Defining the Optimal Extent of Vascular Resection at Pancreaticoduodenectomy (PD): Systematic Assessment of Outcome from Resection Incorporating the Superior Mesenteric Artery (SMA)
M.A. Abdeldayem,1 S. Jegatheeswaran,1 A.K. Siriwardena,1. 1Hepatobiliary Surgery Unit, Manchester Royal Infirmary, Manchester, UK.
Introduction: PD is the treatment of choice for non-metastatic, localized cancers of the head of the pancreas in patients without major co-morbidity. Technically, resection can be prevented when tumour involves the portal/superior mesenteric vein or SMA. Although venous resection is increasingly accepted, SMA resection initially proposed by Fortner in the 1970s remains unestablished. Given the rarity of SMA resection in any given series, this technique is utilized with too low a frequency to be assessable by randomized comparison. Thus this study undertakes an analysis of pooled data on arterial resection in order to assess outcome from all contemporary series.
Methods: Medline and embase were searched for the period 2000–2013 selecting only articles in English which provided complication profile, outcome and survival data allocatable to patients undergoing arterial resection at PD.
Results: During this period 4 manuscripts provided information on 25 patients undergoing PD with SMA resection (24 had concomitant vein resection). Reconstruction (available for 17) was by flap rotation of the splenic artery in 9 (53%), graft in 6 (35%) and primary end-end anastomosis in 2 (12%). Median (range) operating time was 820 (441-1190) minutes, blood loss 6650 (2400-15,900) ml and 30-day mortality was 3 (12%). Median survival was 11 (0-29) months.
Conclusions: Despite widespread use of venous resection the published literature is deficient in the reporting of outcome allocatable to pancreatectomy with venous AND arterial resection. Summating all available outcome data indicates that SMA resection at PD is a prolonged operation associated with prohibitive blood loss but acceptable mortality. Survival seems similar to that of unresected pancreatic cancer. National cancer registries must record outcome on SMA resection at PD.
Natural History of Patients With Predicted Severe Acute Pancreatitis
E. Afghani,1 J. McNabb-Baltar,2 P.A. Banks,2 A. Sinha,1 M. Faghih,1 B.U. Wu,2 D.L. Conwell,2 V.K. Singh,1. 1Division of Gastroenterology, Johns Hopkins Medical Institutions, Baltimore, MD; 2Division of Gastroenterology, Brigham and Women’s Hospital, Boston, MA.
Background: Early scoring systems have been used to predict severe acute pancreatitis (SAP) but the natural history of these patients is not well-studied.
Aims: To describe the natural history of patients with predicted severe acute pancreatitis (PSAP).
Methods: Demographic, clinical, and laboratory data of all consecutive adult (age≥18 years) admitted with a diagnosis of acute pancreatitis was prospectively collected between June 2005 to December 2007. PSAP and predicted non-severe acute pancreatitis (PNSAP) was defined as BISAP≥3 & SIRS ≥2 and BISAP<3 & SIRS<2, respectively, within 24 hours of admission. SAP was defined as persistent (>48 hours) organ failure (POF) using the Marshall score. Pancreatic necrosis (PN) was diagnosed using a contrast enhanced CT.
Results: There were 397 cases, with 52% females, mean age of 52 years, median APACHE II score of 7, and biliary etiology in 27%. There were 58 (15%) and 339 (85%) cases with PSAP and PNSAP, respectively. PSAP cases had higher rates of POF (40% vs. 6.2% p<0.001), longer length of stay (14.9 vs. 7.6 days, p<0.001), higher rates of PN (41.4% vs. 12.4%, p<0.001), and increased mortality (16% vs. 1.5%, p<0.001). Of the 58 cases with PSAP, 23 (40%) did and 35 (60%) did not develop SAP during hospitalization. PSAP cases that developed SAP had significantly longer median ICU stay (5 vs. 0 days, p=0.01) and duration of SIRS (7 vs. 2 days, p= 0.02) as well as higher rates of PN (78% vs. 17%, p<0.001), and mortality (39% vs. 0%, p<0.001) when compared to those who did not develop SAP.
Conclusion: BISAP≥3 & SIRS ≥2 within 24 hours was predictive of SAP in 40% of cases. Among PSAP cases, the duration of SIRS serves as a marker to differentiate patients with and without SAP and could be useful for future clinical trials in acute pancreatitis.
A Large Cohort Study of the Incidence of Severe Post-ERCP Pancreatitis Using the Consensus and Revised Atlanta Criteria
E. Afghani, A. Sinha, A. Storm, Y.A. Patel, V.S. Akshintala, M.A. Khashab, A.M. Lennon, A.N. Kalloo, V.K. Singh. Division of Gastroenterology, Department of Medicine, Johns Hopkins Medical Institutions, Baltimore, MD.
Background: A length of stay (LOS)>10 days defines severe post-ERCP pancreatitis (PEP) but is an arbitrary measure of severity.
Aim: To evaluate the incidence of severe PEP using the consensus and revised Atlanta classification.
Methods: The demographic, clinical, and procedural data for all adult patients (age≥18 years) admitted to our institution with abdominal pain after an outpatient ERCP between 2000-2011 was reviewed. Excluded patients were those with AP within 2 months of ERCP, admitted for non-ERCP related indications, had perforation and/or bleeding,, and those whose records were missing or not available. PEP was defined as ≥2 of following: worsening or new upper abdominal pain post-ERCP, serum amylase and/or lipase ≥ 3 times the upper limit of normal; and/or findings consistent with AP on imaging. Severe PEP was defined using both consensus criteria and revised Atlanta classification.
Results: A total of 2262 patients were identified, of whom 1285 (56.8%) met inclusion criteria. A total of 264 (20.5%) patients developed PEP, of whom 24 (1.9%) met the consensus and 19 (1.5%) the revised Atlanta criteria for severe PEP. There were only 2 patients with LOS>10 and persistent organ failure. There were another 5 patients classified as severe PEP based only on a LOS>10 days, of whom 3 had persistent abdominal pain without changes on imaging, while 2 had prolonged hospitalization due to pulmonary embolism and constipation.
Conclusion: A large number of patients with severe PEP met criteria only due to a prolonged
LOS which, by itself, does indicate clinically severe acute pancreatitis. In this cohort, the consensus criteria overestimated severe PEP by 20%. The consensus definition of severe PEP needs to be re-evaluated.
Strategy for the High-Throughput Analysis of Urine for the Identification of Chronic Pancreatitis Biomarkers
S. Ahmed,1 J. Muntel,1 S. Berger,1 M. Bellin,2 V. Kadiyala,3 S. Suleiman,3 L.S. Lee,3 P.A. Banks,3 D.L. Conwell,3 H. Steen,1. 1Department of Pathology, Boston Children’s Hospital and Harvard Medical School, Boston, MA; 2Department of Pediatrics, University of Minnesota, Minneapolis, MN; 3Center for Pancreatic Disease, Brigham and Women’s Hospital and Harvard Medical School, Boston, MA.
Background: Diagnosing and treating chronic pancreatitis (CP) is hampered by the dearth of diagnostic biomarkers; instead invasive endoscopy as well as radiation- and non radiation-based imaging are currently needed for diagnosing CP. Exploring the potential of alternative modalities for diagnosing CP, we tested the hypothesis that the urinary proteome shows characteristic, i.e. diagnostic disease stage specific changes. Urine has the major advantage that it is quickly, safely and non-invasively collected.
Methods: The Center for Pancreatic Disease at BWH established a urine bank with specimens from a wide range of CP patients and appropriate controls. For the efficient processing, a SDS-PAGE-free strategy was optimized for urine samples. The resulting unfractionated samples are subsequently analyzed by LC/MS using state-of-the-art mass spectrometers, including a Q Exactive (ThermoFisher) and TripleTOF 5600 (AB Sciex). Recently developed stable isotope label free quantification strategies were applied, allowing for simultaneously monitoring of hundreds of proteins.
Results: The optimized sample processing and analysis strategy allowed us to generate the same amount of information with half the processing time, 10 % of the sample amount and 10 % of the instrument time previously required for in depth urine proteome analysis (see e.g. Kentsis et al. Ann Emerg Med. 2010;55:62). In fact, only 300 ul of urine and 2 hours of instrument time per sample now suffice for urine biomarker discovery projects thereby enabling the urine proteome analysis of large cohorts. The ongoing urinary CP biomarker effort has so far resulted in the largest urinary proteome map in the context of pancreatic diseases, providing unprecedented insights into the pathogenesis of CP and novel leads for diagnostic biomarkers.
Conclusions: Novel analytical strategies and mass spectrometry instrumentation enables the quantification of hundreds of proteins in cohorts which are of appropriate statistical power. By being able to monitor many more proteins in many more samples, the entire biomarker discovery process is significantly accelerated and promising biomarkers are more efficiently identified for final validation.
Comparisons of Characteristics of Peritoneal Fibroblasts in Dissemination With Those of Mesothelial Cells and Pancreatic Stellate Cells in Pancreatic Cancer
S. Akagawa,1 K. Ohuchida,1 N. Torada,1 D. Eguchi,1 S. Kozono,1 T. Ohtsuka,1 K. Mizumoto,1,2 M. Tanaka,1. 1Department of Surgery and Oncology, Graduate School of Medical Sciences, Kyushu University, Fukuoka, Japan; 2Kyushu University Hospital Cancer Center, Fukuoka, Japan.
Background: Pancreatic cancer is characterized by desmoplasia. We reported that human peritoneal fibroblasts (hPFs) obtained from disseminated sites of pancreatic cancer enhanced the aggressiveness of pancreatic cancer. Human pancreatic stellate cells (hPSCs) have similar character but the difference between hPFs and hPSCs remain unknown. Moreover, human peritoneal mesothelial cells (hPMCs) are also reported to attain the fibroblast-like character and be involved in dissemination.
Purpose The aim of the present study was to identify differences in the characteristics between hPFs, hPSCs and hPMCs.
Methods: We established three hPFs from disseminated sites, six hPSCs from primary sites and one hPMC from carcinomatous ascites of pancreatic cancer. We investigated the expression of alpha-smooth muscle actin (α-SMA), vimentin and cytokeratin18 (CK18) and morphology in hPFs and hPMCs using immunostaining. We also analyzed the migratory ability of hPFs and hPSCs. Furthermore, we performed microarray analysis to profile gene expression in hPFs.
Results: Immunofluorescent staining showed hPFs expressed α-SMA and vimentin but not CK18 and morphology is spindle shape continuously, suggesting hPFs are activated and different from hPMCs. Migration assay showed hPFs had higher mobility than hPSCs in both situations of mono-culture (P<0.0001) and co-culture with SUIT-2 pancreatic cancer cells (P=0.002). Microarray data showed cartilage oligomeric matrix protein (COMP) is one of the key genes of hPFs.
Conclusion: hPFs are different from hPMCs and have higher migratory ability than hPSCs. Such character of hPFs may contribute to the formation of pancreatic cancer dissemination although more detailed analysis is necessary for further understanding of this phenomenon.
Quantitative Assessment of Solid Debris Within Pancreatic Fluid Collections Using Computed Tomography (CT)
V.S. Akshintala,1 A. Kamal,2 M. Haider,2 A. Sinha,1 V.K. Singh,1 A. Zaheer,2. 1Division of Gastroenterology, 2Department of Radiology, Johns Hopkins Medical Institutions, Baltimore, MD.
Background: Pancreatic pseudocyst and walled-off pancreatic necrosis (WOPN) may require endoscopic drainage but differ in the method and complexity of intervention, due to the difference in amount of solid debris. However, computed tomography (CT), the most commonly available imaging modality, cannot quantify solid debris using conventional methods.
Aim: To differentiate pancreatic pseudocyst and WOPN based on the presence of solid debris and to quantify the amount of solid debris using pixel analysis.
Methods: An administrative database was queried using ICD-9 diagnosis codes to identify patients with pseudocyst or WOPN who underwent both a contrast enhanced CT and magnetic resonance imaging (MRI). Pseudocyst and WOPN were defined based on the revised Atlanta classification. Image pixel analysis based on CT attenuation measured in Hounsfield unit (HU) was performed using a custom made image processing tool based on ImageJ and MATLAB programs. Bayesian methods for image segmentation and thresholding were used for analysis and utilized priors based on corresponding MRI image.
Results: 7 pseudocyst and 29 WOPN collections with both CT and MRI were identified. Among 100 probable attenuation thresholds evaluated, the best threshold to identify WOPN was presence of >5% pixels above 30HU (sensitivity 0.96, specificity 0.28, AUROC 0.76). Using volume of solid debris identified on corresponding MRI, the Bayesian algorithm considered >33 HU for quantification of solid debris on CT. The mean volume% of solid debris was significantly higher in WOPN (26.9% ± s.d. 13.4) compared to pseudocyst (7.4% ± s.d. 4.8) (p<0.0001).
Conclusion: Volume of solid debris relative to volume of fluid has moderate diagnostic value in differentiating pseudocyst from WOPN. However, volume of debris can be quantified using CT and can be used for planning the appropriate procedure prior to drainage.
Triptolide Inhibits NFkB Activity by Inducing ER Stress in Pancreatic Cancer Cell Lines
O. Alsaied,1 S. Banerjee,1 V. Sangwan,1 R. Chugh,1 V. Dudeja,1 S. Vickers,1 A. Saluja,1. University of Minnesota, Minneapolis, MN.
Introduction: Triptolide is thought to induce cell death in pancreatic adenocarcinoma via multiple pathways, including endoplasmic reticulum (ER) stress induction and NFkB signaling inhibition.
Methods: MiaPaCa-2 cells were treated in vitro with 100 nM of triptolide (T100) or 10 microM of chemical NFkB inhibitor Bay 11-7085 (B10). ER stress marker levels (GRP-78, Chop, XBP-1) were assessed over a 24-hour timeline using real time PCR, DNA gel electrophoresis, and western blot. Effects of T100 and ER stress inducers (thapsigargin-TG), and (tunicamycin-TM) on NFkB activity and levels were assessed via luciferase reporter assay for NFkB or real time PCR.
Results: B10 and T100 increased Chop by 8 and 1.3-fold respectively, induced XBP-1 splicing. B10 induced GRP-78 by 2.5, while T100 had no effect on GRP-78 mRNA transcripts. T100 treatment inhibited NFkB activity by 38% and reduced NFkB p50 and p65 subunits by 50% at 12-hour and 90% at 24-hour. NFkB activity was reduced by 80%, 32%, and 22% when pre-treated with tumor necrosis factor-alpha (TNF-a) then treated with T100, TM, or TG. mRNA transcript levels of GRP-78 and Chop were induced by treatment with TM or TG.
Conclusions: Triptolide downregulates NFkB activity and expression in MiaPaCa-2 cells. Inhibiting NFkB induces ER stress. Induction of ER stress with TM/TG independently also inhibits NFkB activity. Induction of ER stress and subsequent NFkB activity inhibition in pancreatic cancer cells is a possible mechanism of action of Triptolide.
Gene Expression Profiling in Acute Pancreatitis
K. Altaf,1 B. Lane,1 L. Rainbow,2 C. Halloran,1 W. Greenhalf,1 R. Sutton,1. 1Liverpool NIHR Pancreas Biomedical Research Unit, Liverpool, UK; 2Centre for Genomic Research, University of Liverpool, UK.
Background: Prognostication in Acute pancreatitis remains a challenging issue. We aimed to measure gene expression of the peripheral blood in patients with acute pancreatitis and identify an expression signature that would accurately stratify patients into either mild or severe disease groups.
Methods: This observational study used Affymetrix HGU133 a2 microarrays. Total RNA extracted from whole blood samples of patients collected at the time of admission was used. A pilot study was conducted in the first instance to enable sample size calculations. Severity was defined in line with Atlanta criteria. Analysis was performed using Partek Genomics Suite software. Expression level data were quantile normalised and ANOVA was used with batch hybridization effects.
Results: 59 patients were included (22 severe, 37 mild). After adjusting for batch effects and setting power at 80%, fold change at 2 and significance at 0.05, 95 genes were identified that were differentially expressed between mild and severe disease. More specifically, 55 genes were up-regulated in severe form and 40 were down-regulated when compared to the mild disease. Canonical pathway analysis revealed signalling in T cells and lymphocytes to be the most significant pathway involved in the process. These were specifically down-regulated in severe form of the disease. While a lot of processes associated with the cellular immune response were down-regulated in severe pancreatitis, the innate response did not change and the humoral response might be stimulated.
Conclusion: Understanding more about the pathophysiology and genomic will provide us with potential prognostic biomarkers and targets for therapy. We have selected a series of gene expression features which could act as biomarkers to accurately stratify patients into mild and severe groups. Appropriate therapy can then be chosen earlier to improve outcomes in the disease.
Micro RNA Profiling in Acute Pancreatitis
K. Altaf,1 B. Lane,1 L. Rainbow,2 C. Halloran,1 W. Greenhalf,1 R. Sutton,1. 1Liverpool NIHR Pancreas Biomedical Research Unit, Liverpool, UK; 2Centre for Genomic Research, University of Liverpool, UK.
Background: MicroRNAs (miRNAs) are small non-coding RNAs that regulate gene expression and play important roles in a variety of cellular functions. These have emerged as major potential biomarkers in a variety of sepsis related disorders and cancers. We explored the prognostic potential of micro RNA at the time of admission in context of acute pancreatitis.
Methods: A pilot study was undertaken to estimate sample size and subsequently, patients were recruited and total RNA were extracted from plasma samples obtained at the time of admission. RNA were hybridized to micro RNA arrays (version 2). Severity was defined in line with Atlanta criteria. Analysis was performed using appropriate packages in R/Bioconductor or using Partek Genomics Suite software. Expression level data were invariant set normalized. Differentially expression of miRNAs in severe compared to mild pancreatitis was detected using ANOVA with batch hybridization effects removed.
Results: Twenty patients were included (severe 10, mild 10). Keeping the FDR p <0.05, 42 micro RNA were found to be differentially expressed between mild and severe pancreatitis. Out of these, only 23 were annotated in IPA – 19 were novel discoveries. Interestingly, 19 small nucleolar RNA (snoRNAs) were identified to be differentially expressed between the two groups. We also explored to identify complication specific signature – 10 miRNA predicted development of organ failure in these patients; five miRNA predicted both the severity of the disease and development of organ failure.
Conclusion: MiRNA and snoRNA can predict severity of acute pancreatitis at the time of admission. These can also be developed to predict specific complications of the disease, including organ failure and pancreatic necrosis, as early as at the time of admission. Once developed, this could fill in the gap that currently exists in prognostication arena in acute pancreatitis.
Trends in Radiographic and Endoscopic Imaging Utilization in Acute Pancreatitis (AP)
V. Antoine-Gustave, V. Kadiyala, P. Banks, D. Conwell. Center for Pancreatic Disease, Division of Gastroenterology, Hepatology and Endoscopy; Brigham and Women’s Hospital, Harvard Medical School, Boston, MA.
Background: Ionizing radiation from radiologic imaging has been associated with increased risk of cancer. The American College of Gastroenterology practice guidelines for the management of AP (2006) suggest abdominal ultrasound (US) over CT during the first 24-48 hrs. Little is known about the impact of these practice guidelines on imaging utilization.
Aims: 1. Report imaging use trends in AP from 2000-2012 in a health care system.
2. Identify changes in imaging utilization on ultrasound and CT scan following publishing of practice guidelines.
Methods: Administrative database search with Partners Healthcare System Research Patient Data Registry (RPDR). We identified in-patients with ICD9-CM diagnoses of AP (577.0) for years 2000 through 2012. Using ICD9-CM and CPT codes we identified patients who had US, CT, MRI, ERCP and endoscopic ultrasound (EUS) during their AP admission. Analysis (SPSS Statistics v21. Armonk, NY): Chi square analysis to compare usage of imaging modality in AP.
Results: AP cases increased from 671 (50% male, 5 pts <18yrs) in 2000 to 1390 (50% male, 29 pts <18yrs) in 2012 (107% change).
Radiographic imaging: There was a marked decrease in use of US (48% vs 38%, p<0.001) and CT (56% vs 34%, p<0.001) and an increase in MRI (3% vs 12%, p<0.001).
Endoscopic imaging: There was a decrease in ERCP (9% vs 2%, p<0.001) use and a non-significant increase in EUS (3% vs 4%, p=0.066).
Imaging utilization changes pre [2000-2005] vs. post [2007-2012] Practice Guidelines: US (39% vs 34%, p<0.001) and CT (50% vs 39%, p<0.001).
Conclusions: 1. Consistent with national trends, admissions for AP are steadily increasing.
2. The use of US and CT scan imaging has significantly decreased.
3. The reciprocal relationship between MRI and ERCP suggests a shift towards non-invasive imaging.
4. US (5%) and CT (11%) use in AP significantly decreased after the 2006 ACG Practice Guidelines.
Significance of Visceral Fat Volume as a Predictive Factor for Postoperative Pancreatic Fistula With Pancreaticoduodenectomy
M. Araki,1 Y. Nakata,1 K. Kamei,1 H. Ishikawa,1 T. Nakai,1 T. Murakami,1 Y. Takeyama1. 1Department of Surgery, Kinki University Faculty of Medicine, Osaka-sayama, Japan; 2Department of Radiology, Kinki University Faculty of Medicine, Osaka-sayama, Japan.
Introduction: Postoperative pancreatic fistula (POPF) is still serious complication after pancreaticoduodenectomy (PD), but there is no definite objective predictive factor. Although pancreatic texture (soft/hard) is generally used for intraoperative predictive factor, its evaluation is subjective and its significance is still controversial. The aim of this study was to detect the preoperative predictive factor of POPF.
Materials & Methods: A prospective analysis was conducted on consecutive patients undergoing PD, from July 2011 to February 2013. POPF was defined as Grade B and C in the criteria proposed by International Study Group on Pancreatic Fistula. CT volumetric measurement of abdominal visceral and subcutaneous fat tissue was performed on the level of the umbilicus, and visceral fat volume (VFV) and subcutaneous fat volume (SFV) were expressed as area (cm2). Preoperative and perioperative data were collected, such as body mass index (BMI), operating time, intraoperative blood loss volume, etc. Statistical analyses were carried out with the use of SPSS for Windows software. Two-tailed (P<0.05) tests of significance were used.
Results: A total of 44 patients were enrolled. The incidence of POPF was 17% (7 cases). The values of VFV, operating time were significantly different between the groups with and without POPF. However, the differences in the values of SFV, BMI and the other parameters between these two groups were not significant. According to the receiver operating characteristic analysis on the VFV as a predictor for POPF, optimal cut-off value was 113cm2. The incidence of POPF was 2.3% in the patients whose VFV were less than 113cm2, and in contrast, it was as high as 13.6% in the other patients.
Conclusion: VFV is a significant predictive factor for POPF in the patients with PD.
Eradication of Drug Resistant Cancer Stem-Like Cells in PDAC
T. Arumugam,1* V. Ramachandran,1 H. Huang,1 T. Fujii,1 C.L. Roland,2 C. Rodriguez-Aguayo,3 G.L. Berestein,1,3,4 A.K. Sood,4,5 J.B. Fleming,2 J.L. Abbruzzese,6 N. Sphyris,7 C.D. Logsdon,1,6. Dept. of 1Cancer Biology, 2Surgical Oncology, 3Experimental Therapeutics,4Center for RNAi and non-coding RNA , 5Gynecologic Oncology 6GI Medical Oncology and 7Molecular Pathology, UT MD Anderson Cancer Center, Houston, TX.
Background: PDAC is resistant to conventional chemotherapy. Cancer Stem-Like Cells (CSLCs) are drug resistant and can act as tumor initiating cells. Evidence suggests that CSLCs can be selected by chemotherapy that targets the more sensitive somatic cancer cells. In the current study we found that Gem treatment can not only select CSLCs, but can also induce transformation of somatic cancer cells into CSLCs. We also observed that p21 is required to maintain CSLCs in a quiescent state.
Methods: Cell growth, cell cycle and apoptosis were analyzed by using MTS and FACS analysis. ALDH and p21 were localized by immunohistochemistry and immunofluorescence. CLSCs were analyzed by ALDH assay, clonogenic in vitro assays and in vivo tumor initiation. Both in vitro and in vivo silencing of p21 was accomplished using nanoparticle coupled siRNA.
Results: Gem treatment induced the p21 and led to a transient cytostasis and chemoresistance in PDAC cell lines. Analysis of resected tumor samples revealed that Gem treatment also induced p21 expression and ALDH positivity in tumors of Gem treated but not untreated patients. In vitro studies indicated that Gem induced the acquisition of a CSLC phenotype and the expression of pluripotent transcription factors (PFTs). Silencing of p21 blocked the both basal and Gem induced CSLC numbers, the maintenance of quiescence, and resistance to Gem in vitro. In vivo, silencing of p21 greatly increased the effectiveness of Gem treatments to inhibit tumor growth and did not increase toxicity.
Conclusion: These data indicate that p21 maintains chemotherapy induced CSLCs quiescence and drug resistance such that targeting p21 in combination with cytotoxic therapies is beneficial in eradicating both basal and Gem transformed CSLCs.
Development of Small Molecule Inhibitors of Cyclophilin D for Treatment of Severe Acute Pancreatitis
M. Awais,1 M.A. Javed,1,2 L. Wen,1 R. Gibson,3 E. Shore,4 N. Kershaw,4 D.N. Criddle,1,2 A. Tepikin,2 N. Berry,4 P. O’Neill,4 L-Y Lian,3 B.K. Park,5 R. Sutton,1. 1NIHR Liverpool Pancreas Biomedical Research Unit, Royal Liverpool University Hospital and University of Liverpool, 2Department of Cellular and Molecular Physiology and 5Department of Molecular and Clinical Pharmacology, Institute of Translational Medicine, 3NMR Centre for Structural Biology, Institute of Integrative Biology and 4Department of Chemistry, University of Liverpool, Liverpool, UK.
Introduction: Mitochondrial dysfunction leading to acinar cell necrosis is a significant feature of pancreatic injury that may be reduced by inhibition of the mitochondrial permeability transition pore (MPTP). Cyclophilin D (CypD), a mitochondrial matrix peptidyl prolyl cis-trans isomerase (PPIase), promotes formation of the MPTP. Cyclophilin D inhibition may prevent pancreatic necrosis and ameliorate severe acute pancreatitis (AP).
Aims: Development of small molecule inhibitors of CypD.
Methods: Small molecules predicted to bind with human CypD were identified using computational molecular modelling. Hits were confirmed and binding affinities estimated by surface plasmon resonance. PPIase assay, isothermal titration calorimetry and nuclear magnetic resonance were used to confirm hits and binding affinities. Candidates with significant affinity were tested in live murine pancreatic acinar cells (PAC) using confocal microscopy to measure mitochondrial membrane potential (Δψm) and necrotic cell-death pathway activation induced by taurolithocholate sulphate (500 μM TLCS).
Results: The KD and Ki of a small molecule inhibitor of CypD (AP-1A02) are 1.0 μM and 1.7 μM, respectively. AP-A102 protected Δψm and significantly reduced necrotic cell-death pathway activation in PACs from TLCS.
Conclusion: We have shown AP-A102 binds with and modulates the PPIase activity of CypD, preserves Δψm and significantly inhibits necrosis in murine PACs. X-ray crystallography and optimization of AP-A102 are underway.
Selective Inhibitors of Nuclear Export (SINE) for Gemcitabine Resistant Pancreatic Cancer
A.S. Azmi,1 M. Kauffman,2 S. Shacham,2 R.M. Mohammad,3,4. Dept. of 1Pathology, WSU; 2Karyopharm, Boston MA; 3HMC, Qatar; 4Dept. of Oncology, KCI, Detroit, MI.
Nuclear export regulates the balanced nuclear expression of tumor suppressor proteins (TSPs). Overexpression of the major nuclear export protein Exportin 1 (XPO1, also called CRM1) that is linked to poor prognosis of pancreatic cancer (PC), results in nuclear exclusion and functional inactivation of TSPs and contributes to the low efficacy of drugs that target TSP activation. Earlier we showed that inhibition of XPO1 by our orally available selective inhibitors of nuclear export (SINEs) can suppress PC cell proliferation and induce tumor growth arrest in mice (Gastroenterology; 2013). Our recent large scale screening of gemcitabine (GEM) refractory PC patient tissues showed consistent over-expression of XPO1 that was concurrent with nuclear exclusion of major TSPs. These important findings suggest that XPO1 inhibition could be an attractive strategy against GEM refractory PC. Here we show that SINEs synergize with GEM (CI<1) in a panel of PC cell lines and spheroid models that overexpress XPO1 [GEM resistant (GR) MiaPaCa-2-GR and AsPC-1-GR]. We also found that SINE-GEM can inhibit the proliferation of two recently identified stem cell models of PC (CD24+;CD44+;EpCam+). Mechanistically, SINEs were found to reverse GEM mediated expulsion of major TSPs and induce nuclear re-alignment of IκB, FOXO3a, p73, Par-4 and p27. Systems analysis of microarray expression datasets from SINE-GEM treated PC cells showed enrichment of gene networks in the nucleus of PC cells. Functional ontology of activated gene networks linked them to pathways that were relevant to stem cell and EMT inhibition and cancer fibroblast cell death associated signaling. Thus, SINE-GEM can interfere with the EMT, stemness and fibroblast promoting signaling. SINE compounds in clinical development for human cancer show preliminary tolerability and antitumor activity. Based on these findings, we are initiating a SINE-GEM based Phase I/bII clinical trial in PC patients.
Minnelide Reduces CD133+ Tumor Initiating Cells in a Syngenic Model of Pancreatic Cancer
S. Banerjee, A. Nomura, V. Sangwan, R. Chugh, V. Dudeja, S.M. Vickers, A. Saluja. Division of Basic and Translational Research, Dept. of Surgery, University of Minnesota, Minneapolis, MN.
Pancreatic ductal adenocarcinoma is currently the fourth most frequent cause of cancer-related death in the United States with poor survival. Tumor recurrence in this devastating disease adds to its grim survival. Relapse of any tumor is largely attributed to the presence of tumor-initiating cells (TIC). These TICs are quiescent cells within a tumor that often evade conventional chemotherapy and result in tumor recurrence either locally or in a distant organ. Thus, these cells are considered as hurdles to cancer therapy. Since no known chemotherapeutic compound is reported to target pancreatic TICs, there is an urgent need to develop a TIC-targeted therapy for pancreatic cancer. The current study is focused on evaluating the role of Minnelide on pancreatic cancer TIC in a immune competent KPC (Kras-p53-Pdx-Cre) mouse model.
KPC cells showed relatively high (7-8% cells) expression of the surface marker CD133. CD133+ cells were sorted from KPC primary tumors and cell lines isolated from these tumors using MACS columns. The sorted cells expressed surface markers like CD24/CD44/ESA (3-4%) and showed ALDH activity (1-2%) where as CD133- cells did not. Further, when implanted subcutaneously in the flank of C57BL/6 mice, as low as 10 CD133+ cells formed tumors where as CD133- cells did not. A similar orthotopic implantation of these cells in the pancreas of the immune competent mice showed that CD133+ cells were tumorigenic while CD133- was not. Further, CD133+ cells showed increased activity of pro-survival pathway like NF-kB pathway (∼4 fold) compared to CD133- cells.
Both CD133+ and CD133- cells showed similar cell death to triptolide (active compound for Minnelide) in vitro (30-40% compared to untreated cells) indicating that triptolide had similar effect on both TIC and non-TIC population. Similarly, tumors derived from CD133+ cells showed 60% decrease in tumor volume in the Minnelide treated group compared to the control group.
In conclusion, our study shows CD133+ derived tumors respond to therapeutic dose of Minnelide and show regression in tumor. As KPC mouse model is considered to be a very close representation of the clinical stages of pancreatic cancer, the identification of TIC from these tumors particularly in a syngenic model that respond positively to a therapeutic compound, is a significant finding in pancreatic cancer research.
Morphine as an Analgesic in Acute Pancreatitis Can Increase Morbidity by Causing Persistence of Local and Systemic Inflammation in Cerulein Induced Pancreatitis
U. Barlass, R. Dutta, S.K. Garg, S. Roy, A. Saluja, R. Dawra. Division of Basic and Translational Research, Department of Surgery, University of Minnesota, Minneapolis, MN.
Background: Analgesia, supportive care, and the treatment of systemic complications are the cornerstones of the treatment of acute pancreatitis. Opioids such Morphine (MS) or its derivatives are widely used for the management of moderate to severe pain. The aim of this study was to evaluate the effect of MS in experimental mouse model of acute pancreatitis severity, gut permeability and systemic inflammation.
Methods: Pancreatitis was induced using Cerulein (Cer) (50ug/kg, hourly × 12), followed by subcutaneous pellet of MS (25mg) or placebo. Mice were sacrificed 48 h after start of experiment.
Results: Gut permeability was significantly increased in animals with acute pancreatitis treated with MS as compared to acute pancreatitis alone. This group also showed an increase in the number of colony forming units in different organs (Lungs, Liver, Spleen, Mesenteric lymph nodes).
The severity of Pancreatitis and its systemic complications were assessed. Myeloperoxidase activity in the lungs and pancreas, a measure of systemic inflammation (neutrophil infiltration) was significantly increased in the pancreatitis + MS group compared to pancreatitis alone, suggesting a persistence of inflammation. Histopathological evaluation of the pancreas again confirmed continuation of inflammation in MS treated group.
Conclusion: MS increases intestinal permeability in mice with acute pancreatitis and causes a persistence of systemic inflammation compared to those with placebo. Clinical implication of this finding is that MS should be cautiously used as analgesia in patients with acute pancreatitis.
Morphine Delays Regeneration of the Pancreas After Cerulein Induced Pancreatitis
U. Barlass, S.K. Garg, Z. Yuan, S. Banerjee, S. Roy, A. Saluja, R. Dawra. Division of Basic and Translational Research, Department of Surgery, University of Minnesota, Minneapolis, MN.
Background: Opioids such as morphine (MS) or its derivatives are widely used for the management of moderate to severe pain. There is up-regulation of different embryonic developmental pathway markers in pancreatic regeneration following injury and these can be used to track the kinetics of tissue restoration.
Aim: To study the effect of morphine on the regenerative capacity of the pancreas secondary to cerulein induced acute pancreatitis.
Methods: Pancreatitis was induced using cerulein (Cer) (50ug/kg, hourly × 12), followed by subcutaneous pellet of MS (25mg) or placebo. Mice were sacrificed 48, 96 and 120 h after start of experiment. Expression of pdx-1, ptf-1 and transcriptional activators of Hedgehog pathway was evaluated.
Results: Our data suggests that treatment with morphine delays the up regulation of pdx-1 and ptf-1 which is an embryonic progenitor marker of the pancreas, inactivated in adult pancreas and up-regulated in de-differentiation following injury and regeneration in mice. Transcriptional activators of the Hedgehog pathway such as Gli-1 together with other markers of regeneration such as Sox-2 were also down regulated with Morphine suggesting a delay in regenerative response secondary to this pathway. Histological analyses revealed less area of normal architecture in the morphine treated mice as compared to the mice with placebo again suggesting a delay in the regenerative response.
Conclusions: This data suggests that morphine use in acute pancreatitis will impede or delay the regeneration process of the pancreas and hence has the potential to prolong or worsen the pathological processes. This can in turn increase morbidity and mortality secondary to the disease.
Is Pancreatic Necrosis With Gas and Absolute Indication for Surgery?
L. Barreda,1 J. Targarona,1 J. Portugal,2 D.M. Arias,3 M. Reynel.11Department of Pancreatic Surgery - Hospital Edgardo Rebagliati Martins; 2Department of Critical Care Medicine - Hospital Edgardo Rebagliati Martins; 3Department of Radiology - Hospital Edgardo Rebagliati Martins, Lima, Peru.
Background: The presence of gas within the necrosis is also called emphysematous pancreatitis and to date is an absolute indication of surgery. There are some reports of patients with gas on tomography managed with medical treatment.
Objective The aim of this work is to determine whether pancreatic necrosis with gas is an absolute indication for surgery or if there is a space for the medical management of this entity.
Materials and Methods: During the period from April 2003 and March 2011 a prospective study was performed in 56 patients with pancreatic necrosis and gas in tomography to determine whether some of these patients can be managed conservatively.
Results: Of the 56 patients 36 (64%) were treated surgically while 20 (36%) were managed conservatively without surgery. It was found that 78% of the operated patients had infected necrosis and that in 22% this was sterile despite having gas in tomography.
83% of the operated patients had organ failure, which did not responded to medical treatment in the intensive care unit. In patients managed medically only 35% presented organ failure and all responded to medical treatment.
Conclusion: Pancreatic necrosis with gas in tomography is a relative indication for surgery, and medical management is still possible in this pathology.
The presence of gas in the necrosis is not always synonymous of infection since at least 22% is sterile.
Isolation of Quiescent and Activated Human Pancreatic Stellate Cells for MicroRNA Profiling
L. Barrera,1 F. Oldfield,1 Q. Nunes,2 F. Campbell,3 B. Lane,2 T. Andrews,3 P. Phillips,4 W. Greenhalf,1 J. Neoptolemos,1 E. Costello,1. 1Liverpool CR-UK Centre, University of Liverpool, UK. 2NIHR Pancreas Biomedical Research Unit, RLUH, UK. 3Department of Pathology, RLUH, UK. 4The University of New South Wales, Sydney, Australia.
Aim: 1) To isolate, culture and phenotype human pancreatic stellate cells (hPSCs) from pancreatic surgical specimens. 2) To detect differential expression profiles of all known miRNAs in hPCSs isolated from adjacent normal, CP and PDAC.
Background: Chronic pancreatitis (CP) and pancreatic ductal adenocarcinoma (PDAC) share the presence of a dense and complex stroma reaction which increases chemoresistance to therapeutic agents. MicroRNAs (miRNAs) are master regulators of the genome, the role of which have not yet been reported in. Although several studies have reported miRNA alterations of pancreatic cancer cells, identifying miRNAs that regulate hPSCs is critical, since they may hold the key to reversing the activity of stellate cells, and thereby retard the growth and spread of cancer cells.
Methods: Isolation of quiescent and activated hPSCs from pancreatic tissues was undertaken from patients undergoing pancreatic surgery (n=5 hPSCs from adjacent normal, CP and PDAC) using the density gradient and outgrowth methods respectively. Total RNA of hPSCs will be hybridized onto GeneChip Affymetrix miRNA 3.0 arrays and Real-Time PCR will be used to validate the data.
Results: To date, isolated hPSCs cultures have been assessed by morphology and by immunocytochemistry using the cellular markers, GFAP, α-SMA, desmin and vimentin. Quiescent hPSCs have been shown to contain vitamin-A lipid droplets, confirming their inactive state. miRNA array experiments are on-going.
Conclusion: An understanding of the impact of miRNAs on the activation of hPSCs may provide effective therapeutic approaches required to target multiple cancer cell-derived signalling pathways within the tumour microenvironment and maximize benefits for patients.
IgG4 Level in Pancreatic Juice to Diagnose Autoimmune Pancreatitis (AIP) – A Pilot Study
M.J. Bartel,1 K.K.B. Kyanam,1 J. Hoyne,2 L. McCrone,1 T.A. Woodward,1 M.B. Wallace,1 M. Raimondo.11Department of Gastroenterology, Mayo Clinic, Jacksonville, FL; 2Laboratory Medicine and Pathology, Mayo Clinic, Jacksonville, FL.
Background: Differentiating between pancreatic cancer (PC) and AIP may be challenging despite well-described diagnostic algorithms. Serum IgG4 is an essential but not optimal test in distinguishing between these diagnoses.
Aim: To assess the feasibility of using IgG4 in pancreatic juice (PJ) collected from duodenum to distinguish between PC and AIP.
Methods: We prospectively collected pancreatic juice between 2007 and 2010 of tissue proven PC (n=17, mean age 68 yrs, SD± 9.4, 53% male), chronic pancreatitis (CP) (n=7, mean age 60 yrs, SD± 7, 43% male), AIP (n=3, mean age 63 yrs, SD± 16.2, 100% male), normal controls (n=6, mean age 66 yrs, SD±6.4, 0% male) and others (IPMN and ampullary cancer) (n=3, mean age 71 yrs, SD±4.5, 66% male). Secretin stimulated PJ was collected during upper endoscopy with a 7F catheter. Protease inhibitor was added and samples frozen at -800C. PJ IgG4 was detected using a commercially available ELISA kit. IgG4 concentration was standardized to a commercially available IgG4.
Results: IgG4 was measured in 36 patients, with the following mean levels [mg/dL]: PC 0.35 (SD±0.37), CP 0.45 (SD±0.36), AIP 0.8 (SD±0.95), normal 0.2 (SD±0), others 0.27 (SD±0.12), all p=NS. Interestingly, serum IgG4 levels correlated poorly with PJ IgG4 levels [mg/dL]: PC 53.2 (SD±68.9), CP 53.0 (SD±18.8), AIP 135.2 (SD±111.2), normal 23.7 (SD±13.3), others 35.5 (SD±14.2); r=0.2. One patient with IgG4 negative AIP had elevated PJ IgG4 (1.9 mg/dL) whereas 2 patients with IgG4 positive AIP proved to have low PJ IgG4 level (0.2 and 0.3 mg/dL).
Conclusions: We proved in this pilot study that IgG4 can be reliably detected in a standardized manner in Secretin stimulated PJ. Given the low number of AIP patients in this study, further investigation with a greater subset of AIP patients is prudent.
Insulinoma of the Pancreas: 60 Years Experience in Diagnosis and Treatment
M. Bezmarevic,1 S. Kikovic,2 D. Mirkovic,1. 1Clinic for General Surgery, 2Clinic for Endocrinology, University of Defence, Belgrade, Serbia.
Introduction: Insulinomas are rare but most frequent endocrine tumors of the pancreas. A spectrum of clinical manifestations is reason for difficult recognition of disease and long time interval between onset of symptoms and diagnosis. The only currative therapy is operative treatment.
Methods: Retrospective study included 42 patients with diagnosis of insulinoma treated in our institution in 60 years duration period. In all patients a demographic and clinical data, types of methods for diagnosis, procedures for insulinoma localization, surgical approach and outcome were analyzed.
Results: A study included 42 patients (m/f:13/29). Median age at diagnosis was 43 (8-77). Median time between onset of symptoms and diagnosis was 3 years (one day to 25 years). The most common symptoms were disturbance of consciousness and abnormal behavior in 73%, confusion and convulsions in 61% of patients. Diagnosis of insulinoma was estimated by Whipple’s triad and 72-hour fast test in 14 patients. Determination of insulinoma localization was assessed by angiography in 16 (36%) patients, by ultrasound (US) in 3 of 16 patients (18.8%), by abdominal computed tomography (CT) in 8 of 18 patients (44.5%), and magnetic resonance imaging (MRI) in 2 of 8 patients (25%). Insulinoma was found in 13 of 13 patients (100%) by arterial stimulation with venous sampling (ASVS) and in 13 of 14 patients (93%) by endoscopic ultrasound (EUS). 38 (90.5%) patients underwent operative procedure. Tumor enucleation was performed in 27 (71%) patients, central pancreatectomy in one (2.6%) patient, distal splenopancreatectomy in 8 (21%) patients and pancreaticoduodenectomy in one (2.6%) patient. Overall postoperative mortality rate was 2.6% (one patient after splenopancreatectomy).
Conclusion: Combining ASVS and EUS as diagnostic procedures ensures high accuracy for preoperative determination of insulinoma localization. Minimal resection such as enucleation should be performed whenever it is possible.
Relationship Between Pancreatic Ductal Adenocarcinoma and Pancreatic Intraepithelial Neoplasia (Panin) in Resected Specimens After Whipple’s Procedure
M. Bezmarevic,1 D. Mirkovic,1 I. Tufegdzic.21Clinic for General Surgery, 2Institute of Pathology, University of Defence, Belgrade, Serbia.
Introduction/Aims: Pancreatic intraepithelial neoplasia (PanIN) has been considered to progress toward ductal adenocarcinoma (DC). The aim was to assess the relationship between patients age, size of DC, grade and number of PanINs in resected specimens and local tumor recurrence (TR) in patients who underwent Whipple’s procedure.
Methods: Forty-four resected specimens after Whipple’s procedure for DC was retrospectively reviewed. Size of DC was measured, PanINs were graded based on the highest grade component of a lesion (PanIN-1A, -1B, -2, -3) and number of PanINs were counted in a 50 areas of the non-neoplastic pancreatic tissue. These data were assessed in regard to the occurrence of TR during follow-up of operated patients.
Results: Median age of patients who had TR was 65 (47-78) and without TR was 59 (31-74). Macroscopic size of DC in patients with TR was 32.4 ± 13.3 mm and in patients without TR was 29.6±11.9 mm (p=0.662). Eleven (25%) patients had PanIN-1A and one of them (9.1%) had TR, 11 (25%) patients had PanIN-1B and two (18.2%) of them had TR, 14 (31.8%) patients had PanIN-2 and 7 (50%) of them had TR, 6 (13.6%) patients had PanIN-3 and two (33.3%) of them TR, and 2 (4.6%) patients were without PanINs and TR. No significant correlation between PanINs grade and TR (p=0.061). Number of PanINs in 50 areas of the non-neoplastic pancreatic tissue was 7 (2-15) in patients who had TR and 3 (0-11) in patients without TR, with significant difference between groups (p=0.009). Significant correlation between DC size and number of PanINs in 50 areas of the non-neoplastic pancreatic tissue was found (p=0.008), and between highest grade of PanIN and DC size (p<0.001).
Conclusion: Number of PanINs in resected specimens after Whipple’s procedure may indicate TR of DC more than the highest grade of PanIN. Larger diameter of DC is associated with higher grade and number of PanINs.
Regulation of Gremlin and Apelin Levels by Ethanol via a PTHrP-Dependent Pathway in Pancreatic Acinar Cells
V. Bhatia,1 Y. Cao,1,2 T.C. Ko,1,2 G.H. Greeley, Jr.1 M. Falzon.11UTMB, Galveston, TX; 2Dept. of Surgery, UTHSC, Houston, TX.
Aim: To define the effect of ethanol-induced parathyroid hormone-related protein (PTHrP) signaling on the bone morphogenetic protein (BMP) and apelin signaling pathways.
Background: Our laboratories have shown that ethanol increases PTHrP levels in acinar cells, and that the ethanol-mediated increase in cytokine release is suppressed by the PTHrP signaling antagonist PTHrP (7-34). Apelin protects against cerulein-induced pancreatic damage in vivo; the BMP antagonist gremlin exerts an opposite effect. BMP-2 regulates apelin levels.
Methods: Mouse acinar cells were treated with PTHrP (1-36) (10-7) for 15-60 min. Acinar cells deficient in PTHrP were isolated from mice with conditional knockout of the Pthrp gene in acinar cells (CKO-PTHrP) and from littermate controls. Cells were treated with ethanol (25 mM) for 15-120 min. Apelin and gremlin mRNA levels were measured.
Results: PTHrP (1-36) reduced apelin mRNA levels (∼3-fold) and increased gremlin mRNA levels (∼2.5-fold). Apelin levels in CKO-PTHrP acinar cells were 8-fold higher than those in controls. Ethanol treatment caused a 3-fold increase in apelin levels in control mice. This effect on apelin was attenuated in CKO-PTHrP cells. Conversely, gremlin levels were ∼2.5-fold lower in CKO-PTHrP cells. Treatment with ethanol caused a 3-fold increase in gremlin levels in control cells, but was ineffective in CKO-PTHrP cells.
Discussion: Previous findings show that pancreatic insult enhances PTHrP signaling, leading to an inflammatory and fibrotic response. Results presented here indicate that increased PTHrP signaling may suppress the BMP and apelin signaling pathways, endogenous pathways which are normally protective.
Conclusion: The BMP and apelin pathways form an endogenous protective network. Inactivation of this protective network by PTHrP prevents the re-establishment of homeostasis following injury, thereby increasing the susceptibility to developing chronic pancreatitis.
S1P Activation of Pancreatic Stellate Cells Promotes MMP9 Dependent Cancer Cell Migration and Invasion
Y. Bi, J. Li, N. Kang, V. Shah. Gastroenterology Research Unit, Department of Gastroenterology and Hepatology. Mayo Clinic, Rochester, MN.
Background and Aims: Microenvironment cells such as pancreatic stellate cells (PSC) are critical for pancreatic cancer cell migration and metastasis. The sphingosine 1-phosphate (S1P) signaling pathway is up-regulated in many cancers. We hypothesized that S1P may activate PSC thereby promoting pancreatic cancer cell migration and invasion.
Methods: Immortalized human or mouse PSC were stimulated with S1P or vehicle. S1P receptors were disrupted by siRNA or pharmacological inhibitors. PSC conditioned media was used to study PANC1 cell migration and invasion in Boyden and Transwell assays. mRNA levels were measured by PCR array and confirmed by real-time PCR. Gelatin Zymography and luciferase assays were used for MMP9 protein and promoter activity, respectively.
Results: Conditioned media from S1P stimulated PSC increased PANC1 cell migration (3.06±0.36 vs 20.44 ±1.57, p<0.01) and invasion (2.83±1.06 vs 16.40 ± 2.62; P <0.05). Targeted PCR array showed that S1P increased MMP9 mRNA levels in PSC, which was confirmed by real-time PCR (4.15±1.1 fold; p<0.05) and MMP9 promoter activity analysis (1.51±0.11 fold; p<0.05). siRNA and pharmacological inhibitor studies showed that S1P activation of MMP9 promoter was mediated by S1P2 but not S1P1 receptor. Signaling studies showed that S1P stimulated c-abl kinase phosphorylation at Y87, Y412 and Y425. C-abl siRNA and the c-abl inhibitor Gleevac diminished S1P induced MMP9 promoter activity and MMP9 activity. Conditioned medium from S1P2 but not S1P1 receptor knockdown cells showed decreased PANC1 migration and invasion. In addition, MMP9 inhibitor blocked PANC1 migration and invasion induced by S1P conditioned PSC media.
Conclusion: S1P can stimulate MMP9 production from PSC through a S1P2 and c-abl dependent pathway that in turn promote pancreatic cancer cell migration and invasion. These mechanisms may be important in pancreatic cancer metastasis and may provide treatment options for pancreatic cancer.
Interaction of BMP, Apelin and PTHrP Pathways in Pancreatic Duct Ligation-Induced Chronic Pancreatitis
Y. Cao,1,2 C. Rastellini,2 S. Han,2 V. Bhatia,2 M. Falzon,2 G. Greeley, Jr,2 T.C. Ko.1,21Dept. of Surgery, UTHSC-Houston, TX; 2UTMB at Galveston, TX.
Background: Our laboratories have shown that pancreatic bone morphogenetic protein (BMP), apelin and parathyroid hormone-related protein (PTHrP) signaling pathways are up-regulated by cerulein-induced chronic pancreatitis (CP) in mice. Furthermore, BMP and apelin are anti-fibrogenic, while PTHrP is pro-fibrogenic. It is unclear whether these pathways interact in CP.
Objective To examine potential interaction of BMP, apelin and PTHrP in pancreatic duct ligation (PDL)-induced CP model.
Methods: CP was induced by PDL in adult male C57/BL6 mice (n=5). The ligated and non-ligated lobes (control) of the pancreas were harvested for histology, protein and mRNA analyses. For in vitro experiment, cultured mouse PSCs were treated with BMP2 (50 ng/ml) or PTHrP (10-7 M). The effect of apelin gene knockout (APKO) on pancreatic PTHrP levels was examined.
Results: In the ligated lobes, PDL-induced CP results in inflammation, acinar degeneration and fibrosis, while the control lobes appear histological normal. Fibronectin protein levels are elevated in the ligated lobes (control 0.27±0.03 vs ligated 1.73±0.30). Pancreatic mRNA levels of BMP2 and apelin are elevated in the ligated lobes (7- and 4 to 10-fold vs control respectively, p<0.05). In vitro, BMP2 induces apelin mRNA expression (vehicle 1.1±0.01 vs BMP2 1.4±0.10), whereas PTHrP inhibits apelin mRNA expression (vehicle 1.0±0.01 vs PTHrP 0.28±0.03). Pancreatic PTHrP mRNA levels are elevated 8-fold in APKO mice compared to wildtype.
Discussion: PDL-induced changes in BMP2 and apelin levels replicate those measured in cerulein-induced CP. BMP2 and PTHrP influence apelin expression implying that BMP2 and PTHrP regulate apelin signaling during CP. Results from APKO imply a feedback axis between apelin and PTHrP.
Conclusion: BMP and apelin interact in CP that may form an endogenous network protecting the pancreas during CP. PTHrP may suppress BMP’s and apelin’s protective effects.
Expression of Glucagon–like Peptide 1 Receptor and Biological Behavior in Pancreatic Neuroendocrine Tumors
A.I. Cases,1 T. Ohtsuka,1 M. Fujino,2 N. Ideno,1 S. Kozono,1 M. Zhao,1 N. Ikenaga,1 K. Ohuchida,1 Y. Oda,2 K. Mizumoto,1 M. Tanaka,1. 1Departments of Surgery and Oncology and 2Anatomic Pathology, Graduate School of Medical Sciences, Kyushu University, Fukuoka, Japan.
Aim: Glucagon-like peptide 1 (GLP-1) is a gastrointestinal hormone secreted by L cells in distal ileum and colon and interacts with its specific high-affinity receptor, glucagon-like peptide 1 receptor (GLP-1R). GLP-1 induces pancreatic β-cells growth and inhibits their apoptosis. This study aimed to investigate the significance of GLP-1R expression in biological behavior of pancreatic neuroendocrine tumors (PNETs).
Materials and Methods: GLP-1R expression was semi-quantitatively evaluated by immunohistochemical staining in 50 PNETs, and the correlation between GLP-1R expression and the clinicopathological features was investigated. Functional differences in proliferation, migration, and insulin secretion were studied in mouse insulinoma cell line MIN6 in the presence or absence of GLP-1.
Results: There were 23 tumors with positive and 27 with negative expression of GLP-1R. Positive expression of GLP-1R was significantly associated with diagnosis (P<0.05), and frequently observed in insulinoma. Nevertheless, expression status of GLP-1R did not affect the prognosis of the patients with PNETs (P=0.82). Most of the metastatic sites such as lymph node and liver (8/11, 73%) showed positive staining for GLP-1R. GLP-1 significantly increased cell proliferation and migration as well as insulin secretion in MIN6 cells (P<0.05).
Conclusion: GLP-1R would be a diagnostic marker of PNETs and would have the possibility to become a molecular target for the treatment of metastatic PNETs and hormonal regulation of insulin, like a somatostatin receptor.
Imaging Modalities to Assay Molecular Biomarkers of Resistance in Pancreatic Cancer
J. Castellanos,1 N. Nagathihalli,1 E. McKinley,2 Y. Beesetty,1 C. Shi,3 H. Charles Manning,2,5 N. Merchant1,4,5. 1Departments of 1Surgery, 2Radiology, 3Pathology, and 4Cancer Biology, 5Vanderbilt-Ingram Cancer Center, Nashville, TN.
Aim: To investigate the utility of imaging modalities to detect molecular biomarkers of resistance in pancreatic ductal adenocarcinoma (PDAC).
Background: PDAC is one of the most difficult malignancies to treat due to its intrinsic chemoresistance. We have previously shown that constitutively activated STAT3 is a biomarker of therapeutic resistance that is dependent on activation of MAPK signaling. Translocator protein (TSPO) expression is mediated through MAPK signaling. We have identified TSPO-positron emission tomography (PET) as a potential imaging modality to detect activation of MAPK signaling.
Methods: Human PDAC specimens were scored for activated STAT3, MAPK and TSPO expression levels. The effects of in vitro STAT3 and MAPK inhibition on the phosphorylation of multiple signaling proteins were assessed. The novel TSPO ligand 18F-VUIIS-1018 was used to detect pancreatic lesions in a genetically engineered mouse model (GEMM) of PDAC (Ptf1acre/+;LSL-KrasG12D;Tgfbr2fl/fl), and tissues were validated with immunohistochemistry. Drug delivery was analyzed by MALDI-MS.
Results: Targeting STAT3 is a novel approach to inhibit tumor stroma, enhance drug delivery and improve therapeutic response in PDAC. STAT3 inhibition leads to immediate activation of MAPK, delayed activation of Src and EGFR, and subsequent reactivation of STAT3 signaling. Combined STAT3 and MAPK inhibition result in sustained inhibition of Src, EGFR, MAPK and STAT3 signaling. TSPO expression is elevated in all stages of PDAC but not in normal pancreas. Changes in TSPO-PET imaging can be seen with targeted inhibition of STAT3 in a GEMM of PDAC.
Conclusions: These findings demonstrate the feasibility of TSPO-PET to detect PDAC and activation of signaling pathways of treatment resistance in vivo. Further studies will investigate the suitability of advancing TSPO-PET to patients with PDAC.
Role of Tobacco in Pancreatic Fibrogenesis: An In-vitro Study in Early and Primary Culture of Pancreatic Stellate Cells
M. Castiñeira-Alvariño,1 M. Luaces-Regueira,1 J.E. Dominguez-Muñoz,1,2. 1Foundation for Research in Digestive Diseases, Santiago de Compostela, Spain, 2University Hospital of Santiago, Department of Gastroenterology and Hepatology, University Hospital of Santiago de Compostela, Spain.
