. Emphasis was placed on bowel ischemia as a complication of predicted severe pancreatitis (expected incidence 1-3%).
Results: 99 consecutive patients with predicted severe pancreatitis were included. Male:female ratio was 1:1, with a median age of 56 years. Median time from hospital admission to the first dose of probiotics and start of enteral nutrition was both 4 days. Sixty patients (60%) developed necrotizing pancreatitis. Infectious complications occurred in 42 patients (42%): 40 patients (40%) were diagnosed with bacteraemia, 11 patients (11%) with pneumonia and ten patients (10%) with infected pancreatic necrosis. Eight patients (8%) died. Bowel ischemia was reported in two patients (2%), one of whom died.
Conclusion: Even with a high percentage of infectious complications, potentially due to the liberal use of parenteral nutrition, the overall mortality in this series is relatively low. The rate of bowel ischemia appears to be within the expected range but selection bias cannot be excluded. Because of the lack of a control group, no conclusions about the effects of the administered probiotics can be drawn.
Clinical Relevance of Fine-Needle Aspiration in Necrotizing Pancreatitis
M.C. van Baal,1 O.J. Bakker,2 M.G. Besselink,2 H.C. van Santvoort,2 T.E. Deelman,3 T.L. Bollen,4 H.G. Gooszen,1 E. van der Harst,3Dutch Pancreatitis Study Group; 1Dept. Operating Room/Evidence Based Surgery, Radboud University Nijmegen Medical Centre, Nijmegen; 2Dept. of Surgery, University Medical Center, Utrecht; 3Dept. of Surgery, Maasstad Hospital, Rotterdam; 4Dept. of Radiology, Antonius Hospital, Nieuwegein, the Netherlands
Background: Fine-needle aspiration (FNA) can be used to detect infection of pancreatic and peripancreatic necrosis. Traditionally, it was used routinely but nowadays most expert-centers use it only selectively, in cases of clinical doubt. However, in the current era of postponing intervention, irrespective of the infection status, the clinical relevance of FNA in necrotizing pancreatitis is uncertain.
Objectives: To determine the impact of FNA on clinical decision making in patients with necrotizing pancreatitis.
Methods: Post-hoc analysis of a prospective database of 639 consecutive patients with necrotizing pancreatitis from 21 hospitals. Patients who underwent FNA were identified. The reference standard was the culture result of intervention (necrosectomy or percutaneous drainage). Two different outcomes were assessed: 1) The proportion of patients with FNA in whom the diagnosis of infected necrosis was confirmed by the reference standard; 2) The proportion of patients with a false negative FNA result.
Results: Between 2004 to 2008, 52 patients underwent FNA, male:female ratio was 2:1, mean age 55 years and mortality 29% (15/52 patients). In 35 patients, infected necrosis was confirmed by intervention (i.e., the reference standard).
There were 34 patients with a positive FNA. Four of these patients were treated without an intervention and all survived. Of the remaining 30 patients, in 2 patients (7%) the culture during intervention following FNA was negative, which means they had a false positive FNA result. Time between FNA and intervention in these patients was 4 and 5 days, respectively.
There were 18 patients with a negative FNA. Sixteen had an intervention, 7 of whom within a week. At intervention, 2/18 patients (11%) were found to have infected necrosis, so false negative FNA results. Time between FNA and intervention in these patients was 2 and 5 days, respectively.
Conclusion: The clinical relevance of FNA in the decision making in necrotizing pancreatitis is questionable as (a) the majority of patients undergoing FNA for suspected infected necrosis turned out to have infection during intervention, (b) almost all patients with negative FNA also underwent intervention and (c) FNA results may be false positive or false negative.
Accuracy of EUS for Diagnosis of Minimal Change Chronic Pancreatitis (MCCP): Correlation with Histopathology in 50 Patients Undergoing Total Pancreatectomy (TP) with Islet Autotransplantion (IAT)
J. Vega-Peralta,1 C. Manivel,1 R. Attam,1 M. Arain,1 J. Mallery,1 D. Radosevich,3 F. Khamis,1 M. Bellin,2 S. Chinnakotla,3 T. Dunn,3 A. Shaukat,1 G. Beilman,3 D. Sutherland,3 M. Freeman,11Gastroenterology Department, University of Minnesota, Minneapolis, MN; 2Pediatrics Department, University of Minnesota, Minneapolis, MN; 3Surgery Department, University of Minnesota, MN
Background: Diagnosis of MCCP is challenging. EUS has been widely used for diagnosing MCCP using 9standard criteria derived from correlation of EUS and histology in conditions other than MCCP. Our aim was to study the correlation of EUS findings and histology in pts undergoing TP-IAT for painful MCCP.
Methods: Retrospective comparison of EUS findings and pancreas histopathology in 50pts with MCCP (no calcifications on CT) from the 141pts undergoing TP-IAT at 1 center from 1/2008 through 7/2010. EUS classified using 9standard criteria. A blinded pathologist evaluated histology from resected pancreas. MCCP defined histologically when fibrosis, atrophy or inflammation was found in at least 1 biopsy.
Results: MCCP was found at histology in 45/50(90%) pts. All 45(100%) pts had fibrosis. Of pts with histologically confirmed MCCP, 27/45(60%) had ≥4/9criteria on EUS. 18/21(85%) pts with ≤3EUS criteria and 4/5(80%) pts with 0/9EUS criteria had MCCP by histopathology. 2/29(7%) pts with ≥4/9EUS criteria had normal histology. AUC 0.593, Kappa <0.11 for all cutoff points. Negative predictive value of a normal EUS was 38%. Positive predictive value of abnormal EUS at ≥6criteria was 72%.
Conclusion: Correlation between EUS and histology of MCCP is poor in pts undergoing TP-IAT. Normal or nearly normal EUS cannot exclude MCCP, and abnormal EUS alone is probably not sufficient for the diagnosis unless ≥6 criteria are present.
Influence of Early Oral Refeeding on Serum Triglyceride in Patients with Mild Acute Pancreatitis
M.H. Wan,1 J. Li,1 G.J. Xue,1 Y.L. Liu,1 X.L. Zhao,1 G.Y. Chen,1 W. Huang,2 W.F. Tang,11Pancreatic Diseases Research Group, Department of Integrated Traditional and Western Medicine, West China Hospital, Sichuan University, Chengdu, China; 2Liverpool NIHR Pancreas Biomedical Research Unit, Royal Liverpool University Hospital, Liverpool, UK
Introduction: We have previously shown early oral refeeding on basis of subjective sensation of hunger was beneficial for patients with mild acute pancreatitis (MAP), compared to normal start of oral refeeding (EPC 2011). We sought to determine whether oral refeeding could affect serum triglyceride (TG) levels in patients with MAP.
Methods: 149 patients diagnosed of MAP were randomly allocated to early refeeding group (n=75) and normal refeeding group (74). Patients in the two groups were comparable in base line characteristics. Numbers of patients with high TG levels were recorded on admission and after oral refeeding, and those whose TG levels decreased below 2.24 mmol/L or 4.48 mmol/L were compared between early refeeding group and normal refeeding group.
Results: On admission there were 31 and 26 patients with TG levels > 2.24 mmol/L, whilst 24 and 12 patients with TG levels > 4.48 mmol/L in the early refeeding group and normal refeeding group, repectively. After oral refeeding, there were significantly more patients whose serum TG levels decreased below 2.24 mmol/L in the early refeeding group compared to those in the normal refeeding group (24/31 vs 13/26, P < 0.05); there were also significantly more patients whose serum TG levels decreased below 4.48 mmol/L in the early refeeding group compared to those in the normal refeeding group (20/24 vs 5/12, P < 0.05).
Conclusions: Early oral refeeding might be beneficial for TG control in patients with MAP.
Activation of Stat3 May Participate in Adaptive Pancreatic Growth
J.Y. Wang,1 G. Gurda,1 L. Guo,1 S.A. Ernst,2 J.A. Williams,1,3Departments of 1Molecular and Integrative Physiology, 2Cell and Developmental Biology, and 3Internal Medicine, University of Michigan, Ann Arbor, MI
Introduction: Adaptive pancreatic growth can be modeled by feeding trypsin inhibitor (TI), which elevates endogenous plasma CCK; CCK then stimulates growth. Activation of Stats (signal transducer and activator of transcription) as part of the growth response was suggested by the up-regulation of Stat-regulated genes after TI feeding.
Aim: To determine the participation of Stat3 in the adaptive growth response, and whether it is directly activated by CCK.
Methods: Male ICR, C57BL/6, and CCK-deficient mice were fed chow containing.1% TI. Induction of phospho-Stat3 was determined by Western blotting and immunohistochemistry. Isolated acini were prepared by collagenase digestion.
Results: When the early (1-8h) response to TI feeding was evaluated, phosphorylation of Stat3 was found to peak at 2h, with a 10-fold increase over fasted control. Immunohistochemistry showed the localization of Stat3 activation to acinar, but not duct or islet, cells. Stat3 activation after TI feeding was blocked in CCK-deficient mice. To determine if Stat3 was increased by feeding alone or required TI, we compared nonfasted, fasted, refed chow, and refed chow with TI. Phospho-Stat3 increased only in mice fed TI, the condition that stimulates pancreatic growth. To determine if CCK had a direct effect on Stat3 activation, isolated acini were stimulated with CCK at various concentrations; minimal response in phospho-Stat3 levels was seen.
Conclusion: These results suggest involvement of Stat3 in the regulation of gene expression in adaptive pancreatic growth. CCK is required for Stat3 activation in vivo, but its action may be indirectly mediated, possibly by a growth factor.
ATDC Synergizes With Oncogenic Kras to Induce Invasive Pancreatic Adenocarcinoma in Transgenic Mice
L. Wang, F. Bednar, H. Yang, G.M. Ney, Pasca di Magliano, M. D.M. Simeone Department of Surgery, University of Michigan, Ann Arbor, MI
Pancreatic ductal adenocarcinoma (PDAC) ranks among the most lethal of human malignancies. We have demonstrated that ATDC is overexpressed in PDAC and required for tumor growth. To determine the role of ATDC during tumor progression, we generated transgenic mice that ubiquitously overexpress ATDC (pCAGGS-ATDC). ATDC overexpression results in acinar atypia with a long latency but is not sufficient to induce PanIN formation. We then analyzed the impact of ATDC overexpression in the context of KrasG12D by crossing of ATDC mice with LSL-KrasG12D;p48-Cre (KC) mice to generate ATDC;LSL-KrasG12D;p48-Cre (AKC) triple transgenic mice. KC mice develop PanINs with long latency, and PDAC is only rarely observed at an age of 15 months or greater. However, ATDC overexpression combined with KrasG12D accelerated PanINs progression and developed invasive and widely metastatic PDAC with a high penetrance. 45% AKC (n=6) (0% KC) mice exhibited high-grade PanIN 2or 3 at as early as 2 months of age. 85% AKC mice (n=6) at 6-8 months of age uniformly developed highly invasive and metastatic (80% liver, 60% lymph node, 40% spleen) PDAC with variable histological features, whereas none of age matched KC mice displayed those lesions. The evolution of PDAC in AKC mice recapitulated the histopathological manifestations of human PDAC, possessing a proliferative stromal component (Ki67 and collagen positive) and glandular architecture (CK19 and E cadherin positive) with a propensity to advance to a poorly differentiated state (CK19, E cadherin and mucin negative). ATDC and β-catenin were up-regulated in these primary and metastatic PDAC lesions. Our observations suggest ATDC promotes the malignant conversion of PanINs into lethal PDAC. This mouse model provides a platform for understanding the pathogenesis of PDAC and for developing targeted treatment strategies.
Clinical Analysis of Vascular Reconstruction Using Artificial Vascular Grafts During Pancreaticoduodenectomy
H. Z. Wang, L.D. Zhang, G. Chen, L. Cai, P. Bie Institute of Hepatopancreatobiliary Surgery, Southwest Hospital, Third Military Medical University Chongqing400038, P. R. China
Introduction: Use of artificial vascular grafts for reconstruction after portal vein (PV) and/or superior mesenteric vein (SMV) resection during pancreaticoduodenectomy (PD) is controversial. The aim of this study was to evaluate whether patients can benefit from vascular reconstruction with artificial vascular graft during PD.