Background: Alcohol has been associated with activation and perpetuation of activated phenotype of pancreatic stellate cells (PSC), but other factors may be required to initiate pancreatic fibrogenesis. Tobacco is recognized as an etiologic factor of chronic pancreatitis (CP), but its effect on fibrogenesis is still unknown.
Aim: To evaluate the effect of tobacco on the PSC activation and production of extracellular matrix (ECM) proteins as a marker of pancreatic fibrogenesis.
Methods: PSC were isolated from rat pancreas Sprague-Dawley and were exposed to tobacco (cigarette smoke condensate) alone (0.01mg/ml) or in combination with increasing concentrations of ethanol (5 to 50mM). PSC activation (α-SMA expression) was measured in both early and primary cell culture by Western blot. ECM proteins were evaluated by expression of fibronectin-1 (FNT-1) and collagen-I. FNT-1 was evaluated by western blot and immunochemistry. Collagen-I was measured by western blot and Masson Trichrome
Results: Tobacco alone (α-SMA 1.83±0.3; p=0.003 versus negative control) or in combination with 50 mM ethanol (α-SMA 1.53±0.2; p=0.04 versus negative control) induced PSC activation in culture. Tobacco in combination with 50 mM ethanol increased the expression of collagen-I (2.3±0.6, p<0.001) and FNT-1 (2.2±0.1, p<0.001) versus negative control and ethanol alone. Tobacco also increased the expression of FNT-1 in combination of 10mM alcohol (2.23±0.1, p=0.007 versus negative control)
Conclusion: Tobacco alone or in combination with alcohol induces the early activation of PSC. Tobacco associated with alcohol increases the expression of ECM proteins. These results support for the first time the synergistic effect of alcohol and tobacco in the pathogenesis of CP.
Prognostic Value of Circulating Tumor Cells in Patients With Pancreatic Adenocarcinoma
C.E. Cauley,1 M. Pittman,2 J. Zhou,1 N. Valsangkar,1 A. Liss,1 C. Fernandez-del Castillo,1 A.L. Warshaw,1 K.D. Lillemoe,1 S.P. Thayer,1. Department of Surgery & Warshaw Institute1; Department of Cytopathology2; Massachusetts General Hospital, Boston, MA.
Introduction: Circulating tumor cells (CTC) likely represent cells in transit with the potential to establish distant metastases. Identifying CTC in peripheral blood of pancreatic ductal adenocarcinoma (PDAC) patients could provide a marker for early metastatic potential. The aims of this study are to determine 1) if CTC could be detected in early stage PDAC patients and 2) if the presence of CTC is a prognostic biomarker for patients with disseminated disease.
Methods: With IRB approval, blood was drawn preoperatively in 68 PDAC patients. CTC were captured for cytologic evaluation using ScreenCell (Paris, France) filtration devices and identified by a pancreatic cytopathologist. Cells were scored as malignant, suspicious, or non-diagnostic. Radiographic, pathologic, and clinical data was reviewed and overall survival (OS) and disease free survival (DFS) calculated.
Results: Thirty of the 68 PDAC patients were found to have malignant CTC on cytologic analysis. Malignant CTC were found in stages IIa-IV PDAC. The presence of CTC did not correlate with advance T, N or M stage. CTC could be detected with equal frequency in early or late stages. Patient identified to have Malignant CTC were not more likely to have poor prognostic markers such as perineural (p=0.23), large/small vessel invasion (p=0.23) or advanced histologic grade (p=0.24). There was no statistical difference in OS or DFS in short term follow-up.
Conclusions: CTC were identified in 44% of PDAC patients using cytologic analysis, including early stage PDAC. Malignant appearing CTC do not correlate with advance stage PDAC or decreased survival. This data suggests that the presence of CTC does not predict patients with clinically occult metastases. Longer term follow-up is needed to more accurately compare survival and recurrence rates between these groups.
Surgery is Associated With Improved Survival in Pancreatic Cancer Patients With Stage 1-2b Tumors: A Population Based Study
S. Chakraborty,1 S. Singh,1,2. 1Department of Internal Medicine; 2Division of Gastroenterology and Hepatology, University of Nebraska Medical Center, Omaha, NE.
Background: Surgery is the only potentially curative treatment currently available for treatment of pancreatic cancer. Large scale studies investigating the impact of surgery in patients with resectable (stage 1,2A and 2B) pancreatic cancer are lacking.
Aim of present study We investigated the impact of surgery on overall survival (measured from the time of diagnosis) in PC patients with resectable tumors. We also investigated factors that determine survival in patients undergoing surgery and sought to investigate effect of demographic and tumor related factors that influence the decision to undergo surgery.
Methods: The Surveillance Epidemiology and End Results (SEER) database was queried for microscopically confirmed cases of stage 1,2A and 2B pancreatic ductal adenocarcinoma diagnosed between 1973-2009. Kaplan Meier univariate and Multivariate analysis using the Cox Proportional hazards model were employed to investigate the impact of surgery on overall survival. Logistic regression was used to study factors that affect the decision to perform surgery in patients with resectable tumors.
Results: Out of 1,759 patients with microscopically confirmed and resectable pancreatic cancer in the SEER database, 92.6% underwent cancer directed surgery. Patients undergoing surgery were significantly younger at the time of diagnosis (mean age 65.8 vs. 69.9 years, p=0.002) and had a longer median survival (18 vs. 7 months by univariate analysis) than those that did not undergo surgery. Surgical resection was a significant predictor of overall survival upon both univariate and multivariate analysis. Age less than 70 years at the time of diagnosis of PC, non-white, non-black race, tumor size <40 mm and tumor located in the tail of the pancreas were factors significantly associated with a chance of cancer directed surgery. Patients with smaller tumors had better survival due to greater percentage undergoing surgery and smaller percentage of patients with tumor positive nodes.
Conclusion: Surgery improves survival in pancreatic cancer patients with resectable tumors. Younger patients with less than 4cm tumors located in the tail of the pancreas have the greatest survival benefit following surgery.
IL1B Mediates NF-kB Activity in Pancreatic Cancer Cells
A. Chandra, A. Nomura, O. McGinn, R. Chugh, V. Sangwan, A. Saluja, S. Banerjee. Division of Basic and Translational Research, Dept. of Surgery, University of Minnesota, Minneapolis, MN.
Background: Pancreatic ductal adenocarcinoma is currently the fourth most frequent cause of cancer-related death in the United States with poor survival and high rate of recurrence. Though the role of inflammatory pathways and cytokines are well accepted in cancer, the molecular mechanisms underlying this association are still unclear. The current study shows that cytokines IL1A and IL1B stimulate the pro-proliferative NF-kB pathway, which results in increased survival of pancreatic cancer cells.
Results: NF-kB pathway was constitutively activated in pancreatic tumors and cell line (MIA-PaCa2, S2-013, AsPC1) compared to normal ductal cells. Suppression of NF-kB by a pharmacological inhibitor BAY-11-7085 or IKK-SR plasmid (super-repressor of NF-kB) resulted in decreased cell viability. Further, pancreatic cancer cells (MIA-PaCa2 and S2013) showed an increased secretion of IL1B in the growth media when assayed by ELISA. Additionally, suppression of IL1 mediated signaling using a IL1receptor antagonist antibody (IL1RA) showed a decrease in NF-kB DNA binding activity in MIA-PaCa2 cells and resulted in decreased viability. Suppression of NF-kB by both a pharmacological inhibitor BAY11-7085 as well as IKK-SR plasmid showed a decrease in the secreted IL1B levels, while enhancing NF-kB using a NF-kB enhancer plasmid (S181E) increased secretion of IL1B.
Conclusion: Our results showed that IL1 regulated NF-kB activity in pancreatic cancer cells, However, once activated, NF-kB also synthesized IL1B, thereby starting a “feedback loop” of the pathway. Our results also showed that blocking IL1 signaling using IL11RA, or inhibition of NF-kB, resulted in a loss in cell viability, indicating that this pathway was of extreme importance in pancreatic cancer.
Obstructive Jaundice Impairs Dynamic Exercise Performance in Patients Undergoing Major Pancreatic Surgery
V.V. Chandrabalan,1,2 L.K. Palani Velu,1,2 N.B. Jamieson,1,2 D.C. McMillan,2 C.J. McKay,1 C.R. Carter,1 E.J. Dickson,1. 1West of Scotland Pancreatic Unit, Glasgow Royal Infirmary, UK; 2Academic Department of Surgery, University of Glasgow, UK.
Aims: Obstructive jaundice (OJ) is common in patients undergoing surgery for periampullary lesions and is associated with adverse postoperative haemodynamic events. We evaluated the effect of OJ on preoperative cardiopulmonary exercise testing (CPET) in patients undergoing major pancreatic surgery.
Methods: Patients who underwent surgery for periampullary lesions and had undergone CPET between August 2008 and April 2013 were studied. Data on demographics, comorbidity, BMI, blood tests and POSSUM score were collected. CPET parameters were compared in patients with and without OJ (bilirubin > 22 μmol/L).
Results: 169 patients underwent pancreaticoduodenectomy (n=140), trial dissection and palliative bypass (n=25), or (sub)-total pancreatectomy (n=4). OJ was present in 83 patients (49%). OJ was associated with lower exercise load (46 vs 37 watts, p<0.05), minute ventilation (24 vs 21 L/min, p<0.05), tidal volume (1.23 vs 1.02 litres, p<0.005) and oxygen consumption (11.1 vs 9.5 ml/kg/min, p<0.001) and increased respiratory rate (19 vs 22 breaths/min, p<0.05) at the anaerobic threshold as well as lower exercise load (95 vs 69 watts, p<0.001), minute ventilation (50 vs 39 L/min, p<0.001), tidal volume (1.94 vs 1.46 litres, p<0.001), peak oxygen consumption (17.9 vs 14.6 ml/kg/min, p<0.001) and oxygen pulse (13.0 vs 10.9 100ml/kg/beat, p<0.005) at peak exercise. OJ was associated with low anaerobic threshold (OR 2.34, CI 1.14-4.80, p=0.020) and peak oxygen consumption (OR 2.08, CI 1.02-4.21, p<0.05) independent of other preoperative factors.
Conclusions: Dynamic cardiopulmonary exercise performance is significantly impaired in jaundiced patients undergoing major pancreatic surgery. Preoperative optimization and postoperative critical care must take into account the effects of jaundice on cardiopulmonary physiology.
Increased Expression of Fibroblast Marker S100A4 in Pancreatic Cancer
K. Chen, C. Chen, G. Cai, M. Hu, Q. Ding. Division of Pulmonary, Allergy and Critical Medicine, University of Alabama at Birmingham, Birmingham, AL.
Introduction: Pancreatic cancer ranks the fourth leading cause of cancer-related death in the US. With a predicted 44,220 new cases in 2013 in the US, most of them will die from the disease. The one-year survival rate is 20%, and the five-year rate is 4% according to National Cancer Institute. Although our lab focuses on pulmonary fibrosis, our recent studies suggested that some of genes we are studying may play roles in oncogenesis of cancers especially pancreatic cancer.
Methods: In this study, we analyzed a public dataset from the NCBI GEO (GSE28735), which is a genome wide gene expression profiling study for 45 matching pairs of pancreatic tumor and adjacent non-tumor tissues from 45 patients with pancreatic ductal adenocarcinoma.
Results: The original study (GSE28735) reveals DPEP1 gene expression inhibition in cancer samples. Our study confirmed the finding and revealed more candidate genes. We improved the statistical comparison of the two groups after variance stabilization, robust multichip analysis (RMA)-normalization and false discovery rate (FDR) p-value correction. S100A4 gene, a fibroblast marker, expressed more in tumor samples. Focal adhesion kinase (PTK2) also increased with statistical significance. The functional analysis of most significantly-changed genes by Ingenuity Pathway Analysis (IPA) revealed that the major changes in gene-gene interaction network of these changed genes are related to cancer, cellular development, cellular growth and proliferation; cell morphology, skeletal and muscular system development and function, which are consistent to our findings. With high significance levels, upstream transcriptional regulators such as TGFB1 (p=1.05E-22), ERBB2 (p=1.23E-18), and HIF1A (p=2.44E-14) are Inhibited based on the prediction model of IPA. Interestingly, caveolar-mediated endocytosis signaling is the top affected signaling pathway (p=1.84E-06).
Conclusion: Our study showed a new role of S100A4 in pancreatic cancer.
Neutrophil Extracellular Chromatic Traps (NETs) May Be Activated in Pancreatic Duct Fluid of Patients With the Worst Prognosis
K. T. Chen, P.D. Kim, K.A. Jones, B.B. Patel, J.P. Hoffman, H. Ehya, K. Devarajan, J.C. Watson, J.L. Tokar, A.T. Yeung. Fox Chase Cancer Center, Philadelphia, PA.
Objectives We found that S100A8 and A9 in duct fluid correlated with poor post-surgery DFS (Disease Free Survival) and OS (Overall survival), the absence of pancreatic enzymes, and the presence of mucins. Next we explored the proteomes deeper to discover other correlations.
Methods: A pool of the duct fluids of 5 PDAC (pancreatic ductal adenocarcinoma) patients of better prognosis was compared with a pool of the duct fluid of 5 PDAC patients of worse prognosis, using iTRAQ quantitative mass spectrometry to quantify 350 proteins.
Results: The better prognosis group of PDAC had an average of 6.6 fold higher expression of 39 pancreatic enzymes than the poorer prognosis group. Interestingly, all of the 20 known proteins of the NETs each averaged 13 fold up-regulated in the PDAC fluid of the poor prognosis group. The coordinated up-regulation supports a stoichiometric relationship previously observed in NETs. NETs are structures from a form of apoptosis (NETosis) in which neutrophils respond to commands and explode. Their chromatins rearrange to form a net-like mesh decorated with globules of antimicrobials. The proteins include: S100A8, A9, A12, histones H1.3, H1.4, H1.5, H2A.V, H2B type 1-C/E/F/G/I, H3.1t, H4, myeloperoxidase, catalase, lactotransferrin, neutrophil defensin 1, neutrophil elastase, myeloblastin, azurocidin, lysozyme C, cytoplasmic actin 1, plastin-2, alpha-enolase, myosin-9, transketolase, keratin type I cytoskeletal 10, and alpha-actinin-1. The histones in NETs are stoichiometrically different from the core histones, are truncated, acetylated and citrullinated.
Conclusions: NETs are normally employed in short-term response to infection but no infection was detected. The histones are also hypothesized for killing eukaryotic cells. Beyond an implication of inflammation, we speculate that NETs may be able to kill wandering cancer cells.
Combination of TRAIL and Triptolide Induces Lysosomal and Mitochondrial Membrane Permeabilization by Modulation of the Death Receptor Pathway in Pancreatic Cancer Cells
Z. Chen, V. Sangwan, S. Banerjee, S.M. Vickers, A.K. Saluja. Department of Surgery, University of Minnesota, Minneapolis, MN.
The tumor necrosis factor related apoptosis-inducing ligand (TRAIL) induces cancer cells death, while leaving normal cells unaffected. However, the resistance to TRAIL occurs in many cancer types. We have previous shown that low concentration of the diterpene triepoxide triptolide, and TRAIL is effective in inducing pancreatic cancer cell death. We have also shown that high concentration of triptolide induces lysosomal membrane permeabilization (LMP).
Aims: To understand the mechanism by which triptolide sensitizes pancreatic cancer cells to TRAIL.
Hypothesis: Low concentrations of triptolide prime the death receptor pathway, thereby allowing activation of this pathway by TRAIL, resulting in pancreatic cancer cell death.
Results: Low concentration of triptolide (50nM) increases levels of Death Receptor 5 (DR5; control=3.77%±1.01%, treated=28.2%±6.43%), and down-regulation of c-FLIP. A combination of triptolide and TRAIL results in activation of caspase-8, leading to lysosomal and mitochondrial membrane permeabilization, and ultimately, apoptosis. Cytosolic cathepsin B activity, a marker for lysosomal membrane permeabilization, increase from 100% in control cells to 831%±193.8% in TRAIL and triptolide treated cells. Blocking LMP using either a cathepsin B inhibitor or cathepsin B specific siRNA results in partial rescue of cell viability (41.17±2.64% vs 56.33±5.27 in LMP-inhibited cells). Additionally, disruption of the death receptor pathway, either using siRNA against DR5 or caspase-8, leads to partial rescue of cell viability in the presence of a combination of TRAIL and triptolide (42.92%±1.00% vs. 64.82%±1.07% with DR5 siRNA and 37.62%±3.94% vs. 61.56%±6.30% with caspase-8 inhibitor).
Conclusions: In this study we show that low concentrations of triptolide sensitize pancreatic cancer cells to TRAIL by increasing levels of DR5 and down-regulating c-FLIP. These events allow the activation of the death receptor pathway, resulting in lysosomal and mitochondrial membrane permeabilization leading to cell death. Minnelide, a triptolide prodrug, will very shortly enter Phase I clinical trials. Since TRAIL is already in use against several cancers, understanding the mechanism by which triptolide sensitizes pancreatic cancer cells to TRAIL may result in a novel therapeutic strategy against pancreatic cancer.
Microscopic and Ultrastructural Changes of Large Bowel in Acute Pancreatitis
S. Chooklin, O. Usach. Department of Surgery, Medical University, Lviv, Ukraine.
Introduction: Mucosa and the entire wall of the large intestine undergo structural and immunological changes in severe acute pancreatitis. Reduction of intestinal blood flow and colonic mucosal ischemia are early events in acute pancreatitis.
Materials and Methods: The microscopic and ultrastructural picture of the colon wall was analyzed in 9 acute pancreatitis patients (4 – mild, 5 – severe).
Results: We identified the decreased counts of colonic lamina propria immune cells. Pathologic thickening of colonic wall close to paracolitis is associated with the same order of lamina propria immune cells changes. In necrotizing pancreatitis the height and number of the microvilli in the epithelial cells decreased. In necrotizing pancreatitis adherens and tight junctions were occasionally open in the colon. Mitochondria in the colon epithelial cells were degenerated. In necrotizing pancreatitis capillaries of the colon showed a broken endothelial lining with narrow lumens.
Conclusion: Severe acute pancreatitis is accompanied by morphological changes in the mucous membrane of the large intestine, typical of the local immunological deficiency. Ultrastructural abnormalities in acute pancreatitis appear early in the colon.
Histone Deacetylase Inhibition (HDAC) Sensitizes Pancreatic Cancer Cells to TRAIL Induced Cell Death
R. Chugh, V. Dudeja, O. Alsaied, S. Banerjee, V. Sangwan, A. Saluja, S.M. Vickers. Dept. of Surgery, Basic and Translational Research Lab, Minneapolis, MN.
Introduction: Pancreatic cancer is one of the most lethal human malignancies with five-year survival of less than 5 % because of its resistance to most conventional chemotherapies like gemcitabine and other novel anti-cancer therapies like TRAIL. Histone deacetylase (HDAC) inhibitors are a new and promising drug family with strong anticancer activity. The aim of the current study was to evaluate whether inhibition of histone deacetylase sensitizes pancreatic cancer to TRAIL induced cell death.
Methods: Highly aggressive metastatic pancreatic cancer cell lines (S2VP10, Capan-1) were treated with the HDAC inhibitor, Vorinostat (0-5μM), TRAIL (0-40ng/ml) or a combination of Vorinostat and TRAIL for 12-72h. The effect on cell viability was evaluated using a WST-8 cell viability assay (Dojindo Labs), apoptosis (caspase 3, 8 and 9 activation) was evaluated using Caspase Glo assay kit (Promega).
Results: HDAC inhibition markedly increased TRAIL induced cell death in both pancreatic cancer cell lines evaluated. Viability, data expressed as % of Control (untreated cells), mean ±SEM. S2VP10 (48h): Vorinostat (5μM) – 64.5 ± 0.1%, TRAIL (20 ng/ml) – 95.13±0.825%, Vorinostat (5μM) + TRAIL (20ng/ml) – 41±0.8%. HDAC inhibition markedly augmented Caspase 3 activation in response to TRAIL. Caspase 3, data expressed as % of Control, mean ±SEM. S2VP10 24h: Vorinostat (5μM) – 206.1±12.07%, TRAIL (20 ng/ml) – 159.6±1.2%, Vorinostat (5μM) + TRAIL (20ng/ml) – 2187.4±77.62%.
Conclusion: Inhibition of Histone deacetylases sensitizes pancreatic cancer cells to TRAIL induced apoptosis and cell death. Combination of HDAC inhibition and TRAIL has immense potential to emerge as novel therapeutic strategy against pancreatic cancer.
World Health Organization (WHO) FRAX Calculator Utility in Chronic Pancreatitis
D.L. Conwell, V. Kadiyala, S.L. Suleiman, S.J. Burton, P.A. Banks. Center for Pancreatic Disease, Brigham and Women’s Hospital, Harvard Medical School, Boston MA.
Background: Chronic pancreatitis (CP) has been associated with metabolic bone disease (MBD) and an increased major osteoporotic fracture (OF) and hip fracture (HF) risk. The WHO FRAX calculator can estimate 10 year fracture risk.
Purpose: To determine
- 10 year probability of fracture in CP patients (pts) referred to a tertiary pancreas center.
- Impact of DEXA BMD data on FRAX risk assessment in CP pts.
Methods: Retrospective study of pts referred for CP (ICD 577.1). Data: age, gender, CP Cambridge imaging, all TIGAR-O etiologies (all that apply), fracture risk factors, DEXA bone mineral density (BMD) and BMI by chart review and phone interviews. FRAX online tool used to calculate 10 year risk of OF and HF both with and without BMD (if available). Analysis: Non-parametric Wilcoxon signed-rank test to compare fracture risk (FRAX score) ±BMD for paired data. (SPSS Statistics v21. Armonk, NY)
Results: FRAX scores calculated for 73 pts with CP findings; median age 52 (IQR 14.0), 61.6% female and median BMI 25.3 (IQR 9.1). TIGAR-O distribution: T(51), I(16), G(1), A(2), R(29) and O(21). Cambridge imaging distribution: I(27), II(12), III(13) and IV(21). Median 10 year risk of OF was 4.5% (IQR 5.3) and that of HF was 0.4% (IQR 1.0). 16 pts met U.S Preventive Services Task Force criteria for DEXA screening.
15/73 pts also had a DEXA scan; median age 51.0 yrs (IQR 14.0), 80% female and median BMI 24.8 (IQR 8.9). Median 10 year risk of OF by FRAX+BMD was 5.2% (IQR 6.0) and FRAX-BMD was 4.7% (IQR 6.5). Median 10 year risk of HF by FRAX+BMD was 0.6% (IQR 0.5) and FRAX-BMD was 0.50% (IQR 1.4). There was no difference in 10 year risk of either OF (p=0.413) or HF (p=0.212) comparing FRAX risk with BMD to that without.
- An estimated 22% of CP pts referred to our tertiary pancreas center are at high risk for fracture.
- The WHO FRAX tool can be used without DEXA scan in the ambulatory setting to screen CP patients for MBD.
Urine Proteomics Identifies Biomarker Candidates for Early Chronic Pancreatitis (CP)
D.L. Conwell,1 J. Muntel,2 S. Ahmed,2 M. Bellin,3 G. Beilman,4 M. Freeman,5 V. Kadiyala,1 S. Suleiman,1 L.S. Lee,1 P.A. Banks,1 H. Steen,2. 1Center for Pancreatic Disease, Brigham and Women’s Hospital, 2Department of Pathology, Boston Children’s Hospital; Harvard Medical School, Boston, MA; 3Department of Pediatrics, 4Department of Surgery, 5Department of Gastroenterology, Hepatology and Nutrition, University of Minnesota, Minneapolis, MN.
Background: CP is currently diagnosed using invasive endoscopy as well as radiation- and non radiation-based imaging. Early stage CP cannot be definitively diagnosed, thereby complicating the application and / or development of appropriate therapies. Here, we explore the potential of proteinaceous urinary biomarkers for diagnosing early CP. Urine is safely and non-invasively collected and as such may offer superior alternative to current CP diagnosis modalities.
Methods: Three patient cohorts were developed for urinary proteomic analysis based on clinical symptoms [pain, RAP], laboratory data [PFT, fecal elastase-1] and [Cambridge / Mannheim] imaging criteria: 1) controls, normal imaging [n=10], 2) early stage CP, equivocal/mild imaging, [n=10], and 3) late stage CP, moderate/ marked imaging [n=10]. The proteomes of 30 clean catch urine samples were analyzed using a gel free sample processing method followed by LC/MS analysis of the unfractionated samples using a Q Exactive mass spectrometer.
Results: 30 patient samples; 17 male, 13 female; median age 48 yr; median peak PFT [HC03- meq/L] was 105 (normal), 78 (early CP) and 50 (late CP), respectively. Proteomics analysis of 300 ul of urine resulted in the identification of >1300 protein groups. Statistical evaluation of the spectral counting-based quantitative proteomics data identified clusters of proteins that were specific for early CP or late CP. For example, a peptidase showed a marked increase in the early CP cohort vs. the normal or late CP cohorts (AUC = 0.95 ± 0.05; p < 0.001). Similarly, a glycoprotein showed a significant increase only in the late CP cohort (AUC = 0.92 ± 0.08; p < 0.001 and p < 0.005, respectively).
Conclusions: Quantitative proteomic analysis of urine specimens showed statistically significant CP stage-specific changes in urinary protein levels. These proteins represent promising biomarker candidates for diagnosing early stage / minimal change CP and will serve as an initial set of targets for further validation studies.
Chronic Pancreatitis (CP) and Fracture: A Retrospective, Population-Based, Veterans Administration Study
*D.L. Conwell,1 *S. Munigala,2 J. Scherrer,3 B. Agarwal,2. 1 Center for Pancreatic Disease, Division of Gastroenterology Hepatology and Endoscopy, Brigham and Women’s Hospital, Harvard Medical School, Boston, MA; 2 Department of Internal Medicine, Division of Gastroenterology and Hepatology, Saint Louis University; 3 St. Louis VAMC, Research Service and Department of Family and Community Medicine, Saint Louis University School of Medicine, St. Louis, MO.
Background: Osteoporotic fractures are a significant burden on healthcare resources.
Female gender and age are major risk factors for development of osteoporosis and fracture. There is increasing evidence that CP is a risk factor for osteoporotic fracture, but data on males with CP and fracture prevalence is sparse.
Aims: Determine the association of gender and age in a large Veterans Administration population database.
Methods: Retrospective cohort analysis of a large VA database developed between 1998-2007 (n > 500,000 US veterans). CP (ICD 577.1) and controls (free of fracture risk) were identified after exclusion of all other known diseases associated with bone loss (National Osteoporosis Foundation). Fracture identified by ICD9 codes for vertebral, hip and wrist. Data collection – age group (0-44, 45-65, > 65 yr), gender (male, female), race (black, white, other), fracture (total, vertebral, hip, wrist) recorded. Statistical analysis. SAS version 9.2, Cary, NC.
Results: A total of 453,912 VA patients identified: 450655 controls and 3257 chronic pancreatitis (CP). Mean age of controls and CP was 53.6 and 54.2 years (p <0.014).
Majority of subjects were male: Controls (88%) and CP (95%), p < 0.0001 and Caucasians: C (65%) and CP (72%), p < 0.0001. CP patients had a higher OR [95% CI] of total fractures 2.35 [2.00, 2.77], vertebral fracture 2.11 [1.44, 3.01], hip fracture 3.49[2.78, 4.38] and wrist fracture 1.68 [1.29, 2.18] when compared to C.
When stratified by age, only males and females in the 45-65 yr and > 65 yr age groups had an increased odds of fracture (p <0.05). Neither gender in the 0-44 age group had increased odds of fracture (p>0.05).
Conclusions: In this population-based, Veterans Administration study, CP patients were identified to be at increased risk of osteoporotic fractures. The risk was higher for hip fracture (> 3 x) in patients with CP compared to controls. All CP patients > 45 years of age, irrespective of gender should be screened for bone mineral density loss. Future directions: development of cost-minimizing screening strategies for metabolic bone disease in CP.
NIS-Mediated Radioiodine Therapy and Imaging for Pancreatic Cancer
J. Davydova,1 M. Trujillo,2 M. Oneal,2 Y. Miura,1 J. Han,1 J. Morris,2 M. Yamamoto,1. 1Department of Surgery, University of Minnesota, Minneapolis, MN; 2Division of Endocrinology, Mayo Clinic, Rochester, MN.
Background: The employment of oncolytic adenoviruses (OAds) constitutes a promising alternative for pancreatic cancer treatment. In this study, we apply the new OAd expressing a therapeutic and imaging transgene, the sodium-iodide symporter (NIS). We hypothesized that NIS expression in pancreatic cancer will induce uptake of radioiodine and allow noninvasive SPECT/CT imaging with 123I, but more importantly, will allow therapy with 131I similar to that practiced for metastatic thyroid cancer. The imaging of NIS-expressing tissues requires only minimal diagnostic doses of radionuclide and can easily be performed in humans. Radioiodine safety has been well established during our ongoing Phase 1 clinical trial employing a first generation NIS-expressing adenovirus for men with prostate cancer (Directed by Dr. Morris).
Vector Design: We designed our NIS-OAds to overcome low efficacy of standard viral vectors. Our viruses are armed with the Ad5/Ad3 fiber to improve poor transduction of pancreatic cancer cells. The NIS-OAds overexpress the Adenoviral Death Protein, which improves virus spread and oncolysis. Viral replication occurs exclusively in tumors through the inclusion of a tumor specific promoter allowing specific delivery of viral genes and NIS. In order to make OAd feasible for systemic administration, we redirected virus from the liver by genetically modifying the Ad hexon protein, which is responsible for interacting with scavenger receptors of liver macrophages.
Results: The new NIS-OAds exhibited selective and greatly improved oncolytic potency in S2O13, S2VP10, AsPC1 and MiaPaca2 pancreatic cancer cells. Radioiodine uptake was shown to be time- and dose-dependent and was significantly greater than that with a replication-deficient control. The therapeutic and imaging abilities were evaluated in a subcutaneous prostate cancer model in mice. A single intravenous injection of NIS-OAd efficiently suppressed tumor growth and resulted in sufficient noninvasive SPECT/CT tumor imaging. Importantly, survival rate was greatly improved when it was combined with 131I. Finally, we confirmed the reduced liver sequestration after systemically-delivered hexon-modified OAd in an immunocompetent hamster model. This indicates the feasibility of our system to treat and visualize cancer upon systemic viral delivery.
Conclusion: Clinical application of this approach will bring the following advances to pancreatic cancer patients: 1) Simultaneous delivery of diagnostic and therapeutic agents may improve preoperative diagnosis; 2) SPECT/CT imaging of selective NIS expression may give more sensitivity and specificity than other imaging modalities; 3) NIS-OAds are designed for systemic administration, providing treatment for metastatic disease; 4) The combination of enhanced oncolytic virus efficacy with the bystander effect of 131I may offer a new approach for inoperable patients.
Mitochondrial Membrane Potential Loss in Pancreatic Acinar Cells in Response to Pathological Insult Is Not Affected By Absence of Trypsin Activation
R. Dawra, S.K. Garg, U. Barlass, V. Dudeja, A. Saluja. Division of Basic and Translational Research, Department of Surgery, University of Minnesota, Minneapolis MN.
Background: Recent studies show presence of significant necrosis of pancreatic acinar cells during pancreatitis even in absence of trypsin activation emphasizing the importance of additional mechanisms. Increased mitochondrial membrane permeabilization and resulting loss in mitochondrial membrane potential have been shown to contribute towards cell death. Both trypsin activation and loss in mitochondrial membrane potential are initial early events observed during initiation of experimental pancreatitis, however, the relationship between these two is not clear.
Aim: To study the relationship between intra-acinar trypsin activation and mitochondrial transition pore permeability in pancreatic acinar cells in response to pathological insult.
Methods: Pancreatic acinar cells were prepared from wild type (WT), Cathepsin B (CB-/-) or trypsinogen7 (T7-/-). Cells were loaded with fluorescent dye JC1 and changes in mitochondrial membrane potential were monitored by determining change in fluorescence ratio in response to treatment with cerulein (0.1μM) or TLCS (200,400μM) or increasing concentrations of Ca2+
Results: There was comparable loss of mitochondrial membrane potential in WT, CB-/- and T7-/- after treatment with cerulein (0.1μM) or TLCS (200, 400μM) or increasing concentrations of Ca2+. There was significant increase in tryspsin after treatment with cerulein or TLCS in WT acini but no increase in CB-/- or T7-/-.
Conclusions: Increase in mitochondrial transition pore permeability in pancreatic acinar cells in response to pathological insult develops independently of trypsin activation. This could contribute towards acinar cell necrosis.
The Safety, Tolerability, and Analgesic Efficacy of Δ9-THC (Namisol®) in Chronic Pancreatitis Patients Suffering From Persistent Abdominal Pain
M. de Vries,1 O.H.G. Wilder-Smith,2 H. van Goor,1. 1Dept of Surgery and 2Dept of Anesthesiology, Pain and Palliative Care, Radboud University Nijmegen Medical Centre, Nijmegen, The Netherlands.
Introduction: Analgesic drug treatment is considered as first choice in pain management of chronic pancreatitis (CP). Opioids are prescribed in the majority of CP patients. The adverse consequences of prolonged opioid use call for an alternative medical treatment. Namisol®, an oral tablet containing purified delta-9-tetrahydrocannabinol (Δ9-THC), provides the opportunity to test the analgesic potential of Δ9-THC, avoiding side effects of traditional formulations.
Aim: To assess the safety and analgesic efficacy of a single dose Namisol® in the treatment of chronic pancreatic pain.
Study design A randomized, single-dose, double-blinded, placebo-controlled, two-way cross-over pilot study. Patients suffering from abdominal pain as result of CP (n=24) were subdivided in opioid (n=12) and non-opioid (n=12) users. Namisoll®l with standardized Δ9-THC content (8 mg) or active placebo (5mg Diazepam non-opioid group/10mg Diazepam opioid group) was administered orally in a double dummy design. The primary outcome was change in Visual Analog Scale (VAS) pain two hours after medication intake compared to baseline. Secondary outcomes, including pharmacokinetics, pharmacodynamics and adverse events (AEs), were measured at baseline and repeatedly after medication intake.
Results: Differences in VASpain at rest were similar between groups (mean diff -.3±1.2 vs -.5±1.4 for Namisol®vs Diazepam; ns). VASpain on movement tended to be more reduced after Namisol® compared to Diazepam (mean diff -.8±1.4 vs -.5±1.8; ns), and particularly in non-opioid users. The number of patients reporting at least one AE was less in Namisol® (71%) compared to diazepam (91%). All AEs were mild to moderate.
Conclusion: Namisol® was well tolerated in CP patients, but a clear analgesic effect on VASpain of a single dose Δ9-THC remained absent.
Effects of Chai-Qin-Cheng-Qi Decoction on Gene Expression Profiles in Cerulein-Induced Acute Pancreatitis
L.H. Deng,1 X.Y. Zhang,1 Z.Q. Lin,1 F.J. Yong,1 W. Huang,1,2 R. Sutton,2 Q. Xia,11Sichuan Provincial Pancreatitis Centre, Dept of Integrated Traditional Chinese and Western Medicine, West China Hospital, Sichuan University, Chengdu, China; 2NIHR Liverpool Pancreas Biomedical Research Unit, Royal Liverpool University Hospital, University of Liverpool, Liverpool, UK.
Background: Chai-Qin-Cheng-Qi decoction (CQCQD) is a multiple Chinese herbal medicine used for treating acute pancreatitis (AP) in China. We sought to determine the effects of CQCQD on pancreas gene expression profiles in murine experimental AP.
Methods: AP was induced with cerulein (50 μg/kg/h IP x 7, CER-AP; saline for controls). CQCQD (4 g/ml, 20 g/kg) or saline was given by gavage at the 3rd and 5th injections. Sacrifice was made at 12 h to assess AP severity with pancreas sampling for mRNA extraction and Affymetrix microarray analyses to investigate gene expression (n = 3 for each group) with P value set at < 0.05.
Results: CQCQD reduced CER-AP severity with markedly reduced biochemical markers and pancreatic histopathology scores (all P < 0.05). CQCQD without cerulein induced 1105 gene expression changes compared to saline, whereas CER-AP compared to saline induced 6153 (3049 up-regulated and 3104 down-regulated) and CER-AP compared to CQCQD without cerulein induced 5146 (2521 up-regulated and 2625 down-regulated) changes. In CER-AP, CQCQD induced 1381 gene expression changes (692 up-regulated and 689 down-regulated) compared to CER-AP without CQCQD, the top 10 pathways being: mTOR signalling, proteasome, ribosome, spliceosome, p53 signaling, glycerophospholipid metabolism, apoptosis, insulin signalling, ubiquitin mediated proteolysis, and Wnt signalling.
Conclusion: CQCQD treatment induced differential expression of genes in related pathways in CER-AP. Candidate genes and pathways can serve as targets for future studies to elucidate therapeutic targets of CQCQD.
Novel Role of Pancreatic Differentiation 2 (PD2/Paf1) in Acinar to Ductal Metaplasia for Pancreatic Cancer
P. Dey, M.P. Ponnusamy, S. Rachagani, A. Vaz, S.K. Batra. Department of Biochemistry and Molecular Biology, University of Nebraska Medical Center, Omaha, NE.
PD2/Paf1 subunit of the PAF complex is overexpressed in poorly differentiated pancreatic cancer (PC) cells and harbors potential oncogenic property. Previous studies demonstrate the role of PD2 in cell cycle and histone modifications in PC cells. In order to decipher its role under in vivo conditions, we analyzed the expression of PD2 in the KrasG12D; PdxCre (KC) mouse model of PC. Our unique observation revealed that it is expressed in the normal mouse pancreatic acinar cells but not in the ductal cells. In contrast, it has gradually increasing expression in the ductal cells of KC mice with increasing age, having highest expression in the CK19 positive neoplastic ductal cells at 50 weeks of age. Interestingly, PD2 specifically colocalized with amylase and CK19 double positive metaplastic ducts, which represent intermediate structures during acinar to ductal metaplasia (ADM). This process involving acinar cell conversion into a ductal phenotype, due to genetic aberrations or inflammatory insult, is considered as an early initiating event in PC. PD2 expression in these transdifferentiated structures was further validated in a cerulein induced murine pancreatitis model that exhibits ADM-like histology. Further in normal mice, inflammatory insult led to decrease in PD2 and amylase mRNA level and increase in CK19 level, which was restored upon recovery. In contrast, mice containing KrasG12D mutation failed to recover and PD2 level continued to decrease with progressive dysplasia and subsequent neoplastic transformation. Finally, knockdown of PD2 in mouse pancreatic acinar cells led to abrogation of amylase, Elastase and Lipase (acinar) transcripts with simultaneous increase in CK19 and CAII mRNA (ductal). Overall our studies indicate loss of PD2 expression during acinar transdifferentiation in PC initiation and PD2 mediated regulation of lineage specific markers.
Pre-SBRT Metabolic Tumor Volume and Total Lesion Glycolysis Predict Survival in Patients With Locally Advanced Pancreatic Cancer Receiving Stereotactic Body Radiation Therapy
A.S. Dholakia,1 M. Chaudhry,2 J.P. Leal,2 S.P Raman,2 A. Hacker-Prietz,1 R.L Wahl,2 D.A Laheru,3 C.L Wolfgang,4 J.M Herman,1. 1Department of Radiation Oncology, Johns Hopkins University, Baltimore, MD; 2Department of Radiology, Johns Hopkins University, Baltimore, MD; 3Department of Oncology, Johns Hopkins University, Baltimore, MD; 4Department of Surgery, Johns Hopkins University, Baltimore, MD.
Rationale: We analyzed the prognostic utility of baseline PET for patients with locally advanced pancreatic cancer (LAPC) undergoing fractionated stereotactic body radiotherapy (SBRT).
Methods: Thirty-two LAPC patients received a baseline PET scan prior to SBRT (6.6 Gy in 5 daily fractions, 33 Gy total) on a prospective clinical trial. Metabolic tumor volume (MTV), total lesion glycolysis (TLG), and maximum and peak standardized uptake values (SUVmax and SUVpeak) on pre-SBRT PET scans were calculated using an in-house software. The threshold of disease measurability was defined as the Livermean + (2 * Liversd). Median values of PET parameters were used as cutoffs for Cox regression analyses.
Results: Of the 32 patients in this study, the majority were male (N=19, 59%), 65 years or older (N=21, 66%), and had tumors located in pancreatic head (N=27, 84%). Twenty-seven patients (85%) received induction gemcitabine (no more than 3 doses) prior to SBRT. Median overall survival for the entire cohort was 18.8 months (95% CI, 15.7-22.0). An MTV of 26.8 cm3 or greater (HR 4.46, 95% CI 1.64-5.88, p<0.003) and TLG of 70.9 cm3 or greater (HR 3.08, 95% CI 1.18-8.02, p<0.021) prior to SBRT were associated with inferior overall survival (OS) on univariate analysis. Both MTV (HR 5.13, 95% CI 1.19-22.21, p=0.029) and TLG (HR 3.34, 95% CI 1.07-10.48, p=0.038) remained independent prognostic factors for OS in separate multivariate analyses.
Conclusion: Pre-SBRT MTV and TLG yield prognostic information on the OS of LAPC patients and may assist in tailoring therapy.
Do Larger Periprocedural Fluid Volumes Reduce the Severity of Post-ERCP Pancreatitis?
M.J. DiMagno,1,2 E.J. Wamsteker,1,2 J.K. Rai,1,2 M.A. Rivera,1,2 J.P. Spaete,1,2 D.D. Ballard,1,2 J.B. Elmunzer,1,2 S.D. Saini,1,2,3. 1Department of Internal Medicine, 2Division of Gastroenterology and Hepatology, University of Michigan School of Medicine, Ann Arbor, MI; 3Center for Clinical Management Research, Health Services Research & Development, Ann Arbor VA Medical Center, Ann Arbor, MI.
Objectives: Fluid therapy is a cornerstone of the early treatment of acute pancreatitis (AP), but data are conflicting as to whether it affects disease severity. According to experimental studies, administering greater fluid volumes (FV) during the initiation of AP preserves pancreatic microvascular perfusion and reduces severity, but without preventing organ damage. We test the hypothesis that administering larger FV during endoscopic retrograde cholangiopancreatography (ERCP) associates with less severe post-ERCP pancreatitis (PEP).
Methods: In a retrospective cohort study, 6505 patients had 8264 ERCPs, 211 had PEP (48 mild, 141 moderate and 22 severe). Data for FVs were available for 173 cases (39 mild, 115 moderate and 19 severe).
Results: In univariable analysis of sixteen variables only one variable was significantly associated with moderate-severe PEP: larger periprocedural FV was protective (0.94+/-0.3 L vs 0.81+/-0.4 L, P=0.0129). Similarly, multivariable analysis identified one independent predictor of moderate-severe PEP: larger periprocedural FV (OR 0.20, 95% CI 0.05-0.83) was protective. Conversely, larger periprocedural FV administered after PEP diagnosis correlated with moderate-severe disease (reflecting treatment decisions).
Conclusions: This hypothesis-generating study suggests that administering larger periprocedural fluid volumes is protective against moderate-severe PEP. Prospective studies are warranted to assess the impact of administering larger periprocedural fluid administration on PEP severity.
Predicting Pancreatic Exocrine Insufficiency (PEI) in Patients With Chronic Pancreatitis (CP) According to the Degree of Pancreatic Fibrosis: Role of Endoscopic Ultrasound (EUS)-Guided Elastography
J.E. Domínguez-Muñoz, M. Castiñeiras, M. Luaces, J. Lariño-Noia, L. Nieto, J. Iglesias-García. Department of Gastroenterology and Hepatology, Foundation for Research in Digestive Diseases. University Hospital of Santiago de Compostela, Spain.
Diagnosis of PEI is hindered by methodological difficulties of pancreatic function tests. The degree of fibrosis in CP may predict the probability of PEI. Pancreatic fibrosis in CP can be quantified by EUS-guided elastography. We aimed at evaluating whether pancreatic elastography can be used to diagnose PEI in patients with CP.
Methods: Prospective observational study including patients consecutively diagnosed with CP based on EUS (≥ 5 criteria) and/or secretin-MRCP findings. PEI was diagnosed by the 13C-mixed triglyceride breath test and defined as a 13C-cumulative recovery rate <29%. EUS-guided elastography was performed by the lineal Pentax echoendoscope and HITACHI-PREIRUS. Two areas were selected for elastographic evaluation: A, representative of the pancreatic parenchyma and B, referring to a soft reference area. The quotient B/A (strain ratio -SR-) was considered as the elastographic result. Data are shown as mean and 95% CI and compared by Student-t test. The probability of PEI according to the SR was calculated.
Results: 115 consecutive patients with CP (mean age 50.2 years, range 21-81, 92 male) were included. 35 patients (30.4%) suffered from PEI. Patients with PEI had a higher SR (4.89; 95%CI 4.36-5.41) than those with a normal breath test result (2.99; 95%CI 2.82-3.16) (p<0.001). A close relationship was found between the SR and the probability of PEI (table).
Conclusion: The degree of pancreatic fibrosis as measured by EUS-guided elastography allows quantifying the probability of PEI and thus the need for enzyme therapy in patients with CP.
Validation Study of Quantitative Endoscopic Ultrasound Elastography (Q-EUS-E) for the Differential Diagnosis of Solid Pancreatic Tumors
J.E. Dominguez-Muñoz, J. Lariño-Noia, J. Iglesias-Garcia. Department of Gastroenterology and Hepatology, Foundation for Research in Digestive Diseases. University Hospital of Santiago de Compostela, Spain.
Q-EUS-E has been reported as a useful tool for evaluating solid pancreatic masses. Aim of the study was to validate and determine the accuracy of a standardize Q-EUS-E in differential diagnosis of solid pancreatic masses.
Methods: Prospective observational study including patients undergoing EUS for the evaluation of solid pancreatic masses. EUS-elastography was performed with the linear Pentax echoendoscope and the Hitachi-PREIRUS processor. Two different areas were selected for quantitative elastographic analysis: Area A, representative of the mass and B referring to a soft reference area. The quotient B/A (strain ratio-SR) was considered as the result of the elastographic evaluation. Final diagnosis was based on surgical histopathology and, in non-operated cases, on imaging assessment, EUS-FNA and clinical follow-up. Data are shown as median and 95%CI. Diagnostic accuracy of Q-EUS-E for detecting malignancy was calculated after drawing the corresponding ROC curve.
Results: 199 consecutive patients (mean age 64 years, 17-90 years, 121 male) were included. Final diagnoses were pancreatic adenocarcinoma (n=135), inflammatory mass (n=38), malignant-NET (n=11), benign-NET (n=4), metastasis (n=4), pancreatic lymphoma (n=3), autoimmune pancreatitis (n=2) and mesenchymal lesions (n=2). SR was significantly higher among patients with malignant tumors (43.4; 95%CI 38.2-48.5) compared with those with benign lesions (9.9; 95%CI 5.6-11.7) (p<0.001). Area under the ROC curve was 0.951. For a cut-off point of 9.5, SR has a sensitivity, specificity, PPV, NPV and overall accuracy in detecting malignancy of 100%, 91%, 97%, 100% and 98%, respectively.
Conclusion: Q-EUS-E performed under a well-established protocol is a very useful tool for the differential diagnosis of solid pancreatic masses.
Pancreatic Enzyme Replacement Therapy and Nutritional Advice are Associated With Longer Survival in Patients With Unresectable Pancreatic Cancer (PC)
J.E. Dominguez-Muñoz,1,2 L. Nieto,2 J. Iglesias-Garcia,1,2. 1Department of Gastroenterology and Hepatology, 2Foundation for Research in Digestive Diseases, University Hospital of Santiago de Compostela, Spain.
Background: PEI is associated with malnutrition, weight loss and poor survival in patients with unresectable pancreatic cancer (PC). We aimed at evaluating the impact of PEI therapy on survival in these patients.
Methods: Retrospective analysis of a prospective database of patients with unresectable PC confirmed by EUS and FNB. Patients with survival <30 days were excluded. All patients were evaluated for palliative chemotherapy and endoscopic bile drainage if needed. Those patients diagnosed in the Dept. Gastroenterology (group 1) were additionally evaluated for PEI and received PERT (Creon®, 50,000 Ph.U. lipase/meal and 25,000 Ph.U. lipase/snack) and nutritional advice, whereas other patients (group 2) were not. Survival (median and 95%CI) was analyzed by Kaplan-Meier test and Cox regression and compared by Log Rank Test.
Results: 66 patients with unresectable PC were included (mean age 69.3 years, range 28-100, 43 male, 50 stage IV), 21 (31,8%) in group 1 and 45 (68.2%) in group 2. Age, tumor stage and PS were similar in both groups. Survival in group 1 (301 days, 95%CI 151-451) was significantly longer than in group 2 (89 days, 95%CI 30-148) (p=0.002). Palliative chemotherapy and PERT+nutritional advice were independent factors associated with longer survival in a multivariate model controlled by age, gender and tumor stage.
Conclusion: PERT and nutritional advice have a relevant impact on survival in patients with unresectable PC. These results should serve as background for future clinical trials in this context.
Comparison of the Natural History, Demographic Features and Progression of Intermediate Risk Unifocal Versus Multifocal IPMNs
V. Dorfman, R. Rosenblatt, J.M. Poneros, A. Sethi, J.D. Allendorf, B.A. Schrope, J.A. Chabot, T.A. Gonda. Department of Medicine, Columbia University Medical Center, New York, NY.
Background: There is insufficient data regarding management of multifocal IPMNs. This study aims to describe the demographic features, natural history and progression of multifocal IPMNs compared to unifocal lesions in intermediate risk patients.
Methods: We retrospectively reviewed the prospectively maintained database of the Pancreas Center Cyst Program and included patients with a clinical diagnosis of IPMN and ≥12 months of surveillance from diagnosis. Based on initial cross sectional imaging or endoscopic ultrasound (EUS) these were categorized as unifocal or multifocal (if ≥2 cysts were identified). Continuous variables were compared using student’s t-test and categorical variables were compared using chi-squared test.
Results: Out of 999 patients that underwent EUS, CT, or MRI, 161 (16%) met criteria: 98 (60.9%) were multifocal (MF) and 63 (39.1%) were unifocal (UF). There was no significant difference in age at diagnosis (67.9 vs 65.1; p=0.123), gender (p=0.402), DM (20.8% vs 33.3%; p=0.577), chronic pancreatitis (10.7% vs 9.3%; p=0.795), family history of pancreatic cancer (15.5% vs 18.4%; p=0.681), or presence of symptoms (19.5% vs 16.7%; p=0.685). The average dominant cyst size at initial presentation was 1.47 cm in the MF group and 1.38 cm in the UF group (p=0.671). In the MF cohort, the dominant cysts showed an average change of +0.14 cm (+20.4%) at 30.6 months surveillance, compared to +0.16 cm (+20%). 15.9% UF patients and 10.2% MF patients required surgery.
Conclusion: The demographics and natural history of IPMNs were not significantly different between the unifocal and multifocal cohorts. This suggests that despite a potentially greater burden of disease, patients with multifocal IPMNs do not represent a higher risk group. It is likely that current surveillance guidelines can be applied based on the characteristics of the dominant cyst in multifocal lesions.
Triptolide Modulates Histone Methylation in Pancreatic Cancer Cells
V. Dudeja, R. Chugh, S. Banerjee, V. Sangwan, R. Dawra, S. Vickers, A. Saluja. Department of Surgery, University of Minnesota, Minneapolis, MN.
Epigenetic changes are critical events underlying pathogenesis of cancer. One of the mechanisms by which epigenetic information is encoded is by covalent modification (e.g. methylation of histones). Alterations in histone methylation can deregulate chromatin-based processes, ultimately leading to oncogenic transformation. In this context, aberrant patterns of histone methylation have been associated with a large number of cancers. Typically, low levels of trimethylated H3K9, H3K27, and H3K36 are observed in cancer tissue. We have previously shown that triptolide, a natural compound from a Chinese herb, is markedly effective in killing pancreatic cancer cells in vitro and in reducing growth and loco-regional spread of pancreatic tumors in vivo. Our understanding of the mechanism by which triptolide induces cell death is still evolving. Whether triptolide induces epigenetic changes in pancreatic cancer cells is not known. The aim of the current study was to evaluate the effect of triptolide on the epigenome of pancreatic cancer cells.
Methods: Pancreatic cancer cell lines MiaPaCa-2 and S2VP10 were treated with triptolide (0-200nM) for 24 hr. and the trimethylation status of H3K9, H3K27 and H3K36 was evaluated by western blotting. The effect of triptolide on the key enzymes involved in epigenetic modulation was evaluated by PCR array.
Results: Triptolide treatment leads to dose dependent increases in the trimethylation levels of H3K9, H3K27, and H3K36 in both MiaPaCa-2 and S2VP10 cell lines. PCR array analysis showed marked alteration in the levels of enzymes which influence epigenetic changes.
Conclusion: Triptolide influences histone tri-methylation status in pancreatic cancer cells. Epigenetic modulation could be one of the mechanisms by which triptolide induces cell death in cancer cells.
Fatty Acids are More Toxic Than Their Ethyl Esters During Pancreatitis
C. Durgampudi,1 P. Noel,2 K. Patel,2 V. Mishra,2 R.N. Trivedi,2 C. Acharya,1 V.P. Singh,2. 1Department of Medicine, UPMC Passavant, Pittsburgh, PA, 2Division of Gastroenterology, University of Pittsburgh, Pittsburgh, PA.
Background: Based on fatty acid ethyl esters (FAEEs) being present in pancreas, adipose tissue of dying alcoholics [Science 231(4737):497-9 (1986)], these are thought to have a causal role in alcoholic AP. We have shown fatty acids (FAs) worsen pancreatic necrosis, cause organ failure in obesity associated severe acute pancreatitis (SAP) [Sci. Transl. Med. 3, 107ra110 (2011)]. We therefore compared the relative toxicities of FAEEs and their parent FAs in pancreatic acini and an in vivo model of SAP.
Methods: Mice pancreatic acini were exposed to FAs (linoleic; LA, oleic; OA, palmitic; PA) and corresponding FAEEs (i.e. LAEE, OAEE, PAEE) at concentrations relevant to human pancreatic debridement fluids. Necrotic cell injury was assessed by LDH leakage, propidium-iodide uptake (PI, both as % total) and ATP levels. Cytosolic calcium levels were also measured. Glyceryl-trioleate (GTO) or the ethyl ester of OA i.e. OAEE were injected intraductally in rats to simulate a model of obesity associated SAP. Serum amylase, lipase, organ failure (blood urea nitrogen; BUN) and mortality were compared between the two groups. Values are reported as mean±SEM.
Results: LA and OA caused more LDH leakage (81±2%, 57±8%) than LAEE, OAEE (18±3%, 11±2%, P<0.003) and also caused more PI uptake (104±5%, 78±11%) than LAEE, OAEE (25±10%, 6±0.5%, P<0.03). FAEEs caused significantly less ATP depletion, and no calcium flux vs. their parent FAs. PA or PAEE did not affect these parameters. Serum amylase (14496±3216 vs. 2574±689 U/L, P<0.006) and lipase (35544±6739 vs. 1574±682 U/L, P<0.001) were higher in the GTO vs. OAEE group. GTO infused rats had gross evidence of hemorrhagic pancreatic necrosis and a 70% mortality in 24 hours, both of which were absent in the OAEE group (0% mortality at 5 days, P<0.004). BUN was higher in GTO infused rats compared to OAEE infused rats (58±2 vs. 22±6 mg/dL, P<0.006).
Conclusions: FAs cause more acinar necrosis, organ failure and mortality than their corresponding FAEEs. Therefore the FAEEs noted to accumulate in the viscera of alcoholics likely play a protective role by detoxifying FAs.
Mouse Models of Pancreatic Cancer Induced by Chronic Pancreatitis and Smoking
M. Edderkaoui,1 P.J. Grippo,2 Y. Ouhaddi,1 H. Benhaddou,1 S. Xu,1 K. Pinkerton,3 H. Tsukamoto,4 B. Knudsen,1 A.S. Gukovskaya,1 S.J. Pandol,1. 1Cedars-Sinai Medical Center, Department of Veterans Affairs & University of California Los Angeles, Los Angeles, CA; 2Northwestern University, Chicago, IL; 3UC-Davis, Davis, CA; & 4University of Southern California, Los Angeles, CA.
Background: Chronic pancreatitis (CP) and smoking are two major risk factors for pancreatic cancer (PaCa). Here we developed mouse models of PaCa by exposing mice to CP and/or smoking.
Methods: Wild type (WT) and Pdx1-Cre;LSL-Kras transgenic mice were subjected to 7-hourly cerulein injections (50μg/kg) twice a week for 3 weeks to induce CP. Another group of mice was exposed to tobacco smoke (80mg/m3) in chambers for 5days/week during 6 weeks. A third group was exposed to both treatments. Mice were then sacrificed and pancreatic tissue analyzed. Pancreatic lesions, proliferation, fibrosis, inflammation, EMT and stem cell markers were analyzed by immunohistochemistry and Western.
Results: CP significantly increased the number and stage of pancreatic neoplastic (PanIN) lesions with fibrosis, stellate cell activation and inflammation in transgenic mice. These changes were significantly less in WT mice. EMT markers (decrease in E-Cadherin and increase in vimentin protein levels) were stimulated by CP in both WT and transgenic mice. Similarly, smoking stimulated PanIN lesion formation, fibrosis and inflammation in transgenic mice, but to a lesser extent than CP model. Expression of EMT and stem cell markers was greater in the smoking model compared to CP. Combination of CP and smoking induced greater stimulation of PanIN lesion formation, fibrosis and inflammation compared to either treatment alone in transgenic mice.
Conclusion: The results indicate that both CP and tobacco smoke promote PaCa progression in transgenic mice. The combination of CP and smoking has greater effects than either treatment alone. EMT and stem cell markers developed in WT as well as in transgenic mice with smoking suggesting that smoking has effects independent of Kras on cell transformation.
A Rapid Computer-Based Automated Algorithm for Assessing Acinar Dropout After Experimental Pancreatitis
J.F. Eisses,1 A. Davis,2 A.B. Tosun,3 Z.R. Dionise,1 C. Cheng,3 J. Guo,3 J.A. Ozolek,2 G.K. Rohde,3 S.Z. Husain1. 1Depts of Pediatrics and 2Pathology, University of Pittsburgh, Children’s Hospital of Pittsburgh of UPMC, Pittsburgh, PA; 3Biomedical Engineering, Center for Bioimage Informatics, Carnegie Mellon University, Pittsburgh, PA.
At present, the histological measurement of pancreatic injury after pancreatitis is tedious and operator-dependent, requiring manual assessment of acinar area on serial pancreatic sections. In this study, we utilized a novel computer-generated learning algorithm to construct a rapid and consistent method of quantifying acinar content. The algorithm works by learning differences in pixel intensities from input examples provided by expert pathologists. H&E-stained pancreatic sections were obtained in mice recovering from a 2-day, hourly caerulein hyperstimulation protocol. For training data, a pathologist carefully outlined discrete regions of acinar and non-acinar tissue in 20 sections at various stages of injury and recovery (termed the “ground truth”). The software results were validated by applying the algorithm to a different group of sections. In normal, non-injured pancreatic sections, the software demonstrated close agreement with the ground truth in identifying acinar tissue area; there was only a 2.4% ± 0.01% difference in area measurements (p=0.43). However, in recovering tissue the software reported 8.8% ± 5.7% less acinar area than the manual method (p=0.04). Surprisingly, on detailed morphological examination, the discrepancy was primarily because the software outlined acini with a tighter fit and excluded inter-acinar edematous stroma with greater precision. Although further reiterations and validation of the computer learning are necessary, the results indicate that the software will be of great potential benefit to both clinicians and researchers in quantifying pancreatic acinar dropout in the injured and recovering pancreas, and it may hold promise in reliably screening specimens for more complex pancreatic processes.
Predictors of Increased Abdominal CT Scanning in Patients with Acute Interstitial Pancreatitis (AIP)
M. Faghih,1 E. Afghani,1 A. Sinha,1 V.S. Akshintala,1 A. Zaheer,2 M.A. Khashab,1 A.M. Lennon,1 A.N. Kalloo,1 V.K. Singh1. 1Division of Gastroenterology, 2Dept of Radiology, Johns Hopkins Hospital, Baltimore, MD.
Background: Patients with necrotizing pancreatitis typically have several CT scans during hospitalization but factors associated with obtaining > 1 CT scan in patients with AIP is not known.
Aim: To determine the predictors of increased (>1) CT scanning in AIP.
Methods: All adults (≥18 years) directly admitted to our institution with acute pancreatitis (AP) between 2010-2012 were evaluated. Demographic, clinical, & laboratory data during the first 7 days of hospitalization was reviewed. Exclusion criteria were: chronic pancreatitis, CTSI≥4 on initial CT or no initial CT within 24 hours of admission. AIP was defined as a CTSI≤3 within 24 hours of admission and either amylase/lipase levels >3X the upper limit of normal or characteristic upper abdominal pain. Multivariable logistic regression was conducted using variables with p<0.05 on univariate analyses and a priori hypotheses.
Results: There were 794 patients with AP, of whom 169 (21.3%) met inclusion with 149 (87%) patients with 1 CT and 20 (13%) with >1 CT. Median BISAP score was 0. Patients with >1 CT had higher rates of persistent SIRS (60.0% vs 15.4%, p<0.001), longer mean length stay (20.1 vs 4.9 days, p<0.001), prolonged narcotic use (4.1 vs 2.5 days, p=0.0009) & acute fluid collection (AFC) on initial CT (50% vs 23.4%, p=0.009). Persistent SIRS (OR 6.5, 95% CI 2.1-20.1, p=0.001), AFC (OR 3.8, 95% CI 1.2, 11.9, p=0.02), & prolonged narcotic use (OR 1.4, 95% CI 1.1, 1.8, p=0.01) were significant predictors of increased CT scanning on multivariable analysis. There were no changes on subsequent imaging that affected management in any patient with >1 CT.
Conclusion: Persistent SIRS, prolonged narcotic use, & AFC on initial CT are predictors of increased CT imaging in patients with AIP. However, additional scans did not change management suggesting they should not be obtained in patients with AIP.
K-Ras Depletion Reduces G2 Cyclins Without Reducing the Ratio of Glycolysis to Oxidative Phosphorylation in Pancreatic Cancer Cell Lines
R. Ferguson, L. Bennett, A. Ware, W. AbuAlainin, J.P. Neoptolemos, E. Costello, W. Greenhalf. Department of Molecular and Clinical Cancer Medicine, Liverpool Cancer Research UK Centre, University of Liverpool, Liverpool, UK.
Introduction: K-Ras mutations occur in 75-90% of pancreatic adenocarcinomas. K-Ras acts upstream of a complex network of pathways. Depletion of K-Ras protein from pancreatic cancer cell lines is cytostatic rather than causing cell death.
Aim: To determine the nature of cytostasis following K-Ras depletion in pancreatic cell lines.
Methods: K-Ras was depleted in pancreatic cancer cell lines using siRNA. Ras activity was analyzed by RAF pull down; metabolic activity by Seahorse Bioanalysis; Cell cycle profile by FACS analysis and cyclin quantification using Western Blotting.
Results: K-Ras depletion caused G2 cyclins to decrease with lesser effects on G1 cyclins. G2 to G1 ratio did not significantly change. Although relative levels of glycolysis to respiration were higher in cells with K-Ras mutations, depletion of K-Ras did not reduce this ratio. Inhibition of respiration by MAPK inhibition was equivalent regardless of K-Ras depletion.
Conclusion: Ras protein is an activator of the MAPK and PI3K pathways, both of which promote G1/S transition via increase in G1 cyclins (D then E). The decrease in G1 cyclins when K-Ras is depleted is less pronounced than the decrease in G2 cyclins (A and B). There does not seem to be a release in suppression of respiration when K-Ras is depleted and cells are equally sensitive to MAPK inhibition (as indicated by rate of respiration). This implies that mutant K-Ras in these cells is acting via a pathway independent of MAPK to reduce G2 cyclins, but that this does not result in promotion of anaphase.
Epigenetic Regulation of the Tumor Microenvironment: A Novel Hedgehog-Independent Role of GLI Proteins
M.G. Fernandez-Barrena, A.L. McCleary-Wheeler, L.L. Almada, E.J. Tolosa, S.L. Safgren, L.D. Mills, A.M. Vrabel, D.L. Marks, W.R. Bamlet, R.R. McWilliams, M.E. Fernandez-Zapico. Schulze Center for Novel Therapeutics, Mayo Clinic, Rochester, MN.
Numerous studies have demonstrated that the desmoplastic reaction, a hallmark of the pancreatic ductal adenocarcinoma (PDAC) tumor microenvironment, plays a major role in the pathogenesis of this tumor by promoting cancer cell survival and proliferation as well as chemoresistance. This cellular compartment is primarily composed of a dense, collagen-rich extracellular matrix and stromal cells. By crossing the LSL-KRASG12D and GLI1 null mice we identified a key role for this transcription factor in PDAC development through the modulation of the tumor microenvironment. Analysis of the mechanism using a combination of expression, luciferase and chromatin immunoprecipitation assays identified Col1a1, a critical component of the desmoplastic reaction, as a new direct target gene of GLI1 in stromal cells. Clinically, expression studies in a cohort of human PDAC samples showed a strong positive correlation between the expression of GLI1 and Col1a1, thus supporting the translational significance of these findings. Next, we showed that GLI1 can function as a downstream mediator of TGFβ signaling, one of the most potent inducers of Col1a1. TGFβ requires GLI1 to promote Col1a1 expression, knockdown of GLI1 in stromal cells abrogated Col1a1 induction by this cytokine. Further, we demonstrated that the regulation of Col1a1 expression by GLI1 was accompanied by an increase in the methylation of the Histone 3 at lysine 4 (H3K4) in the Col1a1 promoter. We have identified components of the H3K4 methylation writer MLL/COMPASS as parts of this GLI1 transcriptional complex. Ongoing experiments are aimed at defining the role of this methyltransferase complex in GLI1-mediated regulation of Col1a1 expression and maintenance of the tumor microenvironment. Thus, together these findings expand the repertoire of proteins involved in the regulation of extracellular matrix proteins, and define a novel epigenetic pathway regulating desmoplasia in PDAC.
CD166 Expression Characterized Tumorigenicity, and Invasive and Migratory Activities of Pancreatic Cancer Cell Lines
K. Fujiwara,1,2 K. Ohuchida,1 S. Akagawa,1 T. Ohtsuka,1 K. Mizumoto,1 M. Tanaka1. 1Departments of Surgery and Oncology, Graduate School of Medical Sciences, Kyushu University, Fukuoka; 2Research Fellow of the Japan Society for the Promotion of Science, Tokyo, Japan.
Aim: CD166, synonymous to ALCAM, is a cell adhesion molecule expressed by epithelial cells in several organs. The role of CD166 is controversial in malignant tumors and is not clear in pancreatic cancer. The aim of this study is to clarify the role and the significance of CD166 expression in pancreatic cancer.
Methods: We performed flow cytometry to analyze expression of CD166 in pancreatic cancer cell lines. After cell separation, we analyzed the functional differences between CD166+ and CD166- pancreatic cancer cells by using invasion assay, migration assay and in vivo mice xenograft model.
Results: In flow cytometry, CD166 was expressed in pancreatic cancer cells in wide range (0-99.5%). We separated cells by using auto MACS Pro Separator (Miltenyi Biotec). In invasion assay and migration assay, CD166– pancreas cancer cells showed stronger biological behaviors in their invasion and migration than did CD166+ cancer cells (p<0.05). Real-time quantitative RT-PCR showed that epithelial-mesenchymal transition activator Zeb1 mRNA was over-expressed in CD166- cells (p<0.001).Meanwhile, in vivo analysis revealed that CD166+ cells showed significantly greater tumor growth than CD166- cells (p<0.05) both in mouse subcutaneous and orthotopic models. By using flow cytometry, percentage of CD166+ cells was greater in tumors formed by CD166+ cells than in tumors formed by CD166- cells.
Conclusion: These data demonstrate that CD166- pancreas cancer cells exhibit strong invasive and migratory activities, and that CD166+ pancreas cancer cells have strong tumorigenicity. CD166 expression is associated strongly with functional differences in pancreatic cancer cell lines. Further investigation is needed, because its mechanisms are still unclear.
Role of Intra-Acinar Trypsin Activation in Pancreatic Injury and Inflammation in Bile Acid Induced Mouse Model of Acute Pancreatitis
S.K. Garg, U. Barlass, J. Ouchveridze, Z. Yuan, R. Dawra, A. Saluja. Division of Basic and Translational Research, Department of Surgery, University of Minnesota, Minneapolis MN.
Background: Using a cerulein and L- arginine induced models of acute pancreatitis, we have recently shown that in the absence of active trypsin, necrosis is significantly reduced but inflammatory response remains unchanged. In initial stages of the bile acid induced acute pancreatitis also, a significant increase in trypsin activity is observed, but this models differs in severity and development from other models.
Aim: To study the role of intra-acinar trypsin activation in a bile acid induced model of acute pancreatitis.
Methods: Acute pancreatitis (AP) was induced by administration of sodium taurocholate (3%, 50 μl) in the pancreatic duct over a period of 10 minutes and animals were sacrificed 24h after bile acid administration. Serum amylase, myeloperoxidase (MPO) in pancreas and lungs and necrosis in the pancreas of wild-type (WT), Cathepsin B knock-out (CB-/-) and trypsinogen isoform T7 knock-out (T7 -/-) mice was measured.
Results: Absence of trypsin activation in CB(-/-) and T7(-/-) mice led to a significant decrease in acinar necrosis in CB(-/-) mice and T7(-/-) compared to WT mice treated with bile acid. Although, both pancreatic and lung MPO were significantly higher in bile acid treated animals vs. saline controls, there was no significant difference between the wild type, CB(-/-) and T7(-/-) groups.
Conclusion: Using the bile acid induced model of acute pancreatitis, we show that loss of trypsinogen activation significantly decreases acinar cell necrosis, but local and systemic inflammation is independent of trypsin activation. This effect is consistent among commonly used models of acute pancreatitis.
Trypsin Activation Is Not Required for Co-Localization of Zymogen Granules and Lysosomal Hydrolases in Acute Pancreatitis
S.K. Garg, U. Barlass, J. Ouchveridze, Z. Yuan, A.K. Dixit, V. Dudeja, R. Dawra, A. Saluja. Division of Basic and Translational Research, Department of Surgery, University of Minnesota, Minneapolis, MN.
Background and significance: Co-localization of lysosomal hydrolases and zymogen granules is observed early during initiation of acute pancreatitis. However, it is not clear whether this event is required for, or is a result of, trypsin activation.
Aim of this study was to understand the relationship between co-localization and trypsin activation.
Methods: Acute pancreatitis was induced in wild type (WT), cathepsin B (CB-/-) and trypsinogen 7(T7-/-) knockout mice by cerulein (50μg/kg i.p.), and animals were sacrificed 30 minutes after injection. Subcellular fractionation of pancreatic tissue was performed to separate the zymogen and lysosomal fractions. Lysosomal hydrolase arylsulfatase activity in both fractions was measured. In order to confirm the absence of trypsin activation in CB-/- and T7-/- animals, trypsin activity in pancreatic tissue was assessed 30 minutes after treatment with cerulein.
Results: Stimulation with cerulein increased significantly the zymogen/lysosome granule ratio of arylsulfatase activity in WT, CB-/- and T7-/- animals compared to saline treated control. After cerulein stimulation Zymogen/lysosomal ratio of arysulfatase activity in WT, CB-/- and T7-/- animals was comparable. Trypsinogen activation at 30 minutes was 7.1±1.0, 1.4±0.2 and 1.2±0.2 fold over saline injected controls in WT, T7-/- and CB-/- animals respectively (p<0.001) confirming lack of significant trypsinogen activation in T7-/- and CB-/- mice.
Conclusions: Supramaximal stimulation with cerulein causes significant redistribution of arylsulfatase activity from the lysosome-enriched to zymogen granule-enriched fractions in WT, CB-/- and T7-/- animals. Absence of trypsin activation does not alter cerulein-induced co-localization, suggesting that co-localization precedes trypsin activation.
The Vacuolar-ATPase (v-ATPase) is Necessary for Wnt-β-Catenin Signal Transduction in PanIN Cells
A.K. Gattu, H. Einwachter, F. Gorelick, R. Schmid, C. Chung. Departments of Medicine, Yale University School of Medicine, VA CT Healthcare System, Technical University of Munich, Munich, Germany.
Background & Aims: The vacuolar ATPase (v-ATPase) is a proton transporter found on intra-cellular organelles and some plasma membranes. We previously reported that the vacuolar-ATPase (v-ATPase) is redistributed during PanIN development and that it confers invasive properties to human pancreatic cancer (PaCa) by activating proteases. The aim of this study was to determine whether the v-ATPase modulates developmental and growth factor-dependent pathways in PanIN cells.
Methods: Pancreas-specific KrasG12D mutations were obtained using Ptf1a-Cre mice. The effect of the v-ATPase on Wnt-β-catenin signaling and the regulation of growth factor receptors such as epidermal growth factor receptor (EGFR) were tested in primary PanIN cells (gift of Dr. Andrew Rhim, University of Pennsylvania School of Medicine). Concanamycin (10-50nM) and bafilomycin (10nM) were used to block v-ATPase function.
Results: Ptf1a-Cre;KrasG12D displayed a PanIN-2 phenotype while Ptf1a; KrasG12D;SOD2 mice mutation resulted in PanIN-1 lesions. These findings were recapitulated in human specimens. Basolateral localization of the V1E subunit was seen in PanIN-2, but not PanIN-1, lesions. Blockade of the v-ATPase in a primary PanIN cells led to an 80% increase in activation of the Wnt receptor, p-LRP6 (p<0.05), and doubled the total LRP6 levels (p<0.01). Despite LRP6 activation, v-ATPase blockade resulted in a 40% decrease in the levels of the effector non-phosphorylated β-catenin (p<0.01), which was reflected in significantly higher levels of its negative regulator GSK3β (p<0.01). EGFR, a necessary component for PanIN progression to PaCa, was significantly reduced by v-ATPase blockade (p<0.01). Functionally, v-ATPase blockade inhibited the proliferative response to EGF ligand (p<0.05).
Conclusions: In primary PanIN cells, a functional v-ATPase is necessary for canonical Wnt-β-catenin function and maintenance of endogenous EGFR levels. The expression pattern of the v-ATPase in advanced PanIN lesions may reflect polarized activation of developmental pathways and regulation of growth factor receptors.
Hereditary Pancreatitis Associated Mutations in the Trypsinogen Activation Peptide
A. Geisz,1,2 P. Hegyi,2 L. Czakó,2 M. Sahin-Tóth,1. 1Department of Molecular and Cell Biology, Boston University Medical Center, Boston, MA; 2First Department of Medicine, University of Szeged, Szeged, Hungary.
Background: Mutations in human cationic trypsinogen cause hereditary pancreatitis by altering its proteolytic regulation by chymotrypsin C (CTRC). CTRC stimulates trypsinogen autoactivation by processing the activation peptide to a shorter form but also promotes degradation by cleaving the calcium binding loop in trypsinogen. Mutations render trypsinogen resistant to CTRC-mediated degradation and/or increase processing of the activation peptide by CTRC.
Objectives: The present study was aimed at clarifying the role of CTRC in the mechanism of action of activation peptide mutations D19A, D22G, K23R and K23_I24insIDK.
Methods: Human pancreatic enzymes were produced recombinantly and purified to homogeneity. Trypsinogen activation was followed by enzymatic assays and SDS-PAGE. Trypsinogen secretion was measured from transfected HEK 293T cells.
Results: Activation peptide mutations robustly increased trypsinogen autoactivation, both in the presence and absence of CTRC. Degradation of activation peptide mutants by CTRC was unchanged and processing of the activation peptide was increased only in the D19A mutant by 4-fold. Surprisingly, however, increased processing had essentially no effect on autoactivation. Finally, the activation peptide mutants exhibited reduced secretion from transfected cells, and secreted trypsinogen levels were inversely proportional with autoactivation rates.
Conclusions: Activation peptide mutations form a mechanistically distinct subset of hereditary pancreatitis associated mutations, which exert their effect primarily through direct stimulation of autoactivation, independently of CTRC. The potentially severe clinical impact of the markedly increased autoactivation is offset by diminished secretion, resulting in a clinical phenotype indistinguishable from typical hereditary pancreatitis.
Clinical and Radiological Follow-Up of Patient with Chronic Asymptomatic Pancreatic Hyperenzymemia
A. Granato, A. Amodio, N. De Pretis, G. De Marchi, T. Tumelero, E. Goni, A. Gabbrielli, L. Benini, L. Frulloni. Department of Medicine, Institute of Pancreas, University of Verona, Verona, Italy.
Background: Chronic asymptomatic pancreatic hyperenzymemia (CAPH) has been described since 1996 as a benign disease. Pathological findings at Magnetic Resonance Cholangio-Pancreatography (MRCP) with secretin stimulation (s-MRCP) in 50% of CAPH subjects, but in 30% their clinical meaning remains undefined.
Aim: To investigate subjects with CAPH during the follow-up to assess the clinical and radiological outcome.
Methods: From January 2012 to March 2013, a cohort of 160 subjects previously studied for CAPH were re-evaluate with s-MRCP and biochemical tests.
Results: Clinical data relative to 109 subjects were collected, whereas 68 (47 males, 21 females, mean age 49.8 ± 12.2 yrs) underwent s-MRCP. Clinically, acute pancreatitis was observed in 1 subject (0.9%) after 5 years from the first detection of hyperenzymemia. Among 68 subjects who underwent s-MRCP, 23 had previous s-RMCP normal and 45 pathological. No radiological alterations in 23 subjects with normal s-MRCP at first evaluation were observed at follow-up, whereas unchanged s-MRCP was documented in 41 out of 45 (91%) subjects with pathological s-MRCP at first evaluation after mean follow-up of 3 ± 2 yrs. 1 out of 11 subject with IPMN had progression of the size of the cyst, 1 out of 11 with suspected SOD showed an increase of Wirsung duct diameter, while a worsening of ductal morphology was observed in 2 out of 20 of subjects with suspected chronic pancreatitis.
Conclusions: CAPH subjects remain asymptomatic during the follow-up. Subjects with previous normal MRCP-s did not show pathological findings at follow-up whereas only a small percentage of those with previous suspected SOD or CP, showed a progression at imaging in a short term follow-up.
Etiologies and Comorbidities Observed in Hospitalized Children With Acute Pancreatitis
A.S. Grover,1 A.J. Deutsch,1 V. Kadiyala,2 P.A. Banks,2 R.J. Grand,1 D.L. Conwell,2 JR Lightdale,1. 1Division of Gastroenterology, Boston Children’s Hospital; 2Center for Pancreatic Disease, Division of Gastroenterology, Hepatology and Endoscopy, Brigham and Women’s Hospital Harvard Medical School, Boston, MA.
Background: There is sparse evidence about factors that may impact hospitalization in pediatric acute pancreatitis (AP).
Aim: The aim of our study was to describe specific etiologies and co-morbidities associated with pediatric AP at a tertiary children’s hospital.
Methods: A retrospective chart review of all children presenting or developing AP (ICD-9 code 577.0) at Boston Children’s Hospital between 1/1/10- 12/31/10. Patients with multi-organ failure were excluded. Data: age, gender, amylase, lipase, BUN, hematocrit (HCT), BMI, length of stay (LOS), co-morbidities, suspected etiology and presence of systemic inflammatory response syndrome (SIRS). Note: Laboratory data adjusted for pediatric age, gender and weight.
Results: Demographics: 57 patients were evaluated; 46% male; median age 14 years (IQR 9). 25% had a BMI > 90th %ile. 12% had multiple admissions for AP during the study period. 70% had pancreatitis as the admitting diagnosis. Median LOS was 7 days (range 1-135). 32% met SIRS criteria at diagnosis.
Laboratory data at time of diagnosis [median (IQR)]: Amylase [342 unit/L (567)], lipase [910 unit/L (1195)], BUN [11 mg/dL (9)], HCT [37.5 % (7.4)]. 24% had elevated BUN; and 35% had elevated HCT.
Etiologic distribution: medications (35%); idiopathic (18%); infectious (14%); gallstones (12%); chronic pancreatitis (12%) and genetic/familial (9%).
Co-morbid illnesses: psychiatric disease (23%); seizure disorders (18%) and prior malignancy (11%).
Mortality: 0/57 died.
Conclusion: Compared to adults, different etiologic factors (medications, idiopathic) and co-morbidities (neuro-psychiatric) are observed in children with AP.
Larger studies are needed to investigate the impact of etiology, co-morbid illness and laboratory measures on prognosis and outcomes in children with AP.
Systemic Inflammatory Response Syndrome (SIRS) at Time of Presentation is Associated With Prolonged Hospitalization in Pediatric Acute Pancreatitis (AP)
A.S. Grover,1 V. Kadiyala,2 P.A. Banks,2 R.J. Grand,1 J.R. Lightdale,1 D.L. Conwell,2. 1Division of Gastroenterology and Nutrition, Boston Children’s Hospital; 2Center for Pancreatic Disease, Division of Gastroenterology, Hepatology and Endoscopy, Brigham and Women’s Hospital Harvard Medical School, Boston, MA.
Background: While there are several adult AP severity prediction models, there are currently none for pediatrics. Severe AP in adults is generally associated with length of stay (LOS)>7 days (revised Atlanta Criteria) and greater resource utilization/costs.
Aim: To identify factors associated with LOS >7 days in pediatric AP.
Methods: Retrospective chart review of all children presenting with AP (Atlanta criteria; ICD-9 code 577.0) to ED at Boston Children’s Hospital from 1/1/10 to 12/31/10. Children developing AP during hospitalization were excluded. Data: age, gender, LOS, BUN, hematocrit (HCT), serum lipase, BMI>90th %tile and SIRS at admission. Analysis (SPSS Statistics v21. Armonk, NY): Univariate analysis (Fisher’s, Mann-Whitney) to identify factors associated with LOS >7 days. Multivariate analysis (logistic regression) to identify independent predictors of LOS>7 days.
Results: 40 pts had AP as admitting diagnosis in ED: 21/40 male, median age 14.5 (IQR 10), median LOS 5 days (IQR 6.0). 14 had a LOS>7 days (median 10.50; IQR 21.5). Univariate analysis (LOS≤7 vs >7): SIRS at admission (p=0.029) and older age (p=0.057) were associated with LOS >7 days. Elevated BUN, elevated HCT, lipase>7x ULN, gender, BMI>90th %ile were not associated with LOS >7 days. Multivariate analysis: Controlling for age, SIRS at admission was associated with LOS>7 days (OR 5.7 [1.1, 28.3], p=0.033).
Conclusions: In our single center, retrospective pediatric AP study, SIRS at admission was associated with prolonged hospitalization (6x greater odds). Hemoconcentration (elevated BUN, HCT) was not associated with longer hospitalization in children with AP. Larger studies needed to identify risk factors and predictors of severity in pediatric AP.
Loss of Mixed-Lineage Leukemia 3 Function in Progression and Metastasis of Pancreatic Cancer
S. Gupta,1 S. Elumalai,1 K. Chawla,1 M. dalMolin,1 J. Lee,2 A. Maitra,1. 1Department of Pathology, Johns Hopkins University, Baltimore, MD; 2Neuroscience Section, Oregon Health & Science University, Portland, OR.
Myeloid/lymphoid or mixed-lineage leukemia 3 (MLL3) is a histone 3-lysine 4 methyltransferase component of the transcriptional co-activating ASCOM complex (ASC-2 complex). Whole genome and whole exome sequencing has revealed MLL3 to be frequently mutated in a variety of solid tumors, including pancreatic ductal adenocarcinoma (PDAC) where it is the fourth most common somatically mutated gene (excluding homozygous deletions). Despite various somatic mutations, the functional role of MLL3 has not been studied in pancreatic cancer or any other solid cancer type. We found, regardless of the mutation status, significantly diminished MLL3 expression in a panel of patient-derived pancreatic tumor cell lines, tumor xenografts and archived patient tumors. Examination of the MLL3 promoter region revealed a CpG island. Methylation of the promoter region was confirmed by methylation-specific PCR in majority of the tumor cell lines and xenografts. Treatment of cell lines with DNA methyltransferase inhibitor relieved the promoter methylation causing increase in MLL3 transcript expression. Further, knockdown of MLL3 by siRNA in cell lines increased anchorage-independent growth, and enhanced the migration/invasion phenotype of cancer cells. Microarray analysis of a pancreatic cancer cell line subsequent to knockdown of residual MLL3 function by siRNA, revealed a battery of potential gene targets and pathways regulated by MLL3. The most common Gene Ontology (GO) categories for differentially expressed genes include those involved in cell cycle, cell migration, and DNA repair. Using transgenic mouse model of pancreatic cancer, we found loss of MLL3 function in exocrine pancreas cooperates with mutant Kras to accelerate the progression of pancreatic intraepithelial neoplasia (PanIN) to invasive neoplasia and distant metastasis.
Pathobiological Implications of Endothelin Axis in Pancreatic Cancer
S. Gupta,1 M.A. Macha,1 S. Rachagani,1 S. Kaur,1 S. Kumar,1 L. Smith,2 S.L. Johansson,3 S.K. Batra,1,4 M. Jain,1,4. 1Department of Biochemistry and Molecular Biology; 2Department of Pathology and Microbiology; 3Department of Biostatistics; 4Eppley Institute for Research in Cancer and Allied Diseases, University of Nebraska Medical Center, Omaha, NE.
Endothelins are group of three 21 amino acids vasomodulatory peptides which along with two GPCRs (ETAR and ETBR) and endothelin converting enzymes (ECEs) form the endothelin (ET) axis. Binding of ET-1 to ETAR activates highly interconnected signaling pathways involved in cell proliferation, invasion, apoptosis, angiogenesis and osteogenesis. Dysregulation of ET-axis promotes tumor development and progression in many cancer types but its role in pancreatic cancer (PC) remains unknown. Expression of ET-1, ET A receptor, ET B receptor was analyzed in a panel of human PC cell lines and in primary and metastatic tissues from human and murine PC.
Expression of ET-1 and its receptors was detected in majority of cell lines; however, both the receptors showed a reciprocal relationship. In primary tumors obtained from UNMC rapid autopsy program, ETAR, ETBR and ET-1 were expressed in 83 %, 44.1% and 49% of the cases respectively. Overexpression of ET-axis was also observed in various metastatic sites including liver, lungs, lymph nodes and diaphragm. In addition to tumor cells, ETAR and ETBR expression was also observed on tumor blood vessels, stellate cells and infiltrating immune cells suggesting a multifaceted role of ET-axis in PC. There was a significant positive correlation between Shh and ETAR expression. In the primary tumor out of 18 spots that were negative for Shh, 94% (17/18) were also negative for ETAR. Similarly of the 37 specimens positive for Shh 92% (34/37) also expressed ETAR (p <0.0001). Similar correlation in Shh and ETAR expression was observed in metastatic lesions. Importantly, analysis of survival data from 43 patients shows Shh and ETAR expression correlates with poor prognosis of the PC patients. Overall our studies suggest a possible role of ET-axis in the pathobiology of PC.
Evolution of Pancreatico-Duodenectomy: Experience of 2055 Consecutive Resections
T. Hackert, U. Hinz, W. Hartwig, O. Strobel, M. Hassenpflug, J. Werner, M.W. Büchler. Dept. of Surgery, University of Heidelberg, Germany.
Background: The aim of the study was to analyze the evolution of pancreatico-duodenectomy (PD) within the last decade in a high-volume and teaching center for pancreatic surgery.
Methods: Patients undergoing PD at the author’s institution between 10/2001 and 09/2012 were identified from a prospective database. Indications, resection techniques, perioperative morbidity and mortality were analyzed in two-year intervals and statistically compared between the first and last study period.
Results: 2055 PD were performed. Initially, five surgeons performed all resections (average case number 27/year) compared to eight surgeons during the last period (27/year), reflecting the increased number of annual resections and the teaching intention. Indications showed an increase in IPMN (9.4% vs. 13.4%, p=0.05). Resections after neoadjuvant therapy remained unchanged at 6.8% vs. 6.7%. Vascular and multivisceral resections increased significantly (8.5% vs. 19.8% and 5.1% vs. 20.8%, p<0.0001). Classical PD (stomach resection) decreased from 23.0% to 16.3% (p=0.05). All other operations were pylorus-preserving PD or PD with pylorus resection (prPD). The rate of prPD was significantly higher during the last observation period (0% vs. 47.5%, p<0.0001). Surgical morbidity was 23.4% and 48.7%, respectively (p≤0.001). Interventional complication management was required in 3.0% vs. 15.6% (p<0.001). In-hospital mortality remained unchanged at 3.9%.
Conclusions: Recent PD evolution includes increasing rates of IPMN, vascular, and multivisceral resections and the introduction of prPD. The increasing morbidity can be attributed to the institutional teaching intention and the higher rate of complex resections. The constant mortality rate shows the importance of perioperative complication management to prevent severe adverse events.
Adenovirus-Delivered Interferon Sensitized Chemo- and Radiotherapy for Pancreatic Cancer
J. Han,1 Y. Miura,1 R. Shanley,2 X. Luo,2 K. Aoki,3 E. Greeno,1 S. M. Vickers,1 M. Yamamoto,1 J. Davydova,1. 1Surgery Department, 2Division of Biostatistics, University of Minnesota, Minneapolis, MN, 3National Cancer Center Research Institute, Tokyo, Japan.
Interferon-α (IFN) in conjunction with chemoradiotherapy has emerged as a promising treatment for pancreatic ductal adenocarcinoma (PDAC). However, despite encouraging survival results, utilization of this regimen has been impeded by systemic toxicity of IFN.
To overcome this challenge, we designed the oncolytic adenovirus expressing human or hamster IFN (OAd-IFN). We hypothesized that combining OAd-IFN with chemoradiation would surmount the major drawbacks of IFN-based regimens. The adenovirus-mediated tumor-selective expression of IFN will eliminate systemic toxicity of the cytokine, while massive IFN expression via replication-competent vector will yield an extended response.
Significance of the design strategy was exemplified through evaluating OAd-IFN as a single agent therapy. In vivo experiments were performed in immunodeficient mice bearing human PDAC and immunocompetent hamster bearing syngeneic pancreatic tumors. The addition of IFN and ADP expression to the OAd structure resulted in remarkably improved oncolysis. Therapeutic effect in immunocompetent hamsters was more evident than in immunodeficient mice, suggesting indirect antitumor effect of IFN as an immunomodulator. In hamster survival studies, a single systemic i.p. OAd-IFN injection exhibited significant improvement in survival rate in an extremely aggressive peritoneal dissemination model.
The multidrug analysis revealed that combination of OAd-IFN with chemotherapeutics (5FU, gemcitabine, cisplatin) and X-ray killed human and hamster pancreatic cancer cells significantly better than either of the single treatments. Furthermore, we established pancreatic tumors in immunocompetent hamsters and showed that combination of OAd-IFN with either 5FU or radiation (8, 20 Gy) resulted in remarkable tumor suppression and was significantly superior to radiation and 5FU alone or both of these combined. The triple-therapy (OAd-IFN+rad+5FU) outperformed all treatment groups. The evaluation of the survival rate showed statistically significant improvement in groups treated with dual (OAd-IFN+rad) and triple (OAd-IFN+rad+5FU) therapies versus conventional approaches (radiation or/and 5FU).
Our results support the impact of OAd-mediated IFN to sensitize chemotherapy and radiation for pancreatic cancer. This strategy may expand clinical use of the robust and promising IFN-based multimodal therapy to meet the pressing need for PDAC treatment.
Apelin Reduces Chronic Pancreatitis-induced Inflammation
S. Han, E.W. Englander, C. Rastellini, G.A. Gomez, G.H. Greeley, Jr. Department of Surgery, University of Texas Medical Branch, Galveston, TX.
Aim: The aim of this study was to define further the influence of apelin exposure on the inflammatory response during chronic pancreatitis (CP) in mice.
Background: Apelin is the endogenous ligand for the APJ receptor. Several studies show that apelin has a broad anti-inflammatory activity. Previous work from our laboratory demonstrates that apelin exposure reduces pancreatitis severity in mice with CP.
Methods: CP was induced in adult male mice by 5 hourly cerulein injections (50 μg/kg, IP; 3 days/week) for 3 weeks in wild-type (WT) and littermate apelin gene knockout mice (APKO). Exogenous apelin-13 (50 μg/mouse, IP, 2x /day, 7days) or vehicle was given during a 1-week recovery period following CP induction. Pancreata were harvested following the 1-week treatment period for measurement of myeloperoxidase (MPO) activity and macrophage inflammatory protein-1 (MIP-1α, MIP-1β) expression levels by real-time quantitative PCR analyses.
Results: Pancreatic MPO levels were elevated significantly (p< 0.05) in mice with CP. MPO levels were reduced significantly by apelin treatment (30-60%) in WT and APKO mice. Pancreatic MIP-1 expression levels were significantly higher in vehicle-treated APKO mice when compared to vehicle-treated WT mice (MIP-1α: WT + vehicle: 0.46±0.14 vs. APKO + vehicle: 1.08±0.33; MIP-1β: WT + vehicle: 0.45±0.17 vs. APKO + vehicle: 1.11±0.23). Apelin administration to either WT or APKO mice reduced MIP-1 expression levels 50-90% (MIP-1α: WT + apelin: 0.23±0.15; APKO + apelin: 0.13±0.03; MIP-1β: WT + apelin: 0.07±0.01; APKO + apelin: 0.07±0.02, p<0.05).
Discussion: Apelin replacement reduced pancreatic MPO activity and MIP-1(α, β) expression levels in CP mice. Results further support an anti-inflammatory role for apelin in the pancreas during CP.
Conclusion: Apelin treatment is a novel approach to reduce pancreatic inflammation in CP patients.
Anti-Diabetic Medications Do Not Affect Survival Following Surgical Resection for Pancreatic Cancer
P. Hart,1 R. Law,1 W. Bamlet,2 P. Burch,2 R. Frank,2 G. Petersen,2 K. Rabe,2 S. Chari,1. 1Division of Gastroenterology & Hepatology, 2Health Sciences Research, Mayo Clinic Rochester, MN.
Introduction: Previous studies have examined the effect of antidiabetic medication use on risk for developing pancreatic cancer (PaC), but not clinical outcomes. We analyzed the effect of these medications on tumor size, cancer stage, and postoperative survival in a cohort of patients who had undergone surgical resection.
Methods: We identified subjects enrolled in the Mayo Clinic Pancreatic Cancer SPORE registry from 2000 – 2010 who had cancer resection (n=488). Data regarding diabetes mellitus (DM) status and medication use were obtained. We evaluated the effect of antidiabetic medications on tumor size, cancer stage, and postoperative median survival using univariate and multivariate comparisons with Kaplan-Meier curves and Cox proportional hazards regression modelling.
Results: A total of 275 (56%) subjects undergoing cancer resection had DM preoperatively. Of the diabetic subjects, 88 (32%) reported taking at least one antidiabetic medication, with insulin (n=48) and metformin (n=43) being the most commonly used agents (both used in 15 patients). For subjects taking DM medications, tumor size (mean 3.6 cm for both groups) and cancer stage at presentation were similar compared to diabetics not on treatment (p =0.97 and 0.98, respectively). The median postoperative survival in those who used metformin (27.1 months) or insulin (25.3 months) was similar to that of diabetics not on treatment (23.8 months, p=0.62) and non-diabetics (26.4 months, p=0.88).
Conclusions: The use of antidiabetic medications in diabetic PaC subjects prior to surgical resection does not affect tumor size or cancer stage at the time of presentation. The use of metformin and/or insulin does not significantly change postoperative survival. Even though antidiabetic medications may influence the risk of developing cancer, they do not appear to affect clinical outcomes after diagnosis.
Does Intra-Tumoral Inflammation Predict Survival Following Pancreatic Cancer Resection?
P. Hart,1 T. Smyrk,2 W. Bamlet,3 S. Chari,1. 1Division of Gastroenterology & Hepatology, 2Department of Pathology, 3Health Sciences Research, Mayo Clinic Rochester, MN.
Introduction: The median survival of resected pancreatic cancer (PaC) is only 18-22 months with nearly a quarter dying within one year of surgery. Tumor-associated inflammation is known to contribute to cancer growth and progression. We hypothesize that tumor-associated inflammation is associated with early death following PaC resection.
Methods: We used the Mayo Clinic Pancreas Cancer SPORE database (2000-2010) to identify patients who had undergone surgical resection for PaC. For each case with early death (within 12 months), we consecutively selected 2 subjects who survived for >36 months. An experienced GI pathologist blinded to clinical outcomes reviewed all slides. Various inflammatory parameters were graded as either high or low on the basis of semiquantitative assessment, and compared using chi-squared and Fisher’s exact t-tests. Odds ratios for early death were calculated using logistic regression modeling.
Results: A total of 62 subjects selected for the study had available tumor specimens to review (21 cases, 41 controls). The groups were similar in regards to gender and age at diagnosis. The density of collagen, stroma, smooth muscle actin, lymphocyte, polymorphonuclear, macrophage (on CD68 stain), and mast cells on tumor specimens was similar between the two groups. The odds of early death were not increased in those with high density of inflammation for each of the findings (p>0.05 for all comparisons), with the exception of higher tumor grade (adjusted OR 201.2, 95% CI 9.4-999.9).
Discussion: Histologic evaluation of the density of inflammatory cells does not identify those with increased odds of early death following PaC resection. Conversely, high tumor grade was associated with early death, even after adjusting for tumor size, margin status, and lymph node status. The composition of inflammatory cells adjacent to pancreatic cancers is not associated with survival following PaC resection.
Diagnostic Performance of Endoscopic Ultrasound-Guided Pancreatic Trucut Biopsy in Autoimmune Pancreatitis
P. Hart,1 L. Zhang,2 C. Lee,2 K. Notohara,2 L. Fujii,1 S. Chari,1 M. Levy.11Division of Gastroenterology and 2Department of Pathology, Mayo Clinic Rochester, MN.
Introduction: Pancreatic histology can definitively diagnose autoimmune pancreatitis (AIP) without need for collateral evidence. However, previous studies suggest that pancreatic core biopsies have a low diagnostic yield. We evaluated the diagnostic sensitivity and prevalence of individual histologic features of endoscopic ultrasound-guided trucut biopsy (EUS-TCB) in AIP.
Methods: We reviewed the Mayo Clinic, Rochester EUS and pathology databases from 2002-2012 to identify 59 patients diagnosed with AIP based on HISORt criteria that underwent EUS-TCB (type 1 (n=37), type 2 (n=19), or type indeterminate (n=3)). Biopsy specimens were independently reviewed by 2 GI pathologists. We evaluated the prevalence of individual features, and diagnostic rate and concordance between pathologists for diagnosis of AIP and subtypes.
Results: A mean of 2 EUS-TCB passes were made per patient, giving a total core specimen length of 14.5 mm with a mean of 3 ducts and veins. Lymphoplasmacytic inflammation, storiform fibrosis, and obliterative phlebitis were identified in 95%, 42%, and 16% of all patients, respectively. Compared to type 2 AIP, in type 1 AIP the density of lymphoplasmacytic inflammation and IgG4+ plasma cells/hpf, and presence of storiform fibrosis was higher (p<0.01). The prevalence of GEL’s, perivenulitis, eosinophils, acinar neutrophils, and foci of lymphoid aggregates was similar. Pathologists made a histologic diagnosis of AIP in 84% and 76% of patients and inter-observer agreement for the AIP subtype was fair (κ=0.52).
Discussion: Core pancreatic tissue obtained using EUS-TCB allows for a definitive diagnosis of AIP to be made in the majority of patients. Dense lymphoplasmacytic inflammation, storiform fibrosis, and IgG4+ plasma cells are more frequently seen in type 1 AIP. Diagnostic agreement between pathologists for AIP subtype is fair, and may be improved by use of threshold values for its histologic features.
Total Pancreatectomy for Cancer: Renaissance of an Unpopular Operation
W. Hartwig, A. Gluth, T. Hackert, U. Hinz, O. Strobel, A. Hector, M.W. Büchler, J. Werner. Department of General Surgery, University of Heidelberg, Germany.
Introduction: Total pancreatectomy may be required in locally advanced or centrally located pancreatic neoplasms to achieve complete cancer clearance, but available data on short- and long-term results is limited. The primary aim of the study was to assess long-term survival in a large cohort of total pancreatectomies. Identification of risk factors for perioperative morbidity and mortality and long-term quality of life were secondary endpoints.
Methods: 602 consecutive total pancreatectomies were performed between 10/01 and 09/12 at the author’s institution and prospectively documented. 431 patients with pancreatic tumors and elective primary resections were analyzed. Long-term outcome was assessed using Kaplan-Meier and quality-of-life analysis (EORTC-QLQ-C30 and PAN26). Uni- and multivariate analysis was performed to identify perioperative risk factors.
Results: Malignant tumors were present in 86% of cases. Vascular resections and extended total pancreatectomies resections were performed in 47% and 19%, respectively, with arterial resections in 15% of cases. Overall 30-day and in-hospital mortality rates were 3.7% and 7.9 %, respectively. In malignant disease, median and 5-year survival was good for standard total pancreatectomies (30.9 months and 23.5%, respectively), and was significantly impaired after vascular resections (p<0.001). Long-term global quality-of-life was comparable to a matched healthy control group. Arterial, porto-mesenteric, and extended resections were associated with increased surgical morbidity (p≤0.02). Perioperative mortality was significantly increased only after arterial resections (p=0.001).
Conclusions: Standard total pancreatectomy, if needed, is associated with good long-term outcome in pancreatic cancer. Marked surgical morbidity and impaired survival associated with arterial resections reflect the invasiveness of extended total pancreatectomies and the underlying advanced malignant disease.
Autophagy in BxPC-3 Human Pancreatic Cancer Cells is Similar to PANC-1 When Treated With Anticancer Drugs and Inhibitors of Autophagy
D. Hashimoto,1,2,3 M. Bläuer,1,2 J. Sand,1,2 M. Hirota,3 J. Laukkarinen,1,2. 1Tampere Pancreas Laboratory and 2Department of Gastroenterology and Alimentary Tract Surgery, Tampere University Hospital, Tampere, Finland, and 3Department of Gastroenterological Surgery, Kumamoto University Graduate School of Medical Sciences, Kumamoto, Japan.
Objectives: Autophagy is a regulated process of degradation and recycling of cellular constituents, participating also in cell-death mechanisms. Previously we have reported autophagy in pancreatic cancer cell line PANC-1 treated with anticancer drugs and inhibitors of autophagy. As some controversy exists about the effect of autophagy in pancreatic cancer cell lines, the aim of this study was to investigate the role of autophagy in another human pancreatic cancer cell line, BxPC-3.
Materials and Methods: BxPC-3 and PANC-1 were incubated with an autophagy inhibitor (chloroquine), two anticancer drugs (5-FU and gemcitabine) and a combination of both drug types. The autophagy status was examined by Western blot analysis of the autophagic marker LC3-II. The effect of the drugs on cell growth was also assessed.
Results: Western blot analysis of LC3-II showed that autophagy is activated in BxPC-3 and PANC-1 under basal culture conditions. This was suppressed by chloroquine. Both 5-FU and gemcitabine induced autophagy in BxPC-3 and PANC-1. When chloroquine, which is known to inhibit the degradation of autophagosomes, was added on the cells together with these anticancer drugs, stronger LC3-II bands were detected. In cell growth experiments, chloroquine greatly increased the growth-inhibiting effects of 5-FU and gemcitabine. Chloroquine alone suppressed cell growth less than in combination with either anticancer drug.
Conclusions: In both BxPC-3 and PANC-1 cells, autophagy contributes to cell growth and has a cytoprotective effect against anticancer drugs (5-FU and gemcitabine). Chloroquine increases the cytotoxicity of 5-FU and gemcitabine by inhibiting autophagy. Possible combination therapy of these anticancer drugs and chloroquine should be further studied.
A High Fat, High Calorie Diet Leads to Inflammation of the Pancreas and Adipose Tissue in the KrasG12D Mouse Model
K.M. Hertzer, A. Moro, D.W. Dawson, O.J. Hines, G. Eibl. Department of Surgery, David Geffen School of Medicine at UCLA, Los Angeles, CA.
Epidemiologic evidence indicates that obesity increases the risk of cancer. However, the driving mechanisms in pancreatic cancer (PaCa) are poorly understood. Our initial studies showed that a high fat, high calorie diet (HFCD) accelerates neoplasia in KrasG12D mice, and that pancreata of HFCD-fed animals had robust signs of inflammation with increased inflammatory cells, COX-2 expression, and desmoplasia. The aim of the current study was to further investigate the mechanisms of PaCa promotion by a HFCD and specifically to characterize the effects of the HFCD on pancreatic and visceral adipose tissue inflammation.
Methods and Results: Both wild type (WT) and KrasG12D mice were allocated to a HFCD or control diet (CD) for 10 weeks. Animals were weighed weekly and imaged at the end of the experiment. Tissues were harvested at sacrifice for histological and molecular analysis. All HFCD-fed mice gained more weight and increased their visceral adipose tissue volume by ∼2-fold. HFCD-fed KrasG12D mice showed an increase in multiple cytokines/chemokines, i.e. G-CSF, M-CSF, MCP-1, LIF, LIX, IL-6 in the pancreas as measured by multiplex assay. Furthermore, peri-pancreatic adipose tissue revealed signs of increased inflammatory cell infiltration in HFCD- vs. CD-fed mice (females: 6.3±1.4 vs. 1.1±0.5 and males: 4.2±1.8 vs. 0.3±0.1 inflammatory structures/HPF).
Conclusion: Our results demonstrate that a HFCD leads to obesity, inflammation in the pancreas and peri-pancreatic adipose tissue, increased infiltration of inflammatory cells, and changes in inflammatory cytokine expression. These changes may underlie the acceleration of pancreatic neoplasia in HFCD-fed KrasG12D mice. Furthermore, our results suggest a critical role of the peri-pancreatic adipose tissue as an important source of inflammatory signals thereby promoting obesity-associated PaCa development and growth.
Mitochondria Regulate Trypsinogen Activation in Pancreatitis
C.T. Holland, I.V. Odinokova, A. Lugea, N. Shalbueva, O.A. Mareninova, I. Gukovsky, A.S. Gukovskaya. VA Greater Los Angeles Healthcare System and University of California, Los Angeles, CA.
Background & Aims: Loss of mitochondrial membrane potential (ΔΨm) is an early pathologic response of pancreatitis leading to acinar cell death. Here we investigate the effects of mitochondrial inhibitors on trypsinogen activation, a hallmark of acute pancreatitis.
Methods: We measured the effects of mitochondrial inhibitors with different mechanisms of action on changes in ΔΨm, ROS, ATP, free cytosolic Ca2+, trypsin and cathepsin B (CatB), induced in mouse pancreatic acinar cells by 30-min incubation with supramaximal (100 nM) CCK-8.
Results: Complex I inhibitors rotenone and DPI, complex III inhibitor antimycin, protonophore FCCP, ATP synthase inhibitor oligomycin (at the appropriate micromolar concentrations), as well as inhibition of mitochondrial respiration with nitrogen, all completely prevented trypsinogen activation. This was associated with a marked decrease in processing and activity of CatB, a key mediator of trypsinogen activation. All the inhibitors decreased cellular ATP by 15-25%; there was no significant decrease in cell viability with these manipulations. Further, the CCK-induced trypsin activity was prevented by iodoacetate, an inhibitor of glycolysis, while it was 40% increased by pyruvate, a mitochondrial substrate. The results indicate that CatB-mediated trypsinogen activation is highly sensitive to ATP level. We did not find the involvement of other possible mechanisms, such as depolarization or changes in ROS or Ca2+. For example, trypsinogen activation was prevented with both FCCP, which fully dissipated ΔΨm, and oligomycin, which slightly increased ΔΨm; with rotenone, which increased the mitochondrial ROS, and FCCP, which blocked ROS production. Moreover, a specific inhibitor of mitochondrial ROS, MitoQ, had almost no effect on trypsin activity.
Conclusion: The results indicate critical energy-dependence of trypsinogen activation and reveal a novel role for mitochondria in the mechanism of pancreatitis.
ERK Activation is Required for Pancreatic Regeneration After Pancreatitis
B.J. Holtz,1 L.E. Watkins,1 S.J. Korpics,1 J.A. Williams,1,2. Departments of 1Molecular and Integrative Physiology and 2Internal Medicine, University of Michigan Ann Arbor, MI.
Introduction: Pancreatic regeneration following acute pancreatitis requires dedifferentiation, proliferation, and redifferentiation and is facilitated by CCK and insulin. ERK signaling has been shown to be involved in many proliferative processes including pancreatic adaptive growth but whether ERK is necessary for pancreatic regeneration following pancreatitis is unknown.
Aim: To evaluate the role of ERK signaling in pancreatic regeneration following pancreatitis.
Methods: Exocrine injury was induced by 8 i.p. doses of caerulein injections, 1 hour apart, over 2 consecutive days in male ICR mice. PD0325901 (PD), a novel Mek inhibitor, was given by oral gavage or mixing with powdered chow. Inhibitors were given 24 h following caerulein injections and tissue was harvested at 7 d to study pancreatic acinar cell recovery. Pancreatic wet weight, DNA, RNA and total protein was measured. Mitogenesis and digestive enzymes were measured by western blot, IHC, and IF at 24 h and 7 d.
Results: The peak of exocrine injury was observed at 24 h after the last dose of caerulein when pancreatic digestive enzyme concentration was 40-60% of controls, TUNEL staining was increased, and protein, DNA, and RNA were reduced. Regeneration occurred at 7 d when acinar cell morphology and pancreatic digestive enzyme concentration were near control levels. Ki-67 and cell-cycle proteins were activated at 24 h. Administration of PD caused a 44% reduction in pancreatic mass and a 30-50% inhibition of protein, DNA, and RNA content at 7 d. Pancreatic amylase and elastase concentration was ∼70% of controls. Morphologically, redifferentiation of pancreatic acinar cells was not affected by ERK inhibition.
Conclusion: Repression of ERK caused an inhibition of recovery to normal pancreatic mass, DNA, RNA, and protein content. ERK signaling is necessary for proliferation and pancreatic mass recovery following pancreatitis.
Follistatin-like 1 Regulates Epithelial to Mesenchymal Transition in Pancreatic Cancer
K. Horioka, K. Ohuchida, D. Eguchi, K. Fujiwara, S. Akagawa, T. Tanaka, M. Sada, K. Mizumoto, M. Tanaka. Department of Surgery and Oncology, Graduate School of Medical Sciences, Kyushu University, Fukuoka, Japan.
Background: In a previous study using the highly metastatic variants of pancreatic cancer cells lines SUIT-2 and PANC-1, we have identified some up-regulated genes relative to parental cells. Follistatin-like 1 (FSTL1) - one of those up-regulated genes - is a secreted glycoprotein belonging to the BM-40/SPARC/osteonectin family. It was reported that FSTL1 has a tumor suppressive function in some cancers, such as lung, endometrial, and ovarian cancer. Here we analyzed the role of FSTL1 on the epithelial to the mesenchymal transition (EMT) process in pancreatic cancer.
Methods: Knockdown of FSTL1 using small interfering RNA (siRNA) was performed in four human pancreatic cancer cell lines and, then, the effect of FSTL1 on cell morphology, proliferation, migration, and invasion ability was investigated. Changes in mRNA and protein level expression, especially those related with the EMT process were evaluated by qRT-PCR, western blotting, and immunofluorescence. Additionally, cell surface markers were checked by flow cytometry analysis in order to identify the effect of FSTL1 on acquiring stem cell-like properties.
Results: After FSTL1 knockdown, the cell morphology was altered to a spindle-shaped mesenchymal appearance. Migration and invasion activity of FSTL1 knockdown cells were significantly increased. However, the proliferation rate of FSTL1 knockdown cells was reduced. FSTL1 knockdown cells expressed low levels of the epithelial markers E-cadherin and cytokeratin19. FSTL1 knockdown decreased the expression of the stem cell markers CD133 and CD44 in some of the pancreatic cancer cell lines studied.
Conclusion: We conclude from these results that FSTL1 plays an important role in the EMT induction and in acquiring stem cell-like properties. These studies suggest that FSTL1 would be a potential therapeutic target against pancreatic cancer metastasis.
Rab3D and Rab27B Cooperatively Regulate Secretion in Mouse Pancreatic Acini
Y. Hou, X. Chen, J.A. Williams. Department of Molecular and Integrative Physiology, University of Michigan, Ann Arbor, MI.
Background: Previous studies have shown that the small G-proteins Rab27B and Rab3D are present on pancreatic zymogen granules and participate in digestive enzyme secretion. However, the relation between these two small GTPases and the mechanism, by which they regulate secretion by binding to downstream effectors are poorly understood. We identified MyosinVc, Slp1 and Slp4 as binding proteins of these two small G-proteins.
Aim: To investigate the interaction between the small G-proteins Rab27B and Rab3D, and their binding proteins.
Methods: Isolated pancreatic acini were prepared from mouse pancreas by collagenase digestion. Over-expression of Rab27B or Rab3D mutant proteins, full-length Myosin Vc and MyosinVc tail was mediated by adenovirus. Effects of over-expression of the mutant proteins on GTP-bound state of Rab27B and Rab3D were assessed by GST-pulldown assay. Protein interaction was determined by co-immunoprecipitation or GST-pulldown assay.