Methods: We retrospectively collected data of all patients who underwent vein reconstruction using polytetrafluoroethylene (PTFE) grafts during PD at Southwest Hospital, Third Military Medical University from January, 2007 to April, 2011. Patient, operative, outcomes, graft patency and survival time were studied.
Results: Forty five patients underwent vein reconstruction using PTFE grafts during PD. Twenty seven patients were male, eighteen patients were female. Median age is 55.7 years old. Median operating time: 372 minutes. Mean blood loss is 1728 ml. Among them, PV reconstruction using PTFE grafts: 5 cases, SMV reconstruction using PTFE grafts: 9 cases, PV and SMV reconstruction using PTFE grafts: 31 cases. Of the 31 cases, 21 cases underwent splenic vein reconstruction simultaneously. UICC Staging: phase III 41 cases, phase IV 4 cases. Reoperative rate: 8.89%. Overall morbidity rate was 30.4%, 30-day mortality was 4.44%. Mean hospital stay: 35 days. No patients developed graft infection or irreversible hepatic necrosis. With mean follow-up of 15 months, overall graft patency was 77.78%. Mean survival time: 7.38 months. One year survival rate: 13.04%. Two year survival rate: 2.17%.
Conclusions: Reconstruction of resected PV/SMV using PTFE grafts during PD has a better life quality than palliative therapy, and similar survival time to palliative therapy. Prospective comparison studies to autologous vein reconstruction and palliative therapy are necessary.
Sirt1 Targets Critical Regulators of the Acinar Cell Programme during Acinoductal Metaplasia
E. Wauters,2 V. Sanchez-Arevalo Lobo,3 A. Vaqueirinho Pinho,1,3 E. Colvin,1 R. Sutherland,1 M. Serrano,4 L. Bouwens,2 F.X. Real,3 A.V. Biankin,1 I. Rooman,1,21Cancer Research Program, Garvan Institute of Medical Research, Sydney, Australia; 2Diabetes Research Center, VUB, Brussels, Belgium; 3Programa de Patología Molecular 4Programa de Oncología Molecular, CNIO, Madrid, Spain
Background: Sirt1, the most studied member of class III histone deacetylases, has broad functions, including being either a tumor suppressor or an oncogene. The function of Sirt1 has been studied in pancreas in relation to endocrine physiology but not to exocrine pathophysiology.
Methodology: The present study examined Sirt1 expression by qRT-PCR, immunostaining, and western blotting, and addressed its function by protein- and chromatin-IP, by siRNA approaches, and by use of specific drugs. Studies were performed in normal pancreas, in experimental models of acinoductal metaplasia (ADM), and included Super-Sirt1 transgenic, and conditional Sirt1 KO mice.
Results: Sirt1 is expressed in the nucleus of acinar cells, where we find it is essential for the cell's differentiation and survival. At onset of ADM, Sirt1 transiently shifts to the cytoplasm and upon redistribution to the nucleus, its inhibitor DBC1 is induced. Changes in Sirt1's subcellular distribution result in it targeting distinct proteins: We have identified Ptf1a and b-catenin as acinar targets for Sirt1 mediated deacetylation. We relate this to Ptf1a being crucial for the acinar differentiation programme and to b-catenin/Wnt signalling being essential for acinar cell regeneration during ADM.
Conclusions: Altered Sirt1 expression and activity impinge on the acinar cell's homeostasis via effects on Ptf1a and b-catenin, two proteins with critical roles in acinar cell dedifferentiation/metaplasia, key events in pancreatitis and carcinogenesis.
A Proposed Threshold for Dispersed-Pancreatic-Tissue-Volume (TV) Infused During Intraportal-Islet-Autotransplantation (IAT) after Total-Pancreatectomy (TP) to Treat Chronic-Pancreatitis (CP)
J.J. Wilhelm, M.D. Bellin, A.N. Balamurugan, G.J. Beilman, T.B. Dunn, D.M. Radosevich, B.J. Hering, D.E.R. Sutherland Schulze Diabetes Institute, Dept of Surgery, University of MN, USA
Background: Complications of IAT after TP to treat CP may be linked to the TV infused. When TV is large, purification is preferred to reduce complication risks such as elevated portal pressure (PP), portal vein thrombosis (PVT), or bleeding. However, purification may result in significant loss of islet-mass, particularly when islets are mantled by exocrine-tissue or of abnormal density. The objective of the current analysis was to determine a "safe" TV threshold, below which complications are minimized.
Methods: The TV infused intraportally during IAT was correlated with immediate PP, detection of PVT and exploration for post-op bleeding in 164 cases from May 2006 to May 2011. All patients received heparin at the time of IAT as prophylaxis against thrombosis.
Results: Increase in PP from baseline (ΔPP) correlated with TV (mL/kg body wgt) infused (R2=0.55, p<0.0001). The incidence of bleeding requiring reoperation was significantly higher when the TV was >0.25mL/kg (18% vs 8%, p=0.05) as well as when the ΔPP was >25 ccH2O (17% vs 7%, p=0.06). Only 6 patients (3.6%) had PVT (benign clinically) detected by ultrasound, with greater incidence in the high ΔPP group (p=0.08) but not with high TV.
Conclusions: We propose that a TV <0.25 mL/kg can be infused intraportally without further purification with a low incidence of complications after IAT. Above this, consideration should be given to volume reduction even though some islets are lost. Options for reducing TV include the Ficoll-gradient-islet-purification or partial-purification technique; or simply transplanting less than the full unpurified islet mass.
c-Met: a New Cancer Stem Cell Marker and Therapeutic Target for Pancreatic Cancer
J-J. Wu,1 C. Li,1 M. Hynes,1 J. Dosch,1 B. Sarkar,1 M.P. Magliano,1,2 D.M. Simeone,1,3Departments of 1Surgery; 2Cell and Developmental Biology; and 3Molecular and Integrative Physiology, University of Michigan, Ann Arbor, MI, 48109
Background and Aims: A subpopulation of self-renewing cells, termed cancer stem cells (CSCs), has been reported to account for the growth of many cancers. c-Met has been recently described as a marker of normal mouse pancreatic stem/progenitor cells. We hypothesized that c-Met functions as a marker and therapeutic target for human pancreatic CSCs.
Methods: Studies were performed in low passage primary human pancreatic adenocarcinoma xenografts established in NOD-SCID mice. Self-renewal capacity of c-Methigh pancreatic cancer cells was assessed using in vitro sphere assays and in vivo tumorigenicity of c-Methigh pancreatic cancer cells was evaluated by implantation in NOD/SCID mice.
Results: c-Methigh cells readily formed spheres, while c-Met- cells did not. Use of the c-Met inhibitor XL184 or c-Met knockdown with shRNA significantly inhibited the tumor sphere formation. Cells expressing high c-Met increased tumorigenic potential in vivo and cells expressing both CD44 and c-Methigh (0.5-5% of pancreatic cancer cells) demonstrated the capacity for self-renewal and the highest tumorigenic potential of all populations studied. Furthermore, c-Met inhibition of established pancreatic tumors by XL 184 in NOD/SCID mice inhibited tumor growth and depleted the CSC population, when used alone or in combination with gemcitabine. Tumors initiated by spheres transduced with cMet shRNA also grew more slowly than the control group. In addition, inhibition of c-Met prevented the metastases using an intracardiac injection model.
Conclusion: c-Met serves as an important marker and therapeutic target for pancreatic CSCs.
Thyroid Hormone Induces Mouse Pancreatic Acinar Cell Proliferation, Polyploidization and Metaplasia In Vivo
J. Xanthopoulos,1 B. Liu,1 C. Nguyen,1 F. Gorelick,1,2 E.S. Swenson,11Internal Medicine, Yale University School of Medicine, New Haven, CT; 2Internal Medicine, Veteran's Affairs Medical Center, West Haven, CT
Background and Aim: Thyroid hormones are major regulators of development, growth and metabolism. Thyroid hormone regulates dramatic remodeling of the Xenopus pancreas during metamorphosis, inducing transient dedifferentiation of acinar cells to a progenitor phenotype, followed by redifferentiation to mature ducts and acini. We hypothesized that adult mouse pancreatic acinar cells stimulated by thyroid hormone would demonstrate characteristics of pancreatic progenitor cells.
Methods: Male C57Bl/6 mice were injected with thyroid hormone (T3, 4 mg/kg i.p. daily for 4 or 7 days) and sacrificed on day 5, 8 or 10. Bromodeoxyuridine (BrdU) was administered 2 hrs prior to sacrifice. Markers of proliferation (Ki67, BrdU), phenotype (amylase, ductal cytokeratins, insulin and glucagon) and differentiation (Sox9, Pdx1) were labeled by immunofluorescence in formalin-fixed paraffin sections. Ploidy was estimated using in situ hybridization for the Y chromosome.
Results: T3 dramatically increased acinar cell proliferation (BrdU and Ki-67) and modestly increased acinar cell ploidy when compared with controls. Zymogen granule content and amylase labeling were markedly reduced in T3-treated mice, but there was no evidence of inflammation or edema. Ductal cytokeratins and the progenitor markers Pdx1 and Sox9 were upregulated in acinar cells after T3. Insulin and glucagon remained restricted to islets.
Conclusion: Increases in circulating thyroid hormone induce acinar cell proliferation and metaplasia toward a ductal/progenitor, but not endocrine phenotype. The reversibility and long-term effects of T3-induced changes are currently under investigation.
Therapeutic Role of Carbon Monoxide in Experimental Acute Pancreatitis
J. Xue, A. Habtezion Department of Medicine, Stanford University School of medicine, Stanford, CA, USA
Acute pancreatitis (AP) is associated with significant morbidity and mortality. Despite the clinical impact of AP, therapy is limited to supportive care. Recent studies show protective and therapeutic benefits of hemeoxygenase-1 (HO-1) upregulation. HO-1 is rate limiting enzyme in the heme degredation pathyway and production of carbon monoxide (CO). CO is increasingly being accepted as cytoprotective and homeostatic molecule with important signaling capabilities in physiologic and pathophysiologic situations. However the role of CO in the on going AP and its potential modulation of pancreatic injury is not clear. The aim of our study is to investigate the role of CO after the onset of AP. We found that CO-releasing molecule 2(CORM-2) has beneficial effect in two independent model of pancreatitis (caerulein and choline deficient dL-ethionine supplemented diet models). Compared with control treatment group, CORM-2 treatment significantly reduced serum levels of amylase, lipase and pro-inflammatory cytokines (TNFα and IL-1β). In addition, pancreatic trypsin activation and lung myeloperoxidase (MPO) level were decreased with CORM-2 treatment. To further determine the COMR-2 effect on infiltrating leukocytes, we isolated leukocytes from inflamed and non-inflamed pancreas. Systemic administration of CORM-2 dramatically decreased leukocyte infiltration in the pancreas. CORM-2 inhibits monocyte differentiation into macrophages. Using intracellular cytokine and flowcytometric analysis, we find decreased macrophage TNFα expression in the pancreas of mice treated with CORM-2. Taken together, our data indicate that CORM-2 has therapeutic effects on AP, and the beneficial effects alteration in the innate immune response in pancreas. CO may offer a future therapeutic option in the treatment of ongoing human AP.
Vincristine Reduces L-Asparaginase Induced Acute Pancreatitis (AP) By Preventing Procathepsin B Trafficking and Trypsinogen Activation
D. Yadav, H. Kim, L. S. Orlichenko, V. P. Singh Department of Medicine, University of Pittsburgh, Pittsburgh, PA
Background: L-asparaginase (LA), which induces autophagy, is sometimes used in combination with the microtubule depolymerizing drug vincristine (V) to treat hematologic malignancies. LA is associated with AP. Our recent work shows Golgi dependence of trypsinogen activation (TA) during autophagy. Thus, using a two phase clinical and in-vitro study, we tested the hypothesis that vincristine may reduce rates of LA induced AP, by interfering with microtubule dependent Golgi trafficking.