Results: 46.7% ± 3.7% of Rab27B proteins are in GTP-bound active form at basal level in isolated pancreatic acinar cells. Over-expression of Rab3D WT and Rab3D QL decreased the endogenous GTP-Rab27B level, while the GTP-bound state of Rab3D was not affected by the overexpression of Rab27B mutants. Rab27B and Rab3D can both bind to Slp1, Slp4 and MyosinVc, among which Slp1 can directly bind to Rab27B and Rab3D. Overexpression of Slp1 inhibited Myosin Vc binding to Rab27B or Rab3D. The interaction between Rab27B and the three binding proteins was decreased by CCK treatment, while the interaction between Rab3D and the three binding proteins was not affected. Rab27B can interact with actin, presumably through the interaction with MyosinVc.
Conclusions: Rab3D functions upstream of Rab27B in mediating secretion. Rab27B and Rab3D regulate both trafficking and docking of zymogen granules, with Rab3D acting in a more constitutive and Rab27B acting in a regulated manner.
Fatty Acid Ethyl Ester Synthase Inhibition Ameliorates Ethanol-Induced Ca2+-Dependent Mitochondrial Dysfunction and Acute Pancreatitis
W. Huang,1,2 D.M. Booth,2 M.C. Cane,2 M. Chvanov,2 M.A. Javed,1,2 V.L. Elliot,1 H. Dingsdale,2 N. Cash,2 Y. Li,1 W. Greenhalf,1 R. Mukherjee,1,2 O.H. Petersen,3 M. Jaffar,4 B.S. Kaphalia,5 A.V. Tepikin,2 R. Sutton,1,2 D.N. Criddle,1,2. 1NIHR Liverpool Pancreas Biomedical Research Unit, RLUH, Liverpool, UK; 2Department of Cellular and Molecular Physiology, University of Liverpool, Liverpool, UK; 3University of Cardiff, Cardiff, UK; 4Morvus Technology Limited, Abergavenny, UK; 5University of Texas, Galveston, TX.
Introduction: Non-oxidative metabolism of ethanol (NOME) produces fatty acid ethyl esters (FAEEs) via carboxylester lipase (CEL) and other enzyme action implicated in mitochondrial injury and acute pancreatitis (AP). The relative importance of oxidative and non-oxidative pathways in mitochondrial dysfunction, pancreatic damage and alcoholic AP was investigated, and whether deleterious effects of NOME are preventable.
Methods: Cytosolic calcium ([Ca2+]C), NAD(P)H, mitochondrial membrane potential and activation of cell death pathways were examined in murine pancreatic acinar cells in response to ethanol and/or palmitoleic acid (POA) in the presence/absence of 4-methylpyrazole (4-MP) to inhibit oxidative metabolism (OME). An in vivo model of alcoholic AP (intraperitoneal ethanol/POA) was developed to assess effects of manipulating alcohol metabolism.
Results: Inhibition of OME with 4-MP converted low ethanol/POA-induced predominantly transient [Ca2+]C rises to sustained elevations, with concurrent mitochondrial depolarization, NAD(P)H decrease and cellular necrosis in vitro, effects prevented by the CEL inhibitor 3-benzyl-6-chloro-2-pyrone (3-BCP). 3-BCP also significantly inhibited pancreatic FAEE rises and ameliorated pancreatic damage and inflammation in the ethanol/POA in vivo experimental model.
Conclusion: A low ethanol/fatty acid combination that did not exert deleterious effects per se became toxic when OME was inhibited. In vitro and in vivo damage was markedly inhibited by CEL blockade, indicating potential for specific therapy of alcoholic AP based on inhibition of FAEE generation.
L-Histidine- but not L-Arginine-Induced Acute Pancreatitis Involves Cyclophilin D-Dependent Opening of Mitochondrial Permeability Transition Pore
W. Huang,1,3 M. Chvanov,1,2 L. Wen,1,3 M.A. Javed,1,2 T. Jin,1,3 A.V. Tepikin,2 D.N. Criddle,2 R. Sutton1. 1NIHR Liverpool Pancreas Biomedical Research Unit, Royal Liverpool University Hospital, Liverpool, UK; 2Deparment of Molecular and Cellular Physiology, University of Liverpool, Liverpool, UK; 3Sichuan Provincial Pancreatitis Centre, West China Hospital, Sichuan University, Chengdu, China.
Background: Intraperitoneal (IP) administration of large doses of amino acids has been shown to induce acute pancreatitis (AP) in rodents, but mechanisms have not been determined.
Objectives: To determine the role of cyclophilin D-dependent opening of the mitochondrial permeability transition pore (MPTP) in amino acid-induced AP.
Methods: Freshly isolated murine wild type (wt) or cyclophilin D knockout (Ppif-/-) pancreatic acinar cells (PACs) were treated with amino acids and cell death assay undertaken with propidium iodide (PI). Cytosolic Ca(2+) concentration was determined by Fluo-4 with confocal microscopy. In vivo, mice received two hourly IP injections of L-Arginine, L-Ornithine, L-Citrulline, or L-Histidine (2.0 or 4.0 g/kg), controls receiving starch solution of identical osmolarity. Mice (6 per group) were sacrificed at designated times to assess AP severity.
Results: 20 mM L-Arginine, L-Citrulline and L-Ornithine induced PI uptake in wt and Ppif-/- PACs at similar rates, whereas 20 mM L-Histidine induced less PI uptake in Ppif-/- cells. Ca(2+) rises in wt PACs were observed in different proportions when responded to individual amino acid. In vivo, starch or low dose amino acid injections did not induce AP. In wt mice L-Ornithine caused rapid mortality, while L-Citrulline caused only pancreatic edema; both L-Arginine and L-Histidine caused AP. AP in Ppif-/- mice induced by L-Histidine was significantly reduced compared to wt, but not following L-Arginine.
Conclusion: A new model of murine experimental AP was developed by IP injection of high dose L-Histidine. L-Histidine-induced murine PAC injury involves Ca(2+) signalling and cyclophilin D-dependent opening of MPTP.
Clinical Significance of GNAS Mutation for Invasive Pancreatic Carcinoma Distinct From/Derived From Intraductal Papillary Mucinous Neoplasm (IPMN) of the Pancreas
N. Ideno,1 T. Ohtsuka,1 K.Tamura,1 T. Aso,1 K. Ohuchida,1 S. Takahata,1 Y. Oda,2 K. Mizumoto,1 M. Tanaka,1. 1Departments of Surgery and Oncology and 2Anatomic Pathology, Graduate School of Medical Sciences, Kyushu University, Fukuoka, Japan.
Background: Although GNAS mutations are specific for IPMN, IPMN without GNAS mutation has not been well characterized. The aims of this study were to investigate the GNAS mutational status in IPMN with and without distinct pancreatic ductal adenocarcinoma (PDAC) and IPMN with an associated invasive carcinoma and to evaluate the significance of GNAS mutation analysis of duodenal fluid (DF) in patients with IPMN.
Methods: Clinicopathologic features of 62 patients with IPMN including 18 with distinct PDAC and 20 with IPMN with an associated invasive carcinoma were reviewed. GNAS mutational status of the tissue samples was assessed by cycle sequencing, high-resolution-melt-curve analysis, and competitive allele specific TaqMan PCR (cast PCR). DF samples were available in 24 patients, and GNAS mutation in DF was investigated by cast PCR.
Results: The rate of GNAS mutations in IPMN with distinct PDAC was significantly lower than that in IPMN without PDAC (5/18, 28% vs 19/24, 79%, P=0.002). By multivariate analysis, the absence of GNAS mutation reached statistical significance as an independent predictor of the presence of distinct PDAC (P=0.018). GNAS mutations were not detected in 12 of 20 IPMNs with associated invasive carcinomas (60%). In DF samples, GNAS mutations were found in 11 of 24 (46%) samples, and the mutant status was consistent with that of tissue samples in 22 of 24 (92%). Of note, in 13 patients whose DF exhibited no GNAS mutation, 5 (38%) had PDAC concomitant with IPMN, and 3 (23%) had IPMN with an associated invasive carcinoma.
Conclusions: The absence of GNAS mutations might be a predictor for the development of invasive pancreatic carcinoma distinct from IPMN, and mutational analysis of GNAS using DF might be clinically useful for the selection of high risk patients.
Inhibition of Pancreatitis-Associated Cell Death Using a Human Pancreatic Acinar Cell Model
M. A. Javed,1,2 R. Mukherjee,1 L. Wen,1 W. Huang,1 M. Chvanov,1, 2 M. Awais,1 D. N. Criddle,1,2 A. Tepikin,2 M. Raraty,1 P. Ghaneh,1 J. P. Neoptolemos,1. R. Sutton,11NIHR Liverpool Pancreas Biomedical Research Unit, Royal Liverpool University Hospital and University of Liverpool and 2Department of Cellular and Molecular Physiology, University of Liverpool, Liverpool, UK.
Introduction: Inhibition of acute pancreatitis (AP) in rodents as a model for human dug development has largely not been successful. We sought to develop an approach adding selective testing of potential drugs for AP using freshly isolated human PACs.
Aims: To evaluate effects of the cyclophilin inhibitors CyclosporinA (CsA) or Debio-025 on human PACs exposed to taurolithocholate sulphate (TLCS).
Methods: Human PACs were isolated by collagenase digestion, mechanical dispersion and low-speed centrifugation from normal pancreatic tissue gifted by consenting surgical patients. Fluorescent confocal microscopy was used to assess mitochondrial membrane potential (ΔΨm measured with 50 nM TMRM, excitation 543 nm, emission >550 nm) and necrotic cell death pathway activation (blind cell uptake count with 1 μM propidium iodide, excitation 488 nm, emission 630-693 nm).PACs were incubated with 500μM TLCS for 30 min in the presence or absence of 10 μM CsAor 100 nM Debio-025 to test the effect of cyclophilin inhibition.
Results: CsAor Debio-025 preserved ΔΨm (P<0.05) and prevented propidium iodide uptake (P<0.05) compared to control cells in response to 500 μM TLCS. ΔΨm and cell death profiles of human PACs were similar to murine PACs in response to this toxin.
Conclusion: CsA and Debio-025 preserved ΔΨm and reduced necrosis in human PACs following TLCS, likely through inhibition of cyclophilin D. Testing of compounds with human PACs may promote evaluation of potential drugs for AP.
TRO40303 Reduces Mitochondrial Injury and Ameliorates Experimental AP
M.A. Javed,1,2 L. Wen,1,2 M. Awais,1,2 M. Chvanov,1,2 P. Ghaneh,1 C. Halloran,1 J.P. Neoptolemos,1 M. Raraty,1 T. Bordet,3 M. Michaud,3 S. Schaller,3 R. Pruss,3 A. Tepikin,2 D.N. Criddle,1,2 R. Sutton,1. 1NIHR Liverpool Pancreas Biomedical Research Unit, Royal Liverpool University Hospital, 2Department of Cellular and Molecular Physiology, University of Liverpool, UK, 3TROPHOS, Marseille, France.
Background & Aims: The mitochondrial permeability transition pore (MPTP) contributes to mitochondrial dysfunction in acute pancreatitis (AP). 3,5-seco-4-nor-cholestan-5-one oxime-3-ol (TRO40303), which binds to the mitochondrial translocator protein 18 kDa (TSPO), inhibits MPTP opening and reduces cardiac ischaemia-reperfusion injury, is in a phase II trial for prevention of myocardial reperfusion injury. We investigated the effects of TRO40303 on toxic injury of murine and human pancreatic acinar cells (PACs) and in murine AP models.
Methods: Changes in mitochondrial membrane potential (ΔΨM; TMRM), cytosolic Ca2+ (Ca2+C; Fluo-4) and necrotic cell death pathway activation (propidium iodide; PI) in response to bile acid (500 μM TLCS) were examined in freshly isolated murine and human PACs by confocal microscopy. AP was induced by cerulein (50 μg/kg/h IP x 7) or intraductal infusion of TLCS (50 μL 3 mM). TRO40303 (3 mg/kg) was given IP after the 3rd cerulein injection (sacrifice 12 h after 1st) or 1 h after TLCS (sacrifice at 24 h). AP was assessed by standard biomarkers and blinded histopathology.
Results: TRO40303 at 1 μM, 3 μM and 10 μM prevented loss of ΔΨM induced by 500 μM TLCS and improved Ca2+C clearance in PACs. TRO40303 reduced necrotic cell death pathway activation in a dose dependent manner with 50% reduction at 10 μM in murine PAC (p<0.05) and reduced necrosis in human PAC (p<0.01). TR040303 significantly reduced serum amylase, pancreatic trypsin, pancreatic and lung myeloperoxidase and histopathology scores in CER-AP and TLCS-AP.
Conclusion: TRO40303 protects mitochondria, reduces necrotic cell death pathway activation and ameliorates the severity of experimental AP. TRO40303 is a candidate drug for human AP.
Beyond Antioxidant Therapy for Chronic Pancreatitis: Systematic Search for Pharmacologic Disease-Modifiers With Evidence of Translational Potential
S. Jegatheeswaran,1 A.K. Siriwardena,1. 1Hepatobiliary Surgery Unit, Manchester Royal Infirmary, Manchester, UK.
Introduction: ANTICIPATE, a randomized, double-blind, placebo-controlled trial of antox (Pharmanord, UK) in chronic pancreatitis (CP) reported a primary endpoint of no difference in clinic pain scores between intervention and placebo. Although at least one other study is still to report, the focus of antioxidant therapy now moves to better identification of target patient populations and to the drug development pipeline. There has been no development of the n-acetylcysteine, vitamin C & selenium-based antox for over 30 years and thus the focus of this study is to undertake a systematic search of reports of drugs with antioxidant-related properties with evidence of translational potential.
Methods: Computerized searches of Pubmed and the web of knowledge™ using the terms “antioxidant” and “experimental chronic pancreatitis” were undertaken with Mesh headings. Studies on acute pancreatitis were excluded. Studies were retained if they examined CP with a prolonged study course. The initial search of 332 articles was reduced to a final population of 9.
Results: There were 8 rat studies and 1 murine model. Modes of induction of CP were: cyclosporine A/caerulein injection, intraductal trinitrobenzene sulfonic acid or dibutylin chloride. Median (range) time course (induction of CP to sacrifice) was 28 (15-56) days. Interventional agents included: α-tocopherol, taurine, pravastatin, ascorbic acid, vitamin E and one compound methionine, selenium tocopherol study. All report reduction in pancreatic injury and fibrosis. Taurine, pravastatin and Tocopherol also ameliorated weight loss.
Conclusion: Experimental models of CP remain relatively simplistic and do not capture the complexities of the clinical disease. Although a range of interventions with anti-inflammatory and anti-oxidant properties modulate injury there is as yet no clear “stand-out” drug to justify the cost of clinical phase I/II evaluation.
iTRAQ Reveals a Serum Marker Significantly Altered in Patients up to 24 Months Prior to Diagnosis With Pancreatic Cancer
C. Jenkinson,1 V. Elliott,1 D. O’Brien,2 A. Evans,1 R. Sutton,1 B. Lane,1 U. Menon,2 S. Apostolidou,2 E. Fourkala,2 I. Jacobs,3 S.P. Pereira,2a W. Greenhalf,1 J. Timms,2 J.P. Neoptolemos,1 E. Costello,1. 1NIHR Liverpool Pancreas Biomedical Research Unit, UK; 2Institute for Women’s Health/2aInstitute for Liver and Digestive Health, University College London, UK, 3University of Manchester, UK.
Aim: To determine serum biomarkers for earlier diagnosis of pancreatic cancer.
Methods: Two sample sets, divided into discrete discovery and validation sets, were used from: 1) patients at diagnosis of pancreatic cancer (PDAC), chronic pancreatitis, benign biliary disease or healthy subjects from Liverpool (n=172 total); 2) participants of the UK Collaborative Trial of Ovarian Cancer Screening (UKCTOCS, www.ukctocs.org.uk) up to 4 years prior to diagnosis of PDAC plus time-matched controls (N=218 total). Pooled discovery samples were labelled with isobaric Tags for Relative and Absolute Quantification (iTRAQ). Validation was by mass spectrometry-based Multiple Reaction Monitoring (MRM), two peptides per MRM, two MRM transitions per peptide. Three aliquots of each sample were each analyzed in duplicate. CA19-9 levels were measured by ELISA.
Results: A total of 225 proteins were detected. A protein dubbed LivT (patent pending), chosen as it was unaffected by biliary obstruction, was downregulated in validation PDAC cases versus healthy controls (p<0.01). In UKCTOCS validation cases, LivT was significantly downregulated in PDAC cases versus controls in the time periods 0-6 (p<0.009), 6-12 (p<0.001) and 12-24 (p<0.004) months prior to diagnosis. Receiver operating characteristic (ROC) analysis of combined LivT and CA19-9 serum levels yielded curve areas (AUC) of 0.83 for PDAC cases (n=46) v controls (n=46) in the 0-12 month time period prior to diagnosis, and AUC=0.74 in the 12-24 month time period prior to diagnosis (n=33 cases and 33 controls).
Conclusions: This study shows the potential of serum biomarkers for pre-symptomatic diagnosis of PDAC.
The Beginning of the End of Nihilism in Pancreatic Ductal Adenocarcinoma (PDAC)?
K. M. Johnson,1 M.A. Firpo,1,3 K.M. Boucher,2,3 C.L. Scaife,1,3 S.J. Mulvihill,1,3. Departments of 1Surgery and 2Oncological Sciences, University of Utah School of Medicine, and 3Huntsman Cancer Institute, Salt Lake City, UT.
Introduction: PDAC has one of the highest death rates of any cancer. Because diagnosis of patients with late stage disease is essentially a death sentence, some clinicians, patients, and families have a nihilistic attitude regarding treatment. We sought to evaluate whether treatment aggressiveness impacts survival for patients with PDAC.
Methods: We collected and analyzed data for PDAC cases using both the SEER Research Database (2004-2010, N=33,472) as well as our own experience (2001-2012, N=704). Gender, age, survival, initial staging, and treatment (resection, radiation) information was abstracted. Additional information regarding chemotherapy was available for our local experience. Contingency analyses and Cox modeling were performed.
Results: In adjusted models using SEER data, resected stage I cases had a median survival of 36 months compared to 7 months for cases that were recommended but refused resection (P<0.0001). Though nodal metastasis is weighted heavily in current TNM staging, IIB cases had longer median survival than IIA cases. In our experience, a greater proportion of IIB cases were resected (P<0.0001) while IIA cases received more neoadjuvant chemotherapy (P=0.04). Causes of potentially resectable cases not undergoing surgery included progression during neoadjuvant therapy 64%, medical comorbidity 20%, patient choice 5%, or unknown 11%. Greater than 50% of stage IV cases in our experience did not receive optimal therapy. Stage IV cases that received any treatment had a median survival of 8 months compared to 2 months for untreated cases (P<0.0001).
Conclusion: Despite the nihilism associated with PDAC diagnosis, our analyses suggest that aggressive treatment improves survival. To reach a goal of doubling of median survival by 2020, we should renew focus on optimizing delivery of effective therapy, including surgery and chemotherapy.
Innumerable Pancreatic Cystic Lesions in a Patient With Lynch Syndrome
T. Kachaamy, R. Pannala, D. Faigel. Department of Gastroenterology, Mayo Clinic, Phoenix, AZ.
An asymptomatic 52-year-old lady was found to have innumerable pancreatic cysts on abdominal CT scan performed during pre-renal transplant work up. Her end-stage renal disease was secondary to scleroderma. She had ovarian cancer at the age of 47 treated with surgery and adjuvant therapy. Her family history was positive for colon and pancreatic cancer in her father at the age of 47 and 69 respectively and colon cancer in her niece at the age of 24. She had no history of pancreatitis. A colonoscopy five years ago was notable for adenomatous polyps though details were not available. MRI of the abdomen confirmed multiple cystic lesions throughout the pancreas with a minimally dilated pancreatic duct. An Endoscopic ultrasound revealed a dominant cyst measuring 16 × 11 mm; there was no mural nodularity. Cyst fluid CEA and amylase were respectively 1,121 ng/ml and 299,500 u/l, suggestive of a side branch intraductal papillary mucinous neoplasm (IPMN). Because of her personal and family history of cancer, we repeated her colonoscopy and she was found to have an ascending colon adenocarcinoma. Genetic testing confirmed it to be colonic primary in origin. Her cancer tested positive for microsatelite instability with loss of MLH1/PMS2. Her family tested positive for germline mutation for Lynch syndrome. While multiple pancreatic cysts in patient with IPMN are common, the finding of innumerable cysts is rare and has never been reported in a patient with Lynch syndrome. The 2012 consensus guidelines recommend management of multicystic IPMNs similar to unifocal IPMN with the acknowledgement that the threshold for total pancreatectomy might be lowered as they might be at increased risk of cancer. A recent study found that patients with 20 or more cysts had 32% chance of invasive carcinoma or high grade dysplasia as compared to 8% in patients with fewer than 20 cysts. The true risk in patients with multiple pancreatic cysts in the setting of Lynch syndrome is not known and thus the patient is being monitored very closely.
Effects of a Combined Cyclooxygenase/Lipoxygenase Inhibitor in Pancreatic Cancer
H.M.A. Kader,1 N. Nalin,1 M. El-Zeiry,1 S. Laufer,2 T.E. Adrian,1. Department of 1Physiology, Faculty of Medicine, UAE University, Al Ain, UAE; 2Department of Pharmaceutical Chemistry, Eberhard-Karls-University, Tübingen, Germany.
Cyclooxygenase 2 (COX-2) and 5- lipoxygenase (5-LOX), which both metabolize arachidonic acid to produce pro-inflammatory eicosenoids, are up-regulated in pancreatic cancer. Both enzymes are already expressed in PanIN 2 and 3 lesions but not in normal pancreas. The products of these enzymes stimulate pancreatic cancer cell growth, enhance the inflammatory and desmoplastic response to the tumor and promote invasion and metastasis. Many studies have shown that inhibitors of either enzyme can inhibit growth, induce cell cycle arrest and apoptosis of cancer cells as well as block invasion and development of metastases of pancreatic cancer. However, a disadvantage of blocking one pathway is that it has been shown to make more substrate available for the other pathway. Clearly, blocking both pathways could provide additional benefit in the treatment of pancreatic cancer.
Licofelone is a dual COX (1 & 2)/LOX inhibitor under clinical development for osteoarthritis that exhibits no gastrointestinal toxicity. We have investigated the effects of a licofelone, a dual COX-2/5-LOX inhibitor on growth and apoptosis in four human pancreatic cancer cell lines (AsPC-1, S2013, HPAF and Panc-1. Licofelone inhibited growth of all four pancreatic cancer cell lines in a concentration- and time-dependent manner with an IC50 of approximately 5 μM in all four cell lines. The effects of the sodium salt and the free acid were identical on a molar basis. Similar concentrations of licofelone induced apoptosis as documented by morphological changes (characterized by cellular blebbing, shrinkage and nuclear fragmentation) by increase in annexin-5 binding and by terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL assay).
These findings suggest that dual inhibition of COX-2 and 5-LOX by licofelone may be of value in the prevention or treatment of pancreatic cancer.
Correlation of Current Fecal Elastase-1 (FE-1) Threshold Values to Pancreatic Secretory Function
V. Kadiyala, S.J. Burton, S.L. Suleiman, L.S. Lee, P.A. Banks, D.L. Conwell. Center for Pancreatic Disease, Brigham and Women’s Hospital, Harvard Medical School, Boston, MA.
Background: The secretin-stimulated endoscopic pancreatic function test (ePFT) evaluates pancreatic secretory function, and is the non-histological standard for chronic pancreatitis (CP) diagnosis. FE-1 testing is a non-invasive, cost effective measure of exocrine function in patients (pts) with suspected steatorrhea.
Aim: 1) Identify relationship between peak pancreatic fluid (PF) bicarbonate concentration ([HCO3]) and fecal elastase-1 concentration ([FE-1]), and 2) determine peak PF [HCO3] corresponding to current thresholds for [FE-1] deficiency.
Methods: Retrospective study of pts referred for suspected CP and with peak PF [HCO3] (by ePFT) and FE-1 testing. Data: age, gender, peak PF [HCO3] and [FE-1]. Abnormal [FE-1] thresholds: <200μg/g stool, <100 and <50. Abnormal ePFT: peak PF [HCO3] <75mEq/L. Analysis: linear regression to determine relationship between [FE-1] and [HCO3]. (SPSS Statistics V21. Armonk, NY).
Results: 72 pts were included in analysis: median age 50yrs (IQR 15.5), median [HCO3] 74mEq/L (IQR 33.8) and [FE-1] 362μg/g stool (IQR 318). [FE-1] and [HC03] were moderately correlated (r=0.628; p<0.001). 50 pts had [FE-1] ≥200μg/g stool; median [HCO3] of 82mEq/L (IQR 22.8). 22 pts had a [FE-1] <200; median [HCO3] of 40.5mEq/L (IQR 36). 15 pts had a [FE-1] <100; median [HCO3] of 39mEq/L (IQR 36). 10 pts had a [FE-1] <50; median [HCO3] of 37mEq/L (IQR 8.8). Regression equation to estimate peak PF [HCO3]: [HCO3] = 45.7 + 0.08[FE-1]. Therefore, a [FE-1] of 200μg/g stool predicts a [HCO3] of 61.7mEq/L, 100μg/g stool predicts 53.7mEq/L, and 50μg/g stool predicts 49.7mEq/L. Conversely, the lower limit of normal [HCO3] (75mEq/L) corresponds to a [FE-1] of 366.3μg/g stool, well within the normal range.
- As expected, acinar exocrine dysfunction is associated with marked ductal secretory dysfunction.
- Current threshold values for [FE-1] correspond to marked [HCO3] deficiency.
Increased Urine Collagen Cross-Linked N-telopeptide (NTX) Is Not Associated With Advanced Chronic Pancreatitis (CP)
V. Kadiyala, S.L. Suleiman, S.J. Burton, P.A. Banks, D.L. Conwell. Center for Pancreatic Disease, Brigham and Women’s Hospital, Harvard Medical School, Boston, MA.
Background: Increased urine NTX has been shown to be a marker of bone resorption. CP has been shown to be associated with increased risk of metabolic bone disease and low impact fractures.
Aim: To compare urine NTX in patients (pts) with 1) definite CP to those without, and 2) exocrine insufficiency to those without.
Methods: Retrospective study of pts referred for evaluation of suspected CP. Data: age, gender, urine NTX, serum 25(OH) Vitamin D (Vit D), fecal elastase-1 (FE-1) and imaging. Exocrine insufficiency defined as FE-1 ≤200μg/g stool. Imaging classified as normal, equivocal/mild and moderate/marked (Cambridge CP criteria). Definite (advanced) CP defined as moderate or marked imaging. Analysis (SPSS Statistics v21. Armonk, NY): univariate analysis (Mann-Whitney and Kruskall-Wallace) to determine association between urine NTX and 1) definite CP 2) exocrine insufficiency.
Results: 53 pts analyzed: median age 58yrs (IQR 16), 57% female. Definite CP vs Non-Definite CP: 18 pts had definite CP (median NTX 28.5nM BCE [IQR 27]) and 35 pts non-definite CP (median NTX 32.0nM BCE [IQR 25]); there was no difference in NTX (p=0.989). NTX was similar across pts with normal, equivocal/mild and moderate/marked imaging (p=0.429). Definite CP pts were younger (p=0.012) and had lower FE-1 (p=0.002), but had similar Vit D (p=0.867) and gender (p=0.249) compared to pts without definite CP. Normal FE-1 vs Abnormal FE-1: 17 pts had normal FE-1 (median NTX 33.0nM BCE [IQR 25]) and 12 pts abnormal FE-1 (median NTX 25nM BCE [IQR 43]); there was no difference in NTX (p=0.913). Pts with abnormal FE-1 had more advanced imaging (p=0.004) but similar age (p=0.263), Vit D (p=0.381) and gender (p=0.060) compared to pts with normal FE-1.
Conclusion: Increased urine NTX was not associated with advanced CP, and was unable to identify these pts as having increased risk for metabolic bone disease and low impact fracture.
A Case of Simple Inflammatory Cyst in the Pancreas: It Does Exist
K. Kamei,1 M. Matsumoto,1 T. Murase,1 M. Araki,1 Y. Nakata,1 H. Ishikawa,1 T. Nakai,1 T. Chikugo,2 Y. Takeyama1. 1Department of Surgery and 2Department of Pathology, Kinki University Faculty of Medicine, Osaka, Japan.
Introduction: Recently, cystic lesions of the pancreas are frequently found owing to the advanced imaging technique. Herein we report a case of enlarged cystic lesion of the pancreas diagnosed as simple inflammatory cyst.
Case: A 42-year-old man was admitted with a growing pancreatic cyst. The cyst was 20mm until 4 years ago, but now growing rapidly as far as 70mm. Abdominal computed tomography and magnetic resonance cholangiopancreatography revealed a multilocular cystic lesion of which the part of cysts were apart from each other. Calcification was found in the pancreatic head. The main pancreatic duct was not dilated. Neither the solid tumor in the cyst nor the connection to the main pancreatic duct was found. Imaging findings suggested branch-duct type of intraductal papillary mucinous neoplasm (IPMN). The cystic lesion was growing rapidly, suggesting malignancy. Thus, subtotal stomach-preserving pancreaticoduodenectomy was performed. Operative findings showed the cystic lesion of the pancreatic head which contained dull white serous fluid. Pathological findings showed that the epithelium of the multilocular cyst was inflamed, fibrotic, and cicatrized. Most of the epithelium changed squamatized and pancreatic parenchyma surrounding cyst was atrophic and furthermore showed chronic pancreatitis. The cyst was not connected to the main pancreatic duct. This case was histologically diagnosed as simple inflammatory cyst. Retention cyst, epidermoid cyst and IPMN were denied.
Conclusion: Cystic lesions of the pancreas are difficult to diagnose in operative indication, and we should not forget that simple inflammatory cyst is one of the differential diagnosis.
Significance of BRCA Mutation Among Patient With Pancreatic Cancer
A.A. Kashintsev,1 N.Y. Kokhanenko,1 E.N. Imyanitov,2 V.M. Moiseenko,3 A.G. Ievleva,2 N.M. Volkov,3 E.N. Suspitsin,2. 1Department of Faculty Surgery, 2Department of Genetics, Pediatric Medical University, 3LDC MIBS, Saint-Petersburg, Russia.
Background: For the people with one kindred’s with pancreatic cancer (PC) risk of morbidity estimates about 2,4-fold higher. Familial pancreatic cancer syndrome is associated with mutated suppressor genes. The role of BRCA1 and BRCA2 remains ambiguous.
Methods: From 2009 to 2012 years 257 patients with PC were included in study. In first part of the study all patients with pancreatic adenocarcinoma ware included and underwent genotyping for BRCA 1 5382insC mutations(n-116). BRCA2 was defined in 22 cases. From September 2011 we selected for genetic examination only patients with breast and/or ovarian cancers in anamnesis and/or with kindred’s with oncological disease(OD) (n-34). As a result we received 4 groups: A – general population (n-257); B –genetic test (n-150); C – patients with kindred’s with OD (n-56); D – patients which has ≥2 kindred’s with OD. Revealing of mutations was carried out by real-time PCR. Chemotherapy (ChT) was administered for patients with somatic status ECOG 0-1 grade. Efficacy of ChT was estimated by RECIST criteria.
Results: Only 3 patients had carriers of BRCA mutations (BRCA1 5382insC-2, BRCA2 5197_5198delTC- 1). Risk of these founder mutations increases with number of first kindred’s with oncological disease (RR=0,34), all of them were in group D. Frequency of BRCA1 is 1/11(9,5%) and of BRCA2 is 1/21(4,8%). Only 1 patient received a ChT. At the 1 line was administered FOLFIRINOX, but after 2 courses it wasn’t effective. In a 4 line ChT by mitomicin C the level of CA 19-9 began decrease and RECIST – stabilization. Time free for the progression is 4,1 months.
Conclusion: Prevalence of BRCA mutations is rare in the case of PC. All these carriers formed certain group of patients, characterized by numerous of kindred’s with OD. We can’t recommend a definite scheme of ChT, but such agent as cisplatin or mitomicin C can improve the results of ChT.
MUC5AC and CA19.9: A Unique Combination for Early Diagnosis of Pancreatic Cancer
S. Kaur,1 M. Menning,1 D. Watley,1 S.R. Krishn,1 L.M. Smith,2 S. Rachagani,1 S.M. Lele,3 A.R. Sasson,4 S. Guha,5 R.E. Brand,6 S.K. Batra,1. Department of 1Biochemistry and Molecular Biology, 2Biostatistic, 3Pathology, 4Surgery, UNMC, NE; 5Gastroenterology, Hepatology, and Nutrition, UTHSC, Houston, TX; 6Gastroenterology, Hepatology and Nutrition, University of Pittsburgh School of Medicine, PA.
Background and Hypothesis: Pancreatic cancer (PC) is a lethal malignancy with death to incident percentile of up to 96%. Lack of sensitive markers and specific symptoms for early malignancy are prime factors behind poor survival rate. Early detection of disease could entirely change the clinical outcome as well as overall survival rate of patient. MUC5AC is highly specific and most differently expressed gene in pancreatic cancer that is absent in normal pancreas, chronic pancreatitis, and normal pancreatic cell lines. In this study, we hypothesized that MUC5AC and CA19.9 combo detection in serum is highly sensitive and specific way for early detection of PC.
Methods: The specificity and early onset of MUC5AC expression was confirmed by assessing its expression in precursor lesion from KRAS G12D mouse model for early pancreatic intraepithelial neoplasias (PanINs). Further, MUC5AC levels were analyzed in tissues, plasma and juice samples from patients with early PC (EPC, n=73), late PC (LPC, n=66) and compared it with those in healthy individuals (n=51) and patients with acute (AP, n= 25) and chronic pancreatitis (CP, n=32). The diagnostic potential was further confirmed in blinded validation set. The diagnostic performance of MUC5AC was compared with that of CA19-9, gold standard for PC. MUC5AC levels were measured using ELISA in plasma and juice samples and via immunohistochemistry in tissues while CA19-9 levels were analyzed by radioimmunoassay.
Results and Conclusion: No expression of MUC5AC was observed in the normal pancreas, while an elevated expression was seen in the increasing grades of neoplasias (PanIN-II<PanIN-III<PDAC) and metastatic tumor tissues of both mouse and human. Novel highly sensitive ELISA-based immunoassay was developed to detect MUC5AC in serum. The mean plasma MUC5AC levels in EPC and LPC patients were 695.27±172.1 ng/ml, and 2,615.19±679.2 ng/ml, respectively that was significantly higher than that in HC [67.3 ng/ml (p=0.000349285, 0.000367)], AP [31.56 ng/ml (p=0.0003, 0.0004)] and CP [43.3 ng/ml(p<0.0003, 0.0004)] cases, respectively. In case of juice samples, it differentiated PC from HC with sensitivity of 85.7% and specificity of 80.9% with AUC of 0.85 (p<0.001). In plasma set, MUC5AC is 75% sensitive and 78.5% specific in differentiating PC from HC while CA19.9 was 81% sensitive and 67% specific in differentiating PC from HC. It differentiated PC from CP cases with the sensitivity and specificity of 79.9% and 80.6% and performed better than CA19.9 (81% sensitive and 61% specific). In conclusion, we identified a potential combination of CA19.9 and MUC5AC (specificity of 92.3% and sensitivity of 68.9%) as early diagnostic markers in differentiating EPC cases from healthy controls for early detection of PC.
Urgent ERCP With Pancreatic Stent Placement or Replacement for Salvage of Post-ERCP Pancreatitis
T. Kerdsirichairat, R. Attam, A.A. Mustafa, Y.G. Bakman, D.M. Radosevich, M.L. Freeman. Department of Medicine, University of Minnesota, Minneapolis, MN.
Introduction: There is only one prior report of salvage ERCP for de novo pancreatic duct (PD) stent placement after onset of PEP, and no prior report of urgently replacing prematurely passed PD stents to attenuate the course of PEP. We report outcomes of a practice of selective salvage ERCP for PEP.
Methods: Cases of PEP were prospectively identified over a 3-year interval. Protocol in high-risk patients (pts) was to measure amylase and lipase at 2 hours post ERCP, observe outpatients for 4 hours, and discharge home if without significant pain or complications. For pts developing PEP who had received a prophylactic PD stent, urgent abdominal Xray was done to evaluate PD stent position. Pts in whom there was concern for evolving PEP who had not received a prophylactic PD stent, those with early PD stent migration or suspected stent occlusion underwent urgent salvage ERCP with pancreatic stenting.
Results: Of 57/3,216 pts (39 female, median age 45) with PEP; 47 had received prophylactic PD stent. 14/57 pts with PEP underwent urgent salvage ERCP to place or replace PD stent (5/10 without prior PD stent, 9/47 with prior PD stent of which 7/9 were prematurely migrated out). Median onset of PEP was at 5 hours in pts with prophylactic PD stent vs 2 hours in pts without prophylactic PD stent. Median pain score before vs 24 hours after salvage ERCP was 10 vs 1 (p<0.001). Median amylase before vs 24 hours after salvage ERCP was 585 U/L vs 367 U/L (p=0.0003). Median lipase before vs 24 hours after salvage ERCP was 4,331 U/L vs 880 U/L (p<0.0001). No pancreatic necrosis or other late complications occurred. After salvage ERCP, 5/14 pts were safely discharged home within 24 hours.
Conclusion: Urgent salvage ERCP with de novo pancreatic stent placement or replacement of migrated stent is a novel and safe approach in the setting of early PEP, and is associated with rapid resolution of elevated lipase, amylase, and clinical pancreatitis after ERCP.
Two-Hour Amylase and Lipase are not Predictive of Post-ERCP Pancreatitis in the Era of Pancreatic Stents
T. Kerdsirichairat, A.A. Mustafa, R. Attam, Y.G. Bakman, D.M. Radosevich, M.L. Freeman. Department of Medicine, University of Minnesota, Minneapolis, MN.
Introduction: Prediction of risk of post-ERCP pancreatitis (PEP) is needed to allow safe same-day discharge. Studies have been inconsistent regarding the usefulness of 2 hour (hr) serum amylase. We evaluated test characteristics of 2 hr amylase and lipase.
Methods: ERCP performed at a tertiary referral center over a 3-year interval were studied. Protocol included clinical evaluation and routine serum amylase & lipase before and at 2 hrs after ERCP in patients at high risk of PEP. A matched case control study was with case-to-control ratio of 1:3 was performed. Criteria for matching were 1) age, 2) gender, 3) diagnosis, 4) year of procedure, and 5) PEP risk score. Multivariate regression analysis was used to identify risk factors. A receiver operating characteristic was used to define the optimal cutoff levels of amylase and lipase.
Results: 3,216 ERCP (1,356 with and 1,860 without PD stent) were performed. 64 pts developed clinical PEP. 7 were excluded due to data lacking at 2 hrs, leaving 57 pts for analysis. Cases were matched with 171 controls, with no difference of PEP risk scores (p=0.2). Median 2-hr amylase and lipase of pts with PEP were 126 U/L and 760 U/L. Median clinical onset of PEP was at 4 hrs (range 0-68) in pts with PD stent vs 1.75 hrs (range 0-4) without PD stent (p=0.002). Multivariate regression for association with PEP was significant for 2 hr amylase at 75% quartile of 321 U/L (p=0.005, CI 1.4-9.9), 2 hr lipase at 90% quartile of 5,265 U/L (p=0.005, CI 1.6-22.9), and pancreatic sphincterotomy (p=0.002, CI 1.5-9.7).
Conclusion: 2 hr serum amylase and lipase are not predictive of subsequent PEP. Only pancreatic sphincterotomy, a markedly elevated (3x uln) 2 hr amylase and a very markedly elevated (21x uln) 2 hr lipase are associated with risk of post-ERCP pancreatitis (p<0.0001). Lack of sensitivity of these tests may partly result from relatively delayed onset of PEP in patients with prophylactic PD stents.
Endoscopic Necrosectomy of Pancreatic Necrosis: Single Center Experience (Update)
J. Kheder, W. Wassef. Department of Internal Medicine, Division of Digestive Disease, University of Massachusetts Medical School, UMass Memorial Medical Center, Worcester, MA.
Background: Walled-off pancreatic necrosis (WOPN) is a known complication of acute or chronic pancreatitis. Abdominal pain, infected necrosis, and gastric outlet obstruction are indications for necrosectomy. We reported previously our experience with 8 patients, now we report a total of 21 patients in our center.
Objective: single center experience with direct endoscopic necrosectomy.
Methods: Nonrandomized observational study conducted using chart review by searching our endoscopy database at UMass Memorial Medical center between December 2010 and June 2013. Primary outcome is resolution of collection without the need for operative or percutaneous intervention.
Results: 21 consecutive patients with WOPN were treated with direct endoscopic necrosectomy (DEN). Mean age 51.6 years; with 76.19% males. 42.8% of WOPN were because of gallstone pancreatitis. Infected necrosis was present in 63%of WOPN. Average WBC before intervention was 10.87± 5.33 th/mm3 with average albumin of 2.4 ± 0.57 g/dL . The mean duration for first intervention was 6.2 weeks after onset of the necrosis, with average endoscopic sessions of 3.
Successful DEN was achieved in 14 patients (93.3%) out of 15. 5 patients are still ongoing treatment, one patient did not want to proceed with treatment. mean time to WOPN resolution was 12.8 ±5.7 weeks. Surgery was needed in one patient due to recurrent pancreatic duct stenosis in the tail of the pancreas. Bleeding as a complication of DEN occurred after three procedures (5.1%) of total 58, first one was oozing from the cystogastrostomy site, stopped spontaneously and did not require transfusion, the second one was an ulcer in site of cystgastrostomy, bleeding in the third patient required blood transfusion and gastroduodenal artery embolization.
Limitation: Single center, retrospective study.
Conclusions: Direct endoscopic necrosectomy is an accepted treatment modality that offers a minimally invasive method of WOPN treatment with good success rate.
Clinicopathological Features of Patients With Intraductal Papillary Mucinous Neoplasms and Simultaneous Pancreatic Neuroendocrine Tumors
D. Koliogiannis,1 S. Fritz,1 M. Klauss,2 F. Bergmann,3 M.W. Büchler,1 J. Werner,1. 1Department of Surgery, University of Heidelberg, Germany; 2Department of Diagnostic and Interventional Radiology, University of Heidelberg, Germany; 3Institute of Pathology, University of Heidelberg, Germany.
Introduction: Both, intraductal papillary mucinous neoplasms (IPMN) and neuroendocrine tumors of the pancreas (NET) are relatively rare tumors. To date little is known about their co-occurrence.
Objective: The present study aimed to analyze prevalence and clinicopathological features of patients with IPMN and simultaneous NETs.
Methods: Patients who underwent surgical resection for IPMN were retrospectively reviewed for presence of histologically proven NETs. Patients’ demographics, imaging findings and histology staging were evaluated.
Results: 486 patients underwent surgery for IPMN of the pancreas between January 2003 and March 2013. Among these, 17 patients (3.5 %) were diagnosed with concomitant NETs (10 male, 7 female; median age of 74 years), with glucagonoma being the most frequent NET (35.3%). Twelve patients had main duct-type IPMN, four branch duct-type IPMN and one patient had a mixed-type IPMN. Concomitant ductal adenocarcinoma of the pancreas (PDAC) was found in three patients and another three patients showed adenocarcinoma derived from main duct-type IPMN. Six patients were diagnosed with neuroendocrine carcinoma, in four cases with lymph node metastases. In all cases, NETs arose in a separate location distant from the IPMN.
Conclusion: The present study represents the largest series of patients with IPMN and coexisting NETs. The median age of patients presenting with IPMN and concomitant NET was significantly higher compared to patients with IPMN only. Thus, the risk for patients with IPMN to develop a concomitant NET over time seems to be higher than expected. Given our results, we need to reconsider whether the pancreas of patients with IPMNs have a field defect with the potential to affect all pancreatic cells.
Genetic Deletion of AMPK Subunits Enhances Pancreatitis Responses in a Cerulein Model of Acute Pancreatitis
T. Kolodecik,1 C. Shugrue,1 E. Akinbiyi,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, CT.
Aberrant activation and retention of digestive zymogens, with reduced apical secretion, is observed in the pancreatic acinar cell during the early stages of acute pancreatitis (AP). We have previously shown that AMP-activated protein kinase (AMPK) may play a protective role in AP; pretreatment of isolated pancreatic acini with pharmacologic activators of AMPK decreases zymogen activation whereas a pharmacologic inhibitor enhances zymogen activation. In our current study, we further validated the role of AMPK in AP using two genetic approaches. AMPK is composed of α1, α2, β1, β2, γ1 and γ2 subunits in most tissues. By qPCR analysis we determined that mouse pancreatic acini express AMPK subunit isoforms in varying amounts: α1=α2, β1>β2, and γ1>> γ2a. From other studies it is known that deletion of α2 reduces AMPK activity and deletion of γ1 inactivates AMPK. Therefore, we used an in vivo approach: C57/BL6 (Wild type or WT) or AMPK-α2-/- mice were given 6 hourly i.p. injections of phosphate-buffered saline (PBS) as a control or the cholecystokinin orthologue, cerulein (CER; 40 μg/kg) to induce pancreatitis, and were euthanized 1h after last injection. Pancreatic homogenates from AMPKα2-/- mice given PBS had the same level of trypsinogen activation as WT mice given CER; furthermore, AMPKα2-/- mice given CER had a nearly 2-fold increase in trypsinogen activation over Wt given CER. Secondly, using an in vitro approach, isolated WT mouse pancreatic acini were incubated with AMPK- γ1 or scrambled siRNA for 24h under conditions that retained cell function. Homogenates from acini incubated with AMPK- γ1 siRNA, but not scrambled siRNA, had enhanced zymogen activation. Together, these studies confirm a protective role of AMPK in acute pancreatitis.
Pancreatic Progenitor Expansion, Mesenchymal Cell Proliferation and Inflammation are Required During Early Carcinogenesis
B. Kong,1 N. Behler,1 P. Bruns,1,2 A.M. Schlitter,3 N. Valkovska,1 S. Fritzsche,1 S. Raulefs,1 I Regel,1 M. Erkan,1 F.J Theis,3 I. Esposito,3 J. Kleeff,1 C.W. Michalski,1,4. 1Department of Surgery, Klinikum rechts der Isar der Technischen Universität München, München, Germany; 2Institute of Bioinformatics and Systems Biology, Helmholtz-Zentrum München, Munich, Germany; 3Institute of Pathology, Technische Universität München, Munich, Germany; 4Oregon Health and Science University, Department of Surgery, Divisions of Surgical Oncology and of Abdominal Organ Transplantation, Portland, OR.
Regeneration of the adult pancreas from inflammation is a coordinated process and a result of sequential proliferation of different cell types. It has been demonstrated that pancreatic regeneration is susceptible to the transforming effect of oncogenic KrasG12D, which diverts the organ regeneration process to early carcinogenesis. In this study, we extensively investigated this biological diversion using a set of mouse models. Briefly, mice (wild type (WT) or p48Cre; KrasG12D (KrasG12D)) were sacrificed at 13 time points (3 hours to 14 days) after induction of pancreatitis. Proliferating cells were analyzed and quantified using combinations of proliferation- and cell-specific markers and expression profiles were analyzed by microarray analysis for 8 time points. This analysis revealed that the regeneration of the adult pancreas is a default process that is divided into three distinctive phases: inflammation (from 3 hours to day 2), organ regeneration (from day 3 to day 7) and refinement (from day 8 to day 14). The inflammatory phase is characterized by immune cell infiltration, proliferation of pancreatic progenitors and mesenchymal cells whereas the organ regeneration phase is predominantly characterized by acinar cell proliferation. In contrast, KrasG12D pancreata were remodeled by pre-neoplastic lesions showing extended phase of inflammation with impaired acinar cell proliferation (e.g. organ regeneration). An extensive bioinformatical analysis of expression profiles confirmed these histological findings. In an inducible mouse system, we further demonstrated that induction of KrasG12D during the inflammatory phase caused formation of pre-neoplastic cells whereas the induction of KrasG12D during the regenerating phase had no such effect. These data provide strong evidence that the expansion of pancreatic progenitors, mesenchymal cells and inflammation are three major prerequisites for oncogenic KrasG12D-deriven early carcinogenesis.
Identification and Characterization of an Aldh1a3-Positive Subtype of Pancreatic Ductal Adenocarcinoma Mediated by Oncogenic Krasg12d/Erk-Mtor Axis
B. Kong,1 W. Wu,1 T. Cheng,1 A.M. Schlitter,2 P. Bruns,1,3 C. Jäger,1 I. Regel,1 S. Raulefs,1 N. Behler,1 M. Irmler,4 J. Beckers,4 M. Erkan,1 F.J Theis,3 I. Esposito,2 J. Kleeff,1 C.W. Michalski.1,51Department of Surgery, Technische Universität München, Munich, Germany; 2Institute of Pathology, Technische Universität München, Munich, Germany; 3Institute of Bioinformatics and Systems Biology, Helmholtz-Zentrum München, Munich, Germany; 4Institute of Experimental Genetics (IEG), Helmholtz-Zentrum München, Munich, Germany; 5Oregon Health and Science University, Department of Surgery, Divisions of Surgical Oncology and of Abdominal Organ Transplantation, Portland, OR.
Pancreatic ductal adenocarcinoma (PDAC) is an aggressive tumor entity with an unfavorable prognosis. This may be partially attributed to the fact that PDAC is a histopathologically-defined tumor entity with great heterogeneity – both on the clinical, the histological and the genetic levels. Recent evidence suggests that the putative molecular subtype is dictated by the dominant oncogenic signal. Various genetic alterations in PDAC signal to the mammalian target of rapamycin (mTOR) via the Tsc1-Tsc2 (tuberous sclerosis) complex. Using a set of compound transgenic mouse models, we dissected different oncogenic mTOR signals in the pancreas. Here, the KrasG12D/Erk-mTOR axis induced aggressive PDACs characterized by tissue hypoxia, necrosis and early metastasis; a PI3K/Akt-mTOR signal induced mainly benign cystic lesions, while the p53-mTOR axis initiated acinar cell transformation. Further analyses identified Aldh1a3 as a specific marker for the KrasG12D/Erk-mTOR-driven mouse PDAC, sensitive to dual Erk- and Akt-inhibition. In humans, ALDH1A3 labeled a distinctive PDAC subtype with a significantly worse prognosis. The current study identifies and characterizes an Aldh1a3-positive subtype of PDAC driven by the oncogenic KrasG12D/Erk-mTOR axis. We provide genetic and functional evidence that define the KrasG12D/Erk-mTOR axis as a therapeutic target for this pancreatic cancer subtype.
Collection Morphology as a Predictor of Failed Endoscopic Necrosectomy for Walled-Off Pancreatic Necrosis
N. Kumar, D. Conwell, C.C. Thompson. Brigham and Women’s Hospital, Harvard Medical School, Boston, MA.
Background: The management of walled-off pancreatic necrosis (WOPN) increasingly incorporates direct endoscopic necrosectomy (DEN). Not all collections may be suitable for DEN. Collections may vary in size, shape, number of compartments, and amount of necrotic material. We analyzed WOPN collection morphology as a predictive factor for WOPN resolution after DEN.
Methods: Patients requiring debridement of symptomatic necrotic collections were retrospectively included. Morphologic characteristics of the collection, including percent necrosis, collection size, bilobed morphology, and extension into the paracolic gutters were recorded. Successful DEN was considered resolution without need for subsequent surgical intervention. Multivariable logistic regression was performed to evaluate predictors of success using STATA 12.
Results: 47 patients (27M/22F, age 52.7 ±2.1) had DEN. Univariable analysis was performed for predictors of successful DEN including percent necrosis, collection size, bilobed morphology, and extension into the paracolic gutters. 17% of patients were considered failure of DEN. Multivariable regression revealed that percent necrosis (coefficient -0.03, 95% CI -0.07 to 0.01, p=0.17) and extension into paracolic gutter (coefficient -2.2, 95% CI -4.49 to 0.07, p=0.058) did not reach independent statistical significance. Bilobed morphology was a significant independent predictor for failure (coefficient -2.6, 95% CI -5.0 to -0.1, p=0.04). Size was reported as collinear.
Conclusions: After controlling for extension into the paracolic gutters, collection size, and percent necrosis, bilobed collection morphology was associated with clinical failure of DEN. This bilobed morphology, due to anatomical impingement by the spine, likely limits endoscopic access for debridement and proper drainage. These collections may necessitate more vigilant management, such as nasocystic catheter lavage, and may demand a more aggressive endoscopic or surgical intervention.
The Microbiome of Walled-off Pancreatic Necrosis: Analysis of Blood and Endoscopic Culture Results
N. Kumar, D. Conwell, C.C. Thompson. Brigham and Women’s Hospital, Harvard Medical School, Boston, MA.
Background: Management of walled-off pancreatic necrosis (WOPN) increasingly incorporates endoscopic techniques. The character and significance of the WOPN microbiome remains unclear. We compared blood culture results and EUS-guided direct WOPN culture results in patients with symptomatic WOPN.
Methods: Patients requiring endoscopic debridement of symptomatic WOPN with available culture data were included. Culture data was obtained from the first endoscopic necrosectomy (DEN).
Results: 55 patients (31M/24F, 53.6 ±1.9 yr) had DEN with microbiologic culture obtained from the WOPN. Two culture bottles were inoculated in the procedure room during DEN. Blood cultures had been obtained on the inpatient floor within 7 days of DEN. Five cultures were polymicrobial. Gram positive organisms (12) were most common: alpha-hemolytic Streptococcus (7), Staphylococcus aureus (4), Enterococcus faecium (4), beta-hemolytic Streptococcus (1), and nonhemolytic Streptococcus (1). Gram negative organisms (7) included Enterobacter cloacae (2), Klebsiella pneumoniae (2), Citrobacter freundii (1), Haemophilus parahemolyticus (1), Haemophilus parainfluenzae (1), Morganella morganii (1), and Escherichia coli (1). Fungal organisms (7) included Candida albicans (5) and yeast, not Candida albicans (2). WOPN culture was positive in 24/55 patients, versus 2/55 blood cultures (OR 20.5, 95% CI 3.0-4.5, p<0.001). Both blood cultures were determined to be skin contaminants by the microbiology lab. 12 patients were on antibiotic therapy prior to DEN. There was no correlation between Gram positive, Gram negative, or fungal organisms and clinical failure of DEN.
Conclusions: Patients undergoing DEN for symptomatic WOPN have diverse culture results with a variety of bacterial and fungal organisms. Blood cultures are insensitive for the detection of infected WOPN. EUS-guided culture of WOPN should be obtained in symptomatic patients at time of DEN to target antimicrobial therapy. No specific organism correlates with DEN clinical outcomes.
Mucin Regulation by Tumor Microenvironment: Impact on Pancreatic Cancer
S. Kumar, S. Joshi, S. Rachagani, S. Chuge, S.K. Batra. Department of Biochemistry and Molecular Biology, University of Nebraska Medical Center, Omaha, NE.
The process of tumorigenesis involves the complex interplay between the cancer cell and the surrounding stroma. The cancer cell initiates the crosstalk between components of tumor microenvironment to progress to the clinical disease. Mucins (Muc1, Muc4, Muc5ac) in the pancreatic cancer (PC) have been shown to contribute significantly to its initiation and progression. The inflammatory conditions created by tumor cell through the secretion of various cytokines have been shown to modulate the mucin expression under various pathological conditions. We profile the cytokine expression during the pancreatic cancer initiation and progression in spontaneous PC mouse model (K-rasG12D; Pdx-1cre and K-rasG12D, TP53 R172H; Pdx-1cre) to delineate the contribution of cytokines in upregulating the mucin expression. The cytokines like Cxcl5, Ccl12, Cxcl3, Ccl2, Cxcl16, IL-1b, IL-rn and IL-23a were upregulated early during pancreatic intraepithelial neoplasia (PanIN). The contribution of tumor stroma in upregulation of these cytokines and their role in mucin regulation was investigated by novel 3D co-culture experiments. The cancer cells derived from tumor, endothelial cells, pancreatic stellate cells, neuronal and immune cells were encapsulated individually in sodium alginate, co-cultured together and the expression of mucins and the cytokines were profiled. The cross talk between tumor and endothelial cells was found to play important role in the mucin upregulation early during PC initiation. The presence of endothelia cells upregulate the expression of Cxcl5 and Cxcl16 in tumor cells which act in an autocrine manner to modulate the expression of Muc4 and Muc5ac and establish a chemotactic gradient for the migration of endothelial cells. The study offers a unique opportunity to understand the role of tumor microenvironment in PC pathogenesis and to develop targeted therapies.
Finnish Binding Pancreaticojejunostomy After Pancreaticoduodenectomy: A Prospective Study of 161 Consecutive Pancreaticoduodenectomies
J. Laukkarinen, I. Nordback, S. Räty, V. Jormanainen, J. Sand. Department of Gastroenterology and Alimentary Tract Surgery, Tampere University Hospital, Tampere, Finland.
Objectives: Post-operative pancreatic fistula (POPF) is considered the most important complication after pancreaticoduodenectomy. Recently, we have developed and described our early results of a modified, Finnish binding (purse string) pancreaticojejunostomy, where the pancreatic stump is slid 2–3 cm inside the jejunal limp with the aid of peripancreatic sutures, after which the bowel is tightened over the pancreas with a purse string. The aim of this prospective trial was to assess the safety of this novel binding pancreaticojejunostomy.
Methods: 163 consecutive patients underwent pancreaticoduodenectomy in TUH. Binding pancreaticojejunostomy was technically achievable in 161 (99%). Two patients (1%) underwent conventional anastomosis. Routine post-operative follow-up included repeated serum and drain output measurements as well as daily urine trypsinogen test. POPF, hemorrhage and delayed gastric emptying (DGE) were defined strictly according to the International Study Group classifications.
Results: 30-day mortality was 1.86% (3/161). The incidence of clinically relevant GrB-C POPF was 8.0% (GrA 5.5% - GrB 5.5% - GrC 2.5%). Hemorrhage occurred in 1.2-0.6-6.8% (GrA-B-C), and DGE in 37-10-3.7% (GrA-B-C) of the cases. 34% of the patients developed clinically relevant post-operative trypsinogen release (suggesting mild pancreatitis), which had a moderate correlation (Pearson 0.53) to GrB-C POPF and a weak correlation (Pearson 0.25) to GrB-C DGE.
Conclusions: Compared with the previous experiences obtained in pancreaticoduodenectomy, Finnish binding pancreaticojejunostomy is a safe and secure technique that may decrease the rate of pancreatic fistula.
Effects of Ethanol and Experimental Alcoholic Pancreatitis on Autophagy
G.E. Lee,1,2,3 J. Ni,2 C.T. Holland,1,2,3 S.W. French,3 A.S. Gukovskaya,1,2,3 I. Gukovsky,1,2,3 O.A. Mareninova.1,2,31VAGLAHS; 2University of California Los Angeles; 3Southern California Research Center for ALPD and Cirrhosis, Los Angeles, CA.
Background & Aims: Alcohol abuse is a major risk factor for pancreatitis; however, the mechanism of alcoholic pancreatitis remains elusive. Autophagy is activated in non-alcoholic pancreatitis but autophagic flux is impaired, underlying key pancreatitis pathologies. Here we investigate the effects of ethanol alone and experimental alcoholic pancreatitis on autophagy in pancreas.
Methods: We used in vivo and ex vivo models of alcoholic pancreatitis. Mice were pair fed for 6 wks ethanol or control Lieber-DeCarli diet followed by injections of low-dose cerulein. Acini were incubated for 1–4 h with ethanol (50-100 mM) and then treated with 0.1 nM CCK-8.
Results: Ethanol feeding induced phosphorylation of AMPK and its target ULK1/Atg1, a protein kinase initiating autophagosome formation. However, ethanol greatly decreased LC3-II, the lipidated form of a protein critical for autophagosome closure. In control acinar cells, blocking lysosomal function with bafilomycin A1 caused LC3-II increase, indicating high basal autophagy; no such increase was observed in ethanol-treated cells. These data indicate that ethanol inhibits autophagosome formation. Accordingly, EM showed fewer autophagic vacuoles in pancreas of ethanol-fed mice. One underlying mechanism can be a marked increase by ethanol of the level and activity of Atg4B, a protease mediating delipidation of LC3-II, which we found in both tissue and cells. By contrast, in ethanol+cerulein pancreatitis Atg4B level decreased while LC3-II and autophagic vacuoles increased. Both in vivo and ex vivo models showed accumulation of p62/SQSTM1, a protein selectively degraded by autophagy, indicating impaired autophagic flux.
Conclusions: Ethanol alone inhibits autophagosome formation due to excessive LC3-II delipidation by Atg4. In contrast, autophagy is stimulated in alcoholic pancreatitis, but autophagic degradation is impaired.
Clinicopathologic Features of Surgically Resected Pancreatic Serous Cystadenoma (SCA)
L.S. Lee,1,2 J. McNabb-Baltar,1,2 S.J. Burton,1 V. Kadiyala,1 S.L. Suleiman,1 P.A. Banks,1 D.L. Conwell.1,21Center for Pancreatic Disease, Division of Gastroenterology, Hepatology and Endoscopy; 2Interdisciplinary Management of Pancreatic Cystic Tumors (IMPACT) Clinic, Fish Center for Women’s Health; Brigham and Women’s Hospital, Harvard Medical School, Boston MA.
Background: Pancreatic SCA is a common benign cystic neoplasm requiring different management than premalignant lesions.
Aim: To 1) describe clinical features of SCA and 2) compare features associated with SCA size (≤3cm vs >3cm) in surgically resected SCA.
Methods: Retrospective study of patients (pts) with surgical resection (4/1995–1/2013) of pancreatic cystic lesion (SCA by pathology). Data: age, gender, race (white vs non-white), symptoms (symptomatic vs asymptomatic), pre-op imaging, imaging-based pre-op diagnosis (SCA vs all other diagnoses) and pathology. Fisher’s/Mann-Whitney tests for association of variables to size (≤3cm vs >3cm) (SPSS Statistics v21. Armonk, NY).
Results: 35 pts with resected SCA analyzed: median age 51.5 yrs (IQR 22), 81% female, 64% white and 69% symptomatic. 27% located in head, 12% in neck, 27% in body and 35% in tail. The most common indication for surgery was to rule out mucinous/malignancy in cyst of unclear etiology (81%). 17 (49%) pts had SCA >3cm: median size 5.2cm (IQR 2.8), age 55 (IQR 27), 77% female, 53% symptomatic (non-specific abd pain most common) and 24% correct pre-op diagnosis. 18 (51%) pts had SCA ≤3cm: median size 1.6cm (IQR 0.9), age 51 (IQR 13), 83% female, 83% symptomatic (non-specific abd pain most common) and no pts with correct pre-op diagnosis. SCA ≤3cm vs <3cm: greater proportion of lesions >3cm correctly identified as SCA (0% vs 24%; p=0.045). Age, gender, race, location and symptoms not associated (p>0.05) with size >3cm.
Conclusion: The most common indication for surgery was to rule out malignant potential. Pts with SCA ≤3 cm are more likely to have symptoms but less likely to be characterized pre-op as SCA. Better pre-op diagnosis of SCA is necessary to guide management.
Development of a Personalized Diagnostic Chronic Pancreatitis Risk Score
L.S. Lee,1 Y. Tabak,2 R.S. Johannes,1,2 X. Sun,2 V. Kadiyala,1 S. Suleiman,1 P.A. Banks,1 D.L. Conwell.1Center for Pancreatic Disease, Brigham and Women’s Hospital, Boston, MA; 2Clinical Research, CareFusion, San Diego, CA.
Background: Diagnosis of early chronic pancreatitis (CP) is challenging. While pancreatic function test is the non-histologic gold standard, EUS is commonly used. Weighting individual EUS CP features is based on expert opinion. Diagnosis of early CP does not account for patient-specific factors.
Aims: 1) To derive a CP prediction model using demographics, patient factors, EUS criteria, and 2) develop a diagnostic CP risk score.
Methods: Retrospective study of pts undergoing EUS and ePFT for suspected CP. Demographics (age, gender, smoking and alcohol status), standard 9 EUS criteria, peak pancreas fluid HCO3- from ePFT recorded. Abnormal ePFT: HCO3- <75 mEq/L. Statistical Analysis (SAS 9.2, Cary, NC): logistic regression model and risk score (0-32) developed. Score validated with 1,000 bootstrap simulations.
Results: 176 patients entered into model; 61% female, median age 48 yrs (IQR 10). 39.2% (69/176) had abnormal ePFT. Variables associated with abnormal ePFT in model: alcohol (p=0.02) or smoking status (p<0.0001), hyperechoicfoci (p=0.03), lobularity (p=0.0007), irregular MPD (p<0.0001), dilated MPD (p<0.0001), dilated branches (p=0.0003), calculi (p<0.0001). Risk Score. 4 variables used to compute: 1) alcohol or smoking status, 2) # parenchymal criteria, 3) # ductal criteria, 4) calculi (Table). Abnormal ePFT in: 10.7% pts with score 0-4, 22.7% score 5-7, 31.7% score 8-10, 73.7% score 11-19, and 92.0% score ≥20. Risk score validation: Model c-statistic 0.78 (95% CI: 0.71, 0.85).
Conclusion: CP risk score for predicting abnormal ePFT was developed including patient features and EUS criteria.
Hyperglycemia and Hyperinsulinemia Promote Pancreatic Stellate Cells Activation and Galectin-3 Expression: Potential Role in Pancreatic Cancer Development
L. Li,1,3 R.T. Waldron,1,2 A. Lugea,1,2 S.J. Pandol.1,21VA Greater Los Angeles Healthcare System, University of California, Los Angeles, CA; 2Cedars-Sinai Medical Center, Los Angeles, CA; 3Department of Endocrinology, Zhongda Hospital of Southeast University, Nanjing, China.
Background and Aims: Epidemiological studies indicate that the risk of pancreatic cancer (PC) is increased in type 2 diabetic patients. Insulin resistance and associated hyperglycemia, hyperinsulinemia, and abnormalities in insulin/IGF receptor pathways have been suggested to be the underlying mechanisms contributing to development of diabetes-associated pancreatic cancer. Activated pancreatic stellate cells (PSC) are key stroma cells responsible for pancreatic fibrogenesis and pancreatic cancer progression. The aim of this study was to investigate the effects of hyperglycemia and hyperinsulinemia on PSC biology.
Methods: In primary mouse PSC and a novel immortalized mouse pancreatic stellate cell line (imPSC), we examined the effects of different culture conditions. Cells were stimulated with high glucose (25 mM) and insulin, either individually or concomitantly. We determined expression of α-smooth muscle actin (α-SMA) and galectin-3, measured matrix synthesis of collagen I (Col-I) and fibronectin (FN), and analyzed the expression of insulin/IGF-1 signaling pathway components and their operation as profibrotic and/or proliferative pathways and assessed the potential contributions of PSC to a procarcinogenic microenvironment.
Results: α-SMA and galectin-3 expression were induced when PSC were incubated with either high glucose or insulin. Col-I and FN production were also increased. PSC express receptors for both insulin (IR) and insulin-like growth factor 1 (IGF-1R), and respond to insulin/IGF-1 stimulation with increased tyrosine phosphorylation at specific autophosphorylation sites. Results in imPSC were in accordance with primary mouse PSC.
Conclusions: Our study suggests that hyperglycemia and hyperinsulinemia are key-factors in the activation of PSC and progression of Fibrosis. Increased expression of galectin-3 and abnormalities in insulin/IGF receptor signaling network may be involved in development and progression of diabetes-associated pancreatic cancer.
Sulforaphane Inhibits Pancreatic Cancer Stem Cell Characteristics and Tumor Growth by Modulating Sonic Hedgehog-Gli Pathway
S.H. Li,1 J. Fu,1 D.N. Watkins,1 R.K. Srivastava,2 S. Shankar.1,31Department of Pathology and Laboratory Medicine, The University of Kansas Cancer Center, The University of Kansas Medical Center, Kansas City, KS, 2Department of Pharmacology, Toxicology and Therapeutics, and Medicine, The University of Kansas Cancer Center, The University of Kansas Medical Center, Kansas City, KS. 3Kansas City VA Medical Center, Kansas City, MO.
Background: Sulforaphane (SFN), a component of dietary cruciferous vegetables has been characterized for its anti-proliferative properties. Pancreatic cancer stem cells (CSCs) display activation of sonic hedgehog (Shh) pathway which regulates stem cell self-renewal. Aims: The objectives of this study were to examine the molecular mechanisms by which SFN regulates human pancreatic cancer stem cell characteristics and tumor growth.
Methods: CSC’s viability and apoptosis were measured by XTT and annexin V-propidium iodide assay, respectively. Gene and protein expressions were measured by quantitative real-time PCR and Western blot analyses, respectively. The effects of SFN on human pancreatic CSC tumor growth were examined in NOD/SCID/IL2Rgammanull mice.
Results: SFN inhibited cell viability, colony and sphere formation, and Gli transcriptional activity and induced apoptosis in pancreatic CSCs. SFN also inhibited pluripotency-maintaining factors, and suppressed epithelial-mesenchymal transition. Furthermore, SFN treatment resulted in a significant reduction in the tumor growth of orthotopically implanted primary pancreatic CSCs isolated from human pancreatic tumors into NOD/SCID/IL2Rgamma mice, which is mediated through the modulation of Shh-GLI signaling. Hedgehog pathway blockade by SFN at a dose of 20 mg/kg resulted in a 45% reduction in growth of pancreatic cancer tumors and reduced expression of Shh pathway components, Smo, Gli 1, and Gli 2 in mouse tissues. Further, SFN inhibited the expression of pluripotency maintaining transcription factors Nanog and Oct-4 and angiogenic markers VEGF and PDGFRα which are downstream targets of Gli transcription. Furthermore, SFN treatment resulted in a significant reduction in EMT markers Zeb-1, which correlated with increase in E-Cadherin expression suggesting the blockade of signaling involved in early metastasis. Interestingly, SFN downregulated the expression of Bcl-2 and XIAP to induce apoptosis.
Conclusions: These data demonstrate that, at a tolerable dose, inhibition of Shh pathway by SFN results in marked reduction in EMT, metastatic and angiogenic markers with significant inhibition in tumor growth in mice. Since aberrant Shh signaling occurs in pancreatic tumorigenesis, SFN may improve the outcomes of patients with pancreatic cancer by targeting CSCs.
30 Day Outcomes Underestimate Endocrine and Exocrine Insufficiency After Pancreatic Resection
P. Lim,1 M. Sullivan,1 W. Wassef,2 J. Zivny,2 G. Whalen,1 J. LaFemina.11Department of Surgery, 2Department of Gastroenterology, University of Massachusetts, Worcester, MA.
Background: The long-term incidence of postoperative endocrine and exocrine insufficiency after pancreatic resection is poorly described. We aim to analyze the long-term risks of pancreatic endocrine and exocrine insufficiency after pancreatic resection.
Methods: Patients who underwent pancreatic resection from 2002–2012 were identified from a prospectively-maintained database. Patients who underwent total pancreatectomy or surgery for pancreatitis were excluded. Development of postoperative endocrine and exocrine insufficiency was defined as the need for new pharmacologic intervention.
Results: In 227 patients, pancreaticoduodenectomy (PD) was the most common operation (n=159), followed by distal pancreatectomy and enucleation. 30 patients (13%) developed new postoperative diabetes (DM) with a median time to diagnosis of 84 days. 33% who required preoperative oral hypoglycemics developed worsening DM requiring insulin. 10% (n=23) of the population developed DM beyond 30 days and 7% (n=15; 50% of those with new DM) after 90 days. 97 patients (43% of population) developed exocrine insufficiency after a median of 84 days. 34% (n=77) of the population developed symptoms after 30 days and 21% (n=48; 49% of those with new exocrine insufficiency) after 90 days. There was no correlation between age, gender, resection type, or pathologic type and postoperative DM. PD (p=0.001) and presence of periampullary adenocarcinoma (p=0.008) were associated with greater risk of exocrine insufficiency.
Conclusion: This is the largest study describing exocrine and endocrine insufficiency after pancreatic resection with extended follow-up. Reporting 30 day outcomes for pancreatic resection is not sufficient, as 50% with new DM and 49% with new exocrine insufficiency were diagnosed after 90 days. 90 day outcome reporting may more reliably assess risk for postoperative exocrine and endocrine insufficiency.
The Harmless Acute Pancreatitis Score in Clinical Use
O. Lindström,1 H. Hartman,2 T. Sippola,3,4 J. Kupcinskas,5 S. Regner,2 C.D. Johnson.6Department of Surgery; 1Helsinki University Central Hospital, Helsinki, Finland; 2Skåne University Hospital, Malmö, Sweden; 3Tampere University Hospital, Tampere, Finland; 4Seinäjoki Central Hospital, Seinäjoki, Finland; 5Department of Gastroenterology, Kaunas University Hospital, Kaunas, Lithuania; 6Department of Surgery, University Hospital, Southampton, UK.
Background: The Harmless Acute Pancreatitis Score (HAPS) is a clinical algorithm for rapid initial identification of patients with nonsevere acute pancreatitis (AP) at admission to the hospital. The parameters of HAPS consist of creatinine and hematocrit levels and signs of peritonitis. Previously the score has been shown to have a specificity of 96% and positive predictive value (PPV) of 98% and 99% in predicting a nonsevere course of AP. The aim of this study was to evaluate the serviceability and reliability of the score in clinical work.
Methods: The study was designed as part of the Pancreas 2000 educational program. Patients with AP were consecutively included at 3 centers between 2010 and 2012, and followed until discharge from hospital. Severe AP was defined according to the old Atlanta classification by the presence of local complications and/or organ failure.
Results: A total of 263 patients were included, and the data for the HAPS analyses were available from 236 patients (152 men, 84 women). 40 cases (17%) were defined as severe. According to HAPS, 113 patients (48%) were predicted to have a nonsevere course of AP. Of these, 7 patients (6%) developed severe AP, but none of these patients were treated in the ICU. The specificity of HAPS in predicting a nonsevere course of AP was 83% with a corresponding PPV of 94%, and the sensitivity was 54%.
Conclusion: The HAPS seems to be a simple, fast and reliable enough algorithm for clinical use in the initial management of patients with AP. By using the HAPS it is possible to early identify patients that will not need treatment in the ICU and target this demanding and expensive care for other patients.
Epigenetic Signaling by HP1 Regulates KRAS-Induced Growth Promoting Gene Networks in Pancreatic Cancer Cells
G. Lomberk, A. Mathison, R. Urrutia. Epigenetics and Chromatin Dynamics Laboratory, Translational Epigenomics Program (CIM), Mayo Clinic, Rochester, MN.
The genetics revolution has significantly advanced the field of pancreatology, although many diseases remain incurable. Fortunately, a new scientific revolution, Epigenetics, promises to further advance the study of pancreatic diseases by generating new tools for their management and treatment. In the last two decades, our laboratory has focused on investigating the epigenetics of pancreatic ductal adenocarcinoma (PDAC). Congruent with this goal, the Objective of the present study has been to unravel novel epigenetic mechanisms that utilize the HP1-Histone Methyl Transferase pathways to extend oncogenic growth signals downstream from KRAS during initiation and pancreatitis-associated cancer promotion. Our Methods: and approach include investigations in human tissues as well as new cells and animal models, performing biochemical, cellular, and in vivo experiments. Our Results: reveal that: 1. HP1γ is upregulated in human pancreatic cancer and in the p48-Cre/LSD-KrasG12D model during initiation and in response to pancreatitis-induced promotion. 2. HP1γ synergizes with KRAS to stimulate cell growth in cultured cells and tumor growth in xenografts. 3. HP1γ is part of a novel pathway that becomes stimulated by EGF in a KRAS-dependent manner, and 4. HP1γ regulates growth-promoting gene expression networks downstream of KRAS. Thus, combined, these results support the Conclusion: that HP1γ functions as an epigenetic regulator downstream from KRAS to promote pancreatic cell growth by regulating the expression of proliferative gene networks. As new drugs targeting both the KRAS and HP1 pathways are being tested in clinical trials, these results build the rationale for applying these tools to the management of patients affected with deadly pancreatic diseases, thereby bearing significant biomedical relevance.
Predictors for Mortality Following Acute Pancreatitis in Children
H. Lu,1 Q. Guo,1 A. Li,1 Z Zhang,1 W. Hu,1 Y. Su,2 Y. Liu.21Hepato-Bilio-Pancreatic Surgery, West China Hospital, Sichuan University, Chengdu; Sichuan Province, China. 2R & D Department, Sichuan ENWEI Pharmacy, CO. LTD. Chengdu, Sichuan Province, China.
Objectives: To identify independent predictors for death following acute pancreatitis in children using a multivariate logistic regression analysis.
Patients and Methods: We reviewed the medical records of all children (ages 0 to 20 years) admitted to West China Hospital with acute pancreatitis between 2002 and 2012.
Results: Biliary diseases (23%), medications (20%), idiopathic (19%), and trauma (11%) were most common etiologies for acute pancreatitis in children. The death rate was 5% and the mean duration of hospital stay was 13 days. Organ failure during admission occurred in 24 of 371 patients and most of them started in 3 days of onset (19/24). Multivariate analysis with logistic regression confirmed that occurrence of organ failure in 3 days of onset (OR=8.0, 95% CI 2.2-12.3, P < 0.001) and idiopathic cause (OR=17.3, 95% CI 2.0-60.5, P < 0.001) were independent risk factors for death in cases with pancreatitis.
Conclusions: Mortality rate and complication rate of acute pancreatitis in children remains low, however, patients with early organ failure or idiopathic cause should be well evaluated because they have higher risk of death. Further research on the pathophysiology of acute pancreatitis in children should be considered in future studies.
Improving the Quantification of Flavonoid Aglycones and Glucuronides in Animal Tissues and Tumor Xenografts
Q.Y. Lu,1 L. Zhang,1 G. Eibl,2 V.L.W. Go.2Department of 1Medicine, and 2Surgery, David Geffen School of Medicine, University of California, Los Angeles, CA.
Background: Flavonoid glucuronides are the main circulating metabolites of flavonoids in human and animals. Many organs and body fluids of the humans and animals exhibit β-glucuronidase (βGC) activity against flavonoid glucuronides. βGC within the tissues hydrolyzes glucuronides to their aglycones during the tissue extraction, leading to artificially higher reported tissue levels of aglycones than actual in vivo concentrations.
Aims: The aims of this study were to estimate the extent by which the aglycones were overestimated and to investigate the use of saccharo-1,4-lactone (SL), a βGC inhibitor, to block the ex vivo hydrolysis of flavonoid glucuronides.
Methods: The pancreatic xenograft mouse model fed with standard diet supplemented with 1% or 5% quercetin, or 0.2% baicalin has been reported previously. Orthotopic tumor xenografts in nude mice were established by implantation of a small piece of subcutaneously grown xenografts of human pancreatic cancer cells (MiaPaCa-2). Aliquots of known concentration of SL were added to frozen tissues which were then homogenized. Products were extracted and analyzed by HPLC.
Results: In mouse liver tissues and pancreatic tumor xenografts, levels of quercetin and methylated quercetin aglycones are over-estimated by >7 fold. The inhibition of de-glucuronidation by SL is dose-dependent. The amount SL used to produce optimal inhibition of the enzyme activity is in the range of 7-24 μmol/g tissue.
Conclusion: The use of SL blocks the ex vivo deconjugation resulting in a much more accurate estimation of tissue levels of selected flavonoid aglycones and conjugates. To differentiate biological activity and to assess efficacy it is essential to accurately determine the levels of flavonoid aglycones and metabolic conjugates in vivo. Our study described here can be extended to other animal models and human studies with different types of substrates of βGC.
Inflammatory Process and Cell Death in Pancreatic Acinar Cells in Response to Tobacco Compared To Alcohol
M. Luaces-Regueira,1 M. Castiñeira-Alvariño,1 J.E. Domínguez-Muñoz.1,21Foundation for Research in Digestive Diseases, 2Department of Gastroenterology and Hepatology, University Hospital of Santiago de Compostela, Spain.
Introduction: Triggering of the inflammatory process through the activation of pro-inflammatory transcription factors and secretion of pro-inflammatory cytokines is a central pathogenic mechanism of chronic pancreatitis (CP). This fact is associated with cell death, both necrosis and apoptosis. Previous studies suggest a role for alcohol in those pathophysiological events, but the effect of tobacco, which is a recognized risk factor for CP, is unknown.
Aim: To analyze the effect of tobacco compared with alcohol in the activation of pro-inflammatory factors, cytokine release and cell death in pancreatic acinar cells in culture.
Methods: Pancreatic acinar cells were isolated from Swiss mouse pancreas. Cells were stimulated over 3 hours with CCK (positive control), alcohol (10 to 100 mM) or tobacco (0.001 to 0.4 mg/ml). NFkappaB activation (translocation of the subunit p65 into the nucleus) was measured by Western blot. Interleukin-1β and TNFα secretion was analyzed by ELISA in supernatant. Acinar cell necrosis was measured by fluorescence assay (propidium iodide). Apoptosis was evaluated by caspase 3 activity by western blot. Data were analyzed by ANOVA.
Results: Tobacco, but not alcohol, induces activation of NFKappaB (2.69±1.05 fold increase of p65 translocation at 0.1 mg/ml over negative control). Neither tobacco nor alcohol induces interleukin-1β and TNFα release. Tobacco stimulates the activation of caspase 3 at 0.01 and 0.1 mg/ml (2.53±0.38 and 1.77±0.12 vs negative control) and induces necrosis at 0.3 y 0.4 mg/ml (14.3% and 19.4%, respectively).
Conclusion: Tobacco, but not alcohol, initiates the inflammatory process through the activation of NFKappaB and induces necrosis and apoptosis in pancreatic acinar cells. These results suggest tobacco as a major pathogenic factor in CP.
Cathepsin D Drives the Fibrogenic Potential of Pancreatic Stellate Cells and Stromal Development
U.M. Mahajan,1 T. Schwaiger,1 F.-U. Weiss,1 M. Löhr,2 W. Halangk,3 M.M. Lerch,1 J. Mayerle.11Department of Internal Medicine A, Ernst-Moritz-Arndt University, Greifswald, Germany; 2CLINTEC, Karolinska Institute, Stockholm, Sweden; 3 Department of Experimental Surgery, Otto-von-Guericke-University Magdeburg, Germany.
Aim: Involvement of the pancreatic cancer tumor microenvironment in remodelled extracellular matrix (ECM) has been implicated in the resistance of cancer cells to therapy. Stromal cell-derived proteases, e.g. cathepsins, are involved in tumorigenesis and cell death but their role in regulating pancreatic stellate cells (PSCs) and thus in modulating fibrogenesis is unknown. We studied whether cathepsin D, an aspartic protease highly expressed in pancreatic cancer, contributes to fibrogenesis by activating PSCs.
Material & Method: We isolated PSCs by Nycodenz gradient from mouse pancreas at 10 weeks of age and used them for Western blotting and immunofluorescence staining. TGFβ1 was measured using microplate sandwich ELISA and proliferation by standard MTT assay.
Results: We found cathepsin D expression to be negligible in quiescent PSCs but it increased in parallel with PSC trans-differentiation to myofibroblasts. Silencing (siRNA) or inhibiting (Pepstatin, N1075) cathepsin D decreased PSC proliferation by 20-30% as well as phenotypic markers of PSC activation such as αSMA, Vimentin and Collagens. Moreover, there was a time-dependent activation of latent TGFβ1 to activated TGFβ1 indicating an increase PSC fibrogenic potential. Cathepsin D cleaved latent TGFb1, activating TGFb1, and inhibition of cathepsin D lead to a decrease in activated TGFβ1.
Discussion: We show that Cathepsin D expression increases during PSC activation in vitro. Cathepsin D is released from lysosomes and modulates ECM as well as membrane receptors such as TGFβ1, which it activates by proteolytic cleavage. In this manner cathepsin D regulates fibrogenesis and stromal development. These findings support a critical role for cathepsin D in PSC activation.
Strong Inhibition of Pancreatic Ductal Bicarbonate Secretion by Ethanol and Fatty Acids
J. Maléth,1 L. Judak,1 L. Kemeny,1 Z. Balla,1 B. Kui,1 P. Pallagi,1 V. Venglovecz,2 A. Somoracz,4 K. Borka,4 A. Balazs,1 I. Nemeth,3 Z. Rakonczay,1 M. Gray,5 P. Hegyi.11First Dept. of Medicine, 2Department of Pharmacology and Pharmacotherapy, 3Department of Dermatology and Allergology, University of Szeged, Szeged, Hungary, 42nd Department of Pathology, Semmelweis University, Budapest, Hungary, 5Institute for Cell & Molecular Biosciences, Newcastle University, Newcastle upon Tyne, UK.
Introduction: Excessive ethanol (EtOH) consumption is one of the most common causes of acute pancreatitis. Pancreatic ductal epithelial cells (PDEC) secrete HCO3- rich fluid via Cl-/HCO3- exchanger (CBE) and cystic fibrosis Cl- channel (CFTR), which is important in the pancreatic pathophysiology. However, no information is available about the effects of EtOH and EtOH metabolites ((fatty acid ethyl esters (FAEE) and fatty acids (FA)) on PDEC.
Methods: We used human pancreatic cell line (Capan-1), guinea pig and human pancreas. The effects of EtOH, FAEE and FA on intracellular pH, Ca2+ concentration ([Ca2+]i), ATP [(ATP)i], mitochondrial membrane potential (ΔΨ) and CFTR Cl- current were measured and the expression and localization of CFTR were analysed in PDEC, guinea pig and human pancreatic tissue.
Results: EtOH (100mM) and palmitoleic acid (POA) (200μM) strongly decreased the activities of the apical CBE and CFTR and inhibited the HCO3- secretion of PDEC in vitro. Moreover they depleted the (ATP)I, decreased ΔΨ and induced sustained [Ca2+]i elevation by releasing Ca2+ from the endoplasmic reticulum and inducing extracellular Ca2+ influx. The prevention of the sustained [Ca2+]i elevation abolished the inhibitory effect of EtOH and POA. The expression of CFTR was significantly decreased by EtOH and POA after 48h in PDEC. In addition, ip injection of EtOH and FA decreased the luminal expression of CFTR in guinea pig pancreas. Importantly, we observed the same changes in the pancreatic tissue of acute and chronic alcoholic pancreatitis patients.
Conclusion: These results suggest that EtOH and FA inhibit pancreatic ductal HCO3- secretion via sustained [Ca2+]i elevation and decrease the expression of CFTR, which can contribute to the development of acute or chronic pancreatitis.
Inhibition of GSK3 Reduces p70S6K Activity and Promotes Autophagy in Pancreatic Cancer Cells
B. Marchand, M.-J. Boucher. Div. of Gastroenterology, Dept. of Medicine, Université de Sherbrooke, Québec, Canada.
We previously demonstrated that inhibition of GSK3 activity induces JNK-dependent cell death in human pancreatic cancer (PC) cells. Interestingly, GSK3 inhibition was recently shown to induce autophagy in other cell types suggesting a link between GSK3 activity and autophagy. Besides, inhibition of autophagy sensitizes diverse cancer cells to a wide array of stress conditions. The Aim: of the study was thus to evaluate whether GSK3 controls autophagy in PC cells.
Methods: Experiments were performed using the PC cell lines PANC1, BxPC-3 and MIAPaCa2. GSK3 activity was inhibited by treatment with GSK3 inhibitors (SB216763, CHIR99021) or by specific shRNAs targeting GSK3alpha/beta. Apoptosis was measured by assessment of PARP and caspase-7 cleavages. Autophagy was evaluated by the detection of the membrane-bound LC3B-II isoform and was inhibited by treatment with bafilomycin A1 or transfection of a siRNA targeting ATG5.
Results: 1) Inhibition of GSK3 induced both an apoptotic and autophagic response. 2) Inhibition of autophagy sensitized PC cells to the GSK3 inhibition-induced cell death. 3) GSK3 inhibition correlated with the activation of the JNK-cJun pathway and reduced levels of phosphorylated p70S6K and S6. 4) While activation of the JNK-cJun pathway was required for the apoptotic response, it was dispensable for the autophagic response following GSK3 inhibition. Also, shRNA-mediated cJun downregulation did not prevent the reduced levels of phosphorylated p70S6K following GSK3 inhibition. These results suggest that GSK3 might act on the mTOR/p70S6K pathway to modulate autophagy.
Conclusion: Our results provide evidence that, in addition to cell death, GSK3 inhibition induces autophagy in PC cells. The autophagic response protects PC cells against GSK3 inhibition-induced cell death. Our observations suggest a potential role for the mTOR/p70S6K signaling pathway in the GSK3-dependent regulation of autophagy.
IPMN Involving the Main Pancreatic Duct: Biology, Epidemiology, and Long-Term Outcomes Following Resection
G. Marchegiani,1 M. Mino Kenudson,2 V. Morales-Oyarvide,2 S. Thayer,1 C. Ferrone,1 A.L. Warshaw,1 K. Lillemoe,1 C. Fernandez-del Castillo,1Departments of Surgery1 and Pathology2, Massachusetts General Hospital, Boston, MA.
IPMNs that involve the main pancreatic duct are known to harbor a risk of malignancy and different patterns of epithelial subtypes. The appropriateness of including in MD group combined cases with only minimal MPD involvement has been challenged. We sought to describe the characteristics of a large cohort of resected IPMNs with either exclusive involvement of the MPD or extensive involvement of the MPD with additional branch duct disease.
Methods: Retrospective review of demographics, pathology, and survival after surgical resection for MD-IPMN from 1990 to 2013. All slides from specimens were reviewed by a single pancreatic pathologist and classified based on histological type, invasive component and type of invasion.
Results: A total of 209 patients underwent surgical resection for either MD or combined-IPMN. Of these, 48 were excluded because of minimal MPD involvement. Among the 161 patients analyzed, median age was 68 years (range 37 to 87) and 54% were male. Predominant epithelial phenotype was intestinal (51%), followed by gastric (32%), oncocytic (9%) and pancreatobiliary (8%). 48 patients had low- or intermediate-grade dysplasia, whereas the 70% was high grade.
Invasive IPMN was found in 58 cases (36%) and more often presented with jaundice (RR 5.7, 95% CI 2.2-14.7). Among invasive tumors, there were 29 tubular carcinomas (50%), 22 colloid (38%), and 7 oncocytic (12%); superficial invasion was observed in 24 (41%), and macroscopic in 34 (59%). Lymph node metastases were seen in 20 patients (12% of total, 34% of invasive cases), including 37% of tubular carcinomas, 27% of colloid and 14% of oncocytic (P=NS). Median overall survival was 14 years, and 5-year and 10-year disease free survivals were 82% and 76%, respectively. Recurrence of IPMN in remaining pancreas was 19%. For the 58 pts with invasive cancer, overall median survival was 94 months, and the 5-and 10-year disease specific survival 60% and 49%, respectively.
Conclusions: IPMN with predominant involvement of the main pancreatic duct is mainly intestinal-type and malignant. Following surgical resection, it has a very favorable prognosis, especially in the absence of invasive carcinoma.
Mechanisms of Action That Define Opposing Effects of Omega-3 & 6 Fatty Acids in Pancreatic Neoplasia
W.E.T. Mascarinas, L.J. Zhu, R. Chiu, D.R. Principe, K. Adrian, P.J. Grippo. Northwestern University Feinberg School of Medicine, Chicago, IL.
Environmental factors, particularly diet, have been associated with influencing the development of various cancers including pancreatic, though the underlying mechanisms remain largely unexplored. We previously showed in murine models that diets rich in omega-3 fatty acids (ω-3) decrease whereas diets high in omega-6 fatty acids (ω-6) facilitate both the size and number of mutant Kras-induced neoplastic lesions. These events were paralleled in vitro, where cultured pancreatic ductal epithelial cells exposed to ω-3 decreased pAKT levels, whereas treatment with ω-6 led to an increase in pAKT. We now show that changes in pAKT leads to subsequent downstream phosphorylation events in the pro-apoptotic factor, BAD, and the anti-proliferative factor, FOXO3a. However, when treated with VO-OH, a pTEN inhibitor, these cells did not show fluctuations in levels of pAKT with respect to control, indicating pTEN is required for polyunsaturated fatty acid alteration of pAKT protein levels. We now demonstrate in vivo that these effects may likewise be directed, in part, by PI3K/AKT signaling. When compared to an isocaloric diet, increases in pAKT and BrdU uptake were observed in mice fed a high ω-6 rich diet, though mice on a high ω-3 rich diet showed no significant changes in BrdU uptake or expression of pAKT and subsequently pBAD and pFOXO3a. These data suggest that the benefit of ω-3 may not lie in a sustained decrease in proliferation/AKT signaling, but rather in a delay in lesion onset, which supports claims of the chemoprotective nature of ω-3 fatty acids in early pancreatic neoplasia. Also, these findings further substantiate the procancer effects of a western diet heavy in ω-6 fatty acids and the potential role of PI3K and pTEN in tipping the balance between these fatty acids. Hence, further studies are required in order to assess pTEN as a chemopreventative target against pancreatic neoplasia in vivo.
Preclinical Testing of a New Combinatorial Therapy Against Aurora A and G9a for the Treatment of Pancreatic Cancer
A. Mathison, M. Fernandez-Zapico, R. Urrutia, G. Lomberk. Epigenetics and Chromatin Dynamics Laboratory, Translational Epigenomics Program (CIM), Mayo Clinic, Rochester, MN.
Aurora Kinase A (AurkA) is overexpressed and functions as an oncogene for many tumors, including pancreatic adenocarcinoma (PDAC), where it is being clinically tested as a target for chemotherapy. We found that AurkA mediates the phosphorylation of HP1γ, a member of the Heterochromatin Protein 1 (HP1) family of non-histone proteins, at S83. In addition, we find that this phosphorylation induces the formation of a complex between HP1γ and its Histone Methyl Transferase, G9a. Interestingly, high levels of P-S83- HP1γ and G9a correlate with the upregulation of AurkA in PDAC compared to normal controls. Thus, we have begun to study the role of the Aurora A- HP1γ−G9a pathway complex in the regulation of PDAC growth by both genetic, as well as pharmacological inhibition of AurkA and HP1γ-G9a. We find that treatment of PDAC cells with Aurora A and HP1γ−G9a inhibitors, alone or in combination, inhibit cell growth in culture. Moreover, similar treatments lead to a reduced tumor size in xenografts. Thus, our results confirm a novel role of the HP1γ˜G9a complex as a downstream mediator of AurkA. More importantly, since this pathway can be targeted with two enzyme inhibitors (against AurkA and G9a), we are further investigating how these small drugs modulate PDAC cell growth, which may serve as the foundation for the development of new combination therapeutic approaches to treat this dismal disease.
Discovery of New Metabolic Biomarkers for the Diagnosis of Pancreatic Discovery of New Metabolic Biomarkers for the Diagnosis of Pancreatic Ductal Adenocarcinoma (PDAC)
J. Mayerle,1 H. Kalthoff,2 R. Reszka,3 B. Kamlage,4 B. Schniewind,2 C. Pilarsky,5 R. Grützmann,5 F.-U. Weiss,1 M. M. Lerch.11Department of Medicine A, University Medicine Greifswald, Ernst-Moritz-Arndt-University, Greifswald, 2Institut für Experimentelle Tumorforschung (IET), UKSH, Campus Kiel, Kiel, 3Metanomics Health GmbH, 4metanomics GmbH, Berlin, 5Clinic and Outpatient Clinic for Visceral-, Thorax- and Vascular Surgery, University Hospital Carl Gustav Carus, Dresden, Germany.
Introduction: Early symptoms of pancreatic cancer (PDAC) are rare and nonspecific and this burdens PDCA with a especially dismal prognosis. Although modern imaging techniques have pushed the detection limit combination of diagnostic imaging and lab-based biomarkers can, under the best of circumstances, distinguish between pancreatic cancer and chronic pancreatitis in only 67%. We have therefore conducted discovery and confirmation studies to identify metabolite plasma biomarkers for the detection of pancreatic cancer and the differentiation from chronic pancreatitis.
Aims & Methods: The study was conducted in three phases: An initial pilot study (n=200) on plasma samples was followed by analysis of a second plasma sample collection and a serum sample collection from pancreatic cancer, chronic pancreatitis and liver cirrhosis patients as well as matched healthy blood donors (in total n=320). Metabolomic profiles of plasma and serum samples were generated applying high quality polar and lipid GC-MS and LC-MS/MS technology (MxP™ Broad Profiling). In addition, targeted platforms for steroids and lipids (MxP™ Steroid, MxP™ Lipids) were applied. Up to 477 metabolites were analyzed semi-quantitatively or quantitatively. Statistical data analysis was done by linear models (ANOVA) on log10 transformed data considering age, gender, BMI and sample storage time as fixed effects. A selection of 10 metabolites was done via classification by using the Random Forest (RF) algorithm with forward feature selection. The predictive ability of the biomarker was evaluated through the estimation of ROC characteristics and AUC values from Bootstrap-based Cross-Validation.
Results: Within the three consecutive studies, a multimarker panel identified by Random Forest consisted of 10 metabolites and provided an AUC=0.85 when discriminating between pancreatic cancer and pancreatitis. When CA19-9 serum levels (AUC=0.82) were included in the analysis, an AUC of 0.94 was reached. The NPV was determined with 99.68%.
Conclusion: The results indicate that a plasma metabolite biomarker panel may be used to distinguish between pancreatic cancer and chronic pancreatitis with a high degree of accuracy. The most discriminating metabolites showed robustness with respect to transferability of their diagnostic potential from plasma to serum. A diagnostic assay based on this biomarker would help in guiding suspected PDCA patients and chronic pancreatitis patients burdened with an increased PDCA risk towards more or less invasive diagnostic procedures.
Chronic Pancreatitis and Low-Trauma Fracture: Analysis of the Nationwide Emergency Department Sample
J. McNabb-Baltar, E. Afghani, L. Lee, P. Banks, D. Conwell. Division of Gastroenterology, Hepatology, and Endoscopy, Brigham and Women’s Hospital, Harvard Medical School, Boston, MA.
Background: Chronic pancreatitis (CP) is associated with an increased risk of low-trauma fracture. Vertebral, wrist and hip fracture are all low-trauma fracture, but lead to very different morbidity and healthcare costs.
Aim: Examine patients with CP presenting to the Emergency Department in the US with low-trauma fractures, assess if they are more prone to a certain type of fracture, and draw comparison with patients with other “high-risk” gastrointestinal (GI) disease.
Methods: Within the Health Care Utilization Project Nationwide Emergency Department Sample (NEDS), we focused on patients, 18 years and older, presenting to the emergency department (2006-2009) with low-trauma fracture (hip fracture, vertebral fracture, and wrist fracture). Within that cohort, patients with CP, Crohn’s disease (CD), cirrhosis (CR), and celiac sprue (CS) were compared. Models were fitted to predict the likelihood of wrist, hip and vertebral fractures.
Results: Overall, weighted sample of 3,820,179 patients with low-trauma fracture were identified, of which 2,967 (0.08%) had CP, 17,776 (4.65%) had CR, 6,251 (0.16%) had CD, and 1,662 (0.04%) had CS. The most common fracture site was hip, affecting 57.6% of patients with CP, 53.7% with CD, 57.4% with CR and 63.2% with CS. The least common site was wrist, affecting 12.8% of patients with CP, 12.8% with CR, 12.6% with CS, and 21.5% with CD. Multivariable analysis revealed that compared to the overall low-trauma fracture population, CP (OR=1.51 p<0.001); CD (OR=2.10 p<0.001), and CR (OR=1.49 p<0.001) were independent predictors of hip fracture.
Conclusion: Similar to other high-risk GI diseases, CP patients are associated with an increase odds of hip fracture. Attention needs to be directed towards in-office screening assessment and modification of risk factors in this vulnerable population.
Chronic Pancreatitis Outcomes in the Emergency Department
J. McNabb-Baltar,1 L. Lee,1 P. Banks,1 D. Conwell.11Divison of Gastroenterology, Hepatology, and Endoscopy, Brigham and Women’s Hospital, Harvard Medical School, Boston, MA.
Background: Chronic pancreatitis (CP) is a debilitating chronic illness.
Aim: We sought to assess the most common reasons patients with CP visit the Emergency Department (ED), the outcomes of ED visit [admission, discharge, death], and evaluate predictors of admission.
Methods: Within the Health Care Utilization Project Nationwide Emergency Department Sample (NEDS), we focused on patients, 18 years and older, presenting to the emergency department with CP (ICD-9 code 577.1) as a diagnosis (2006-2009). Model was fitted to predict the likelihood of admission.
Results: Overall, a weighted sample of 638,310 patients visits for CP were identified, of which 399,559 (62.6%) were admitted, 228,523 (35.8%) were discharged from the ED, 5572 (0.9%) discharged against medical advice, and 4370 (0.7%) died during the hospital visit. The most common associated diagnoses at the time of ED visit were abdominal pain (25.4%), acute pancreatitis (22.5%), cardiac complication (11.1%), infection (10.6%), and dehydration (8.8%). Multivariable analysis revealed that older (OR=1.02 p<0.001), sicker patients (OR=2.44 p<0.001), patients presenting with acute pancreatitis (OR=6.60 p<0.001), alcoholic etiology of chronic pancreatitis (OR=1.67 p<0.001) were associated with increased odds of hospitalization. In contrast, ED visit on the weekend vs. weekday (OR=0.96 p<0.001), and abdominal pain as an associated diagnosis (OR=0.25 p<0.001) were associated with a lower admission rate.
Conclusion: CP is a debilitating condition associated with a high incidence of ED visits. Our study demonstrates that abdominal pain, followed by acute pancreatitis and cardiac complications, were the most common associated diagnoses for these ED visits. Whilst the majority of patients presenting to the ED with CP are admitted, very few die as a result of their hospitalization.
Female Gender Impacts Mortality and Length of Stay in Acute Pancreatitis in the United States
J. McNabb-Baltar,1,2 L. Lee,1,2 P. Banks,1 D. Conwell.1,21Divison of Gastroenterology, Hepatology, and Endoscopy, 2Fish Center for Women’s Health, Brigham and Women’s Hospital, Harvard Medical School, Boston, MA.
Background: Acute pancreatitis (AP) is the most common gastrointestinal admission, and thus represents a significant burden on healthcare expenditure. Racial and gender differences have been reported by our group in the emergency ward but it is unclear if inequalities exists after hospital admission.
Aim: Using a population-based US database, the aim of this study was to assess the impact of gender on in-hospital mortality and prolonged length of stay (LOS) in patients presenting with AP.
Methods: Within the Health Care Utilization Project Nationwide Inpatient Sample (NIS), we focused on patients, 18 years and older, admitted from the emergency department with AP as primary diagnosis (1998-2010). Models were fitted to predict the likelihood of in-hospital mortality, and LOS (hospital stay beyond the 75th percentile of 7 days).
Results: A weighted sample of 2,844,497 patients visits were identified, of which 1,423,328 (50.1%) were women. A total of 75,716 (2.7%) patients died during the admission; 48.2% were women. The etiology of acute pancreatitis differed according to gender; alcohol was the most common etiology of pancreatitis in men (23.0 vs. 7.5%, p<0.001), while biliary pancreatitis was more common in women (32.5 vs.18.3%, p<0.001). Male patients were more likely to have a secondary diagnosis of chronic pancreatitis (8.4 vs. 5.5%, p<0.001), but women had more comorbidities (CCI ≥ 3; 63.5 vs. 55.0%, p<0.001). Multivariable analysis revealed that relative to men, women had improved survival (OR=1.34, p<0.001), and decreased length of stay (OR=1.03, p<0.001).
Conclusion: Gender represents an independent predictor of in-hospital mortality and prolonged length of stay in patients admitted with AP. Disease severity, biological differences or unequal care could potentially explain these gender-specific inequalities, and deserves further assessment.
Temporal Trends of Nutritional Therapy in Acute Pancreatitis
J. McNabb-Baltar, L. Lee, V. Kadiyala, S.L. Suleiman, P. Banks, D. Conwell. Division of Gastroenterology, Hepatology, and Endoscopy, Brigham and Women’s Hospital, Harvard Medical School, Boston, MA.
Background: In recent years, parenteral nutrition (TPN) has fallen out of favor as a means for nutritional support in patients with acute pancreatitis (AP).
Aim: Assess the use of TPN, and enteral nutrition (EN) in patients admitted with AP in the United States, and evaluate factors associated with use of nutritional therapy (NT).
Methods: Within the Health Care Utilization Project Nationwide Inpatient Sample (NIS), we focused on patients, 18 years and older, admitted between 1998 and 2010 from the emergency department with AP as primary diagnosis (ICD-9 code 577.0). Models were fitted to predict the likelihood of NT use. Covariates included cause of pancreatitis, socio-economical status and other patient and hospital characteristics. Temporal trends of NT, TPN and EN use were assessed using estimated annual percentage change (EAPC), based on linear regression methodology.
Results: Overall, a weighted sample of 2,844,497 patients admitted with acute pancreatitis was identified. A total of 112,076 (3.9%) NT: 100,312 (3.5%) received TPN, and 14,083 (0.5%) received EN. Multivariable analysis to predict use of NT showed that patients with more comorbidities (CCI≥3 OR=1.26, p<0.001), secondary diagnosis of chronic pancreatitis (OR=1.33, p<0.001), highest annual income (OR=1.33, p<0.001), in large (OR=1.22, p<0.001), urban hospitals (OR=1.45, p<0.001) were more likely to be treated with NT. Conversely, patients admitted with biliary pancreatitis were less likely to receive NT than those with alcoholic AP (OR=0.88, p=0.001). Temporal trends to assess the use of NT showed a modest increase (EAPC = 0.4%, p<0.001) between 1998 and 2010; TPN use remained stable (EAPC=-0.2%, p=0.09), and EN use increased during the study period (EAPC=5.8%, p<0.001).
Conclusion: In the United States, the use of NT and EN is increasing in patients admitted with AP, while that of TPN remains stable.
Concerning Trends in the Management of the Metastatic Pancreatic Cancer
J. McNabb-Baltar,1 J. D. Sammon,1 V. Trinh,1 Q.D. Trinh,1 D. Conwell.11Divison of Gastroenterology, Hepatology, and Endoscopy, Brigham and Women’s Hospital, Harvard Medical School, Boston, MA.
Background: A recent study showed an increasing rate of admissions from the Emergency Department (ED) over the past decade for many common diseases, which may reflect ineffective and inadequate referral pathways between primary care providers and hospital specialists. Testing such a hypothesis is difficult to perform in the context of acute conditions. However, it would be intuitive to believe that care of patients with metastatic cancer could theoretically be organized in a manner that would minimize the use of the ED, as most metastatic malignancies follow an expected and protracted course.
Methods: Within the Nationwide Inpatient Sample (NIS), we focused on patients 18 years and older admitted with pancreatic cancer (PC) as a primary diagnosis (1998-2006). Metastatic disease was ascertained by the presence of concomitant diagnostic codes for metastatic sites. Temporal trends in incidence were reported per 100,000 person-years, and assessed using estimated annual percentage change (EAPC), based on linear regression methodology adjusted for clustering.
Results: Overall, a weighted sample of 131,370 hospital admissions with metastatic PC was identified. Between 1998 and 2006, the incidence of overall admissions for metastatic PC slightly increased from 5.06 to 5.08 per 100,000 person-years (EAPC +2.45% p<0.001). Concurrently, the incidence of admissions from the ED significantly increased from 1.68 to 2.45 per 100,000 person-years (EAPC +6.64% p<0.001). The proportion of admissions from the ED relative to all admissions for metastatic PC increased from 33.1 to 48.1% (EAPC +4.12% p<0.001).
Conclusion: The incidence and proportion of ED admissions for metastatic PC have increased significantly in the past decade. This may reflect ineffective and inadequate referral pathways between primary care providers and hospital specialists, and requires further investigation.
CA19-9 Is a Significant Prognostic Factor Following Pancreatoduodenectomy for Intra-Pancreatic Bile Duct Cancer (Cholangiocarcinoma)
J. Melling, A. Thomas, P. Sykes, O. Jones, R. Jackson, P. Whelan, F. Campbell, M. Raraty, C. Halloran, R. Sutton, J. Neoptolemos, P. Ghaneh. NIHR Liverpool Pancreas Biomedical Research Unit, University of Liverpool, Liverpool, UK.
Aim: We aimed to identify clinicopathological factors associated with survival following pancreatoduodenectomy for intra-pancreatic bile duct cancer (distal cholangiocarcinoma). Previous studies have identified resection margin status and tumor differentiation, amongst others, as being significant survival factors.
Methods: A prospectively maintained database was interrogated to identify all patients undergoing pancreatoduodenectomy for cholangiocarcinoma from 1997 to 2011. Perioperative blood tests, pathological findings and survival data were collected. Kaplan-Meier survival curves were produced, and differences assessed using the Log-Rank test. Multivariate analysis was performed using a Cox Proportional Hazard model.
Results: There were 104 (60 male, 44 female) patients with a median age of 65 (IQR 57-70) years. There were three (2.9%) post-operative deaths. Median overall survival was 17.9 (95%CI 14.6-21.3) months with a 5-year survival of 18%. Univariate analysis revealed resection margin status (p<0.001), tumor differentiation (p<0.001), number of positive margins (p<0.001), tumor stage (p=0.002), lymph node involvement (p=0.002) and raised post-operative CA19-9 (p=0.014) to be significant predictors of survival. On multivariate analysis, positive resection margin status (HR=2.27, 95%CI 1.32-3.85; p=0.003), poor tumor differentiation (HR=2.04, 95%CI 1.20-3.50; p=0.009) and raised post-operative CA19-9 (HR=1.93,95%CI 1.15-3.25, p=0.013) remained independently significant predictors of survival.
Conclusion: This study is the first in a UK population and the largest in a European population and confirms the prognostic significance of resection margin status and tumor differentiation. The significance of postoperative CA19-9 is however a novel finding and may assist the design of future randomized adjuvant trials for intra-pancreatic bile duct cancer.
Development of Pancreatitis in MMP13 Deficient Mice
A. Mendonsa,2 L. Gorden,1,2 M.N. VanSaun.1,21Department of Surgery, Vanderbilt University, Nashville, TN; 2Department of Cancer Biology, Vanderbilt University, Nashville, TN.
Acute pancreatitis, or inflammation of the pancreas, represents a pancreatic disease that leads to the hospitalization of over 200,000 people per year in the United States. Repetitive acute episodes culminate in chronic pancreatitis with accompanying fibrosis and permanent damage of the pancreas. Identification of factors influencing the development and/or progression of pancreatitis is necessary to stop progression and aid in the recovery from this disease. In a subset of human normal versus pancreatitis plasma samples we were able to detect and increase in Matrix MetalloProteinase 13 (MMP13) through ELISA based analysis. MMP13 is an interstitial collagenase which is known to be important in the fibrotic response of pancreatic stellate cells. Subsequently, we detected an increased number of alpha smooth muscle actin positive cells in the MMP13 deficient murine pancreas. We hypothesize that altered modulation of MMP13 levels in the pancreas leads to dysregulation of matrix turnover by pancreatic stellate cells and under chronic conditions enhances development and severity of pancreatitis. Preliminary analysis demonstrated that MMP13 deficiency in mice leads to the development of focal areas of pancreatitis. MMP13 deficient mice aged 6 months additionally exhibited increased plasma amylase levels when compared to wildtype mice of similar age or to younger three month old MMP13 deficient mice. Current studies are directed at testing how stellate cells contribute to onset, severity and recovery from cerulein induced acute and chronic pancreatitis in the absence of MMP13. The results from this study suggest an important role for MMP13 in stellate cell function and in the development of pancreatitis. Future studies in this area will provide mechanistic insight into the development and progression of pancreatitis.
A Plea for Biological Resectability Criteria in Surgery of Locally Advanced Pancreatic Cancer
C. W. Michalski,1 C. Jäger,2 B. Kong,2 H. Friess,2 M. Erkan,2 J. Kleeff.21Dept. of Surgery, Oregon Health and Science University, Portland, OR; 2Dept. of Surgery, Univ. of Technology, Munich, Germany.
Background: We hypothesized that – in contrast to published reports and current clinical practice – not all patients suffering from pancreatic ductal adenocarcinoma (PDAC) with presumed invasion of the portal/superior mesenteric vein may be good candidates for surgical resection.
Patients and Methods: 212 patients who were resected for PDAC between 2007 and 2011, were stratified into a group with a survival of <12 months (n=44) versus >24 months (n=67). Because this analysis yielded resection of the portal/superior mesenteric vein (PV/SMV) as a prognostic factor, we compared the subgroup of patients with PV/SMV resection (n=54) and those without (n=102).
Results: Multivariate analysis of the short- and long-term survivors demonstrated N, G and venous resection as prognostic of survival. While venous resections could be performed safely, there was a clear trend towards shorter survival in the PV/SMV resection group (median survival 22.7 vs. 15.9 months, p=0.157). These tumors were significantly larger (4.3 vs 3.5 cm; p=0.026) and margin-positivity was more frequent (34.8% vs 52.2%, p=0.05). Preoperative imaging predicted true invasion of the vein at a low sensitivity and specificity (70% and 30%, respectively). Though patients with G1/2 PDACs lived significantly longer (G1/2 vs. G3, median survival 39.9 vs. 24.5 months, p<0.001), such a stratification was not seen in the venous resection cohort (median survival 15.5 vs. 12.5 months).
Conclusion: The shorter survival of the venous resection patients and the irrelevance of the tumor grade in this group, lead us to the conclusion that for patients in whom an invasion of the PV/SMV is suspected on imaging, an scientific effort is necessary to define biological rather than technical resectability criteria. The current standard approach of claiming that venous invasion is no contraindication for up-front resection should be revisited.
Risk Factors Of Postoperative Cholangitis and Biliary Strictures of Pancreaticoduodenectomy
A. Miki, Y. Sakuma, H. Sasanuma, Y. Kaneda, N. Sata, Y. Yasuda. Department of Surgery, Jichi Medical University, Tochigi, Japan.
Background: Recent progress in surgery and perioperative care contributes to the improved outcome of pancreaticoduodenectomy. In this study, incidence and outcomes of biliary complications after pancreaticoduodenectomy (PD) were evaluated.