Methods: We systematically reviewed all published clinical trails and compared AP rates of LA alone with those using LA+V. Vincristine's (5μM) effect on microtubules and Golgi (by immunostaining), 100nM caerulein (CER) induced TA (kinetic assays), cathepsin B activity were studied in acinar cells. Subcellular localization of Procathepsin B and amounts in lysates were studied using western blotting.
Results: The LA+V combination significantly lowered AP rates(mean 1.1% [range 0-18%], in 118 trials with 22409 patients) compared to LA alone (mean 3.9% [range 0-19%] in 39 trials with 2061 patients, p<0.001). Post Grubb's correction these differences remained significant (LA; 1.8% vs. LA+V: 1.1%, p=0.0124). V disrupted microtubules, dismantled the Golgi and prevented caerulein induced loss of procathepsin B and TA (29% of CER). Procathepsin B resided in the GM130 (Golgi) enriched fraction and its propeptide inhibited cathepsin B activity.
Conclusions: Vincristine induced disruption of the microtubules perturbs post Golgi trafficking of procathepsin B resulting in its retention and consequent inhibition of cathepsin B mediated trypsinogen activation. The effect may underlie the protection by vincristine from L-asparaginase induced AP in humans.
Natural History After First-Attack of Acute Pancreatitis (AP)
D. Yadav, M. O'Connell, G. Papachristou University of Pittsburgh, Pittsburgh, PA
Background: Although AP is common during the course of chronic pancreatitis (CP), data on natural history after first-attack of AP are scarce. We determined the risk of recurrent AP (RAP) and subsequent CP diagnosis after first attack of AP in Allegheny County, Pennsylvania, USA.
Methods: Pennsylvania Health Care Cost Containment Council (PHC4) is authorized to collect information on all hospitalizations in Pennsylvania. Using PHC4 dataset we identified all unique White and Black Allegheny County residents who received first-time primary inpatient discharge diagnosis of AP from years 1996-2005. AP etiology was determined using associated diagnoses codes. We noted if any patient had readmission for AP and/or received CP diagnosis until 3rd quarter 2007.
Results: 7456 unique residents (mean age 58±20 yrs, 45% male, 80% White) with incident AP admission were identified. The common etiologies were- biliary (28%), alcohol (19%) and idiopathic (36%). Alcoholic AP patients (vs. biliary and idiopathic AP) were significantly younger and more likely to be male and Black. Among survivors (98.1%) and those without pancreatic cancer, follow up (median 40 mths, IQR 18-69) was available in 84%. Readmissions for primary or any AP diagnosis occurred in 22 and 29%; while subsequent primary or any CP diagnosis was assigned to 6 and 12.8% patients. About one-third RAP patients received CP diagnosis and three-quarter of patients with CP had RAP. On Cox-regression analyses, risk of RAP (OR 2.1, 95% CI 1.9-2.4) and CP (OR 1.5, 1.1-2.0) were significantly higher among patients with alcoholic etiology (vs. non-alcoholic). History of tobacco abuse was an independent risk factor for RAP and CP.
Conclusion: Readmissions after first-attack of AP are common. Progression to CP is infrequent and usually occurs in the setting of RAP, alcohol and smoking.
Pancreatic Duct Glands (PDG) Are the Origin of Gastric-Type IPMN
J. Yamaguchi,1 M. Mino-Kenudson,2 A.S. Liss,1 C. Fernández-del Castillo,1 A.L. Warshaw,1 S.P. Thayer,1Warshaw Institute & Departments of 1Surgery and 2Pathology, Massachusetts General Hospital and Harvard Medical School, Boston, MA
Background: Pancreatic duct glands (PDG) are mucinous gland-like outpouches of major pancreatic ducts that function as progenitor stem cell niches responsible for epithelial regeneration. Our previous work in a mouse model of pancreatitis revealed PDG undergo a gastric GI mucinous metaplasia and cystic hypertrophy in response to injury. These PDG have a mucinous character and location consistent with side branch IPMN (SB-IPMN). Here we investigate the role of PDG as the compartment of origin for SB-IPMN.
Methods: The morphological features of PDG were analyzed in human specimens from normal organ donors, chronic pancreatitis and SB-IPMN. Expression of MUC5ac, MUC6, TFF1/2 and proliferative activity were assessed by IHC.
Results: PDG are characterized by MUC6 and TFF2 expression. In pancreatitis specimens PDG are noted to undergo a mucinous hypertrophy with enhanced proliferative activity. IHC reveals an expansion of TFF2 and MUC6 which remains contained within the PDG. There is also the de novo expression of TFF1 and MUC 5ac in the epithelium emanating from the PDG. SB-IPMNs are also noted to be MUC5ac and TFF1 positive throughout the cyst wall. However, MUC6/TFF2-positive PDG cells were found at the bases between large papillary projections and comprise the basal segment of the cyst walls. Proliferative activity was noted to be located predominantly in these basal segments.
Conclusion: SB-IPMNs are a complex two layer structure. PDG integrate and form the basal segments and are the likely origin of neoplastic epithelial cells that line the cyst wall. These data suggest that PDG are the origin of SB-IPMN.
Pancreatic Duct Glands (PDG), a Progenitor Stem Cell Niche Responsible For Pancreatic Epithelial Renewal and Repair in Response To Inflammatory Injury
J. Yamaguchi,1 M. Mino-Kenudson,2 A.S. Liss,1 C. Fernández-del Castillo,1 A.L. Warshaw,1 S.P. Thayer,1Warshaw Institute & Departments of 1Surgery and 2Pathology, Massachusetts General Hospital and Harvard Medical School, Boston, MA
Background: Pancreatic duct glands (PDG) are a novel compartment that histologically appears as gland-like outpouches of major pancreatic ducts. These glands have molecular features known to mark stem cell niches, but their function remains to be determined. The aim of this study was to investigate the role of PDG in pancreatic epithelial regeneration.
Methods: PDG were analyzed in normal and inflammatory pancreata in humans and mice. Proliferative activity and the expression of trefoil factor (TFF), a family of secreted factors thought to play an essential role in cell migration, were assessed by IHC. Migration of PDG cells was evaluated by BrdU tag-and-chase method in a pancreatitis mouse model.