Methods: Data from patients underwent PD from January 2006 to December 2011 at Jichi Medical University were analyzed regarding preoperative laboratory data, imaging studies and postoperative outcomes.
Results: Total 140 patients including 121 malignant and 19 benign diseases were analyzed. Fourteen patients (10%) developed postoperative cholangitis and nine patients (6.5%) developed biliary stricture. The median time to cholangitis onset was 14 months (range 3 – 43 month). Preoperative drainage and type of disease were not related to cholangitis and biliary stricture. By univariate analysis, the value of preoperative Ca19-9, type of pancreatic duct reconstruction (pancreaticogastrostomy), operation time, and postoperative pancreas fistula were associated with cholangitis. The value of Ca19-9, the type of pancreatic duct reconstruction, and postoperative pancreas fistula were associated with biliary strictures. All cholangitis patients were treated with antibiotics at the onset. Biliary strictures were initially treated with double balloon endoscopy (DBE) and percutaneous transhepatic cholangiodrainage was performed for one patient whose endoscopic treatment was failed. No surgery was performed. Recurrent neoplastic disease was discovered in one patient of 9 patients (11%).
Conclusion: The significant univariate predictors for cholangitis were preoperative Ca19-9, type of pancreas duct reconstruction, and postoperative pancreas fistula. All patients with biliary stricture were managed with intervention using DBE or PTCD without surgery.
Transgastric Hypothermia Achieves Temperatures that Synergistically Slow Multiple Signaling Pathways Relevant to Acute Pancreatitis (AP)
V. Mishra,1 R.N. Trivedi,1 K. Patel,1 P. Noel,1 C. Durgampudi,2 C. Acharya,2 J.A. Holmes,3 S. Narla,4 V.P. Singh.1,5Department of Medicine University of Pittsburgh1, UPMC Passavant2, Swanson Center for Product Innovation3, Graduate School, Lake Erie College of Osteopathic Medicine4, Division of Gastroenterology, University of Pittsburgh, Pittsburgh, PA1,5.
Background: Multiple deleterious signaling cascades are simultaneously activated in AP, which may limit the success of pharmacologic approaches. Moreover drug toxicity may be higher in AP with ongoing organ failure. We therefore explored the feasibility of successfully cooling down the pancreas and learning what temperatures could be achieved without causing generalized hypothermia, and studying if temperatures in this range can inhibit deleterious signaling in acinar cells.
Methods: A collapsible gastric balloon attached to a dual lumen tube was perfused with ice cold water in sedated rats. Rectal, pancreas body and tail temperatures were noted. Caerulein (100nM) induced trypsinogen activation (TGA), CXCL1, 2 mRNA levels, cell injury (LDH leakage, propidium iodide uptake) were studied at 37°C, 34°C, 31°C, 28°C and 25°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, which normalized rapidly on discontinuing cooling. In acini, a >80% reduction in TGA, CXCL1, 2 mRNA increase were noted at 29°C with a similar reduction in cell injury at 34°C compared to 37°C. While trypsin and cathepsin B activities at 23°C were 53% and 64% of those at 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 pancreatic temperatures achieved slow mechanistically deleterious phenomena by >80% and synergistically slow sequential steps relevant to AP. Therefore Transgastric Hypothermia may be a better option than drug therapy for AP.
Reduced Anterior Gradient 2 Expression is Induced by EMT and Correlated With Poor Outcome in Pancreatic Ductal Adenocarcinoma
Y. Mizuuchi,1 S. Aishima,1 K. Ohuchida,2 K. Shindo,1 K. Mizumoto,2 M. Tanaka,2 Y. Oda.11Department of Anatomic Pathology, Graduate School of Medical Sciences, Kyushu University, Fukuoka, Japan; 2Department of Surgery and Oncology, Graduate School of Medical Sciences, Kyushu University, Fukuoka, Japan.
Anterior gradient 2 (AGR2) was reportedly overexpressed and acted as a pro-oncoprotein in many human cancers. We investigated the clinical importance of AGR2 expression in pancreatic ductal adenocarcinoma (PDAC) by immunohistochemical analysis and confirmed these results in vitro. While AGR2 expression was not seen in normal pancreas, all the pancreatic precursor neoplastic lesions were positive for AGR2 even in the earliest precursor lesions such as PanIN-1A and IPMN with low grade dysplasia. In PDAC, AGR2 expression was reduced in 27.7% (54/195 cases), and AGR2 downregulation correlated with epithelial mesenchymal transition (EMT) markers (cytoplasmic vimentin overexpression and reduced membranous E-cadherin expression), high UICC stage, high cellular grade and poor prognosis. In vitro, AGR2 was variously expressed among assessed 10 pancreatic cancer cell lines in mRNA and protein levels. AGR2 knockdown in Aspc-1 cells using small-interfering RNA significantly reduced cell invasiveness and migration, but not altered EMT markers. To confer more aggressive phenotype and induce EMT in PDAC cells, we co-cultured Aspc-1 cells with primary cultured cancer associated fibroblasts (CAFs) and showed that AGR2 was downregulated in co-cultured Aspc-1 cells, compared with monocultured cells in accordance with EMT induction and increased cell invasiveness and migration. ELISA showed that TGF-beta1, which was known as EMT inducer, was detected in co-cultured cells supernatant. Treatment of Aspc-1 cell with recombinant TGFbeta1 induced EMT and decreased AGR2 expression. In summary, it was suggested that AGR2 expression could be useful prognostic marker and reduced by EMT. EMT by TGF-beta1 may partially contribute to the AGR2 downregulation in PDAC patients.
The Conundrum of Groove Pancreatitis
E. Morandi, L. Turati, M. Castoldi, D.A. Merlini. Department of General Surgery, Rho Hospital, Rho (MI), Italy.
Introduction: Groove pancreatitis is a rare condition characterized by inflammatory fibrosis of the space between the dorsocranial part of the pancreatic head, duodenum and common bile duct, determining chronic abdominal pain, weight loss, nausea/vomiting, often without jaundice, with worsening in quality of life. Pancreaticoduodenectomy is curative.
Case report: We report a case of a 44 years male patient with an history of strong alcohol intake, presenting with epigastric pain for 6 months not responding to analgesic drugs and a 8 kilos weight loss. An abdominal US evidenced an hypoechoic mass of the head of the pancreas. CT confirmed a 4x5 cm mass with duodenal infiltration and two little ipodense cystic areas in the lesion, mimicking adenocarcinoma. An EUS showed duodenal wall thickening, with three cysts into the duodenal wall; the pancreatic mass, determining neither Wirsung nor common bile duct dilation, was assessed to be inflammatory; EUS-FNA didn’t show neoplastic cells. A gastro-duodenoscopy evidenced a 6 cm lesion in the second part of the duodenum involving the papilla, histologic examination of the biopsies found only signs of chronic inflammation. Patient underwent Whipple procedure. Histologic examination demonstrated signs of chronic inflammation in the groove area, with a great hyperplasia of the muscular propria of the duodenum wall, containing cysts. The patient underwent CDP. 6 months after the operation the patient is asymptomatic and gained 4 kilos.
Conclusion: The diagnosis of groove pancreatitis is demanding. Symptoms are not specific: chronic abdominal pain, often epigastric and postprandial with possible dorsal irradiation, weight loss, nausea/vomiting can also be present in patients with cancer.
Considering anamnestic datas (especially alcohol intake), the young age of the patient, the worsening in quality of life and the impossibility to exclude malignancy at the preoperative study, the pancreaticoduodenectomy is the treatment of choice, with a prompt resolution of the disease.
Can Antisense Oligonucleotides Specific to K-Ras Gene Inhibit the Tumor Growth, Invasion and MMP-2 and MMP-9 Expression in Hamster Pancreatic Cancer Model In Vitro and In Vivo?
C.Y. Morioka,1-4 J.P. Otoch,3 S. Saito,4 M.C.C. Machado,3 A. Watanabe,4 C.C. Huang.21MoriokaÇs Center for Integrative Care; 2Advantage Health; São Paulo, Brasil; 3Depart of Surgery, University of São Paulo, São Paulo, Brazil; 3rd Depart of Internal Medicine, Toyama University, Toyama, Japan.
Matrix metalloproteinases (MMP), especially MMP-2 and MMP-9, are thought to play major roles in pancreatic cancer growth and metastasis. Ras activates a multitude of downstream activities with roles in cellular processing, including invasion and metastasis. Therefore, antisense oligonucleotides (ASO) targeting this K-ras gene may be a therapeutic approach.
Aim: To elucidate the effectiveness of this gene therapy in hamster experimental cancer model.
Materials and Methods: HaP-T1, a cell line derived from BHP-induced pancreatic cancer was used. Transfection with ASO were performed. MTT and MTT agarose assays were done. Chemoinvasion assay was performed. MMP-2 and MMP-9 production by the cell lines was determined by gelatin zymography. For in vivo experiments, subcutaneously resected tumors were implanted orthotopically in Syrian golden hamsters, which were divided in 3 groups: A) Positive control (PC), B) Sense treated hamsters (STH), and C) Antisense treated hamsters (ATH). Oligonucleotides were administered for 2 weeks. Follow up was done. Five animals of each group were sacrificed at Days 10, 17, 24, 31, and 38, to study the local response and metastatic sites. Five animals of each group were left to study the survival time. Specimens were studied histopathologically. Orthotopic pancreatic tumor MMP production was measured by gelatin zymography.
Results: ASO inhibited the tumoral growth and invasiveness. They downregulated active forms of MMP-2 and MMP-9 in a dose dependent manner in vitro. Positive controls, STH, and ATH survived in average 72.7, 74.3, and 82.7 days, respectively. Spontaneous lymph node metastases were found from 31 days in ATH group, while PC and STH groups showed metastases and direct invasion to adjacent organs from 17 days. After death, metastatic sites were similar in the 3 groups. ASO downregulated the activation of MMP-9, more than MMP-2 in vivo.
Conclusions: These experiments suggest that ASO targeted K-ras gene may be a good choice in the management of pancreatic cancer because of the suppression of tumor growth and invasiveness in vitro and in vivo. ASO also downregulated the activation of MMP-9 and MMP-2 in vivo.
Higher Overall Risk of Pancreatic Cancer in Patients With Pancreatic Cyst
S. Munigala,1 S. Javia,1 A. Gelrud,2 D.L. Conwell,3 J. Scherrer,4 B. Agarwal,1. 1Department of Internal Medicine, Division of Gastroenterology and Hepatology, Saint Louis University; 2Center for Pancreatic Disorders, Section of Gastroenterology, Hepatology and Nutrition, University of Chicago; 3Center for Pancreatic Disease, Division of Gastroenterology Hepatology and Endoscopy, Brigham and Women’s Hospital, Harvard Medical School, Boston; 4St. Louis VAMC, Research Service and Department of Family and Community Medicine, Saint Louis University School of Medicine.
Background: Certain pancreatic cysts (MCN and SB-IPMN) have malignant potential and require surveillance. However, overall long term risk of pancreatic cancer (PaCa) in patients with pancreatic cysts is still not known.
Patients and Methods: This is a retrospective study with data from Veterans Health Administration (VHA) national medical care data sets from Fiscal Year (FY) 1998/1999-2007. Patients noted to have pancreatic cyst on CT/MRI (n=1050) were identified using ICD 9 codes. Patients diagnosed to have PaCa within 1 year of identification of cyst (n=102) were excluded. Remaining 948 patients were divided into two groups: 1) group A- 402 patients without h/o acute or chronic pancreatitis 2) group B- 546 patients with new diagnosis chronic pancreatitis or episode of acute pancreatitis within 1 year of the diagnosis of pancreatic cyst. These patients and remaining patients in the database without cysts (group C, n=533481) were followed from 1998 to 2007.
Results: The mean age of patients in group A, B and C was 60 ± 12.5, 54 ± 10.5 and 54 ± 14.1 years respectively. During the following-up of 96 months, in group A, B and C respectively PaCa was diagnosed in 7, 10 and 1406 patients and the annual incidence rate of PaCa was17.41, 18.31 and 2.63 per 1000 patient-years. In comparison to group C, the relative risk of PaCa in group A was 6.61 (95% CI 3.16, 13.80, p<0.0001) and in group B was 6.61 (3.16, 13.80, p<0.0001). The incident pancreatic cancers were distributed throughout follow-up period.
Conclusion: Patients with pancreatic cysts have a significantly higher overall risk of PaCa. Since only a subset of pancreatic cyst types have malignant potential, the present study is limited due to lack of information about the type of the pancreatic cyst in study patients. This would underestimate the risk of PaCa in patients with mucinous cystic neoplasm (MCN) and SB-IPMN. Future studies need to evaluate the risk of PaCa in patients with known etiologic types of pancreatic cysts.
Differential Patterns of Abdominal Adipose Tissue Loss in Pancreatic Cancer vs Post Gastric Bypass Surgery
S.J. Nagpal,1 R.P. Sah,2 N. Takahashi,3 J.M. Miles,4 G.M. Petersen,5 D. Mukhopadhyay,6 S.T. Chari,1. Divisions of 1Gastroenterology and Hepatology, 2Internal Medicine, 3Radiology, 4Endocrinology, 5Epidemiology, 6Biochemistry and Molecular Biology, Mayo Clinic, Rochester, MN.
Background/Aims: Visceral adipose tissue (VAT) is metabolically active. Increase in VAT is associated with the development of insulin resistance and type 2 diabetes (DM); improvement in insulin sensitivity following weight loss from lifestyle change, weight loss medication, and Roux-en-Y gastric bypass (RYGB) is associated with significant loss of VAT. Pancreatic cancer (PaC) is paradoxically associated with development of DM in the face of ongoing weight loss. To explain the lack of improvement in DM despite weight loss in PaC we hypothesized that VAT is relatively preserved compared to subcutaneous adipose tissue (SAT). Using paired abdominal CT scans we compared the relative changes in SAT and VAT in PaC and RYGB.
Methods: We identified patients with PaC (n=151, mean age 71±1) and morbidly obese patients who had undergone RYGB (n=68, mean age 48±1) who had 2 abdominal CTs available for review: CT1 done > 6 months prior to diagnosis of PaC (median 1190 days) or RYGB (median 678 days) and CT2 done at PaC diagnosis (median 2 days) or after RYGB (median 220 days). We measured SAT and VAT at the L3/L4 vertebral level using a semi-automated protocol in the iNtuition software (TeraRecon Inc, USA);
Results: While the prevalence of DM at CT1 was similar in PaC and RYGB groups (33.8% vs 33.3%, p=ns), it was significantly higher in PaC at CT2 (64.3% vs 41.0, p<0.001). Between CT1 and CT2, while PaC patients preferentially lost SAT over VAT (14.4% vs 6.0%, p=0.026*), RYGB patients lost both SAT and VAT equally (26.7% vs 30.0%, p=0.22). In PaC this differential loss of SAT over VAT was seen only in patients who had impaired fasting blood glucose/diabetes (n=70) (16.2% vs 1.2%, p=0.0038*), but not in those with normal fasting glucose (n=40) (7.0% vs 4.2%, p=0.51).
Conclusion: The paradoxical worsening glucose tolerance in patients with PaC despite loss of adipose tissue is associated with the unique phenomenon of relative preservation of VAT over SAT. The fact that this observation is limited only to patients with elevated fasting blood glucose points to the presence of a PaC specific mediator responsible for diabetes and adipose tissue loss.
First Jejunal Vein-Oriented Mesenteric Excision and Reduction of Intraoperative Bleeding During Pancreatoduodenectomy
M. Nakamura, T. Abe, H. Nakashima. Department of Digestive Surgery, Kawasaki Medical School, Kurashiki, Japan.
Background: Operative bleed loss is one of the risk factors of complications after pancreatoduodenectomy (PD). SMA first technique with ligation of IPDA is one of the common solutions to decrease operative blood loss, although early detection of IPDA is not guaranteed. Furthermore, IPDA is duplicated in some cases, or sometimes branching into several small arteries. Early total transection of mesoduodenum between SMA and SMV is an ideal method to resect all the branches arising from SMA into pancreas head. However the first jejunal vein (FJV) exists in the mesoduodenum, and impatient dissection of the mesoduodenum without detection of FJV causes massive bleeding from FJV. We applied a new surgical approach, first jejunal vein-oriented mesenteric excision (FME) for PD. This study aimed to clarify the effect of FME on reduction of bleeding during PD.
Methods: We retrospectively analyzed the perioperative outcome of 54 patients (FME32/conventional PD21) who underwent resection of the pancreatic head, PD and total pancreatectomy (TP) from 2010 through May 2013 in Kawasaki Medical College.
Results: The FJV was posterior to the SMA in the majority of the patients but was anterior to the SMA in 16.7% of patients. IPDA was placed cranial to FJV except for one patient whose FJV was anterior to the SMA. Additionally, IPDA was within 15mm cranial to the FJV in 70% of the patients. FME-based PD significantly reduced intraoperative blood loss compared with conventional PD (571 ml, SD 324 (FME group) versus 1234 ml, SD 905 (standard group), P=0.006). Operation time of two groups showed no statistical significant difference (394 min, SD 114 (FME group) versus 418 min, SD 71 (standard group), p=0.125).
Conclusions: FJV can be an indicator of IPDA and FME is useful for reducing intraoperative bleeding
Salinomycin Induced Autophagy and Is Related With Its Killing Effect For Pancreatic Cancer Cells
K. Nakata, M. Zhao, K. Ohuchida, Y. Miyasaka, R. Maeyama, T. Ohtsuka, K. Mizumoto, M. Tanaka. Department of Surgery and Oncology, Graduate of Medical Sciences, Kyushu University, Fukuoka, Japan.
Background: Salinomycin, which is widely used as an anticoccidial drug, is now increasingly recognized as a novel and effective anti-cancer agent. However, the molecular mechanism underlying salinomycin-induced cell death still remains unclear. In the present study, we investigated the mechanisms of cell death after salinomycin treatment of pancreatic cancer cell lines.
Methods: We examined the effect of salinomycin on cell viability with colony formation assay using pancreatic cancer cell lines CAPAN-2, AsPC-1 and SUIT2. We also investigated the autophagy levels of pancreatic cancer cell lines using GFP-LC3 transfection and immunoblotting. Further, we investigated the effect of autophagy inhibition on cell killing effect of salinomycin using pancreatic cancer cell lines.
Results: Salinomycin treatment reduced the pancreatic cancer cell viability in a time and concentration dependent manner. The number of colonies was significantly lower in the salinomycin treatment group than in control group. We also found salinomycin treatment inhibited AKT/mTOR signaling pathway. Furthermore, the elevation of LC3-II levels was observed after salinomycin treatment, suggesting autophagy induction. These results were confirmed by GFP-LC3 transfected pancreatic cancer cell lines. After the inhibition of autophagy-related genes ATG5 and ATG7, the salinomycin-induced cell killing effect was inhibited.
Conclusion: The present findings provide the evidence that the killing effect of salinomycin treatment on pancreatic cancer cell lines is related with autophagy induction.
Initial Blood Urea Nitrogen as an Independent Predictor of Inpatient Morbidity in Acute Pancreatitis: A Retrospective Observational Study in Minority Populations
S.K. Nayudu,1,2 H. Chelimilla,1,2 J. Makker,2 A. Sinha,2 H. Hashmi,2 C. Chandrala,2 A. Sendilnathan,2 K. Kumbum,1,2 S. Chilimuri.1,21Division of Gastroenterology, 2Department of Medicine, Bronx Lebanon Hospital Center, Albert Einstein College of Medicine of Yeshiva University, Bronx, NY.
Background: There has been evolving data correlating Blood Urea Nitrogen (BUN) with mortality in acute pancreatitis. We hypothesized that initial BUN can serve as a single marker of severity of acute pancreatitis and can predict inpatient morbidity.
Methods: We conducted retrospective observational study between January 1, 2009 and June 30, 2011 including all individuals admitted to our institution with acute pancreatitis. We collected demographic, laboratory data including BUN on admission and Length of Stay (LOS) as a measure of inpatient morbidity. BUN of more than 25 milligrams/deciliter was considered abnormal. We analyzed data using standard statistical tests and JMP10 software.
Results: There were 209 individuals who met the inclusion criteria. Men and women represented equally. There were 51% Hispanics and 49% non-Hispanics of which 80% were African Americans. There were no significant differences in LOS between women and men (9.36 vs. 11. 25 days, p 0.17), between Hispanics and non-Hispanics (9.79 vs. 10.82, p 0.46). However we found significant difference between LOS among individuals with initial abnormal BUN (15.34 days) compared to normal BUN (9.28 days, p 0.019). On multiple regression analysis higher BUN levels directly correlated with longer LOS.
Conclusion: Initial BUN can independently predict LOS in acute pancreatitis reaffirming BUN as a single marker of severity. Our study is unique in associating BUN with LOS and including minority populations. We recommend that initial BUN can be used as a single marker of severity in acute pancreatitis and early interventions targeting at normalizing BUN may reduce LOS there by reducing health care expenditure.
Phenocopying Hereditary Pancreatitis Associated Mutations in Mouse Trypsinogens
B. C. Németh, M. Sahin-Tóth. Department of Molecular and Cell Biology, Boston University Medical Center, Boston, MA.
Introduction: Hereditary pancreatitis associated PRSS1 mutations cause increased autoactivation of human cationic trypsinogen in the presence of chymotrypsin C (CTRC). Introduction of the clinically most frequent p.R122H mutation in mouse trypsinogens does not recapitulate this pathogenic biochemical phenotype, because mouse Ctrc regulates mouse trypsinogens differently. To develop mouse models mimicking the human disease, we need to identify or design mouse trypsinogen variants that phenocopy the human mutants. Recently, we demonstrated that mutations p.D19A, p.D22G and p.K23R in the activation peptide of human cationic trypsinogen increase autoactivation in a CTRC-independent manner.
Aims: Here we investigated the effects of mutations p.D19A, p.D23A and p.K24R in the context of the mouse T7 trypsinogen isoform. These mutations are either analogous or similar to those found in human cationic trypsinogen.
Methods: Mouse trypsinogens and Ctrc were expressed recombinantly. Trypsinogen activation was studied by enzyme activity assays and SDS-PAGE.
Results: Mutation p.D23A resulted in strikingly (>20-fold) accelerated autoactivation of T7 trypsinogen, whereas autoactivation of the p.K24R mutant was increased 4-fold relative to wild-type T7 trypsinogen. In contrast, mutation p.D19A had no effect on autoactivation. Mouse Ctrc did not modify autoactivation of the mutants to a significant extent.
Conclusions: Mutations p.D23A and p.K24R cause increased autoactivation of T7 mouse trypsinogen in a manner that resembles the effect of similar mutations in human cationic trypsinogen. Thus, these mutations offer a viable approach for the development of mouse models of human hereditary pancreatitis.
Fully Covered Through-The-Scope Esophageal Metallic Stents for Endoscopic Transluminal Drainage and Necrosectomy For Walled- Off Necrosis
Y. Nemoto, J. Roat, M. Arain, M. Freeman, R. Attam. Department of Medicine, University of Minnesota, Minneapolis, MN.
Background: Endoscopic transluminal drainage and necrosectomy have been increasingly utilized for infected or symptomatic walled-off necrosis (WON) in necrotizing pancreatitis, but are limited by the size of cystenterostomy provided by use of conventional plastic or biliary (10mm) covered stents. Use of large diameter (18mm) fully covered esophageal self-expandable metallic stents (CSEMS) may potentially provide more efficient and safer drainage and minimize number of repeat interventions. A newly available through-the-scope (TTS) 18mm fully covered esophageal stent offers the largest diameter removable TTS CSEMS to date.
Objective: To evaluate the efficacy and safety of endoscopic treatment of WON using fully covered TTS esophageal metallic stents.
Methods: Following initial endoscopic transmural drainage, patients underwent regular sessions of DEN as needed. Percutaneous catheter drainage and sinus tract endoscopy were used as adjunctive treatments for patients with very extensive extrapancreatic WON.
Results: 61 patients had endoscopic drainage/DEN for WON from 2009-2013 at one center. Median size of initial balloon dilation was 15mm. Combined endoscopic duodenal + gastric cystenterostomies were performed in 9 (15%) of 61. Stents placed were plastic only in 42, plastic + metallic <18mm in 10, >18mm enteric only in 2, plastic + >18mm enteric in 6; TTS 18mmx6cm CSEMS were placed in 6; mean WON 16cm (range 13-19); etiology gallstones (3), alcohol (1), post-surgical (1), and medication-induced (1). Median # sessions of DEN was 3 (range 2-4). One pt required adjunctive PCD. Five patients (83%) achieved complete resolution. There were no complications during endoscopic insertion of stents, necrosectomy, or difficulty removing TTS stents.
Conclusions: Endoscopic transmural drainage using large diameter (18mm) fully covered TTS esophageal stents seems to be effective for DEN of very large WON. Randomized control trials are required to compare esophageal stents with plastic stents.
Step Up Approach to Interventions for Necrotizing Pancreatitis Using Primarily Endoscopic Approach: Outcomes in a Large Cohort of Patients
Y. Nemoto,1 J. Roat,1 M. Arain,1 S. Mallery,1 M. Freeman,1 G. Beilman,2 R. Attam. 1 Department of 1Medicine and 2Surgery, University of Minnesota, Minneapolis, MN.
Background: Minimally invasive step up approach involves drainage followed by necrosectomy as needed, and is superior to primary surgical necrosectomy for infected walled off necrosis (WON). Reported series of direct endoscopic necrosectomy (DEN) include highly selected patients. We report outcomes of a large series of patients with necrotizing pancreatitis managed by a step up approach based primarily on endoscopy when feasible with adjunctive percutaneous techniques as needed.
Methods: Consecutive patients with necrotizing pancreatitis managed by a multidisciplinary team at one center from 2009-2013, with intervention only for infected or persistently symptomatic WON; Endoscopic drainage/DEN was primary technique when feasible with percutaneous catheter drainage (PCD) plus additional sinus tract endoscopy (STE) for deep extrapancreatic extension and surgery only for failures.
Results: Of 76 patients with necrotizing pancreatitis intervention was required in 69 (91%), including endoscopic in 61 (alone in 48, and combined with PCD in 13, of which 5 had STE); dual duodenal + gastric cystenterostomy in 9 (15%) of 61; metallic stents in 18 (16%); pancreatic duct stenting in 39 (64%); and mean # endoscopic interventions 3.6 (range 1-9). Complications in 9 (14.7%) were minor bleeding (7), major hemorrhage (1), air embolism (1, prior to CO2). Cystenterostomy stents were left in indefinitely for disconnected pancreatic duct in 23 (38%) of 61. PCD was used primarily in 8 (11%). Persistent fistula in 3 all after PCD. 5 (7%) of 76 pts failed step up approach and required open necrosectomy. All-cause mortality was 4 (5.5%) including 3 (60%) of those requiring surgery.
Conclusions: A step up approach for interventions in necrotizing pancreatitis using primarily endoscopic techniques with percutaneous as adjuncts was highly effective with minimal open surgery, fistulae and mortality. Approximately 2/3 patients requiring intervention could be managed purely endoscopically.
Optimal Duration and Timing of Adjuvant Chemotherapy After Definitive Surgery for Ductal Adenocarcinoma of the Pancreas: Ongoing Lessons from the ESPAC-3 Study
J. Neoptolemos,1 J. Valle,2 D. Palmer,1 R. Jackson,1 T. Cox,1 P. Ghaneh,1 C. Bassi,3 M. Büchler.4 for the European Study Group for Pancreatic Cancer. 1Cancer. Research U.K. Liverpool CTU, University of Liverpool, UK; 2Christie Hospital, University of Manchester, UK; 3Surgical Department, University of Verona, Italy; 4Department of Surgery, University of Heidelberg, Heidelberg, Germany.
Purpose: Adjuvant chemotherapy improves patient survival following resection for pancreatic adenocarcinoma but the optimal duration and time to initiate chemotherapy is unknown.
Patients and Methods: Patients with pancreatic ductal adenocarcinoma treated within the international, phase III, ESPAC-3(v2) study were included if they had been allocated chemotherapy (gemcitabine or 5-FU). Overall survival analysis was performed on an intention-to-treat basis retaining patients in their randomized groups, adjusting the overall treatment effect by known prognostic variables as well as the start time of chemotherapy.
Results: There were 985 patients, of whom 486 (49%) received gemcitabine and 499 (51%) had 5-FU; 674 (68%) completed all six cycles of chemotherapy (full course) and 294 (30%) completed one to five cycles. Lymph node involvement, resection margins status and tumor differentiation as well as completion of therapy, were all shown by multivariate Cox regression to be independent survival factors. Overall survival favored patients who completed the full six courses of treatment versus those who did not (HR=0.516, 95% CI=0.443-0.601, p<0.001). Time to starting chemotherapy did not influence overall survival for the full study population (HR =0.985; 95% CI=0.956 – 1.015). Chemotherapy start time was an important survival factor only for the sub-group of patients who did not complete therapy, in favor of later treatment (p=0.004).
Conclusion: Completion of all six cycles of planned adjuvant chemotherapy rather than early initiation was an independent prognostic factor following resection for pancreatic adenocarcinoma. There was no survival disadvantage from delaying chemotherapy up to 12 weeks thus allowing adequate time for post-operative recovery.
Is the Association of the p.N34S SPINK1 Variant Explicable by a High Risk Haplotype Rather Than the Polymorphism?
J.A Nicholson,1* J. Grant,2* M. Johnstone,1 S. Harrison,1 C.M. Halloran,1 M.G.T. Raraty,1 P. Ghaneh,1 R. Sutton,1 R. Choudouri,3 J.P. Neoptolemos,1 R. Mountford,2 W. Greenhalf,1 (*Joint First Author). 1NIHR Pancreas Biomedical research Unit, Royal Liverpool University Hospital, Liverpool, UK; 2Regional Molecular Genetics Unit, Liverpool Women’s Hospital, Liverpool, UK; 3Department of Bioinformatics, University of Liverpool, Liverpool, UK.
Background: The p.N34S polymorphism of the SPINK1 gene is associated with idiopathic chronic pancreatitis however there is no clear functional effect of the sequence variant. It has been suggested that the polymorphism is associated with a high risk haplotype but this is widely disputed.
Aim: To identify whether a haplotype exists that distinguishes pancreatitis cases from controls where both cohorts have the p.N34S variant.
Methods: DNA from patients with chronic pancreatitis of no known etiology (idiopathic) and controls were analyzed by pyrosequencing to test for the p.N34S variant. A 2MB region (146Mb and 148Mb of chromosome 5) surrounding the SPINK1 gene was targeted using a custom designed liquid based capture system and sequenced using next generation sequencing in 5 pancreatitis and 5 control patients, heterozygous for the p.N34S variant. Identified sequence variants were then filtered based on their increased frequency within chronic pancreatitis patients, revealing 7 haplotype segments. Seven variants (Single Nucleotide Polymorphisms), one selected within each haplotype segment, were then screened in 38 pancreatitis patients and 20 controls (both groups heterozygous for p.Asn34Ser). Haplotypes based on 7 single nucleotide polymorphisms within this region were constructed. These were analyzed using the “haplo.stats” package.
Results: 29 haplotypes were identified; only haplotype 13 was significantly associated with pancreatitis (p=0.0009, hapscore: -3.31).
Conclusion: It is possible that a high-risk haplotype rather than a simple base variant is responsible for SPINK1 associated pancreatitis.
Circulating Nucleosomes as Predictors of Organ Dysfunction in Severe Acute Pancreatitis
A. Nieminen,1 A. Rouhiainen,2 L. Kylänpää,1 P. Puolakkainen,1 H. Repo,3 H. Rauvala.21Department of Surgery, Helsinki University Central Hospital, Helsinki, Finland; 2Neuroscience Center, University of Helsinki, Helsinki, Finland; 3Department of Bacteriology and Immunology, University of Helsinki, Helsinki, Finland.
Objectives: Circulating nucleosomes are released from apoptotic cells, and elevated serum levels have been shown to have predictive value for sepsis and organ dysfunction in critically ill patients. The aim of this study was to evaluate the prognostic value of circulating nucleosomes in severe AP with persistent organ dysfunction.
Methods: Eighty-two prospectively collected patients with AP admitted to Helsinki University Central Hospital within 72 hours after onset of symptoms, between June 2003 and December 2007, were included in the study. The severity of AP was assessed according to the revised Atlanta criteria into mild, moderately severe and severe. The serum levels of circulating nucleosomes were measured on admission and between days 5-7, if the patient was still hospitalized. Statistical analysis was performed using IBM SPSS Statistics version 19.
Results: Fifty-seven (69.5 %) patients had mild or moderately severe AP and 25 patients (30.5 %) had severe AP with persistent organ dysfunction. On admission the serum levels of circulating nucleosomes were higher in patients who already had or later developed persistent organ dysfunction (p<0.05). During hospitalization (days 5-7), the levels of circulating nucleosomes declined, but still remained higher in patients with severe AP (p<0.05). Using receiver operating characteristic (ROC) curve with the optimal cut off point, the specificity of circulating nucleosomes for predicting organ dysfunction on admission was 88 % and the sensitivity was 40 %. Positive predictive value was 62.5 % and negative predictive value 74.6 %.
Conclusion: We demonstrate that elevated serum levels of circulating nucleosomes on admission predict the development of persistent organ dysfunction in severe AP.
Unsaturated Fatty Acids Determine Severity of Pancreatic Necrosis and Inflammatory Response in Human Severe Acute Pancreatitis (SAP)
P. Noel,1 C. Durgampudi,2 S. Navina,3 K. Patel,1 K. Lee,4 R. Brand,1 J. Chennat,1 A. Slivka,1 G.I. Papachristou,1 A. Khalid,1 D.C. Whitcomb,1 J.P. DeLany,1 R.A. Cline,1 V. Mishra,1 R.N. Trivedi,1 C. Acharya,2 D. Jaligama,2 F. Murad,5 D. Yadav,1 V.P. Singh.1Departments of 1Medicine, 3Pathology, 4Surgery, University of Pittsburgh Medical Center, University of Pittsburgh, Pittsburgh, PA; 2Department of Medicine, UPMC Pasavant, Pasavant, PA; 5Department of Medicine, Washington University, Saint Louis, MO.
Background: The distinction between markers and potential drivers of SAP associated necrosis and systemic inflammation is unclear. This distinction is important since targeting a driver, but not a marker could improve outcomes in SAP. Potential targets include cytokines, proteases or unsaturated fatty acids (UFAs).We aimed at characterizing the major players present in human pancreatic fluid collections, and used animal models of acute pancreatitis (AP) to investigate causality underlying SAP.
Methods: Post-inflammatory collections (PICs) [i. e. post necrotic fluid collections (PNFCs) or pseudocysts (PCs)] and non-inflammatory collections (NIC) were analyzed for DNA, amylase, trypsin, cytokines and UFAs. Glyceryl tri-linoleate (GTL) with or without the lipase inhibitor orlistat were infused into the pancreatic ducts of rats and outcomes were compared to caerulein pancreatitis alone or with IL-1β and KC/GRO (the rat IL-8 homolog).
Results: Amylase and trypsin were higher in PCs. Consistent with increased necrosis, PNFCs had significantly more DNA. PNFCs also had higher UFAs, IL-8 and IL-1β. Additionally, PICs had higher UFAs, resistin and IL-1β concentrations than NICs. Rats administered GTL had similar serum amylase, pancreatic trypsin to caerulein pancreatitis. IL-1β and KC/GRO did not worsen caerulein pancreatitis. However GTL infused rats had elevated serum UFAs, IL-1β and KC/GRO, 70-80% necrosis and 100% mortality within 1 day. All these and serum lipase were reduced or improved in rats receiving GTL with orlistat.
Conclusions: While high UFA, IL-1β and IL-8 concentrations in PNFCs suggests these to be potential drivers of necrotizing pancreatitis, their reduction by orlistat along with serum lipase, pancreatic necrosis and mortality, and the inability of IL-1β, KC/GRO to worsen mild pancreatitis supports lipolytic generation of UFAs to drive inflammation and necrosis. Overall our data suggest that lipotoxicity from UFAs determines severity of inflammation and necrosis in human SAP.
Minnelide Decreases Pancreatic Cancer Invasion Through NF-kB
A. Nomura, S. Banerjee, V. Sangwan, S.M. Vickers, A.K. Saluja. Division of Basic and Translational Research, Department of Surgery, University of Minnesota, Minneapolis, MN.
Introduction: Triptolide, a compound derived from the traditional Chinese herb, Tripterygium wilfordii Hook F, has been demonstrated to effectively induce cell death within pancreatic cancer cell lines and several animal models. In this study, we aimed to determine the effect of Minnelide on the Epithelial-Mesenchymal Transition (EMT) and invasion and the mechanism by which this compound limits the invasive potential of pancreatic cancer cells.
Methods: Human pancreatic tumor samples propagated in SCID mice and pancreatic tumors derived from KPC (LSL-KrasG12D/+; LSL-Trp53R172H/+; Pdx-1-Cre) mice were utilized for these experiments. Several established human pancreatic cancer cell lines were used for in vitro experiments. Migration was measured by ECIS, an automated cell proliferation assay by Applied Biophysics and Boyden chamber invasion inserts were used to determine cellular invasion. Quantitative PCR and western blot were used to determine expression of markers involved in EMT and invasion. Triptolide and Minnelide (a water soluble analog) were used for in vitro and in vivo treatment, respectively. NF-kB activity was determined by p50 binding ELISA and pharmacological inhibition was achieved using BAY11-7085.
Results: Invasion and migration in vitro decreased with triptolide treatment within several established pancreatic cancer cell lines. Markers of EMT were downregulated both in vitro and in vivo upon treatment with triptolide and Minnelide, respectively. This effect was determined to be mediated through the modulation of NF-kB activity. Inhibition of NF-kB activity through pharmacological or IKK inhibition resulted in the downregulation of EMT markers and attenuation of in vitro invasion.
Conclusion: Minnelide downregulates EMT marker expression resulting in lower cellular invasion in vitro and metastasis in vivo mediated by decreasing NF-kB activity in pancreatic cancer.
Racial Disparities in the Treatment and Survival of Resectable Pancreatic Cancer Can Be Reduced
Z. Nwe, N. Anand, B.U. Wu. Center for Pancreatic Care, Southern California Permanente Group, Department of Gastroenterology, Kaiser Permanente, Los Angeles, CA.
Background: Racial disparities in treatment and survival have been reported in pancreatic cancer. The aim of the present study was to determine whether these disparities persist in an equal access health care setting.
Methods: We performed a retrospective cohort study of data collected from patients with stage I to IIB pancreatic cancer from an integrated healthcare system in Southern California from 2005 to 2010. Case identification was performed using a prospective internal cancer registry. Race was defined according to self-reporting as either white, black, American Indian, Asian and unknown. We compared the proportion of patients that underwent surgical resection within each racial/ethnic group (Chi-square test) and the survival of patients in each racial/ethnic group (Cox proportional hazard regression), adjusted for age, gender, stage of cancer and year of cancer diagnosis.
Results: 666 patients with stage I-IIB pancreatic cancer were identified (65.3% white, 12.5% black, 8.0% American Indian, 13.4% Asian and 0.9% unknown). Median age at diagnosis was 68 years (IQR 60-77) with 55% women. Median time of follow up was 288 days (125-517 days). 382 patients (57%) had surgery. Rates of surgery were similar among all races (55.2% white, 62.7% black, 66.0% American Indian, 56.2% Asian, 83% Unknown; P value - 0.27). All races had similar crude rates of mortality (77.0% white, 68.7% black, 69.8% American Indian, 68.5% Asian, 66.7% unknown; P value -0.25). There was no significant difference in survival based on race/ethnicity (black vs. white adjusted HR 0.796, 95% CL 0.59-1.06).
Conclusion: Racial disparities in the treatment and survival of resectable pancreatic cancer were reduced in an equal access care setting. Previous findings of racial disparities in survival for resectable pancreatic cancer are likely related to health care access issues rather than differences in cancer biology.
Intraoperative Irrigation Cytology to Detect Unexpected Significant Lesion of the Remnant Pancreas in Patients with Intraductal Papillary Mucinous Neoplasm Undergoing Partial Pancreatectomy
T. Ohtsuka, Y. Mori, K. Tamura, N. Ideno, T. Aso, J. Ueda, S. Takahata, Y. Oda, K. Mizumoto, M. Tanaka. Departments of Surgery and Oncology and Anatomic Pathology, Graduate School of Medical Sciences, Kyushu University, Fukuoka, Japan.
Background: Patients with intraductal papillary mucinous neoplasm (IPMN) of the pancreas may have distinct pancreatic ductal adenocarcinoma (PDAC) in the same pancreas. We evaluated the safety and usefulness of intraoperative irrigation cytology of the remnant pancreas (IICP) during pancreatectomy to detect unexpected significant lesions in the pancreas planned to be left in place in patients with IPMN.
Methods: Medical records of 60 patients with IPMN who underwent IICP during partial pancreatectomy between April 2007 and May 2013, were retrospectively reviewed. After division of the pancreas, a 4-French tube was inserted from the cut edge into the main pancreatic duct of the remnant pancreas, and fluid for cytological examination was obtained by saline irrigation through the tube. IICP was repeated 3 times and if the third IICP was positive or suspicious, then the patients underwent additional pancreatic resection. Clinicopathological outcomes were evaluated.
Results: The third IICP was positive in 5 of the 60 patients (8.3%). Postoperative pathological examination showed that all these patients had distinct PDAC in the additionally resected specimen, which was not detectable on preoperative imaging. There were 4 patients with stage 0 PDAC and one with stage III. Two other patients required additional resection of the remnant pancreas because of a positive margin by frozen section histology, although IICP was negative. No procedure-related complication was observed, and there was no patient with peritoneal recurrence which might be caused by seeding during IICP.
Conclusions: IICP appears to be a safe and useful method for detection of early stage PDAC concomitant with IPMN which cannot be detected by preoperative imaging.
Alcohol Metabolizing Enzyme Gene Polymorphisms in Alcoholic Chronic Pancreatitis from India
P. Ola Rajendra, R. Satyavati, K. Bhasin Deepak, S. Rana Surinder. Department of Super Specialty of Gastroenterology. Postgraduate Institute of Medical Education and Research. Chandigarh, India.
Background: Susceptibility to organ damage induced by alcohol may be related to genetic polymorphisms in alcohol metabolizing enzyme genes. Alcohol dehydrogenase (ADH) and aldehyde dehydrogenase (ALDH) are principal enzymes responsible for metabolism of ethanol. Alcohol is also metabolized in extra hepatic tissues, by the enzymes cytochrome P450 (CYP2E1) and catalase (CAT). CYP2E1 activity is expressed in the liver and pancreas, where it is induced by chronic alcohol consumption. Functional polymorphisms of ADH2, ADH3, ALDH2, CYP2E1 and CAT genes occur among racial populations.
Aim: The aim was to compare the genotype frequencies of ADH2, ADH3, ALDH2, CYP2E1 and CAT genes in patients with alcoholic chronic pancreatitis to healthy subjects from North India.
Materials and Methods: Genotyping analysis of ADH2, ADH3, ALDH2, CYP2E1 and CAT genes were conducted in 105 alcoholic chronic pancreatitis (ACP) patients and 110 healthy volunteers by the PCR-RFLP assay.
Results: Polymorphic alleles distribution in ACP was 25.7% CYP2E1*c2, 0.0% ALDH2*2, 23.8% ADH2*2, 61% ADH3*1 and 21% CAT TT while in controls, 10% CYP2E1*c2, 0% ALDH2*2, 12% ADH2*2, 43.6% ADH3*1 and 17.3% CAT TT. Polymorphic alleles of CYP2E1*c2, ADH2*2 and ADH3*1 genotype frequency were significantly higher (p < 0.05) in patients with ACP as compared to controls.
Conclusions: This study indicates that CYP2E1*c2, ADH2*2 and ADH3*1 alleles are significantly higher in alcoholic chronic pancreatitis patients from North India and thus they may play an important role in manifestation of alcohol induced pancreatitis.
High-Throughput Next Generation RNA Sequencing of Pancreatic Cancer Transcriptome Identifies Novel NcRNA Biomarkers That Uniquely Associates With the Disease
L. Orlichenko, R. Brand, A.E. Lokshin. Department of Medicine, University of Pittsburgh Cancer Institute, Hillman Cancer Center, Pittsburgh, PA.
Pancreatic cancer is the eighth leading cause of cancer-related death in men and the ninth leading cause of death in women worldwide. With no clinically approved biomarkers for the early detection of pancreatic cancer, the majority of patients is diagnosed with advanced disease and has a median survival with treatment of 6 months. To discover better non-invasive molecular markers, we assessed the potential of non-coding ncRNAs as new biomarkers for pancreatic cancer screening using high-throughput next generation RNA-sequencing. NGS RNA-seq is a novel digital approach to gene expression studies that offers rapid profiling of the cancer transcriptome allowing detection of intragenic and antisense transcripts as well as expressed pseudogenes.
To reveal ncRNAs uniquely associated with pancreatic cancer cases, we conducted a differential analysis of the RNA sequences present in urine samples pooled from 15 pancreatic cancer and from 15 non-neoplastic lesional cases. Whole transcriptome RNA-seq generated about 1.8 million bases of pair-end reads from the pancreatic cancer and 1.6 million from the benign samples. A total of 9,727 confidently mapped transcripts were expressed in urine from pancreatic cancer patients and 3,431 from benign cases. The most abundant RNA sequences expressed uniquely in the pooled pancreatic cancer urine included a previously known pancreatic cancer antigen MUC4, as well as LOC389787, a tumor protein translationally controlled 1 pseudogene, a small nucleolar RNA SNORA58, and RNA transcripts mapped to BCYRN1, TNFRSF10D, and PFDN5 genomic sequences. Interestingly, BCYRN1, the brain cytoplasmic RNA1, expressed at high levels in invasive carcinomas, is rarely detectable in normal tissue or in benign tumors. The up regulation of ncRNA BCYRN1 in pancreatic cancer may indicate its role in the early process of neoplastic transformation. This is the first report of urinary ncRNA profiling associated with pancreatic cancer suggesting that BCYRN1 along with other ncRNAs may provide a useful panel of biomarkers for early detection of cancer and to follow progression of the disease.
Serum Amylase on the Night of Surgery Predicts Clinically Significant Pancreatic Fistula After Pancreaticoduodenectomy
L.K. Palani Velu,1,2 V.V. Chandrabalan,1,2 S. Jabbar,1 D.C. McMillan,2 C.J. McKay,1 C.R. Carter,1 N.B Jamieson,1,2 E.J Dickson.11West of Scotland Pancreatic Unit, Glasgow Royal Infirmary, U.K; 2Academic Department of Surgery, University of Glasgow, U.K.
Introduction: Drainage after pancreaticoduodenectomy (PD) remains a controversial topic with the risk of uncontrolled postoperative pancreatic fistula (POPF) balanced against the potential morbidity associated with prolonged and possibly unnecessary drainage. We investigated the utility of serum amylase on the night of surgery (postoperative day [POD] 0 serum amylase) to predict POPF after pancreaticoduodenectomy, in an attempt to identify patients suitable for early drain removal.
Methods: 185 patients who underwent pancreaticoduodenectomy between 2008 and 2013 were studied. POPF was graded using the ISGPS classification. Receiver operating characteristic (ROC) analysis identified an optimal threshold value of POD 0 serum amylase associated with clinically significant POPF (ISGPS grades B/C) from a test cohort of 50 patients. A validation cohort (n=135) was used to test the accuracy of this threshold value in predicting clinically significant POPF. Multivariate regression analysis was carried out to identify independent risk factors for POPF.
Results: 63 patients (34.1%) developed POPF; 43 patients (23.2%) developed clinically significant POPF. The optimized threshold value of POD 0 serum amylase for identification of clinically significant POPF using ROC analysis was ≥130IU/L (P<0.001). Serum amylase <130IU/L had a negative predictive value of 90.8% for clinically significant POPF (P=0.001). Soft pancreatic parenchyma, age > 60 years, BMI ≥ 25 kg/m2 and POD 0 Serum Amylase ≥130IU/L were independent risk factors for clinically significant POPF.
Conclusion: POD 0 Serum Amylase <130 IU/L allows early and accurate categorization of patients at least risk of clinically significant POPF and may identify patients suitable for early drain removal, thus avoiding potential drain associated morbidity.
Serum CRP on the Second Postoperative Day Predicts Clinically Significant Infective Complications After Pancreaticoduodenectomy
L.K. Palani Velu,1,2 V.V. Chandrabalan,1,2 C.J. McKay,1 C.R. Carter,1 N.B. Jamieson,1,2 D.C McMillan,2 E.J Dickson.11West of Scotland Pancreatic Unit, Glasgow Royal Infirmary, UK; 2 Academic Department of Surgery, University of Glasgow, UK.
Introduction: Innovations in surgical technique and improved patient selection have reduced mortality after pancreaticoduodenectomy (PD) however postoperative morbidity rates remain high even at high volume centers. Biochemical markers of inflammation are associated with infectious complications after PD, but to date, no reliable thresholds have been identified to guide clinical management.
Methods: 195 consecutive patients undergoing PD between January 2008 and April 2013 were included in the analysis. Routine serum investigations, including C-reactive protein (CRP), albumin, and white cell count (WCC), were performed from the pre-operative day. Postoperative lower respiratory tract infection (LRTI), wound infection, post-operative pancreatic fistula (POPF) and infected intra-abdominal collections were classified as infective complications. Complications of Grade II and greater on the Clavien-Dindo scale were defined as clinically significant. Receiver operating characteristic (ROC) curve analysis was used to identify threshold values of these markers with infective complications as outcome measures.
Results: 117 patients (59.7%) experienced infective complications of which 96 patients (49.0%) had clinically significant complications. Serum CRP on postoperative day (POD) 2 identified the occurrence of clinically significant infective complications. ROC analysis identified POD 2 serum CRP of 180 mg/L as the optimal threshold value (P=0.05). This was also associated with a prolonged hospital stay (P=0.006), prolonged stay in critical care (P=0.017), and requirement for invasive postoperative intervention (P=0.035).
Conclusion: A serum CRP of ≥180mg/L on POD 2 should raise the clinician’s awareness of an otherwise occult process that could lead to a clinically significant infective complication post-PD.
Further Results of Prevention of Post-ERCP Pancreatitis With Multimodality Techniques in Fragile Pancreatic Cancer Patients
A. Pap. Dept of Gastroenterology, National Institute of Oncology, Budapest, Hungary.
Introduction: Etiology of post-ERCP pancreatitis is multifactorial, involving mechanical injury to papilla and pancreatic duct with impaired drainage of pancreatic juice, chemical and hydrostatic damage by infection, thermal injury due to electrosurgical current during biliary and/or pancreatic sphincterotomy. Multifactorial pathomechanism needs combined measures for prevention. There are 3 established techniques for prevention of post-ERCP pancreatitis: pancreatic stent placement, rectal NSAIDs, and sublingual (with/without transdermal) NO.
Aims: Early jejunal feeding is on the horizon for severe pancreatitis, it is time to use it for prevention of post-ERCP pancreatitis at least in the most fragile cancer cases when diagnostic purposes, intraductal cytology and operative maneuvers are equally important. At the same time jejunal route is the most physiological way of early fluid resuscitation and supplementary enteral feeding can be beneficial preoperatively or at the first period of chemotherapy.
Patients & methods: A combination of at least 2 preventive measures were applied: the most frequent technique used was jejunal tube placement at the end of ERCP with rectal NSAID and/or sublingual plus dermal NO.
Results: In the last 5 years we have performed 278 ERCPs in 236 patients with intraductal cytology, with/without papillotomy and stenting for biliary and pancreatic cancer cases. Post-ERCP amylase elevations occurred in 17 pts with pancreatitis in 7 pts (2.52%); 5 (71.4%) with mild, 1 (14.3%) moderate, 1 (14.3%) severe form. Death happened in 1 case with mild pancreatitis complicated by COPD causing pulmonary insufficiency in two days.
Conclusion: Post-ERCP pancreatitis is less frequent even in the most fragile pancreatic cancer patients if a combined preventive measure is used. A prospective randomized study is needed to select the best combination with the more rational jejunal feeding to prevent definitively post-ERCP pancreatitis in these unfortunate patients.
A Non-Invasive Method of Assessing Mouse Pancreatic Volume in Experimental Models Using Micro-MRI
J. Paredes,1 T. Ahmad,1 K. Lemon,1 T. Javed,1 A. Orabi,1 K. Tobita,2 S. Husain.11Department of Pediatrics, University of Pittsburgh, Children’s Hospital of Pittsburgh of UPMC, Pittsburgh, PA, 2Children’s Hospital Imaging Core, University of Pittsburgh, Children’s Hospital of Pittsburgh of UPMC, Pittsburgh, PA.
Aim: The purpose of the current study was to test the hypothesis that the non-invasive imaging modality of micro-MRI is an accurate and reliable method of evaluating changes in pancreatic volume in experimental mouse models. This is a gross parameter that changes during severe pancreatic injury.
Background: Experimental pancreatitis and the resulting dynamic changes in pancreas size are primarily assayed by having to euthanize a large cohort of animals at varying time points. However, in clinical practice, patients with pancreatitis routinely undergo non-invasive cross-sectional imaging of the pancreas. MRI is a noninvasive, safe, and painless procedure that generates accurate images.
Methods: Using a powerful 7Telsa MRI system, we performed abdominal imaging in whole-fixed mouse with continuous slices in three standard planes: axial; transverse; and coronal.
Results: The contour of the pancreas was traced using Vitrea software and then transformed into a 3D reconstruction, from which volumetric measurements were calculated. The volumes were compared to a gold standard, which was a 3D reconstructed image from the dissected pancreas ex vivo. Remarkably, the volumetric calculations between the in situ and ex-vivo gold standard differed by less than 5%.
Conclusion: The mouse pancreas is easily identified by MRI and, using a fat attenuation protocol, could be well-differentiated from peri-pancreatic fat. These initial results point to the feasibility of reliably tracking changes in pancreatic size using MRI in experimental models of pancreatic disease, with the use of fewer animals. The bedside to bench approach, once perfected, can be expanded to yield novel, non-invasive imaging modalities for pancreatic disease to take back to the bedside.
Lipotoxicity Converts Caerulein Pancreatitis into a Lethal Model in Obese Mice
K. Patel,1 P. Noel,1 C. Durgampudi,2 C. Acharya,1 R. Cline,1 S. Navina,3 V. Mishra,1 R. N. Trivedi,1 V. P. Singh.1Department of 1Medicine, 3Pathology, University of Pittsburgh, Pittsburgh, PA; 2Department of Medicine, UPMC Passavant, Passavant, PA.
Background: Caerulein induced acute pancreatitis (AP) is not lethal in lean mice. Obesity is associated with Severe Acute Pancreatitis (SAP) and increased mortality in humans. We previously showed the lipase inhibitor Orlistat to improve SAP outcomes, mortality in IL-12/IL-18 induced SAP in obese mice, via reduction of unsaturated fatty acid (UFA) mediated lipotoxicity (Sci Transl Med 3, 107ra110(2011). We therefore aimed to study this phenomenon in caerulein pancreatitis.
Methods: AP was induced in lean (c57b6) and obese (ob/ob) mice by hourly caerulein injections (50μg/kg, 12 doses on 2 consecutive days), with/without Orlistat or its vehicle. Outcomes measured were survival, local injury, lung injury (TUNEL positive cells), renal failure (blood urea nitrogen, BUN), serum calcium, UFAs, IL-6 and TNF-α levels. Values are reported as mean± SEM. A P<0.05 was considered significant.
Results: Obese mice with AP had a 100% mortality by day 3 associated with gross intraperitoneal saponification, lung injury (3.7±1.0 cells/HPF), elevated BUNs (72±8 mg/dl), hypocalcaemia (4.4±0.4 mg/dl) and elevated UFAs (965±184μM), IL-6 (204653±12613.9 pg/ml), TNF-α (118±4.89 pg/ml). Orlistat at day 2 significantly reduced lung injury (0.47±0.1 cells/HPF), BUN (45±7 mg/dl), which normalized by day 5, and improved survival (2/10 mortality at 5 days) along with normalization of serum calcium (8.2±0.9 mg/dl), UFAs (370.6±37.9μM), reduction in fat necrosis (6.3±1.9% vs. 15.5±2%), acinar necrosis (2.9±1.1% vs. 6.2±0.9%), IL-6 (19748±8321 pg/ml) and TNF-α (21.2±4.5 pg/ml) compared to those without orlistat. AP was non-lethal in lean mice with significantly less IL-6, TNF-α than ob/ob mice, a transient BUN increase and no increase in UFAs over controls.
Conclusions: Obesity modifies outcomes of AP via UFA mediated lipotoxicity. The reduction in mortality, IL-6, TNF-α, lung and renal injury, and normalization of serum calcium and UFAs by lipase inhibition in both IL12/18 and caerulein induced AP supports this concept.