Results: Normal pancreatic epithelium has a low rate of proliferative activity. In response to inflammatory injury, however, robust proliferative activity was identified within the PDG in both humans and mice. BrdU was used to tag proliferating cells in a mouse model of pancreatitis. Tagged cells were initially found in PDG. In mice harvested 5 days after BrdU tagging, the BrdU-positive cells were present in the pancreatic ductal epithelium, indicating that newly generated PDG cells migrate to renew the epithelium. The migratory factor TFF1 is upregulated in response to inflammation in these PDGs and is found in these migrating cells. In vitro assays reveal that transfection and overexpression of TFF1 promotes migration of HPDE (Human Pancreatic Duct Epithelial cells).
Conclusion: PDG are a progenitor stem cell niche responsible for epithelial regeneration and migration.
Nestin Inhibits Proliferation of Vascular Endothelial Cells in vitro And Tumor Angiogenesis in Pancreatic Cancer in vivo
K. Yamahatsu,1,2 Y. Matsuda,1 M. Hagio,1 T. Aimoto,2 Y. Nakamura,2 E. Uchida,2 Z. Naito,1 T. Ishiwata,11Departments of Pathology and Integrative Oncological Pathology, 2Surgery for Organ and Biological Regulation, Graduate School of Medicine, Nippon Medical School, 1-1-5 Sendagi, Bunkyo-ku, Tokyo 113-8603, Japan
Background: Nestin, a class VI intermediate filament protein, has been reported to express in various progenitor cells. In this study, we investigated whether nestin could be useful as a marker of angiogenesis in pancreatic ductal adenocarcinoma (PDAC), and analyzed the roles of nestin in vascular endothelial cells using a gene silencing strategy.
Methods: PDAC tissues were immunostained with anti-nestin antibody, and antibodies against vascular and lymphatic endothelial cell markers. Wedetermined the numbers, dimensions and PCNA-labeling indices of nestin-positive vessels. Furthermore, the effects of nestin siRNA transfection on vascular endothelial cell growth and migration were analyzed. To evaluate the effects of reduced nestin expression on tumor growth, we performed in vivo administrations of nestin siRNA in the subcutaneous tumors of nude mice.
Results: Nestin was expressed in proliferating small blood vessels of PDAC tissues, but not in lymph vessels. A decrease in the nestin expression suppressed vascular endothelial cell growth, while the migration ability of siRNA-transfected cells were not altered. In vivo administration of siRNA targeting nestin inhibited subcutaneous tumor growth in nude mice, as compared with control groups.
Conclusion: It is suggested that nestin is a marker of newly formed tumor vessels in PDAC, and might be a novel therapeutic target in pancreatic cancers via inhibition of tumor angiogenesis.
ATDC (TRIM29) Interacts with RNF8 and Protects Pancreatic Cancer Cells from DNA Damaging Agents
H. Yang,1 L. Wang,1 E. Kim,1 J. Prasad,3 D. Misek,1 M. Ljungman,3 D.M. Simeone,1,2Depts of 1Surgery, 2Physiology, and 3Radiation Oncology, University of Michigan
The ataxia-telangiectasia group D complementing gene (ATDC), also known as TRIM29, is a member of the tripartite motif (TRIM) protein family. We have previously shown that ATDC, a nucleocytoplasmic shuttling protein, is highly expressed in pancreatic cancer and promotes cell proliferation via β-catenin signaling. We have also observed that ATDC expression leads to resistance to multiple forms of DNA damage, including ionizing radiation (IR). To elucidate the role of ATDC in IR protection, we performed a screen to identify ATDC binding partners and found it binds to the E3 ubiquitin ligase RNF8, a DNA damage response protein. Co-immunopreciptation experiments using full length and truncation mutants of ATDC transfected into HEK293 cells (no endogenous ATDC) identified the C-terminal portion (aa 348-588) of ATDC and the ring domain of RNF8 as interaction domains. We found the coil coiled domain of ATDC is responsible for nucleocytoplasmic shuttling, as an ATDC mutant lacking this domain was unable to shuttle into the nucleus. ATDC significantly promoted cell survival in HEK293 ATDC+, BxPC-3 and Panc-1 cells after IR treatment in comparison to HEK293 ATDC-, and ATDC shRNA-treated BxPC-3 and Panc-1 cells. Interestingly, HEK293 cells expressing the ATDC truncation mutant lacking the C terminus (ATDC Δ348-588) had significantly reduced survival rates after IR. In addition, IR induced ATDC, but not ATDC (Δ348-588), loading to chromatin in a RNF8 dependent manner. The ATDC-RNF8 interaction was critical for regulating IR-induced γ-H2AX ubiquitination, recovery of H2AX phosphorylation (a measure of DNA double strand breaks), 53BP1 phosphorylation, and DNA damage foci recovery.
Conclusion In the study we identify a molecular mechanism by which ATDC, a highly expressed oncogene in pancreatic cancer, may protect cancer cells against IR-induced DNA damage through an interaction with RNF8. These studies better define the role of ATDC in mediating pancreatic cancer cell resistance to DNA damage and may allow the development of more effective therapeutics to target ATDC.
A Case of the Pancreatic Duct Stenosis at the Body with Focal Atrophic and Fatty Change of the Adjacent Parenchyma
G. Yoshimatsu,1 N. Sakata,1 M. Mizuma,1 K. Ishida,2 S. Ottomo,1 K. Nakagawa,3 H. Hayashi,1 F. Motoi,1 T. Rikiyama,1 Y. Katayose,3 S. Egawa,1 A. Kanno,3 J. Masamune,3 T. Shimosegawa,3 M. Unno,11Division of Hepato-Biliary-Pancreatic Surgery, Tohoku University, Graduate School of Medicine, JAPAN, 2Department of Pathology, Tohoku University Hospital, JAPAN, 3Division of Surgery and Oncology, Tohoku University Graduate School of Medicine, JAPAN, 4Division of Gastroenterology, Tohoku University, Graduate School of Medicine, JAPAN
Introduction: Stenoses of the main pancreatic duct with diffuse atrophic changes in distal parenchyma were shown commonly in many pancreatic diseases. Recently we encountered a patient who had local narrowness of the main pancreatic duct with focal atrophic and fatty change of adjacent parenchyma.
Case report: A 61-year-old woman was admitted to our hospital for repeated left flank pain. She had only a slight spontaneous pain. Laboratory data were as follows: CEA 2.7 ng/dl, CA19-9 2.0 U/ml. On CT examination, a low density area was shown as atrophy at the pancreatic body, but tumors were undetectable in the pancreas. Endoscopic retrograde cholangio pancreatography showed that smooth narrowness of the main pancreatic duct at the body and the dilatation of the distal duct. There were no malignant cells in pancreatic juice by cytological examinations. PET-CT showed no abnormal hot spot. Ultimately we performed distal pancretectomy, because we could not deny malignancy. In the resected specimens, local atrophic change was detected in parenchyma of the pancreatic body. Histological findings revealed the stenosis of the main pancreatic duct with wall thickness due to fibrosis, increasing of fibroblasts and slight infiltration of neutrophil. Focal atrophy with fatty change was detected in the adjacent distal parenchyma of the stenosis. In addition, PanIN 1A-B was recognized at the margin of the stenotic duct. There was no sign of adenocarcinoma.
Discussion and Conclusion: We considered that fatty change was caused by chronic pancreatitis involving the pancreatic duct stenosis, not by epithelial proliferation of pancreatic duct because fatty change required longer time than neoplastic changes. Epithelial proliferation was considered as secondary change. Recently autoimmune pancreatitis was noted as idiopathic duct-centric chronic pancreatitis (IDCP) and lymphoplasmacytic sclerosing pancreatitis (LPSP). IDCP was characterized much neutrophil infiltration around the pancreatic duct, but in our case, demonstrated only slight neutrophil infiltration. LPSP was characterized high level of the IgG4, but it was normal range, so our case was untypical as autoimmune pancreatitis. We experienced a case with a rare type of pancreatic duct stenosis, who underwent surgery finally for risk of malignancy, but operative indication for the similar cases might be well considered.
90Y DOTATOC and Multidisciplinary Therapies for a Patient with Pancreatic Neuroendocrine Carcinoma with Systemic Metastases
G. Yoshimatsu, S. Egawa, N. Sakata, F. Motoi, T. Rikiyama, Y. Katayose, M. Unno Div. Hepato-Biliary-Pancreatic Surgery, Dept. Surgery, Tohoku University, Sendai, Japan
Case Report: A 56-year-old female who had an upper abdominal pain and a high serum amylase level was diagnosed as pancreatic neuroendocrine tumor (NET) with multiple liver metastases. Needle biopsy of the liver tumor revealed that Ki67 was positive 15.6% and glucagon. Somatostatin receptor (SSTR) 1, 2A, 2B, 5 were also positive but there were no positive cells in insulin, somatostatin, and gastrin. Due to the diagnosis of a Grade 3 NET, we started octreotide treatment to the patient at first, but both primary and metastatic tumors did not reduced in size. The second line, octreotide and S-1 was also ineffective. Therefore she underwent 90Yttrium radio-labeled DOTATOC (90Y DOTATOC) treatment in Europe as the third line therapy. After twice administrations of 90Y DOTATOC, the primary tumor was almost vanished (considered as CR), and the sizes of the liver tumors were markedly reduced (considered as PR). While primary and liver tumors maintained significant diminish more than a year, bone metastases developed in the scull and the spine. The tumor of scull bone was resected and treated by γ-Knife irradiation,Third administration of DOTATOC and systemic S-1 chemotherapy has been effective so far to control bone metastases. Now she is alive with no weakness for 4 years.
Conclusion:90Y DOTATOC is one of promising treatments for pancreatic NET with unresectable multiple liver metastases even when octreotide and chemotherapies are not effective. During the course of treatment, systemic metastases with different biological behavior may appear. Continuous and multidisciplinary treatment can achieve the survival with good quality of life in NET patients.
Heat Shock Proteins 27 and 47 Inhibit Platelet-Derived Growth Factor (PDGF)-Induced Pancreatic Stellate Cell Proliferation: Implication in Pancreatic Cancer Progression
J. Youkhana,1 J. Liu, N. Kiriella,1 L. Yang,1 Z. Xu,1 A. Biankin,2 D. Goldstein,1 R. Pirola,1 J. Wilson,1 M.V. Apte,1 P.A. Phillips,11Pancreatic Research Group, University of New South Wales and 2Garvan Institute of Medical Research, Sydney, Australia
Heat shock proteins (HSPs) can regulate cell migration and proliferation and are known to be overexpressed in human pancreatic cancer (PC) tissue. The stromal reaction of PC is produced by activated pancreatic stellate cells (PSCs) which interact with PC cells to facilitate cancer progression. Growth factors (including PDGF) produced by tumour cells induce proliferation and migration of PSCs. We have also previously shown that PDGF induces the expression of several HSPs (27, 47, 70 and 90) in human cancer associated-PSCs (CA-hPSC). However, little is known about the role of HSPs in PSC function.
Aim: To determine the effect of silencing HSPs (27, 47, 70 or 90) in CA-hPSCs on cell migration and proliferation.
Methods: CA-hPSC were isolated from resected pancreatic tissue from PC patients and treated (n=4) with siRNA targeting HSP27 (protein 1 or 2), HSP47, HSP70, HSP90 (>90% protein silencing achieved) or non-silencing siRNA (ns-siRNA). 48h post transfection CA-hPSCs were incubated ± PDGF (10ng/ml) for 48h. Cell proliferation assessed by the cell counting kit-8 and migration assessed using modified Boyden chambers with PDGF as a chemotactic agent.
Results: PDGF stimulated CA-hPSC proliferation and migration in ns-siRNA treated PSCs. Notably, silencing HSP27 (p1 and p2) or HSP47 (but not HSP 70 or 90) inhibited PDGF-induced proliferation (% of ns-siRNA without PDGF: ns-siRNA+PDGF 154.7±5.9#, HSP47 siRNA+PDGF 115.1±9.3*, HSP27(p1)-siRNA+PDGF 125.0±12.4*, HSP27(p2)-siRNA+PDGF 127.5±3.2*; #p<0.001 vs ns-siRNA; *p<0.01 vs ns-siRNA+PDGF). However, silencing HSP27 (p1 and p2) and HSP47 had no effect on basal or PDGF-induced PSC migration.