A History of Acute Recurrent Pancreatitis (HARP) Protects Against Post-ERCP Pancreatitis (PEP) in Large Duct Chronic Pancreatitis (LDCP)
Y.A. Patel, E. Afghani, A. Sinha, A.C. Storm, M.A. Khashab, V.S. Akshintala, A.M. Lennon, A.N. Kalloo, V.K. Singh. Division of Gastroenterology, Department of Medicine, Johns Hopkins Medical Institutions, Baltimore, MD.
Background: Chronic pancreatitis is associated with a reduced risk of PEP, but it is not known whether this is due to clinical or morphological features of this disease.
Methods: The demographic, clinical, and procedural data for adult patients (age≥18 years) with symptomatic LDCP (PD dilation≥5mm on CT and/or MRCP) and with their first ERCP at our institution between 1/00–7/12 were evaluated. The presence of calcifications was recorded. HARP was defined as ≥2 episodes of AP occurring ≥6 months prior to ERCP. Exclusion criteria were: acute pancreatitis within 2 months of ERCP, pancreatic/bile duct cancer, IPMN, autoimmune pancreatitis, and post-ERCP perforation or bleeding. PEP was defined as ≥2 of the following: worsening/new upper abdominal pain; amylase and/or lipase ≥3 times upper limit of normal; and/or acute pancreatitis on abdominal imaging. Multivariable logistic regression was used to identify risk factors for PEP.
Results: 478 patients were identified, of whom 364 (76.1%) met inclusion criteria and 23 (6.3%) developed PEP. A total of 271 (74.5%) patients had HARP and, of these, only 9 (3.3%) developed PEP. Multivariate analysis revealed a reduced risk of PEP for HARP (OR=0.28, CI 0.10-0.77, p=0.01), but increased risk in those with a history of PEP (OR=7.94, CI 1.98-31.70, p=0.003) after adjusting for calcifications and procedural maneuvers (pancreatic injection, pancreatic manometry, failed pancreatic cannulation, pancreatic brushing, pancreatic sphincterotomy, pre-cut pancreatic sphincterotomy, pancreatic dilation, pancreatic stone extraction, and/or pancreatic stenting).
Conclusion: PEP is significantly lower in LDCP patients with HARP suggesting that the protective mechanism is early fibrosis. The low incidence of PEP in these patients suggests that multiple endotherapy sessions may be safely pursued.
Antimicrobial Peptide Defensin Alpha 1 Promotes Inflammatory-Driven Malignant Transformation in the Pancreas
T. Pausch, S. Schneider, K. Felix, N. Giese, M.W. Büchler, J. Werner, W. Hartwig. Department of General-, Visceral- and Transplantation-Surgery, University of Heidelberg, Germany.
Background: Antimicrobial peptides defensins contribute to innate immunity and epithelial cell-regeneration and play a role in carcinogenesis of various inflammation-triggered malignant tumors. The aim of our study was to analyze the role of defensins in the carcinogenesis of ductal adenocarcinoma of the pancreas (PDAC) and its suspected association with CP.
Methods: Defensins alpha 1, 2, 3, and 5 were detected in human tissue specimen of healthy pancreas, CP, and PDAC by immunohistochemical staining. Expression levels of defensin alpha 1 (hAD1) were quantified using mass spectrometry (SELDI-TOF). Furthermore, hAD1 expression in several pancreatic cancer cell lines (COLO 357, T3M4, PANC-1) and in immortalized normal pancreatic duct epithelial cells (HPDE) was studied in vitro, and the effects of proinflammatory cytokines (TNF-α, IL-1ß, and IFN-γ) on this expression were investigated.
Results: Immunohistochemistry showed accumulation of defensins alpha 1 in malignant pancreatic ductal epithelia. In SELDI-TOF analysis expression levels of hAD1 tended to be higher in CP than in healthy pancreas, and levels were significantly increased in PDAC (p=0.0029). hAD1 expression was found in pancreatic cancer cell lines at a significantly higher level than in HPDE (p=0.04 for lysates, p=0.0004 for supernatants). After incubation of cancer cells with proinflammatory cytokines multiple significant elevations of the protein levels in cell culture supernatants were seen (with Il-1ß p=0.0079 for Colo 357, p=0.0013 for Panc-1, p=0.0028 for T3M4; with IFN-γ p=0.0069 for Panc-1, p=0.027 for T3M4) combined with significant decreases of DEFA1 expression in cell lysates (with TNF- α p=0.0258; with IL-1ß p=0.0357 for T3M4). Stimulation of HPDE with cytokines led to decreased levels of hAD1 in supernatants and lysates (p=0.0044 and p=0.0062).
Conclusion: Defensins may be a link between chronic inflammation and malignant transformation in the pancreas. Since hAD1 is highly expressed in PDAC tissue and in pancreatic cancer cell lines it could play a role in the carcinogenesis of PDAC triggered by inflammatory stimuli as seen in CP. Clarification of this role may help to establish new diagnostic and therapeutic options in PDAC.
Gpbar1 Expressing Non-Bone Marrow Derived Cells Regulate the Severity of Mouse Biliary-Induced Acute Pancreatitis
G. Perides, J. Louhimo, M. Field, M.L. Steer. Dept. of Surgery, Tufts Medical Center, Boston, MA.
Background: Global deletion of G-protein coupled bile acid receptor 1 (Gpbar1) ameliorates the severity of biliary-induced mouse pancreatitis, thus indicating that Gpbar1activation exerts a deleterious effect on pancreatitis severity.
Aim: To determine whether this effect is afforded by Gpbar1receptors expressed by bone marrow or non-bone marrow derived cells.
Methods: Gpbar1+/+ and Gpbar1-/- mice were exposed to lethal total body irradiation and rescued by transplanting them with bone marrow obtained from either Gpbar1+/+ or Gpbar1-/- donor mice. Four groups of animals were generated: (1) global Gpbar1+/+ mice; (2) global Gpbar1-/- mice; (3) mice with Gpbar1+/+ bone marrow but Gpbar1-/- non-bone marrow-derived cells; and (4) mice with Gpbar1-/- bone marrow but Gpbar1+/+ non-bone marrow-derived cells. Pancreatitis was induced by retrograde pancreatic duct infusion of 10mM TLCS. Mice were sacrificed 18 hours after pancreatitis induction and pancreatitis severity was quantitated as follows: edema, by measuring pancreatic water content; inflammation, by quantitating pancreatic myeloperoxidase levels; and acinar cell injury by histologically quantitating acinar cell necrosis.
Results: Pancreatic edema, inflammation, and acinar cell injury were evident in all animals with biliary pancreatitis compared to control animals but these parameters of pancreatitis severity were each lower in animals with either global Gpbar1deletion or mice with Gpbar1deletion confined to non-bone marrow derived cells. The reduction in edema and acinar cell injury was statistically significant (p<0.05) but the reduction in inflammation did not quite achieve statistical significance (p>0.05).
Conclusions: Pancreatic inflammation during pancreatitis is not dependent upon Gpbar1 expression by bone marrow cells. The Gpbar1-mediated worsening of pancreatitis severity is mediated by non-bone marrow derived, Gpbar1-expressing cells and those cells may be pancreatic acinar cells.
Can Circulating Tumor Cells be Used to Diagnose Pancreatic Ductal Adenocarcinoma in Surgical Candidates?
J. Perkins,1 M. Pitman,2 C. Fernández-del Castillo,1 A.L. Warshaw,1 K. Lillemoe,1 A.S. Liss,1 S.P. Thayer.11Dept. of Surgery & Warshaw Institute; 2Dept. of Pathology, Massachusetts General Hospital/Harvard Medical School, Boston, MA.
Introduction: Endoscopic ultrasound guided fine needle aspiration remains the gold standard for diagnosing pancreatic ductal adenocarcinoma (PDAC). This method is invasive and expensive. In this study we propose that the detection of circulating tumor cells (CTCs) by liquid biopsy is a less invasive and cost efficient means to diagnose PDAC.
Methods: A total of 85 patients had blood samples drawn pre-operatively. Blood samples were filtered using devices from ScreenCell® (Paris, France). Captured cells were analyzed by cytology by a pancreatic cytopathologist (M. Pitman) and, immunofluorescence for detection of nuclear (DAPI), WBC specific (CD45), or pancreatic cancer specific (plectin) markers.
Results: Cytology was categorized as positive, suspicious, or non-diagnostic. Positive cytology was defined as clusters of large cells with visible nuclei and cytoplasm or as single cells with well defined cytoplasm, hyperchromatic nuclei, and eight times larger than the pores on the filter. Suspicious cytology was defined as aggregates of small cells with scant cytoplasm. Non-diagnostic samples included blood cells, lymphocytes, and filter pores. Of the 85 patients enrolled, 72 were found to have pathologically confirmed PDAC, 32 (44%) had malignant cytology, 12 (17%) were suspicious, and 28 (39%) were negative. Of the 13 non-PDAC patients, 10 (77%) had negative cytology and 3 (23%) were suspicious. CTC cytology was found to have a sensitivity of 44% and a specificity of 100%. Immunofluorescence confirmed these malignant cells were CD45 negative, DAPI and plectin positive.
Conclusion: Here we demonstrate that PDAC can accurately be diagnosed by a simple liquid biopsy in 44% of our surgical patients. Thus we propose CTC cytology should be considered as a first diagnostic modality that would decrease time, cost, and risk.
Activating Transcription Factor 3 Is a Key Regulator of Acinar Cell Transcription During Pancreatitis
C. Pin,1-3,5 W. MacDonald,4,5 R. Mehmood,1,5 C. Johnson,1,5 S. Laing,1,5 P. Swan,2,6 S. Cregan,2,6 E. Fazio.2,5Departments of 1Paediatrics, 2Physiology and Pharmacology, 3Oncology, and 4Biochemistry, University of Western Ontario, 5Children’s Health Research Institute, 6Robart’s Research Institute, London, Ontario, Canada.
Introduction: Pancreatitis affects more than 100,000 people annually in North America. Experimental models of pancreatitis have identified changes in acinar cell gene expression and differentiation immediately following injury. A key signalling pathway activated by injury is the unfolded protein response (UPR), which consists of three pathways that mediate their downstream effects on gene transcription. In this study we have focused on Activating Transcription Factor 3 (ATF3), since it is expressed only after induction pancreatic injury.
Hypothesis: ATF3 alters the acinar cell phenotype by affecting expression of genes required for differentiation.
Methodology: Cerulein-induced pancreatitis (CIP) was initiated in C57 Bl6 mice and ATF3 expression assessed by qRT-PCR and western blot analysis. The relationship of ATF3 to acinar cell gene expression was assessed by chromatin immunoprecipitation analysis followed by targeted qPCR or Next Generation sequencing. Loss of function experiments were performed in mice harboring a deletion of the Atf3 gene.
Results: During CIP, ATF3 targets a number of genes involved in acinar cell function and differentiation including Mist1. Recruitment of ATF3 to the Mist1 promoter coincides with its rapid decrease in expression and involves recruitment of histone deacetylase 5. In the absence of ATF3, MIST1 accumulation and the acinar cell phenotype are maintained, suggesting ATF3 promotes loss of the acinar cell phenotype.
Conclusions: We have identified similar ATF3 accumulation in tissue samples from patients with chronic pancreatitis suggesting that the UPR, and particularly ATF3, promotes loss of the acinar cell phenotype during disease.
Epigenetic Reprogramming Promotes a Differential Response to Experimentally-Induced Pancreatitis in Mice
C. Pin,1-4 G. Varga,5 S. Mohanty,5 S. Laing,1,4 A. Kharitonenkov,5 R. Mehmood.1,4Departments of 1Paediatrics, 2Physiology and Pharmacology, and 3Oncology, University of Western Ontario, 4Children’s Health Research Institute, London, Ontario, Canada; 5Lilly Research Laboratories, Eli Lilly and Company, Indianapolis, IN.
Introduction: Gene expression is partly regulated by modifications to histone core proteins within chromatin. Recent evidence suggests that changes in histone modifications, or epigenetic reprogramming, dictates cell fate and promotes susceptibility to disease. In the mouse pancreas, trimethylation of lysine residue 4 on histone 3 (H3K4Me3) correlates with changes in cell differentiation.
Goal: To compare global H3K4Me3 enrichment in acinar cells from normal C57Bl6 mice and congenic mice lacking Mist1 (Mist1-/-), a model for chronic pancreatic injury.
Methods: Chromatin immunoprecipitation for H3K4Me3 was followed by Next Generation sequencing to identify novel regions of H3K4Me3 enrichment in the pancreatic genome and correlate enrichment to gene expression in both genotypes.
Results: While the majority of H3K4Me3-enrichment occurred at transcriptional start sites, a significant number of marks (>25%) existed outside of this region. Less than 10% of H3K4Me3 marks were changed in Mist1-/- acini with most changes not reflecting alterations in gene expression. Ingenuity Pathway Analysis revealed increased representation of genes associated with pancreatitis and pancreatic ductal adenocarcinoma in Mist1-/- tissue suggesting these genes may be primed for activation. Induction of acute experimental pancreatitis resulted in significantly higher levels of gene expression in Mist1-/- mice compared to WT mice for Ripk3 and Ptgs2, two genes linked to increased severity of pancreatitis.
Conclusions: We propose that epigenetic reprogramming of H3K4Me3 occurs prior to changes in gene expression and dictates the molecular response to external events promoting disease.
Hepatocyte Growth Factor Inhibition: A Therapeutic Approach Superior to Gemcitabine in Pancreatic Cancer?
S. Pothula, Z. Xu, S. Arun, D. Goldstein, A. Biankin, R. Pirola, J. Wilson, M. Apte Pancreatic Research Group, UNSW, Sydney, Australia.
Standard chemotherapy e.g. gemcitabine (Gem, targeting cancer cells alone) is of limited benefit in pancreatic cancer (PC). The stroma is now known to play a major role in PC progression. Pancreatic stellate cells (PSCs, which produce PC stroma) interact with cancer cells to facilitate PC growth. A candidate factor that may mediate this interaction is the hepatocyte growth factor (HGF).
Aims: To compare the effects of HGF inhibition with those of gemcitabine on PC progression in an orthotopic model.
Methods: Orthotopic PC produced by implantation of AsPC-1 (human PC cell line) + hPSCs (human PSCs) into mouse pancreas. Mice treated (IP twice weekly x 6 wks) with anti-HGF monoclonal antibody Rilotumumab AMG102 (Amgen Inc, 300ug), or relevant IgG, or Gem (75mg/kg BW), (n=9 per group; doses based on preliminary studies). 7 weeks later, tumor volume and metastasis assessed. A subset of tumors (n=4/group) assessed by IHC for CD-31 (angiogenesis) and αSMA (PSC activation) and by Sirius red staining (collagen).
Results: Compared to IgG treated mice,
- a) AMG102 inhibited tumour growth (516.7±73.3 vs 1355±164.4mm3, p<0.01) and metastasis to liver (30% vs 80%), diaphragm (0% vs 40%), mesentery (40% vs 80%), retroperitoneum (10% vs 50%) (p<0.05) and kidney (0 vs 60%) (p<0.01). Histologically, AMG102 reduced CD-31 expression (12±0.9 vs 16±1.3 +ve cells/HPF, p<0.02), but did not alter αSMA or collagen expression.
- b) Gem inhibited tumour growth (587.8±94.2 vs 1355±164.4mm3, p<0.01), but not metastasis to liver (70% vs 80%), diaphragm (30% vs 40%), mesentery (70% vs 80%), retroperitoneum (30% vs 50%) and kidney (40 vs 60%). Histologically, Gem had no effect on angiogenesis, αSMA or collagen expression.
Conclusions: We show for the first time that while HGF inhibition is as effective as standard chemotherapy in inhibiting local tumour growth, it is significantly more effective than Gem in reducing tumour angiogenesis and local & distant metastasis.
Implication: Targeting the stromal reaction with relevant specific inhibitors may represent a novel treatment modality in PC.
Therapeutic Implications of Compartmental TGFβ Signaling
D.R. Principe,1 J. Bauer,1 W. Mascarinas,1 R. Hwang,2 B. Jung,1 P.J. Grippo,1. 1Northwestern University Feinberg School of Medicine, Chicago, IL, 2MD Anderson Cancer Center, Houston, TX.
A prominent event in pancreatic tumorigenesis is desensitization of TGFβ signals. TGFβ is a potent inducer of growth arrest, and many tumors harbor mutations affecting this pathway. Yet, TGFβ signaling can elicit a pro-survival response in developing cancer. In PDAC, SMAD4 deletion can both ablate growth arrest and promote pro-cancer processes. This is evident in models with conditional deficiency of TGFβ signaling, which significantly exacerbated KRAS-induced neoplasia. Yet, globally Tgfbr1 haploinsufficient mice present with far less lesions than controls. This suggests strategies targeting TGFβ may be efficacious in the clinic, though such an approach may lead to worse cancer in some patients. To identify patient populations more likely to respond, a more complete mechanism of TGFβ-induced growth arrest was refined in vitro. In TGFβ-induced expression of the anti-proliferative marker, p21, both pERK and SMAD4 are required. Co-culture experiments with human pancreatic stellate cells revealed that TGFβ (1) induced p21 in normal epithelial cells in the presence of stromal cells, (2) had little effect in the same cells harboring mutant KRAS, and (3) repressed p21 in PANC1 cells. These data suggest that TGFβ-mediated epithelial-stromal interactions become increasingly detrimental throughout carcinogenesis, further substantiating p21 as a relevant biomarker. Finally, since TGFβ facilitates tumor evasion from immune surveillance, in vivo reduction of TGFβ signals in the hematopoietic compartment promoted targeted destruction of metaplastic lesions, evident by GranzymeB/Perforin colocalization. This phenomenon was paralleled by a decrease in Foxp3+ T cells, which impede T cell-mediated cytotoxicity. In light of these data, patients with PDAC harboring SMAD4 deletions, high desmoplasia, and poor GranzymeB staining may benefit from TGFβ inhibition without risk of further aggravation of the cancer phenotype.
Loss of ADAM17 Promotes Rampant LPS-Induced Pancreatic Tumorigenesis Despite Restoration of Tumor Suppressive Signaling
D.R. Principe,1 P.J. Grippo,1 H.C. Crawford,2. 1Northwestern University Feinberg School of Medicine, Chicago, IL & 2Mayo Clinic, Jacksonville, FL.
Due to its promotion of EGFR-mediated mitogenic signaling, the membrane bound metalloproteinase, ADAM17, has emerged as a potential therapeutic target in several cancers. ADAM17 has also been linked to endodomain shedding of the Type 1 Transforming Growth Factor-β-Receptor (TGFBR1), abrogating its tumor suppressive properties in epithelial cells and further substantiating the case for clinical ADAM17 inhibition. Additionally, ADAM17 is responsible for liberation of the inflammatory cytokine Tumor Necrosis Factor (TNF). Despite its role in catalyzing these detrimental events, Pdx1-Cre/LSL-KRAS/ADAM17flox/flox (KCA) mice treated with lipopolysaccharide (KCA-LPS) present with a more aggressive phenotype compared to ADAM17 wild type littermates (KC-LPS). This was highly unexpected, as KCA animals frequently have a lower incidence of pancreatic neoplasia than their Pdx1-Cre/LSL-KRAS (KC) counterparts. Immune profiling revealed that in LPS treated-KRAS animals, the absence of ADAM17 leads to mass granulocyte infiltration coupled with a near complete absence of a cytotoxic response. Our data suggests that this is partially due to a combination of both T helper 17 (Th17) and Foxp3+ regulatory T cell (Treg) expansion, establishing a unique pro-inflammatory microenvironment with respect to granulocytes, while simultaneously suppressing lymphoid cells. The resultant imbalance between innate and adaptive immunity is sufficient to overcome the resistance of the epithelium to tumorigenesis, leading to a more advanced disease phenotype. In the setting of inflammation, ADAM17 inhibition may serve to exacerbate cancer phenotypes via selective immune remodeling, suggesting that such therapeutic strategies must be carefully considered and arguing for continued work in improving drug delivery to a specific target.
A Kinase Inhibitor Specific for DCLK1 Demonstrates Therapeutic Potential Against Human Pancreatic Adenocarcinoma Cells
D. Qu, N. Weygant, W. Berry, R. May, D. Owen, C. W. Houchen. Department of Medicine, University of Oklahoma Health Sciences Center, Oklahoma City, OK.
Aim: To determine whether kinase inhibitors specific for DCLK1 demonstrate therapeutic effects against pancreatic cancer.
Background: Doublecortin-like kinase 1 (DCLK1) is a putative tumor stem cell marker overexpressed in human pancreatic tumor tissues. It regulates key oncogenes, pluripotency factors, and EMT related transcription factors. Thus DCLK1 may be a novel therapeutic target in PDAC. Using siRNA mediated DCLK1 knockdown, we have shown antitumor effects in pancreatic cancer cells. However, the effects of DCLK1 kinase inhibition on these processes are unknown. The small molecule kinase inhibitor LRRK2-in-1 has a highly specific binding affinity for DCLK1 (Kd=5 nM). In this study, we assessed the effects of LRRK2-in-1 on pancreatic cancer cells.
Methods: in vitro kinase assay was performed in the presence of LRRK2-in-1. AsPC-1 cells were treated with various amounts of LRRK2-in-1 and MTT, Live/Dead, Matrigel invasion, Caspase-3/7 activity assays, real-time RT-PCR, and western blot were performed. Nude mice were injected with AsPC-1 cells to generate xenografts, and treated with LRRK2-in-1 every other day. Tumor sizes were measured for 3 weeks.
Results: DCLK1 kinase activity was inhibited by LRRK2-in-1 (IC50=2.9nM). Cell proliferation was inhibited (IC50=1.69nM) as a result of increased cell morbidity confirmed by Live/Dead Assay. Bcl2 expression was decreased more than 50% compared to control. Caspase3/7 activity was significantly increased and exponentially associated with cell morbidity (R2=0.97). mRNA and protein analysis revealed a significant loss of DCLK1, stemness related genes, oncogenes, and EMT related transcription factors. Invasive potential was decreased more than 50% by LRRK2-in-1. There was a more than 50% reduction in AsPC-1 xenograft tumor volume in LRRK2-in-1 treated mice.
Conclusion: These results suggest that DCLK1 kinase inhibitors may have therapeutic potential in PDAC.
Predictors of Surgical Utilization for Low Risk Pancreatic Cystic Lesions
S. Y. Quan,1 B. Visser,2 G. Poultsides,2 J.A. Norton,2 A. Chen,1 S. Banerjee,1 S. Friedland,1 W.G. Park,1. 1Department of Medicine, 2Department of Surgery, Stanford University, Stanford, CA.
Background: Consensus-based guidelines for pancreatic cysts have existed since 2006 to guide clinical management. The primary objectives of this study were to evaluate the proportion of surgeries performed for pancreatic cysts outside of these guidelines and to identify associated factors.
Methods: A 5-year retrospective review between July 1, 2007 and June 30, 2012 was performed of consecutive patients who underwent endoscopic ultrasound or surgery for a pancreatic cyst at a single tertiary medical center. Relevant clinical variables and cyst characteristics were collected.
Results: Of 486 patients identified with pancreatic cysts, 148 patients (30%) underwent surgery. Of the surgery group, 23 (16%) cases had no high-risk criteria as defined by the 2006 Sendai criteria. These cases included: 14 (61%) branch duct IPMNs, 3 (13%) MCNs, 1 (4%) SCA, 1 (4%) pseudocyst, 1 (4%) squamous cyst, 1 (4%) area of focal pancreatitis and 2 (9%) neuroendocrine tumors (NET). Besides NETs, there were no high-grade dysplastic or cancerous lesions. A high cyst CEA (35%), patient preference (26%), and physician suspicion (22%) were explicit reasons identified in the medical record to recommend surgery. Cyst CEA was highest in this group (mean 64,961 ng/ml (95% CI 0 – 195,948)) compared to those observed (mean 2904 ng/ml (95% CI 165 – 5,645)) and those meeting Sendai criteria (mean 4,870 (95% CI 1567 – 8175)) (p < 0.0001). By multivariate analysis, an elevated CEA level > 192 ng/ml was the most significant factor (OR 5.14 (95% CI 1.47 – 18.0)).
Conclusion: Surgery performed outside of consensus-based criteria is uncommon. The majority of these cases were branch duct IPMNs, but cystic NETs and MCNs were also identified. A high cyst fluid CEA level was the most significant factor associated with surgical utilization outside of consensus-based guidelines. This may influence patient anxiety and physician uncertainty to proceed to surgery.
Background and Design of Controlled Randomized Trial: Pancreaticogastrostomy versus Pancreaticojejunostomy in Reconstruction after Cephalic Duodenopancreatectomy in Patients with Soft Pancreas and Small Pancreatic Duct- PANAM Study
D. Radenkovic,1 D. Bajec,1 N. Ivancevic,2 P. Gregoric,2 V. Jeremic,2 A. Antic,1 I. Pejovic,1 S. Kmezic,1 M. Levicanin,1 A. Bajec,2. 1Clinic for Digestive Surgery, Clinical Center of Serbia, School of Medicine, Belgrade, Serbia; 2Clinic for Emergency Surgery, Clinical Center of Serbia, School of Medicine, 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 anastomosis 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, biliary 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 anastomosis 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.
Basic Amino Acids Cause Mitochondrial Injury in Pancreatic Acinar Cells
Z. Rakonczay Jr.1 E. Végh,1 G. Kovács,1 B. Iványi,2 T. Wittmann,1 T. Takács,1 P. Hegyi,1. 1First Department of Medicine, 2Department of Pathology, University of Szeged, Szeged, Hungary.
Introduction: Large i.p. doses of basic amino acids (L-arginine, L-ornithine and L-lysine) are known to induce acute pancreatitis (AP) in rodents, but the mechanisms mediating pancreatic toxicity remain unknown. Mitochondrial injury is thought to play a role in the pathomechanism.
Aim: To get insight into the mechanisms by which basic amino acids damage the exocrine pancreas.
Methods: Pancreatic acinar cells were isolated from male Wistar rats by enzymatic digestion. Isolated acinar cells were treated with different concentrations (20-60 mM) of L-arginine, L-lysine or L-ornithine. The morphology of acinar mitochondria was monitored with transmission electron microscopy. We measured intracellular Ca2+ concentration ([Ca2+]i) by microfluorometry using the Ca2+-sensitive fluorescent dye FURA-2-AM. The effect of basic amino acids on basal and cerulein-stimulated amylase secretion was tested.
Results: We observed the swelling of acinar mitochondria after incubating the cells for 2 hours with 20-60 mM basic amino acids. However, we did not detect any change in the [Ca2+]i of cells in response to administration of basic amino acids, whereas marked Ca2+ signaling was found in response to 100 μM carbachol. Basal and cerulein-stimulated amylase secretion was not influenced by basic amino acids vs the control group.
Conclusion: Our data suggest that basic amino acids are unlikely to cause pancreatitis via calcium signaling, they do not alter amylase secretion, but they injure mitochondria. Further experiments are needed to investigate the exact pathomechanism.
This study was supported by OTKA, MTA and NFÜ (TÁMOP).
E2F1, the Transcription Factor Related to Proliferation and Cell Death, Mediates Activation of Vmp1 Gene Promoter Inducing Autophagy in Pancreatic Tumor Cells
A. Ropolo, M. Giovenco, A. Lo Re, M.I. Molejon, C. Catrinacio, V. Boggio, M.I. Vaccaro. Institute for Biochemistry and Molecular Medicine, CONICET, University of Buenos Aires, Argentina.
Autophagy is an evolutionarily preserved degradation process of cytoplasmic cellular constituents, which participates in cell response to disease. We characterized VMP1 (Vacuole Membrane Protein 1) as an essential autophagy related-protein that mediates autophagy in pancreatic diseases. Gemcitabine treatment induced early expression of VMP1 and autophagy in pancreatic cancer cells. However, the regulatory pathways that control vmp1 gene expression and autophagy in pancreatic tumor cells are not fully understood. The retinoblastoma pathway is often inactivated in human tumors resulting in deregulated E2F activity that can induce both proliferation and cell death. E2F1 expression has been correlated with higher tumor grade and worse patient survival in PDAC. The activation of E2F1 was reported to up-regulate the expression of three autophagy genes (LC3, ATG1 and ATG5) and a damage-regulated autophagy modulator (DRAM). The aim of this work is to evaluate the effect of E2F1 on VMP1 expression in pancreatic tumor cells. We found that overexpression of E2F1 transcription factor in Panc-1 and MiaPaCa-2 pancreatic tumor cells induces VMP1 expression. We also demonstrated that E2F1 expression induces autophagy analyzed by the increasing number of RFP-LC3 positive cells with a punctate staining. Moreover, we showed that p300, a classical co-activator transcription factor, cooperates with E2F1 in VMP1 promoter regulation. Conversely, RNAi knockdown of p300 impairs E2F1 induced activation of this promoter. Chromatin immunoprecipitation assays demonstrated that E2F1 binds to VMP1 promoter when cells were submitted to gemcitabine treatment. Together these data provide evidence that E2F1 is a new regulatory factor modulating autophagy in pancreatic tumor cells. The knowledge of the mechanism of response to Gemcitabine treatment by tumor cells will improve disease therapeutics.
Clinical Significance of Stromal CD90 and α-SMA Expression in Pancreatic Cancer
M. Sada,1 K. Ohuchida,1 K. Fujiwara,1 K. Horioka,1 T. Tanaka,1 B. Zheng,1 A.I. Cases,1 S. Akagawa,1 K. Shindo,1,2 T. Ohtsuka,1 S. Takahata,1 Y. Oda,2 K. Mizumoto,1 M. Tanaka,1. 1Department of Surgery and Oncology; 2Anatomic Pathology, Graduate School of Medical Sciences, Kyushu University, Fukuoka, Japan.
Background and Aims: Activated pancreatic stellate cells are myofibroblast-like cells which play a central role in desmoplasia, phenomenon that contributes to pancreatic cancer progression. Recent evidence has suggested CD90(+) fibroblasts in other organs have the ability to differentiate into myofibroblasts but not those CD90(-). The aim of this study was to investigate the clinical significance of stromal CD90 and α-SMA expression in pancreatic cancer.
Methods: The CD90 and α-SMA expression were immunohistochemically examined in 66 resected specimens of pancreatic ductal adenocarcinoma. Final immunoscores were obtained by staining intensity and percentage, and the clinicopathological correlations were statistically evaluated. In addition, pancreatic stellate cells isolated from fresh surgical resected tissues of pancreatic cancer were examined for their expression of CD90 via flow cytometric analysis, and sorted into two populations based on their cell surface expression for further analysis.
Results: Immunohistochemistry showed CD90 and α-SMA expression were highly detected in stromal tissue surrounding tumor cells. The group with low CD90-low α SMA index was significantly associated with longer survival (P=0.02). Absence of nodal metastasis was more frequent in the group with low CD90-low α SMA index (P=0.02). Flow cytometric analysis revealed more than 93% of isolated pancreatic stellate cells were CD90(+). The proportion of CD90(+) cells increased in a time-dependent manner after sorted CD90(-) cells had been maintained under culture conditions.
Conclusions: These data suggest stromal CD90 could be a useful prognostic marker of pancreatic cancer as well as α-SMA, and may be a potential therapeutic target for its treatment.
Efficacy of Endotherapy in the Treatment of Chronic Pancreatitis Pain: Systemic Review and Meta-Analysis
J.N. Sadiq,1 M. Jafri,1,2 E. Grossman,1 D. Lee,1 Y. Taur,3 A. Goodman,1,4 F. Gress,1. 1Department of Gastroenterology, SUNY Downstate, NY; 2Department of Gastroenterology, St. Luke’s Hospital, NY; 3Department of Infectious Disease, Memorial Sloan-Kettering Cancer Center, NY; 4Department of Gastroenterology, New York University, NY.
Background: Endotherapy is important in the management of chronic pancreatitis pain. However, its use as the primary modality is controversial due to limited available data. The aim of this study is to assess the efficacy of endotherapy for alleviating pain in patients with chronic pancreatitis through a systematic review and meta-analysis.
Methods: A search of Medline, Pubmed, and Embase databases between 1988 to December 2012 for studies that analyzed the use of endotherapy for pain relief in chronic pancreatitis. We included large prospective blinded studies, randomized controlled trials (RCT) and retrospective analyses. Exclusion criteria included: studies not in English or those with less than 10 patients, case series/reports and studies that enrolled patients in dual therapies including surgery or celiac plexus neurolysis. In addition, a subgroup analysis was conducted on studies that included only patients with pancreatic duct strictures. A model of meta-analysis was developed and the data on pain relief was subsequently extracted, pooled, and analyzed. I2 estimates were calculated to test for heterogeneity across the included studies. The results were calculated and based on logits with inverse-variance weights and back-transformed normal-theory confidence intervals.
Results: Our final analysis included sixteen studies, comprising 1498 patients. Eleven studies included data for immediate relief of pain after endotherapy while twelve studies had data available for both immediate and sustained pain relief on follow-up (mean 47.4 months). The compiled result of the sixteen studies for immediate pain relief demonstrated 88% efficacy (95% NT CI [81.0%, 94%]). Similarly, analysis for long term follow-up showed a 67% efficacy (95% NT CI [58%, 76%]).
Conclusion: Endotherapy is beneficial in the immediate and long term relief of pain due to chronic pancreatitis. In contrast, patients with pancreatic duct strictures had a better immediate pain relief as compared to long-term results. Large prospective multicenter trials are warranted to further evaluate the benefit of endotherapy for managing chronic pancreatitis pain and decreasing the heterogeneity of currently published data.
3.0-Tesla Diffusion Weighted MRI (DW-MRI) Differentiates Healthy Controls from Equivocal/Mild Chronic Pancreatitis (CP)
V.A. Sahni,1 N.I. Sainani,1 V. Kadiyala,2 L.S. Lee,2 S.L. Suleiman,2 P.A. Banks,2 D.L. Conwell2. 1Division of Abdominal Imaging and Intervention, 2Center for Pancreatic Disease, Division of Gastroenterology, Hepatology and Endoscopy; Brigham and Women’s Hospital, Harvard Medical School, Boston, MA.
Aim: Determine the mean apparent diffusion coefficient (ADC) in the pancreas of healthy controls and CP patients. Measurement of ADC by DW-MRI is potentially an indirect marker of pancreas fibrosis/inflammation.
Methods: Retrospective study of patients (pts) who underwent 3.0-Tesla DW-MRI for evaluation of CP. Demographics, clinical and imaging data collected. Subjects were classified according to Cambridge/MANNHEIM criteria as equivocal/mild or moderate/marked CP. ADC was measured in the pancreas head, body and tail, and the mean gland ADC calculated. Statistical analysis: non-parametric inter-cohort comparisons (SPSS version 20, Armonk, NY).
Results: Thirty six (36) pts met inclusion criteria and were included in the CP imaging cohorts: 9 equivocal/mild CP imaging (median peak [HCO3-] 82mEq/L) and 27 moderate/marked CP imaging (median peak [HCO3-] 64mEq/L). A control cohort of 11 healthy volunteers was included for comparison (median peak [HCO3-] 105mEq/L). The median ADC was 1.25x10-3 mm2/s (IQR 0.24) in healthy controls, 1.07x10-3 mm2/s (IQR 0.10) in the equivocal/mild cohort and 1.14x10-3 mm2/s (IQR 0.17) in the moderate/marked cohort. ADC differentiates healthy controls from all CP (p=0.003), equivocal/mild (p=0.002) and moderate/marked (p=0.013). ADC was similar in equivocal/mild and moderate/marked (p=0.403). Of note, ADC measurement is technically limited by glandular atrophy in moderate/marked CP, resulting in falsely elevated intensity.
- 3.0-Tesla DW-MRI may be useful in differentiating healthy controls from pts with CP.
- ADC may aid in the evaluation of pts with mild/equivocal imaging changes for CP.
- DW-MRI may be limited in pts with moderate/severe CP imaging changes due to ADC measurement technique.
Not All Combined Intraductal Papillary Mucinous Neoplasms (IPMNs) Behave Like Main-Duct Lesions: Implications of Minimal Involvement of the Main Pancreatic Duct (MPD)
K. Sahora,1 C. Fernandez-del Castillo,1 S.P. Thayer,1 C. Ferrone,1 W. Brugge,2 D. Sahani,3 A.L. Warshaw,1 K. Lillemoe,1 M. Mino-Kenudson,4. Department of 1Surgery, 2Gastroenterology, 3Radiology and 4Pathology, Massachusetts General Hospital, Boston, MA.
Background: The malignant potential of IPMN is closely associated with involvement of MPD. Because neoplasms involving both main and side branches (combined IPMN) are thought to behave like main duct (MD)-IPMN, resection is recommended. However, the biological nature of combined IPMN with only minimal involvement of MPD (min-Com-IPMN) may be different.
Methods: A prospective database of 407 IPMNs resected at a single institution was queried to identify patients with combined and branch duct (BD)-IPMN. Minimal involvement was defined as absence of gross abnormality (except for dilation of MPD) and non-circumferential microscopic involvement of the MPD limited to one or few histologic sections. Clinicopathological features and patient outcomes were compared between groups.
Results: 171 patients had combined IPMN, of which 45 were found to have min-Com-IPMN and 126 extensive involvement (ex-Com-IPMN); 173 patients had BD-IPMN. The median diameter of the MPD was 9mm (2-40mm) in ex-Com-IPMN vs. 3mm (2-12mm) in min-Com-IPMN (p= .001) and 2mm (2-9mm) in BD-IPMN (p= .001). Cysts > 10mm were present in 63.5% of ex-Com-IPMN versus 95.6% in min-Com-IPMN (p= .001). Most significantly, the vast majority of min-Com-IPMN (90%) exhibited gastric-type epithelium, like BD-IPMN, not intestinal-type epithelium, like MD-IPMN. The prevalence of high-grade dysplasia (11.1% vs. 40.5%) and invasive carcinoma (6.7 % vs. 31.7%) was significantly lower in min-Com-IPMN compared to ex-Com-IPMN (p= .001).
Conclusion: min-Com-IPMN often presents with no MPD dilation and is an incidental finding by microscopic examination. It shares the pathological features and less aggressive biology of BD-IPMN. We propose that min-Com-IPMN be categorized and treated differently than ex-Com-IPMN.
The Radiologic Assessment of Pancreatic Disease Project (RAPiD): Predicting Pancreas Function with Qualitative MRI Features
N.I. Sainani,1 V. Kadiyala,2 L.S. Lee,2 S.L. Suleiman,2 P.A. Banks,2 D.L. Conwell,2. 1Division of Abdominal Imaging and Intervention; 2Center for Pancreatic Disease, Brigham and Women’s Hospital, Harvard Medical School. Boston, MA.
Aim: To identify qualitative MRI/sMRCP features which must be present to predict abnormal secretin-stimulated endoscopic pancreas function test (ePFT) in patients (pts) evaluated for chronic pancreatitis (CP).
Methods: Retrospective study of pts with MRI/sMRCP and ePFT to evaluate suspected CP. Data: age, gender, peak pancreatic fluid (PF) bicarbonate concentration ([HCO3-]) and MRI features (parenchymal and ductal) were recorded. ePFT peak PF [HC03-]: abnormal (<75meq/L), mild deficiency (74-65meq/L), marked deficiency (<65meq/L). Analysis: (SPSS Statistics v21. Armonk, NY) Fisher’s exact test to identify MRI features associated with abnormal ePFT. ROC analysis to determine number of MRI features predictive of abnormal ePFT.
Results: 93 pts included; mean age 49 yrs (range 18–78) and 35.5 % male. 60% had normal ePFT and 40% abnormal. In the abnormal cohort (n=37) 32% had mild [HC03-] deficiency, 68% marked deficiency. 9 MRI features (4 parenchymal and 5 ductal) were associated with abnormal ePFT. Parenchymal features: atrophy (p<0.001), loss of glandular lobularity (p=0.037), decreased T1 signal (p<0.001), delayed enhancement (p=0.034). Ductal features: pancreatic duct (PD) irregularity (p<0.001), PD narrowing/stricture (p<0.001), calculi/PD filling defects (p<0.001), side-branch dilatation (p=0.031), PD non-compliance after secretin administration (p=0.030). The number of MRI features identified was correlated to [HCO3-] (Pearson r = -0.629; p<0.001). MRI features did not predict mild deficiency (AUC=0.389), but a threshold of ≥6 features was 64% sensitive and 94% specific for marked deficiency (AUC=0.876).
Conclusion: MRI/sMRCP qualitative features are associated with marked secretory deficiency (peak [HC03] < 65 meq/L). MRI features are not associated with mild secretory deficiency.
Change in the Attitudes of Surgical Leaders Regarding the National Centralization of Whipple Procedures
J. Sand,1 J. Laukkarinen,2 I. Nordback,2. 1Division of Surgery, Gastroenterology and Oncology, Tampere University Hospital, Finland; 2Department of Gastroenterology and Alimentary Tract Surgery, Tampere University Hospital, Finland.
Background: In 1990-94, we found in a nationwide survey in Finland based on individual patient records that hospital mortality and re-operations were independently associated with annual hospital volumes. After that we have repeatedly promoted the centralization of Whipple procedures.
Aim: To analyze current attitudes of surgical leaders, the centralization and the national results of Whipple procedures.
Materials & methods: The same questionnaire was send to all surgical leaders in the country in 2002 and 2012. Whipples and hospital mortalities in different hospitals were searched from national databases in 2000-2001 and 2010-2011 and compared to the earlier results.
Results: 93% of the surgical chiefs in 2002, compared to 100% in 2012 were aware about the international results about centralization of Whipple procedures. 75% compared to 77% were aware about the similar national results. Centralization to all or only few university hospitals increased popularity in the attitudes (69% vs. 96%; p<0.05). Centralization has occurred slowly in Finland, with low nationwide hospital mortality (1990-1994 10%, 2001-10 4%, 2010-11 3%). Hospital mortality has remained lowest in the highest volume hospitals. Hospital mortality in volume >10/yr hospitals was 2%, 2% and 4% (in 1990-1994, 2001-10 and 2010-11) compared to 13%, 8% and 5 % in volume <5/yr hospitals. The 150 annual Whipple operations in 2010-11 were performed in a total of 14 hospitals (compared to 24 hospitals for 110 annual operations in 2000-2001 and 33 hospitals for 70 annual operations in 1990-94). In 2011, 55% of the annual Whipple operations were performed in just two hospitals.
Conclusion: Despite of good knowledge about benefits and favorable attitude in questionnaires, national centralization is a very slow process in a large European country with sparse population.
Loss of HSP70 Does Not Prevent Pancreatic Cancer Tumor Initiation or Progression
V. Sangwan, K.M. Jensen, U. Mahasahit, S. Banerjee, V. Dudeja, S.M. Vickers, A.K Saluja. Department of Surgery, University of Minnesota, Minneapolis, MN.
Background: Spontaneous pancreatic tumors arising in the KRasG12D; Trp53R172H; Pdx-1 Cre (KPC) animal model mimic all stages of human disease, and therefore represent an important tool for the understanding of pancreatic cancer. We have previously shown that HSP70 is over-expressed in pancreatic tumors, and loss of HSP70 results in pancreatic cancer cell death. HSP70 is a target of Minnelide, a prodrug of the Chinese herb triptolide, slated for Phase I clinical trials against pancreatic cancer. Preclinical studies with Minnelide have demonstrated its efficacy in preventing tumor formation and causing tumor regression in multiple animal models of pancreatic cancer. We have therefore developed a novel mouse model in which KPC mice were crossed to HSP70-null mice in order to investigate the mechanisms of pancreatic tumor progression in the absence of HSP70.
Aim: To evaluate the role of HSP70 in pancreatic cancer using the KPC; HSP70-null (KPCH) animal model.
Results: KPC mice develop pancreatic tumors with a median survival of ca. 4 months. Loss of HSP70 in this background (KPCH) did not significantly affect survival (KPC, 111 days; KPCH, 124 days; p=0.9; n=37 each) or penetrance (100% for both), and tumors from KPCH animals do not show increased HSP90 expression. However, loss of HSP70 results in perturbation of the angiogenic response, as seen by an increase in CCL2 expression and decrease in F3 expression. Intriguingly, triptolide-treated cell lines derived from either KPC and KPCH tumors undergo apoptotic cell death as demonstrated by decreased cell viability (100nM triptolide, 24 h: KPC, 29.3 ± 3.8%; KPCH, 34.2 ± 2.6%) and increased activation of caspase-3 (100nM triptolide, 24 h: KPC, 1367 ± 13.5%; KPCH, 1189 ± 22.8%).
Conclusion: Loss of HSP70 alone is not sufficient to prevent pancreatic tumor initiation or progression in the KPC animal model. However, investigation of the KPCH model will reveal mechanisms through which Minnelide inhibits pancreatic tumor growth beyond those downstream of HSP70.
Outcome of the Patients With Pancreatic Ductal Adenocarcinoma and Borderline Operable Vascular Invasion
H. Seppänen,1 A. Juuti,1 H. Mustonen,1 C. Haapamäki,1 S. Nordling,2 J. Sirén,1 C. Haglund,1 T. Kiviluoto,1. 1Department of Surgery, 2Department of Pathology, Helsinki University Central Hospital Helsinki, Finland.
Background: The portal or superior mesenteric vein resection and reconstruction has become more common when operating pancreatic ductal adenocarcinoma (PDAC). The aim of our study was to evaluate the immediate outcomes from pancreatic operations with vascular reconstruction (VR) and long term survival.
Material and Methods: The patients were operated for PDAC at the Helsinki University Hospital 2000-2009. The operations were performed in an extended manner to those with malignant disease. When the infiltration to the superior mesenteric or portal vein was detected they were excised and reconstructed by either end to end anastomosis or by a spiral graft from great saphenous vein.
Results: There were 546 patients undergoing a pancreatic operation during the ten year time period. Histological diagnosis was PDAC in 187. There were 43 (23 %) patients who had VR. There were no 30-day or in-hospital deaths. One (2 %) patient had grade C, none had grade B and 3 (7 %) had grade A fistula postoperatively. There were no significant differences in post-operative complications with VR. The tumor status was T3 in 74 % of the patients with VR when it was 59 % for those with no VR. The tumor size was also bigger median 40 vs. 30 mm respectively, p<0.001. There were 5 (12 %) patients who had neoadjuvant therapy and 26 (60 %) who had adjuvant oncological therapy of the VR patients. Only 25 (60 %) of the VR patients and 120 (88 %) of the non VR patients had R0 resection, p<0.001. The lymph node status, cancer grade or perineural invasion did not differ significantly between the groups. Survival of the patients with VR was poorer from those with no VR (mean 1.9 vs. 2.8 months, p=0.039). Yet of the patients with VR those who had R0 vs. R1 resection the 3 year survival was 32 % vs. 7 %. However the median survival did not differ significantly (1.7 vs. 1.5 months). Those patients who had VR and status was R0N0 the 3 year survival was 55 %.
Conclusions: Patients can be operated safely and acquire higher survival when they have no lymph node metastasis and R0 resection can be achieved.
Pain in Established Chronic Pancreatitis is not Associated With a Systemic Inflammatory Response: Evidence for Non-Inflammatory Nociceptive Mechanisms From a Subgroup of the ANTICIPATE Study
N. Shah, A.K Siriwardena. Hepatobiliary Surgery Unit, Manchester Royal Infirmary, Manchester, UK.
Introduction: The ANTICIPATE study undertook a randomized, placebo-controlled trial of Antox (Pharmanord, UK) based antioxidant therapy in radiologically-confirmed, moderately-exocrine impaired, pain-stable chronic pancreatitis (CP). A detailed cytokine profile analysis was undertaken in a severity-matched subset to assess whether pain in CP was associated with an elevated systemic inflammatory response (SIRS).
Methods: A subgroup of 20 patients from ANTICIPATE were recruited after ethics committee-approved protocol amendment. Antioxidant levels comprising selenium, vitamins C and E and glutathione were measured at baseline, study mid-point and at 6 months. A range of cytokines were measured at baseline and at 6 months: interleukin 1β, interleukin 6, tumor necrosis factor alpha (TNFα); anti-inflammatory cytokines: interleukin 4, interleukin 10; vascular endothelial growth factor (VEGF) and epidermal growth factor (EGF). Non hemolyzed, non-lipemic samples were collected into non-absorbent containers, stored at -70° C and measured by enzyme-linked immunosorbent assay (ELISA).
Results: Allowing for losses, 14 patients (7 antioxidant; 7 placebo) were available. At recruitment, with stable pain scores >4 on a numeric rating scale (NRS) of 0-11 all 14 had normal or low pro-inflammatory cytokine levels. After 6 months there were significant elevations in antioxidant levels in the intervention group: vitamin C in intervention group: 17.6 (12.8-29.3) µg/ml vs 4.8 (1.6-9.1) µg/ml in placebo [P<0.001; 95% CI 9 to 20.2] with similar trends in selenium. There was no elevation in any measured cytokine at 6 months.
Conclusions: In patients with stable CP, with opiate-dependent abdominal pain, circulating cytokine levels are low suggesting that pain is not simply a manifestation of inflammation and supporting current concepts that non-inflammatory nociceptive mechanisms are important.
βIII-Tubulin is a Novel Therapeutic Target for the Treatment of Pancreatic Cancer
G. Sharbeen,*1 J. Liu,*1 J. McCarroll,*2 J. Youkhana,1 J. Teo,1,2 N. McCarthy,2 D. Goldstein,1 M. Kavallaris,2 P.A. Phillips,1. *Equal authors 1Pancreatic Cancer Translational Research Group, Lowy Cancer Research Centre, University of New South Wales (UNSW), Sydney, Australia; 2 Children’s Cancer Institute Australia, Lowy Cancer Research Centre, UNSW, Sydney, Australia.
Introduction: Pancreatic cancer (PC) is the 4th leading cause of cancer-related deaths. Chemoresistance and metastases are common in PC. Hence, new therapies are needed. We have shown that the microtubule protein βIII-tubulin is highly expressed in PC cells and silencing its expression using siRNA increases their sensitivity to chemotherapy. However, the role of βIII-tubulin in regulating PC growth and metastases is unknown.
Aim: To determine the effect of silencing βIII-tubulin on PC growth and metastases both in vitro and in vivo.
Methods: In vitro: MiaPaCa-2 and HPAFII PC cells were transfected with βIII-tubulin or control (Ctrl) siRNA. Following transfection clonogenic potential was assessed by counting colonies. Apoptosis was assessed using caspase activity assays and Annexin V staining.
In vivo: MiaPaCa-2 cells were stably transfected with βIII-tubulin or Ctrl shRNA and were injected into the pancreas of mice. Eight weeks post-implantation, primary tumors and metastases were harvested and measured.
Results: Silencing βIII-tubulin reduced colonies in MiaPaCa-2 and HPAFII cells compared to Ctrls [MiaPaCa-2: Colony number (%): Ctrl siRNA 93.2 ± 4.0, βIII-tubulin siRNA 36.2 ± 6.2, p<0.001]. Silencing βIII-tubulin in PC cells also increased apoptosis. Stable suppression of βIII-tubulin in PC cells reduced tumor size (Ctrl: 145.0 ± 30.5mm3, βIII-tubulin shRNA: 80.6 ± 21.9mm3, p<0.05) and metastases (59% reduction, p<0.05).
Conclusions: We show for the first time that silencing βIII-tubulin: 1) reduced PC cell clonogenicity and increased apoptosis in vitro; and 2) decreased pancreatic tumor growth and metastasis in vivo. Therapeutic targeting of βIII-tubulin may significantly improve disease outcome for PC patients.
PI3K/AKT/mTOR and Sonic Hedgehog Pathways Cooperate Together to Inhibit Human Pancreatic Cancer Stem Cell Characteristics and Tumor Growth in NOD/SCID/IL2Rgammanull Mice
N. Sharma,1 Y. Ma,2 W. Yu,2 M. Rodova,1 S. Shankar,2 R.K. Srivastava,1. 1Department of Pharmacology, Toxicology and Therapeutics, and Medicine, The University of Kansas Cancer Center, The University of Kansas Medical Center, Kansas City, KS. 2Department of Pathology and Laboratory Medicine, The University of Kansas Cancer Center, The University of Kansas Medical Center, Kansas City, KS.
Background: Cancer stem cells (CSCs) regulate tumor initiation, progression, recurrence, and resistance to conventional therapy. PI3K/AKT and Sonic hedgehog (Shh) signaling pathways play significant role in pancreatic cancer development.
Aim: The objectives of this study were to examine the molecular mechanisms by which NVP-BEZ-235 (AKT/mTOR dual inhibitor) and NPV-LDE-225 (Smoothened inhibitor) regulate human pancreatic cancer stem cell characteristics and tumor growth.
Methods: CSC’s viability and apoptosis were measured by XTT and annexin V-propidium iodide assay, respectively. Gli translocation and transcriptional activities were measured by immunofluorescence and luciferase assay, respectively. Gene and protein expressions were measured by quantitative real-time PCR and Western blot analyses, respectively. The interactive effects of BEZ-235 and LDE-225 on pancreatic CSC tumor growth were examined in NOD/SCID/IL2Rgammanull mice.
Results: BEZ-235 and LDE-225 inhibited cell viability, colony and sphere formation, and Gli transcriptional activity and induced apoptosis in a synergistic manner. BEZ-235 cooperated with LDE-225 in inhibiting pluripotency-maintaining factors Nanog, Oct4, Sox2, and cMyc. BEZ-235 suppressed epithelial-mesenchymal transition by upregulating E-cadherin and inhibiting N-cadherin, Snail, Slug, and Zeb1, and these effects were further enhanced by LDE-225. Furthermore, BEZ-235 and LDE-225 alone inhibited CSC’s tumor growth in NOD/SCID/IL2Rgammanull mice. Finally, the combination of BEZ-235 and LDE-225 was more effective in inhibiting CSC’s tumor growth than single agent alone.
Conclusion: These data suggest that inhibition of PI3K/AKT/mTOR and Shh pathways cooperate together to inhibit pancreatic CSC characteristics and tumor growth, and thus combination of BEZ235 and LDE225 can be used for the treatment of human pancreatic cancer.
Beneficial Effect of Garlic (Allium Sativum) on L-Arginine Induced Chronic Pancreatitis in Rats
S.K. Sharma,1 S.V. Rana,1 D.K. Bhasin,1 S. Rana,1 R. Nada,2 S. Malhotra,3. 1Department of Gastroenterology; 2Histopathology; 3Pharmacology, PGIMER, Sector-12, Chandigarh, India.
Background: Protective role of Garlic (Allium sativum) has been reported in various chronic diseases in human as well as animals. However its role in chronic pancreatitis has not yet been studied.
Methods: 36 Wistar rats of either sex were divided into 6 groups. Group 1(Control): Rats were given IP injections of normal saline on day 1,4,7,10,13,16 & 19 and water intragastrically daily 2 days before starting IP injections. Group2: L-arginine hydrochloride (250mg/100g bw/day) IP injections in 2 repeated doses of 1 hr interval on day 1 and single dose on day 4,7,10,13,16 & 19. Group 3-5: Rats received freshly homogenized Garlic in water intragastrically at different doses i.e. 0.1, 0.25, 0.50 gm/kg b.wt daily 2 days before starting of IP injections of L-arginine as per group 2. Group6: Animals received freshly homogenized garlic alone at highest dose of 0.5 gm/kg b.wt. Levels of serum amylase, lipase, TNF-α and pancreatic glutathione (GSH) & lipid peroxidation (LPO) were studied on day 21. H&E and Masson’s trichrome stain were used for histology.
Results: Levels of serum amylase & lipase were significantly higher in arginine group as compared to control group and they significantly decreased with supplementation of 0.25, 0.5 gm/kg bwt. of garlic. Levels of LPO in pancreatic tissue were significantly higher where as GSH levels were significantly lower in arginine group. In garlic treated groups, GSH levels were significantly increased (p<0.05) while LPO decreased significantly (p<0.05) as compared to L-arginine group. Histologically, fibrosis, fat infiltration and acinar atrophy scores were significantly higher in arginine group as compared to garlic treated group. Levels of serum TNF-α found significantly higher (p<0.05) in L-arginine group as compared to garlic groups.
Conclusion: Garlic supplementation (0.25gm/kg bw/day) significantly reduced severity of chronic pancreatitis in experiment model of rats.
Pathological Wall Thickness of Intraductal Papillary Mucinous Neoplasm of the Pancreas Is a Predictor of Malignancy, and Stromal Podoplanin Expression is a Poor Prognostic Indicator
K. Shindo,1 S. Aishima,1 M. Tanaka,2 Y. Oda,1. 1Departments of Anatomic Pathology; 2Department of Surgery and Oncology, Graduate School of Medical Sciences, Kyushu University, Fukuoka, Japan.
Aim: The aim of this study was to clarify the correlation between wall thickness of intraductal papillary mucinous neoplasm (IPMN) of the pancreas and its malignancy in terms of histopathology, and to investigate whether stromal podoplanin (PDPN) expression was a prognostic indicator.
Background: Wall thickness on image findings is regarded as one of “worrisome features”, but the evidence of pathological findings has not been reported to date. PDPN was expressed by stromal fibroblasts of pancreatic ducal adenocarcinoma in association with poor outcome.