Conclusion: This is the first study to show that suppression of HSP27 or HSP47 inhibits PDGF-induced PSC proliferation.
Implication: Modulation of HSPs in PSCs may represent a novel approach to influence PSC function and PSC interactions with cancer cells.
The Interaction of PSC with Carcinoma Cells Promotes Angiogenesis
Z. Zhang,1,2 H. Habisch,1 S. Zhou,1 M. Siech,3 M. Bachem,11Clinical Chemistry, University Hospital Ulm, Germany; 2General Surgery, 1st Affiliated Hospital of Anhui Medical University, Hefei, PR. China; 3Surgery Dept. Ostalbklinikum Aalen, Germany
Background: PSCs are the main source of extracellular matrix in pancreatic cancer. The role of PSCs in angiogenesis associated with pancreas carcinoma is unclear. This study investigated the interaction of PSCs with pancreatic carcinoma cells (PCCs) regarding angiogenesis.
Methods: Supernatants (SN) of cultured PSC and PCC (mono- and co-culture) were added to cultured HUVECs. Proliferation, apoptosis, migration and tube formation of HUVEC were measured. Migration was quantified by single cell tracking and Boyden chamber assay. Production of MMPs was demonstrated by zymography. To study a potential role of MMPs on migration of HUVECs the MMP-inhibitor GM6001 was added to cultured HUVEC together with PSC-SN and PSC-PCC-SN.
Results: PSC-SN and PCC-SN stimulated dose dependent proliferation (1.92-fold and 2.42-fold of control) and migration (1.41 to 2.11-fold and 1.49 to 1.95-fold of control) of HUVEC while reducing apoptosis. The pro-migratory effect of PSC/PCC coculture-SN was higher compared to the mixture of monoculture SNs (coculture 1.14-fold of mixture) indicating a synergistic effect of both cell types. In addition, MMP2 was much higher in coculture SN. PSC-SN stimulated tube formation of HUVEC (tube number 3.27-fold, total tube length 3.30-fold, branch points 6.87-fold of control). GM6001 inhibited the pro-migratory effect of PSC-SN (34.4% less) and PSC-PCC-SN (23.8% less).
Conclusion: Through interaction of PSC with PCC proliferation, migration and tube formation of HUVECs are stimulated. MMPs seem to play an important role in stimulated HUVEC migration.
Early Oral Refeeding Wisdom in Severe Acute Pancreatitis
X.L. Zhao, J. Li, G.J. Xue, Y.L. Liu, M.H. Wan, G.Y. Chen, W. Huang, R. Sutton, W.F. Tang The Pancreatic Diseases Research Group, Department of Integrated Traditional and Western Medicine, West China Hospital, Sichuan University, Chengdu, China; Liverpool NIHR Pancreas Biomedical Research Unit, Royal Liverpool University Hospital, Liverpool
Purpose: In severe acute pancreatitis (SAP), oral refeeding after initial fasting is suggested to start as soon as possible. But the optimal time of starting oral refeeding is not well defined. To ascertain the safety and efficacy for early oral refeeding on basis of subjective sensation of hunger, compared with normal oral refeeding with relieve of abdominal pain and distension and normalization of pancreatic enzymes.
Method: A prospective randomized controlled trial was conducted. The total and post-refeeding length of hospitalization (LOH), recurrence of abdominal pain, transitional abdominal distension, and related serum biomarkers were recorded.
Results: Six-eight patients were randomized to early oral refeeding (subjective sensation of hunger) or normal oral refeeding group with 34 in each. Patients in early refeeding group started to receive oral refeeding significantly earlier than those in normal refeeding group (8.3±3.5 versus 12.3±7.7, p < 0.05), with shorter total LOH (14.7±6.71d versus 18.4±7.2d, p < 0.05). There were no significant differences of time intervals between the pain onset and admission, post-refeeding LOH, pain relapse, transitional abdominal distension and biomarkers between two groups. No apparent gastrointestinal side effects were observed and no patients needed cessation of diet in both groups.
Conclusions: Early oral refeeding with the subjective feeling of hunger was safe with shorter LOH for SAP. The remission of symptoms and normalization of pancreatic enzymes were not imperative for oral refeeding in patients with SAP.
Chymotrypsin C (CTRC) Mutants and Endoplasmic Reticulum Stress
J. Zhou, M. Sahin-Tóth Department of Molecular and Cell Biology, Boston University, Boston, MA
Background and Aims: Mutations in the chymotrypsinogen C (CTRC) gene have been identified as risk factors for chronic pancreatitis. We have previously found that the p.A73T CTRC mutant elicits endoplasmic reticulum (ER) stress in pancreatic acinar cells. Our present aim was to extend these studies to other CTRC mutants, and examine whether induction of ER stress is a general property of CTRC mutants.
Methods: Dexamethasone-differentiated AR42J rat pancreatic acinar cells were infected with recombinant adenovirus carrying wild-type CTRC or mutants p.Q48R, p.G61R, p.A73T, p.R254W, and p.K247_R254del. CTRC secretion was measured by activity assays and SDS-PAGE. Messenger RNA levels of ER chaperones BiP and calreticulin were quantified by real time PCR. Splicing of XBP1 was detected by PCR and agarose gel electrophoresis.
Results: Mutants p.Q48R, p.G61R and p.A73T were poorly secreted from AR42J cells and caused a significant increase in ER stress markers including BiP and calreticulin mRNA levels and XBP1 splicing. The magnitude of ER stress seemed to correlate with the extent of the secretion defect (p.G61R>p.A73T>p.Q48R). Mutants p.R254W and p.K247_R254del were secreted at moderately reduced levels relative to wild-type CTRC, and elicited no ER stress.
Conclusions: Induction of ER stress seems to be associated with CTRC mutants that exhibit a secretion defect (p.Q48R, p.G61R and p.A73T). CTRC mutants p.R254W and p.K247_R254del that are statistically associated with chronic pancreatitis in European populations do not induce ER stress.