Methods: Immunohistochemical staining for αSMA was performed to detect the pathological wall of IPMNs, and average thickness between minimum and maximum was examined. Then, we compared the wall thickness of 122 IPMNs (35 low, 23 intermediate, and 33 high grade dysplasia, and 31 invasive carcinoma). Same assessment was performed for PDPN expression in the wall. We also evaluated the rate of PDPN-expressing fibroblasts in invasive areas of IPMN with invasive carcinoma.
Results: Pathological wall recognized by αSMA staining increase the thickness along with the adenoma-carcinoma sequence (P<0.0001). Furthermore, the wall thickness was associated with existence of mural nodule considered as a strong predictive factor for malignancy (P=0.013). PDPN expression was frequently seen in the luminal layer of thickened wall of IPMN and associated with its malignancy (P=0.009). In addition, high ratio of PDPN-expressing fibroblasts (>50%) in the invasive areas was an indicator of poor outcome (P=0.021).
Conclusion: Pathological thickened wall and thick layer of PDPN expression in the wall of IPMNs indicates its malignancy. PDPN expression in stromal fibroblasts of IPMN with invasive carcinoma is a reliable indicator of poor outcome.
AMPK Activators (Salicylate and Metformin) Reduce Pancreatitis Responses in an In Vivo Cerulein Model of Acute Pancreatitis
C. Shugrue,1 E. Akinbiyi,1 M. Shitia,1 V. Patel,1 E. Thrower,1 F. Gorelick,1,2. 1Department of Internal Medicine, Section of Digestive Diseases, 2Department of Cell Biology, Veterans Administration Connecticut Healthcare, West Haven, CT, Yale University School of Medicine, New Haven, CT.
Early events of acute pancreatitis (AP) include the activation and retention of digestive zymogens, reduced apical secretion, NFκB activation and inflammatory cell infiltration of pancreatic tissue. We have previously shown that AMP-activated protein kinase (AMPK) may play a protective role in AP; pretreatment of isolated pancreatic acini with pharmacologic activators of AMPK decreases zymogen activation. In an accompanying abstract [Kolodecik et al] we have confirmed this using genetic approaches. The anti-diabetes drug, metformin (MET), and the aspirin metabolite, salicylate (SAL), are known AMPK activators. We hypothesized that these drugs would ameliorate pancreatitis responses in the intact animal. C57/BL6 (Wild type or Wt) mice were given daily i.p. injections of either phosphate-buffered saline (PBS) as a control, MET (200 mg/kg) or SAL (120 mg/kg) for 7 days. On the 8th day, mice were given 6 hourly i.p. injections of PBS or the cholecystokinin orthologue, cerulein (CER; 40 μg/kg) to induce pancreatitis, and were euthanized 1h after last injection. Pretreatment with MET followed by CER resulted in decreased trypsinogen activation and inflammatory cell infiltration. SAL pretreatment completely abrogated trypsinogen activation and inflammatory cell infiltration. Given this result, we tried an acute 1h pretreatment with SAL: Wt mice were given a single i.p. injection of SAL 1h before 6 hourly injections of CER as before. Again, SAL pretreatment eliminated trypsin activation. Serum IL-6 was reduced but no decrease in serum IL-1β and neutrophil count were observed, suggesting longer pretreatment may be necessary. In conclusion, our studies suggest that AMPK activators may provide potential prophylactic therapy for treating AP.
Experience With Intraduodenal Hydrochloride Acid Infusion and Intravenous Injection of Secretin for Facilitation of Cannulation of the Dorsal Pancreatic Duct at ERCP in Patients With Pancreas Divisum—A Non-Randomized, Single Center Study
Q. Shu, S. Yarandi, K. Woods, S. Keilin, F. Willingham, Q. Cai. Division of Digestive Diseases, Department of Medicine, Emory University, School of Medicine, Atlanta, GA.
Background: Dorsal pancreatic duct (PD) cannulation at endoscopic retrograde cholangiopancreatography (ERCP) is an important step in diagnosis of pancreas divisum (PD) and in prevention of pancreatitis attacks for patients with recurrent acute pancreatitis secondary to PD by performing sphincterotomy or stent placement in the dorsal duct. To achieve a successful cannulation of the dorsal PD may be very difficult. Intravenous injection of biological porcine secretin or synthetic porcine secretin has significantly increased the dorsal PD cannulation rate. However, secretin is not readily available and expensive. Intraduodenal acid infusion is a physiologic method for inducing endogenous secretin secretion. We report our experience with the effect of secretin and acid infusion on the dorsal PD cannulation in the last few years.
Methods: This was a non-randomized study compared the effect of intraduodenal hydrochloride acid infusion with that of intravenous injection of secretin on dorsal PD cannulation. Patients with PD in whom minor papilla cannulation was initially unsuccessful were enrolled. Repeated cannulation was attempted after either intraduodenal hydrochloride acid solution infusion or intravenous injection of secretin. Success or failure was recorded and compared.
Results: Twenty one patients were enrolled after failed to cannulate the dorsal PD. Thirteen of them had hydrochloride acid solution infusion and eight had secretin injection. Repeated cannulation was successful in 9 patients (69%) in the acid group and 6 (75%) in the secretin group. There was no statistical difference between those two groups. There were no adverse events associated with the study.
Conclusions: The results demonstrated that intraduodenal hydrochloride acid infusion was safe during ERCP and had similar effect to secretin in increasing the minor papilla cannulation rate in patients with pancreas divisum in whom cannulation was difficult. A multiple center, randomized trial is needed for further study.
Covered Self-Expanding Metal Stents (CSEMS) May Offer Improved Clinical Success Compared to Multiple Plastic Stents (PS) in Strictures Secondary to Chronic Pancreatitis: A Systematic Review and Meta-Analysis
A. Siiki,1 M. Helminen,2 J. Sand,1 J. Laukkarinen,1. 1Dept. of Gastroenterology and Alimentary Tract Surgery, Tampere University Hospital, Tampere, Finland; 2Science Center, Pirkanmaa Hospital District and School of Health Sciences, University of Tampere, Tampere, Finland.
Background: CSEMS are being increasingly used in the endoscopic treatment of benign biliary strictures (BBS). Despite promising results, there is not yet solid evidence to support their routine use.
Aim: To evaluate feasibility of CSEMS compared to PS in BBS in terms of clinical success and complications.
Materials and methods: A systematic search of Medline, Scopus and Embase database for studies published 2000-2012 combined to hand-search of reference lists resulted 4977 articles. Out of 99 potentially relevant studies selected for full-text review, 12 CSEMS (376 patients) and 13 PS studies (570 patients) met the final inclusion criteria. A systematic review was made using proportion meta-analysis.
Results: A tendency to successful use of CSEMS in strictures related to chronic pancreatitis (CP) was shown: clinical success of 77% and 33% (95%CI 61-94% vs. 4-63%, p=0.06) was achieved with CSEMS and PS at 12 months follow-up, respectively. There were no differences in the success rates of other etiologies except CP or in the early complications. In CSEMS, incidence of late adverse events was lower in CP related strictures (3% vs. 67%, 95%CI 0-13% vs. 17-99%, p=0.02). The median number of ERCPs was lower with CSEMSs (1.5 vs. 3.9, p=0.002).
Conclusions: Improved clinical success with fewer endoscopic sessions and corresponding complication rate may be achieved with CSEMS compared to PS in BBS secondary to chronic pancreatitis.
A Simple Risk Stratification Score for Predicting Post-ERCP Pancreatitis
A.C. Storm, E. Afghani, A. Sinha, Y.A. Patel, M.A. Khashab, V.S. Akshintala, A.M. Lennon, A.N. Kalloo, V.K. Singh. Div of Gastroenterology, Dept of Medicine, Johns Hopkins Medical Institutions, Baltimore, MD.
Background: Prior studies weigh risk factors of post-ERCP pancreatitis (PEP) equally and assume no increase in risk of PEP in the presence of multiple risk factors.
Aims: To develop a risk stratification score for predicting PEP.
Methods: Adult patients admitted after outpatient ERCP to our institution between 1/2000-12/2011 were evaluated. Exclusion criteria were: non-ERCP related indications for admission, perforation/bleeding, AP within 2 months, and missing records. Cases were divided randomly into development and validation cohorts with 4:1 ratio. Demographic, clinical and procedural risk factors for PEP were analyzed independently using χ2 test. A forward stepwise regression was used to identify risk factors to enter into a final regression model. The medians of β-coefficients were weighted and used to develop an integer-based propensity risk score. This score was examined in the validation cohort using area under receiver operating curve (AUROC). PEP and severe PEP were defined using the clinical definition.
Results: A total of 2262 patients were evaluated, of which 1285 (56.8%) met inclusion criteria. PEP was found in 20.5% and severe PEP 1.9%. Development and validation cohorts had no statistically significant differences in distribution of the risk factors. Risk factors with significance were assigned integer scores: history of PEP = 3, age<60 years = 2, female sex=1, and pancreatic injection (PI) = 1, with max score of 7. This 4 variable score had AUROC 0.67 (95% CI 1.37,1.72) and AUROC 0.63 (95% CI 1.07, 1.62) for predicting PEP in the development and validation cohorts, respectively. There was no statistically significant difference in prediction ability of our score vs. a model with all 15 risk factors weighed equally.
Conclusions: Risk of PEP can be predicted by a simple score of age<60, female, history of PEP, and PI as accurately as all 15 risk factors weighed equally.
Adequate Number of Examined Lymph Nodes for Staging and Prediction of Prognosis after Pancreatoduodenectomy for Pancreatic Adenocarcinoma
O. Strobel,1 U. Hinz,1 A. Gluth,1 T. Hank,1 W. Hartwig,1 T. Hackert,1 F. Bergmann,2 M. Büchler,1 J. Werner,1. 1Department of General Surgery and 2Institute of Pathology, University Hospital Heidelberg, Heidelberg, Germany.
Background: There are no clear guidelines for the minimum number of examined lymph nodes (LN) after pancreatoduodenectomy for pancreatic adenocarcinoma. Studies based on the SEER database suggest different LN numbers between 11 and 15 to be adequate. However, the heterogeneous SEER data is based on non-standardized lymphadenectomy and low numbers of examined LN (median: 7). We aimed to determine the adequate number of examined LN based on a single high-volume center series with standardized lymphadenectomy and pathological workup.
Methods: Between 10/2001 and 06/2012 1009 patients underwent pancreatoduodenectomy for pancreatic adenocarcinoma at our institution. Survival data was available in 916 patients. Median overall survival of N0/N1 cases was analyzed dependent on the numbers of examined LN.
Results: Of 916 patients with a median LN number of 23 (IQR: 17-31), 205 patients were staged N0 and 711 patients N1. In N0 cases, median and 5-year survival were significantly shorter if ≤11 LN were examined compared to >11 LN (median survival: ≤11: 25.5 months vs >11: 38,6 months; 5-year survival: ≤11: 19% vs >11: 41%; p<0.0084). The significance was lost at 15 examined LN (≤15: 28.0 months vs. >15: 36,8 months; 5-year survival: ≤15: 26.1% vs >15: 32.7%; p=0.1276). In N1 patients, the lymph node ratio was significantly associated with survival if >15 LN were examined (LNR<0.2: 24.6 months, LNR 0.2-0.4: 17.9 months, LNR>0.4: 16.2 months, p<0.001) but failed to be significant if ≤15 LN were analyzed.
Conclusion: If an adequate lymph node dissection is performed during pancreatoduodenectomy for pancreatic adenocarcinoma, examination of >15 lymph nodes appears to be necessary to correctly stage N0 and to properly predict prognosis in N1 cases.
The Atp2c2 Gene as Transcribed From a Novel Transcriptional Start Site in Pancreatic Acini
C. Sullivan,1,2,3,4 R. Mehmood,1 C. Pin,1,2,3,4. Department of 1Paediatrics, 2Oncology and 3Physiology and 4Pharmacology, Western University Canada, Children’s Health Research Institute, London, ON, Canada.
Background: A key initiator in pancreatitis is aberrant Ca2+accumulation, regulated in part by pumps that shuttle Ca2+ out of the cytosol. Our laboratory identified a novel isoform of Secretory Ca2+ ATPase 2 (SPCA2) containing only the carboxy terminus. SPCA2 is a 103 kDa protein encoded by the Atp2c2 gene. Pancreatic SPCA2, which is approximately 17-20 kDa, is completely absent in Mist1-/- acini. Mist1-/- mice exhibit deregulated Ca2+ signaling and increased severity to pancreatic injury. The goals of this study were to define the transcriptional regulation of Atp2c2 in the pancreas and during pancreatitis.
Hypothesis: Atp2c2 is transcribed from an alternative transcriptional start site (TSS) regulated by MIST1.
Methodology: Using chromatin immunoprecipitation (ChIP), epigenetic modifications consistent with the TSS were examined in the Atp2c2 gene. Quantitative (q) RT-PCR was used to define exons contributing to the Atp2c2 transcript, and potential alternative promoters for Atp2c2 were examined using promoter-luciferase reporter assays. Bioinformatic and comparative analysis between species identified putative regulatory regions for Atp2c2.
Results: Bioinformatic analyses identified a truncated Atp2c2 isoform initiated within the 23rd intron of the Atp2c2 gene. Comparative analysis suggested this isoform differs between humans and mice. QRT-PCR analysis identified increased amplification of regions 3’ to the putative TSS relative to 5’ regions of Atp2c2. ChIP-seq analysis identified enrichment for trimethylated (Me3) histone 3(H3) at lysine 4 (K4) in intron 23 in pancreatic tissue, which was confirmed by ChIP-qPCR. ChIP-qPCR also identified enrichment of MIST1 and RNA PolII within this region.
Conclusions: Our results identify a unique TSS for Atp2c2 within the pancreas that is regulated by MIST1. This suggests an important role for MIST1 in affecting acinar cell Ca2+ levels.
Regulation of Angiogenic Factors by DCLK1 in Pancreatic Cancer
S.M. Sureban,1,2 R. May,1,2 N. Weygant,1 D. Qu,1 C.W. Houchen,1,2. 1Department of Medicine, University of Oklahoma Health Sciences Center, Oklahoma City, OK; 2Veterans Affairs Medical Center, Oklahoma City, OK.
Background: VEGFR1 and VEGFR2 play a significant role in tumor angiogenesis, progression and metastasis and are regulated by miR-200 family members. DCLK1, a putative pancreatic stem cell marker is upregulated in pancreatic ductal adenocarcinoma (PDAC). siRNA mediated knockdown of DCLK1 results in the inhibition of c-Myc, KRAS and Notch1 via miRNA related mechanisms. In this study, we aim to determine whether DCLK1 regulates the angiogenic factors (VEGFR1 and VEGFR2) via miR-200 dependent mechanisms.
Methods: Human pancreatic tumor xenografts (generated utilizing AsPC-1 cells), following treatment with PLGA nanoparticles encapsulated DCLK1 siRNA (NP-siDCLK1) were subjected to immunohistochemical analysis for protein and realtime RT-PCR for mRNA expression (DCLK1, VEGFR1 and VEGFR2) and miRNA expression (pri-miR-200a, 200b and 200c) analysis. Additionally, AsPC-1 cells were transfected separately with plasmids encoding the firefly luciferase gene with a miR-200a, 200b and 200c binding site at the 3’UTR to measure miR-200 expression. Following DCLK1 knockdown, cells were subjected to invasion assay using BD biosciences Matrigel invasion chambers.
Results: NP-siDCLK1 treatment resulted in AsPC-1 tumor xenograft growth arrest, downregulation (˜50%) of DCLK1, VEGFR1 and VEGFR2 mRNA and protein with a related ˜2-fold increase in pri-miR-200a, 200b and 200c miRNA. This increase was confirmed by a corresponding reduction in miR-200a, 200b and 200c specific luciferase activity in AsPC-1 cells treated with NP-siDCLK1. Furthermore DCLK1 knockdown resulted in decreased invasiveness of AsPC-1 cells.
Conclusions: These data taken together demonstrate a potential central regulatory role for DCLK1 in angiogenic factor expression in PDAC via miR-200 miRNA dependent mechanisms. Thus DCLK1 may represent a novel target for anti-angiogenesis based therapeutic strategies for PDAC and perhaps other solid tumors.
Superparamagnetic Iron Oxide Nanoparticles (SPIONs) as Targeted Drug Delivery Vehicles in Pancreatic Cancer Cell Lines
P. Sykes,1 E. Hasan,2 D. Lydon,2 C. Rubbi,1 J. Neoptolemos,1 R. Sutton,1 E. Costello,1 M. Rosseinsky,2 C. Halloran,1. 1Liverpool NIHR Pancreas Biomedical Research Unit, UK; 2Department of Chemistry, University of Liverpool, UK.
Aim: To develop SPIONs capable of bimodal targeting through either conjugation of a targeting antibody, or exploitation of their superparamagnetic properties and are capable of intracellular delivery of a chemotherapy payload into pancreatic cancer cell lines.
Methods: Micellar iron oxide nanoparticles conjugated to an anti-CA19.9 antibody (NP:CA19.9) were manufactured using a self-assembly methodology. The cellular uptake process was assessed with TEM imaging and co-localization fluorescent microscopy. Modified gemcitabine molecules were incorporated via acetal bonds sensitive to low pH levels. Antibody targeted delivery was assessed using EZ4U cytotoxicity assay in BxPC-3 (CA19.9 +ve) and SUIT-2 (CA19.9 –ve) cell lines. Magnetic field targeting was assessed in SUIT-2 cell line over a 6mm2 area using quantitative fluorescent microscopy and computer mosaic stitching.
Results: Endocytosis was confirmed by the presence of SPIONs in endosomes and corroborated by co-localization analysis (Manders coefficient=0.92). The IC50 of NP:CA19.9 was significantly improved by antibody targeting in BxPC-3 cells (904nM vs 4,122nM; p=0.046). This effect was absent in SUIT-2 cells (2,221nM vs 4,108nM; p=0.387). Magnetically targeted drug release showed significant reduction in cell count at the area of greatest magnetic force (3 cells/mm2, IQR 2-5 vs 92 cells/mm2, IQR 62-124; p<0.001).
Conclusions: We have developed a novel nanoparticle capable of targeting antigen expressing pancreatic cancer cells using a specific antibody tag or non-antigen expressing cells using a focused external magnetic field. When loaded with modified gemcitabine molecules, these SPIONs act as pH-triggered delivery vehicles capable of intracellular drug release. This could provide the opportunity to reduce off-target effects and increase the efficacy chemotherapy agents.
The Significance of Cationic Trypsinogen Mutation p.E79K in Chronic Pancreatitis
A. Szabó, M. Sahin-Tóth. Department of Molecular and Cell Biology, Boston University Medical Center, Boston, MA.
Background: Clinically frequent hereditary pancreatitis-associated PRSS1 mutations p.R122H and p.N29I increase activation of cationic trypsinogen in the presence of chymotrypsin C (CTRC). The relatively rare p.E79K mutation was reported both in subjects with chronic pancreatitis (9 carriers) and in healthy controls (6 carriers), suggesting that this variant may have lower penetrance or may not be disease associated at all.
Aim: Our goal was to classify the p.E79K mutant as pathogenic or non-pathogenic on the basis of its biochemical phenotype.
Methods: Cationic trypsinogen and CTRC were expressed recombinantly and purified with affinity chromatography. Trypsinogen activation was followed by activity measurements and gel electrophoresis.
Results: When assayed in the absence or presence of CTRC, mutation p.E79K reduced the rate of trypsinogen autoactivation, which stands in contrast to the stimulatory effect of mutations p.N29I and p.R122H. In the presence of CTRC, however, mutant p.E79K autoactivated to markedly higher trypsin levels than wild-type cationic trypsinogen. This effect was due to increased resistance to CTRC-mediated trypsinogen and trypsin degradation.
Conclusions: The peculiar biochemical phenotype of the p.E79K mutant, exhibiting lower rates of activation with higher final trypsin levels, is consistent with p.E79K acting as a low penetrance risk factor for chronic pancreatitis.
Clinicopathological Feature of the Slight Change of Branch and Main Pancreatic Duct Induced By Intraductal Low Papillary Neoplasm without Mass Forming and Mucin
Y. Tada, K. Yoshida, R. Ugaji, Y. Nakashima, T. Iwao. Aizu Central Hospital. Okayama, Japan.
In order to the progress of diagnostic imaging modality, we can detect small pancreatic cyst less than 10mm and slight stricture of main pancreatic duct without mass forming. Most of the patients which have these slight change of pancreatic duct were asymptomatic, and suspected pancreatic disease during the follow-up of other diseases or on routine medical check up. But in some cases there exist cancer lesion. We treated 16 cases with slight change of pancreatic duct. 11 patients were carcinoma in situ (CIS), 4 were minute invasion (MI) to stroma before forming mass, and PanIN2. In order to reveal the clinicopathological feature of the change of pancreatic duct, we investigate the location of carcinoma, the degree of desmoplastic change around carcinoma, and the character of mucin . We define the degree of the desmoplastic change as the grade 1 to 3. In 7 of 11 CIS and 3 of 4 MI and PanIN2 , tumor cells mainly located in the branch pancreatic duct. There exists no IPMN next to these tumor cells. The degree of the desmoplastic change is stronger around MI than CIS or PanIN2. These tumor cells are low papillary (ILPN) with less mucin and have the character of mucin of the gastric type. It is difficult to distinguish ILPN as IPMN and PanIN, but we must recognize that there may exist carcinomas in the slight changes of pancreatic duct. So we have to investigate and carefully follow up these slight change of pancreatic duct especially in the high risk individuals of pancreatic cancer, such as a strong family history of pancreatic cancer, non-O blood group, obesity, diabetes mellitus, metabolic syndrome, smoking, alcohol consumption and hepatitis B viral infection.
Urgent Surgical Treatment of Chronic Relapsing Pancreatitis
S. Takahata, T. Otsuka, J. Ueda, K. Mizumoto, S. Shimizu, M. Tanaka Department of Surgery and Oncology, Kyushu University, Fukuoka, Japan.
Background: Chronic pancreatitis is often refractory to medical treatment and needs surgical operation. Most of the patients are operated because of uncontrollable pain, however, urgent surgical therapy is needed in some rare conditions like bleeding or infection.
Patients and Methods: From 1992 to 2012, we treated 112 patients diagnosed chronic pancreatitis by surgical operations. Among 112 cases 14 needed emergent treatment. Patient’s backgrounds, treatment and prognosis of these emergent cases were retrospectively assessed.
Results: Backgrounds of urgent admission were spontaneous bleeding (10 cases,71%), infection on pseudocyst (2 cases, 14%) and formation of pseudoaneurysm (3 cases, 21%). Performed operation procedures were distal pancreatectomy (9 cases), side to side pancreato-jejunostomy (3 cases) and pancreatoduodenectomy (2 case). Urgently operated patients (n=14) resulted in long mean operation time (403min vs. 331min) and massive intraoperative blood loss (2340g vs. 643g) compared with non-urgent group (n=96).
Follow up CT scan revealed enlarged pancreatic pseudocyst in 5 out of 8 patients with hemorrhage before urgent admission. In 12 patients with hemorrhage or pseudoaneurysm formation, 5 had undergone arterial embolization for ruptured or enlarged pseudoaneurysm and successfully reduced intraoperative bleed loss (1291g vs. 2171g compared with 4 non-preoperatively embolized patients). All the urgently operated patients were with uneventful postoperative clinical course.
Conclusion: Patients with chronic pancreatitis forming pseudocyst should be carefully followed up and increasing size of the pseudocyst is one of the reliable predictor of bleeding. Preoperative arterial embolization is a useful approach to perform surgical operation safely.
Chronic Pancreatitis Associated With Congenital Pancreatic Abnormalities
Y. Takeyama, Y. Nakata, H. Ishikawa, K. Kamei, M. Araki, M. Matsumoto, T. Nakai. Department of Surgery, Kinki University Faculty of Medicine, Osaka-sayama, Osaka, Japan.
Introduction: Chronic pancreatitis (CP) is sometimes accompanied with congenital pancreatic abnormality. Recently, we experienced two interesting cases of CP associated with complicated abnormalities.
Case 1: 47 y.o. female suffered from upper abdominal pain. At the age of 20 y.o., extrahepatic bile duct resection and hepaticojejunostomy were performed elsewhere under the diagnosis of congenital choledochal cyst. Magnetic resonance cholangiopancreaticography (MRCP) and computed tomography (CT) showed pancreaticobiliary maljunction with complete pancreatic divisum. The pancreatic duct of the ventral pancreas was filled with pancreatic stones, and the stones were also found in the remnant bile duct. On the other hand, the dorsal pancreas seemed quite normal and neither calcification nor pancreatic duct dilatation were not found. In the operation, the dilated pancreatic duct of the ventral pancreas was cut open from the back side, and pancreaticojejunostomy was performed after stone removal.
Case 2: 37 y.o. male consulted for upper abdominal pain. Two weeks after birth, duodenojejunostomy was performed due to complete obstruction of the second portion of the duodenum. On the CT and MRCP images, the pancreas did not exist in the left side of the portal vein, and dilated pancreatic duct, which was filled with the stones, encircled the duodenum. We diagnosed that annular pancreas occurred in combination with the defect of the dorsal pancreas, and that CP was developed in the ventral pancreas. In the operation, the pancreatic duct of the pancreas head was cut open from the font side and pancreaticojejunostomy was performed after stone removal.
Discussion: Both cases had previous operations due to congenital abnormalities, and thereafter CP was developed. In the treatment of such cases, it is important to figure out the physical relationship of ducts and organs on the basis of the knowledge of pancreatic embryology.
Treatment Strategy for Main Duct Intraductal Papillary Mucinous Neoplasms of the Pancreas Based on the Assessment of Recurrence in the Remnant Pancreas After Resection: A Retrospective Review
K. Tamura,1 T. Ohtsuka,1 N. Ideno,1 T. Aso,1 K. Shindo,2 Y. Oda,2 K. Ohuchida,1 S. Takahata,1 K. Mizumoto,1 M. Tanaka,1. Departments of 1Surgery and Oncology, 2Anatomic Pathology, Graduate School of Medical Sciences, Kyushu University, Fukuoka, Japan.
Background: The most appropriate resection line for MD-IPMNs remains an unresolved issue. The aims were to clarify the recurrence pattern after resection of main duct intraductal papillary mucinous neoplasms (MD-IPMNs) using molecular analyses and to determine the most adequate treatment strategy.
Methods: Medical records of 56 patients with pancreatectomy were retrospectively reviewed. Histological subtypes and Kras/GNAS mutations were assessed in patients with recurrence in the remnant pancreas.
Results: Forty-nine patients underwent partial pancreatectomy, and 7 total pancreatectomy (TP). Thirty-six patients (64%) had malignant MD-IPMNs. Recurrence was observed in 7 of 49 patients (14%), including 6 with malignant IPMNs and 1 with pancreatic ductal adenocarcinoma (PDAC), all of whom underwent remnant pancreatectomy. The cumulative survival rate of patients with pancreatic recurrence was greater than that of patients with extrapancreatic recurrence (P<0.001). Although the pancreatic margin status at the initial operation did not affect the pancreatic recurrence rate, all 4 recurrent IPMNs examined had subtypes and Kras/GNAS mutations identical to those of the initial lesions. Four patients experienced recurrence in the remnant pancreas (n=3) or systemic recurrence (n=1) after resection of high-grade dysplasia (HGD). Three of the 56 patients had concomitant PDACs and MD-IPMNs.
Conclusions: One-step TP can be avoided, and remnant TP would lead to favorable outcomes in patients with pancreatic recurrence, some cases of which seem to involve residual lesions. Postoperative surveillance of HGD should be performed as if malignant, and close attention should be paid to the occurrence of concomitant PDACs even in patients with MD-IPMNs.
What’s the Impact of Duodenopancreatectomy on the Elderly Patient?
J. Targarona, D. Callacondo, C. Pino, C. Rodriguez, G. Coayla, R. Garatea, D.C Barreda, L. Barreda. Department of Pancreatic Surgery - Hospital Edgardo Rebagliati Martins, Lima, Peru.
Introduction: Many reports point to a significantly higher mortality rate in the elderly patient undergoing duodenopancreatectomy; the ages used to substantiate this statement are, nevertheless, very diverse.
The purpose of this study is to find out the age related to the highest mortality and complication rates in duodenopancreatectomy patients.
Material and Methods: A retrospective study of 314 duodenopancreatectomy patients was carried out using data from the period between October 2002 and June 2012. Statistical analysis of data was used to determine the age at which mortality was significantly higher after duodenopancreatectomy.
Results: There wasn’t significant morbility difference between any age group (p=0.6).
Mortality, however, was significantly higher in patients older than 75 years (2.9% vs. 9.4%, p< 0.017).
Conclusions: From this study we can say that being older than 75 years significantly increases the mortality rate in patients undergoing duodenopancreatectomy. However, being in this age group doesn’t proscribe surgical approach, as it’s the only choice to attempt curative treatment in these patients.
Inhibition of βIII-Tubulin Using a Novel Nanoparticle-siRNA Approach Sensitizes Pancreatic Cancer Cells to Chemotherapy
J. Teo,1,2,3 C. Boyer,3 J. Liu,1 G. Sharbeen,1 R. Erlich,2,3 J. Youkhana,1 H.T. Duong,2 D. Goldstein,1 T.P. Davis,3 M. Kavallaris,2,3 J. McCarroll,2,3 P.A. Phillips,1. 1Pancreatic Cancer Translational Research Group, Lowy Cancer Research Centre, University of New South Wales (UNSW), Sydney, Australia; 2Tumour Biology and Targeting Program, Children’s Cancer Institute Australia, Lowy Cancer Research Centre, UNSW, Sydney, Australia; 3Australian Centre for Nanomedicine, UNSW, Sydney, Australia.
Background: Pancreatic cancer (PC) is a devastating disease due to its high level of chemoresistance. Recently, we showed for the first time that targeting the microtubule protein βIII-tubulin using siRNA sensitizes PC cells to chemotherapy. However, there are no pharmacological inhibitors for βIII-tubulin and delivery of siRNA as a therapeutic agent requires a vehicle.
Aim: To design and synthesize a nanoparticle which can self-assemble with siRNA against βIII-tubulin for delivery to PC cells.
Methods: Star polymers were synthesized by reverse addition fragmentation transfer polymerization. Star polymers ± siRNA size was determined by electron microscopy and dynamic light scattering. Confocal microscopy, qPCR and western blots were used to assess cell uptake and silencing efficiency of star-siRNA complexes in PC cells (MiaPaCa-2, HPAF-II). Effects of silencing βIII-tubulin on PC cell survival ± paclitaxel were assessed by clonogenic assays.
Results: Star–siRNA nanoparticles were 35nm in size and non-toxic. Star-βIII-tubulin siRNA complexes potently silenced βIII-tubulin mRNA and protein levels in MiaPaCa-2 cells after 72h compared to cells treated with control siRNA. Silencing βIII-tubulin expression in MiaPaCa-2 significantly reduced colony numbers by 53.21±7.19% (p<0.001, n=4), and in the presence of paclitaxel (1.25nM) reduced colonies by an additional 30.04±6.64% (p<0.05, n=4). Similar results were obtained with HPAF-II.
Conclusion: These promising results support the development of star polymers as a delivery vehicle for βIII-tubulin siRNA and other targets for the treatment of PC.
Cholesterol Polyps in Common Bile Duct Mimicking Malignant Biliary Tract Obstruction
D. Thirabanjasak. Department of Pathology, Faculty of Medicine, Chulalongkorn University, Bangkok, Thailand.
Introduction/Background: Cholesterolosis or cholesterol polyps are the condition of small polypoid mass from lipid-ladened macrophages deposit in the lamina propria. The polyps are mostly found in the gallbladder with or without accompanying gall stones. This condition is benign however when it comes to a matter of common bile duct location, it can be a problematic one.
Method: A retrospective study of a case Thai female patient presenting with thickening distal common bile duct that suspicious for malignancy is present. The radiologic investigation cannot exclude the malignant lesion of the common bile duct so the Whipple’s operation was done.
Results: After the Whipple’s operation, there is a circumferential yellow lesion with fine granular cut surface at the distal common bile duct and narrowing ampulla of Vater on the macroscopic examination. The microscopic examination demonstrates no evidence of malignancy or premalignant ductal lesion. There are several cholesterol polyps at the distal part of common bile duct with the very thick mucosa. There is a degree of mixed inflammation.
Discussion/Conclusion: The cholesterolosis or cholesterol polyp could be the cause of misleading diagnosis of malignancy in the biliary tract because there is thick duct and the leading symptoms suggestive for biliary obstruction. At first the malignancy was concerned but later it was the cholesterol polyps in the common bile duct. This condition is very rare and had been report once in the archive. This is so called the unawared case of common bile duct lesion.
Characteristics and Outcomes of Borderline Resectable Pancreatic Cancer Compared with Resectable Disease
A. Thomas,1 J. Melling,1 R. Jackson,2 P. Whelan,3 P. Sykes,1 O. Jones,1 R. Smith,1 F. Campbell,3 J. Evans,3 M. Raraty,3 C. Halloran,1 R. Sutton,1 J. Neoptolemos,1 P. Ghaneh,1. 1NIHR Pancreas Biomedical Research Unit and CR-UK Pancreas Cancer Centre, Liverpool, UK; 2CR-UK Liverpool Cancer Trials Unit, Liverpool, UK; 3Royal Liverpool and Broadgreen University NHS Hospital Trust, Liverpool, UK.
Aim: To compare survival in patients with pancreatic ductal adenocarcinoma 9PDAC) defined preoperatively as resectable (RS) versus borderline resectable (BR) using standardized criteria.
Background: Although 5-20% of patients with PDAC may undergo resection with a 5-year survival rate of 25% with adjuvant therapy, survival of BR cases with major vessel involvement and without neoadjuvant therapy is poorly described.
Methods: Patients with PDAC who had not had neoadjuvant therapy and were planned for surgery were classified preoperatively as RS or BR (1997-2010). Resection rate, survival outcomes and clinical-pathological prognostic factors were analyzed.
Results: From 366 patients planned for resection 90 (26.8%) were categorized as BR. The resection rate was 39/90 (39%) for the for BR group vs 243/276 (88%) for the RS group (p<0.001). The overall median survival was 6.3 (95% CI 4.7-8.0) months for all of the 90 BR patients versus 14.5 (12.8-16.2) months for the 276 RS patients (p<0.001). The overall survival in the BR patients who just underwent resection was 10.9 (8.4, NA) months, versus 15.1 (CI 13.4-16.8) months in the RS group (p=0.096). Multivariate analysis showed that age, bilirubin-corrected serum CA19-9 levels, lymph node status, resection margin status and tumour grade were independent prognostic factors for both groups.
Conclusions: BR patients have lower resection rates and poorer survival compared with RS patients. BR patients who undergo a resection demonstrate better survival but this needs considerable improvement. Randomized studies of neoadjuvant therapy are required to determine whether this may improve outcome.
Role of Pancreatic Duct (PD) Stenting for “Smoldering” Pancreatitis
G. Trikudanathan, S. Gupta, R. Attam, M. Arain, M. Freeman. Division of Gastroenterology, University of Minnesota, Minneapolis, MN.
Background and Aim: “Smoldering” pancreatitis refers to a syndrome wherein patients with acute pancreatitis experience unremitting abdominal pain, intolerance to oral feeds, weight loss and recurrently elevated pancreatic enzymes with minimal or no changes in CT scan. One small study, reported 91% resolution of symptoms with the placement of pancreatic duct (PD) stent. We report our experience with the use of PD stents in these patients.
Methods: From our endoscopic database, we identified all patients who underwent ERCP with PD stenting from May 2009 to May 2013. Patients were included if they had ongoing abdominal pain, on daily narcotics, weight loss, persistently elevated pancreatic enzymes. Those with necrosis or peripancreatic fluid collection or >3/9 EUS changes of chronic pancreatitis were excluded.
Results: 25 patients (males-7 and females-18, mean age 34.8) with “smoldering” pancreatitis underwent ERCP with PD stent placement. Of these 6 were on TPN/ NJ tube feeds. Etiologies were (idiopathic-21, autoimmune-1, pancreatic divisum-2 and neuroendocrine tumor-1). Duration of symptoms from onset of first episode of pancreatitis until PD stenting ranged from 3 weeks to 2 years. A PD stent (5-7 Fr) was placed in all 25 patients. It provided pain relief and allowed discontinuation of narcotic analgesics in 16/25 (64%) of patients. TPN/NJ feeds were discontinued in 2/6 (33%) of patients.
Conclusion: “Smoldering” pancreatitis has been postulated to result from functional obstruction, potentiated by edema or spasm of the papillary orifice. This is the largest reported case series of this unusual entity. Unlike the previous series, there was only a modest response. Nevertheless, there may be a role for PD stenting in those who do not respond to conservative treatment measures. Further studies are mandatory to justify the role of PD stenting in functional “smoldering” pancreatitis.
Comparison of IgG4-positive Plasma Cell in Type 1 Autoimmune Pancreatitis and Pancreatic Ductal Adenocarcinoma
K. Uchida,1 Y. Fukui,1 T. Ikeura,1 Y. Sakaguchi,1 M. Shimatani,1 T. Fukui,1 M. Matsushita,1 M. Takaoka,1 A. Nishio,1 S. Satoi,2 A.H. Kwon,2 K. Okazaki,1. 1Department of Gastroenterology and Hepatology, 2Department of Surgery, Kansai Medical University, Hirakata, Japan.
Background: Infiltration of IgG4-positive cells is characteristic of type 1 autoimmune pancreatitis (AIP). We previously reported that regulatory T cells (Tregs) may regulate IgG4 production in type 1 AIP. It is reported that an increased serum IgG4 is observed in some patients with pancreatic ductal adenocarcinoma (PDA). In this study, we examined the infiltration and correlations of IgG4-positive cells and Tregs in patients with PDA.
Subjects and methods: A total of 21 PDA and 9 type 1 AIP patients were enrolled in this study. The numbers and ratios of Tregs, IgG4-, and IgG-positive cells immunohistochemically stained with anti-Foxp3, IgG4, and IgG antibodies, were counted in three areas of resected pancreata in PDA, peritumoral pancreatitis (PT), and obstructive pancreatitis (OP).
Results: In PDA, PT, OP area, the number of IgG4-positive cells (5.183 ± 1.061, 2.250 ± 0.431, 4.033 ± 1.018, respectively; p\0.05) and the ratio of IgG4/IgG (0.391 ± 0.045, 0.259 ± 0.054, 0.210 ± 0.048, respectively; p\0.05) were significantly lower than those in type 1 AIP (21.667 ± 2.436 and 0.306 ± 0.052, respectively). The numbers of IgG4-positive cells did not differ significantly among the three areas of resected pancreata. However, the IgG4/IgG (0.391 ± 0.045) and Foxp3/monocyte (0.051 ± 0.008) ratios in PDA area were significantly (p\0.05) higher than those in OP area (IgG4/IgG: 0.210 ± 0.048; Foxp3/monocyte: 0.0332 ± 0.005), but not in PT area. Of the 21 cases of PDA, the ratio of IgG4/IgG was 40 % in 9 (43 %), 6(29 %) and 3 (14%) cases in PDA, PT and OP area, respectively. Foxp3 and IgG4 were positively correlated in OP area, but not in PDA and PT area.
Conclusions: It should be caution of a differential diagnosis of PDA and type 1 AIP based on the numbers of IgG4-positive cells and the ratio of IgG4/IgG.
Differential Plasma Metabolites in Pancreatic Cancer–Associated Diabetes
S. Urayama, V. Tolstikov. Department of Internal Medicine/Gastroenterology; Genome Center, University of California, Davis, Sacramento, CA.
Background: Pancreatic cancer has been associated with development of glucose intolerance and dysregulation. Based on our previous report of strong potential for existence of metabolite biomarkers for PDAC detection, in this study, we have pursued initial characterization of plasma metabolites for discriminating PDAC cases among diabetic patients.
Methods: IRB-approved prospectively collected database and corresponding plasma from UC Davis Pancreas Registry was searched for appropriate cases: PDAC patients with recent-onset diabetes (≤3yrs prior to the index PDAC diagnosis) (Grp 1) and non-PDAC patients with long history of diabetes mellitus (>3 yrs) (Grp 2).
The plasma was processed and non-targeted GC/MS and targeted LC/MS were performed to assay for identifiable plasma metabolites. The data was processed and the peak intensity levels were normalized, scaled and processed via metabolomics specific multivariate analysis & bootstrap methods as well as ROC evaluation (Metaboanalyst & ROCCET) for visualization and biomarker development.
Results: We have identified 36 patients in Grp 1 and 22 patients for Grp 2. The metabolite specific multivariate and ROC analyses revealed specific features such as elaidic acid, uric acid, 2,3-propanediol, arachidonic acid, docosahexanoic acid, 5-oxo-EET, lysine, LysoPC(18:2), 9(10)-EpOME, LysoPC(16:0), sphingosine-1-phosphate were identified as a strong discriminators for recent-onset diabetic patients with pancreatic cancer. Such set yielded AUC of 0.964 with 15 features [CI=0.876-1].
Conclusion: We have demonstrated that a set of candidate plasma metabolite biomarkers for discriminating developing diabetic patients with pancreatic cancer. These features could be utilized in a larger set of population for further validation in near-future study.
Pre-operative Mutational Analysis of Fine Needle Aspirates is Feasible Using Next Generation Sequencing: Steps Toward Personalized Treatment of Pancreatic Cancer
V. Valero, III,1 T.J. Saunders,2 J.M. Herman,3,4 J.L. Cameron,1 C.A. Iacobuzio-Donahue,1,2,3 C.L. Wolfgang,1,2. 1Department of Surgery, 2Department of Pathology; 3Department of Oncology; 4Department of Radiation Oncology, The Johns Hopkins University, Baltimore, MD.
Background: A personalized approach to the treatment of pancreatic cancer that is based on genetic status depends on tissue for genetic analysis. Fine needle aspiration (FNA) is the most commonly used method for obtaining tissue prior to surgical resection, or for patients in whom surgical resection is not indicated. Potentially limiting factors of FNA include the paucity of malignant cells within a fibroblast-rich stroma and tumor heterogeneity. Therefore, we sought to determine: the ability to confidently detect mutant alleles in low cellularity samples, the concordance between the FNA and the primary tumor and the feasibility of identifying driver mutation in clinical FNAs.
Methods: We determined the ability to detect cancer-specific mutations in a wild-type stromal background in an in vitro model of Panc1 cells diluted into the fibroblast cell line, CAF 35, and the genotypic concordance between FNAs and their matched resected human adenocarcinoma tissues using next-generation sequencing by Ion Torrent PGM. Finally, the feasibility of driver mutation identification via FNA was explored by sequencing clinical FNA samples.
Results: The limit of confident detection occurred at a 1:25 ratio of malignant to stromal cells for both the KRAS and TP53 mutant alleles corresponding to a hypothetical carcinoma with 4% neoplastic cellularity. The concordance rates between the FNA and tumor samples ranged from 63% to 100%, although there was 100% concordance for driver mutations in both samples. Sequencing of clinical FNAs revealed driver mutations of KRAS, TP53 and SMAD4 genes.
Conclusions: Confident genotyping of low cellularity FNA samples is possible and accurately detects the driver mutations in primary tumors. Genotyping tumors in the neoadjuvant setting will enable physicians to offer personalized treatment paradigms according to chemosensitivity, overall survival and outcome data.
Abdominal Compartment Syndrome in Acute Pancreatitis: A Systematic Review
S. van Brunschot, A.J. Schut, S.A. Bouwense, M.G. Besselink, O.J. Bakker, H. van Goor, H.S. Hofker, M.A. Boermeester, H.G. Gooszen, H.C. van Santvoort, for the Dutch Pancreatitis Study Group.
Background: There is no effective drug therapy for Acute pancreatitis (AP). Experimental evidence suggests that tumor necrosis factor alpha (TNF- α) contributes to the pathogenesis of multiple organ failure. We performed a randomized, double-blind, placebo-controlled study using Pentoxifylline (a TNF- α blocker) in patients presenting with predicted severe AP.
Methods: All consecutive patients with AP, admitted to Mayo Clinic Rochester hospitals between 2009 and 2012,who met any one of the criteria of predicted severe AP ( age > 60 years, BMI >30, APACHE-II score >8, admission hematocrit >45, SIRS score ≥2, abnormal chest X ray (pleural effusion, infiltrates, atelectasis), >30% pancreatic necrosis or Balthazar Grade D (single fluid collection) or E (2 or more fluid collections) on admission CT scan, were approached within the first 72 hours of onset of AP. 28 patients who agreed to participate were randomized to “treatment group” (pentoxifylline 400 mg three times daily by mouth from the time of enrollment for a maximum of 3 days or 9 doses) and the “placebo group. The levels of TNF-α, Interleukin 6 (IL-6), Interleukin 8 (IL-8) and C-reactive protein (CRP) were measured at the time of enrollment, and every other day until 7 days or till the dismissal date whichever occurred earlier.
Results: The baseline characteristics were similar between the two groups. Changes in serum levels of IL-6, IL-10 and CRP from Day 0 to Day 1 and Day 3 were similar in two groups. There was a significant decrease in the need for ICU transfer in pentoxifylline group. The length of hospitalization (at a cutoff of 4 days and 10 days) was also less in the pentoxifylline group.
Conclusions: There was a significant decrease in the need for ICU stay, length of ICU stay and a decrease in the rate of prolonged hospitalizations in the Pentoxifylline group. These results call for a larger study with pentoxifylline preferably targeting the first 24 hours and following inflammatory markers for longer time.
Pentoxifylline Treatment in Severe Acute Pancreatitis
S. Vege, T. Atwal, S. Chari, R. Pearson, C. Loftus, F. Enders, M. Clemens Mayo Clinic, Rochester, MN.
Background: There is no effective drug therapy for Acute pancreatitis (AP). Experimental evidence suggests that tumor necrosis factor alpha (TNF- α) contributes to the pathogenesis of multiple organ failure. We performed a randomized, double-blind, placebo-controlled study using Pentoxifylline (a TNF- α blocker) in patients presenting with predicted severe AP.
Methods: All consecutive patients with AP, admitted to Mayo Clinic Rochester hospitals between 2009 and 2012,who met any one of the criteria of predicted severe AP ( age > 60 years, BMI >30, APACHE-II score >8, admission hematocrit >45, SIRS score ≥2, abnormal chest X ray (pleural effusion, infiltrates, atelectasis), >30% pancreatic necrosis or Balthazar Grade D (single fluid collection) or E (2 or more fluid collections) on admission CT scan, were approached within the first 72 hours of onset of AP. 28 patients who agreed to participate were randomized to “treatment group” (pentoxifylline 400 mg three times daily by mouth from the time of enrollment for a maximum of 3 days or 9 doses) and the “placebo group. The levels of TNF-α Interleukin 6 (IL-6), Interleukin 8 (IL-8) and C-reactive protein (CRP) were measured at the time of enrollment, and every other day until 7 days or till the dismissal date whichever occurred earlier.
Results: The baseline characteristics were similar between the two groups. Changes in serum levels of IL-6, IL-10 and CRP from Day 0 to Day 1 and Day 3 were similar in two groups. There was a significant decrease in the need for ICU transfer in pentoxifylline group. The length of hospitalization (at a cutoff of 4 days and 10 days) was also less in the pentoxifylline group.
Conclusions: There was a significant decrease in the need for ICU stay, length of ICU stay and a decrease in the rate of prolonged hospitalizations in the Pentoxifylline group. These results call for a larger study with pentoxifylline preferably targeting the first 24 hours and following inflammatory markers for longer time.
Ethanol and Its Non-Oxidative Metabolites Profoundly Inhibit CFTR Function in Pancreatic Epithelial Cells Which is Prevented by ATP Supplementation
V. Venglovecz,1 L. Judák,1,2 Z. Rakonczay, Jr.2 J. Maléth,2 M.A. Gray,3 P. Hegyi,2. 1Department of Pharmacology and Pharmacotherapy, 2First Department of Medicine, University of Szeged, Szeged, Hungary, 3Institute for Cell and Molecular Biosciences, University Medical School, Newcastle upon Tyne, UK.
Introduction: Excessive alcohol consumption causes acute pancreatitis but the mechanism involved is not well understood. Recent investigations suggest that pancreatic ductal epithelial cells (PDECs) are involved in the pathogenesis of pancreatitis. Because the cystic fibrosis transmembrane conductance regulator (CFTR) Cl- channel plays a major role in PDEC anion and fluid secretion, and that dysfunction of CFTR is often associated with pancreatitis, we therefore tested the hypothesis that ethanol exerts a direct effect on CFTR to impair ductal function.
Methods: The dose- and time-dependent effects of ethanol, its oxidative and nonoxidative metabolites (acetaldehyde (Ac) and palmitoleic acid ethyl ester (POAEE), respectively) and palmitoleic acid (POA) were investigated on CFTR activity on freshly isolated guinea pig PDECs and Capan-1 cell line, using the whole cell configuration of the patch clamp technique. Changes in intracellular ATP (ATPi) were measured by spectrofluorometry.
Results: Ethanol (10 and 100 mM) significantly increased the basal, but reversibly blocked forskolin-stimulated CFTR currents. The inhibitory effect of ethanol was mimicked by POAEE and POA, the latter being produced by fatty acid ethyl esterase hydrolase. Ethanol, POAEE and POA caused depletion of intracellular ATP (ATPi) linked to CFTR inhibition, since their inhibitory effects were almost completely abolished if ATPi depletion was prevented.
Conclusion: We propose that ethanol causes functional damage of CFTR through an ATPi-dependent mechanism. Furthermore, we suggest that the maintenance of ductal ATPi may represent a therapeutic option in the treatment of the disease.
This study was supported by OTKA, MTA and NFÜ/TÁMOP.
Ethanol Feeding Alters the Redox Status of the Endoplasmic Reticulum Proteome in Mouse Pancreas
R.T. Waldron,1,2 J. Capri,3 R. Gong,1 J.P. Whitelegge,3 K.F. Faull,3 S.J. Pandol,1,2 A. Lugea,1. 1Department of Medicine, VAGLAHS, 2Cedars Sinai Medical Center, 3Semel Institute, UCLA, Los Angeles, CA.
Background: Early events linking ethanol and its metabolites to alcoholic pancreatitis remain unclear. We hypothesize that redox changes within endoplasmic reticulum (ER) and sustained ER stress in the acinar cell as early pathologic events. The unfolded protein response (UPR) protects acini from modest redox imbalance, but sustained ethanol exposure sensitizes to severe redox insult involving modifications to key ER targets including chaperones, calcium regulators, redox-dependent folding proteins, and zymogens.
Methods: Redox changes were studied in wild-type and Xbp1+/- mice fed ethanol using the Tsukamoto-French intragastric model, and in vitro in AR42J cells.
Results: Ethanol fed mice exhibited selectively reduced total GSH in pancreas tissue extracts and GSH/GSSG in ER fractions. More severe pancreas pathology in pancreas slices, and more distended and vacuolated ER and enlarged autophagic vacuoles in the acinar cells were found in ethanol fed XBP+/- mice, a recently developed model to assess the protective role of UPR. The redox status of protein thiols was measured in ER fractions from control- and ethanol-fed mice using OXICAT, a novel proteomic approach. Whereas many of the same proteins were detected between control and ethanol-fed mice, several proteins had either higher or lower percentages of oxidable cysteines in the ethanol-fed group. AR42J cells were cultured with 50-100 mM ethanol for up to 72 h. Biotin switch to measure exposed cysteine residues indicated that ethanol selectively induced redox modifications in proteins including PDI and amylase. Staining for protein aggresomes revealed large cytoplasmic aggregates in ethanol-treated AR42J cells.
Conclusions: These data suggest ethanol-induced early events involved in alcoholic pancreatitis pathogenesis include modification at specific cysteine residues within key proteins and enhanced protein aggregate formation.
Pancreatitis Quality of Life Instrument (PANQOLI): Validation of the Electronic Version
W. Wassef,1 C. Bova,2 B. Barton,3 C. Hartigan,1. 1Division of Gastroenterology, 2 Graduate School of Nursing, 3Department of Quantitative Health Science, University of Massachusetts Medical School, Worcester, MA.
Purpose: PANQOLI is a disease specific instrument developed for the evaluation of quality of life in chronic pancreatitis patients. Preliminary reliability and validity of the paper and pencil version has been reported for patients (n=159) with severe disease. The goal of this study was to examine its psychometric properties when administered electronically to patients with variable degrees of disease.
Methods: An invitation to chronic pancreatitis patients was placed in the National Pancreas Foundation newsletter. Interested subjects were asked to log on to a website where they were provided with a unique password and asked to consent. Those who consented were asked to complete the following instruments on-line: 1) demographics, 2) PANQOLI, 3) Short Form 12 (SF-12) and 4) EORTC-PAN. Once completed, the information was transmitted electronically to our REDCap database. Statistical evaluation of the instrument included: 1) its internal consistency using Cronbach’s alpha, 2) multi-trait-multi-test analysis to evaluate its validity, and 3) ANOVA to test its generalizability.
Results: 179 patients completed the survey: mean age 47.49 (SE +/- .884) years, 76% were female, 93.9% were white, 28.5% were working, and 70.9% were on pain management. In this group, only 94/179 (52.5%) received treatment at tertiary care centers, and 23/179 (12.8%) lived outside the United States. The Cronbach’s alpha for the PANQOLI was .900. It correlated very well with the SF-12 (PHC- .560, MHC-.554) as well as the EORTC PAN (pf .502, rf .455, ef .502, qol .635). Using single ANOVA analysis, there was no significant difference in results based on sex, race, work status or pain management.
Conclusion: PANQOLI is just as useful in an electronic on-line format in a mostly female Caucasian population, as it was in paper and pencil format. Its role in patients with other ethnicities has yet to be determined. Nevertheless, enough validation has been conducted to permit its implementation in future studies aimed at evaluating the impact of therapeutic interventions on chronic pancreatitis patients.
Orai1 Inhibition Prevents Calcium Toxicity and Acute Pancreatitis
L. Wen,1,2 D. Collier,2 S. Voronina,2 M.A. Javed,1,2 M. Awais,1 M. Begg,3 J. Barrett,3 D.N. Criddle,1,2 A. Tepikin,2 R. Sutton,1. 1Liverpool NIHR Pancreas Biomedical Research Unit, Royal Liverpool University Hospital, 2Department of Cellular and Molecular Physiology, University of Liverpool, 3Respiratory Therapy Area, GlaxoSmithKline, Stevenage, UK.
Background: Prolonged elevations of intracellular calciumtrigger pancreatic acinar cell (PAC) injury contributing to experimental acute pancreatitis (AP). The elevations depend on external Ca2+influx through store-operated Ca2+entry channels that may include Orai1 but the role of this channel remains to be defined.
Objective: To determine the effect of Orai1 inhibition with GSK-7975A on PAC injury and of its pro-drug GSK-6288B on experimental AP.
Methods: TIRF and confocal microscopy were used to measure cytosolic Ca2+(Fura-2), vacuole formation (Lucifer yellow) and necrotic pathway activation (PI) changes in freshly isolated murine and human PACs following toxin (TLCS, CCK-8, thapsigargin) administration. AP was induced by cerulein (50 μg/kg/h IP x 7) or intraductal infusion of TLCS (50 μL 3 mM). GSK-6288B was administered SC by osmotic mini-pump (2001D) and pharmacokinetic (PK) profiles was measured. AP severity was assessed by standard biochemical parametersand blinded histopathology.
Results: GSK-7975A inhibited calcium influx, vacuole formation and necrotic pathway activation in murine (at10µM or 30µM, p<0.05) and human (30µM or 100µM, p<0.05) PACs after toxin administration.TRPC3 inhibition did not further increase inhibition of calcium influx. PK confirmed GSK-6288B consistently achieveddrug levels (circa 10µM or 30µM GSK-7975A) in blood and pancreas. GSK-6288B significantly reduced all AP severity parameters in both models (all p<0.05).
Conclusion: Orai1 plays a major role in PAC Ca2+entry and injury in response to toxins. This work confirms the role of calcium overload in AP and suggests GSK-7975A has potential in the treatment of AP.
IFN-α Promotes Pancreatitis-Associated Lung Injury in Murine Acute Pancreatitis
L. Wen,1,2 W. Huang,1,2 T. Jin,2 Q. Xia,2 D.N. Criddle,3 R. Sutton,1. 1NIHR Liverpool Pancreas Biomedical Research Unit, Royal Liverpool University Hospital, 2Sichuan Provincial Pancreatitis Centre, Western China Hospital, Sichuan University, Chengdu, China, 3Department of Cellular and Molecular Physiology, University of Liverpool, UK.
Background: Interferon alpha (IFN-α) or pegylated IFN-α have been associated with drug-induced acute pancreatitis (AP), but mechanisms have not been determined.
Objective: To determine the effect of IFN-α on the severity of experimental AP.
Methods: Murine IFN-α (0.1, 1.0, 3.0 or 10.0 MIU/kg SC) was given one day and 30min before AP induction in male CD1 mice (six per group)by (i) cerulein (50 μg/kg/h IP x 7,CER-AP)(ii)intraductal infusion of TLCS (50 μL 3 mM, TLCS-AP] (iii) palmitoleic acid (150 mg/kg/h IP × 2) and ethanol (1.35g/kg/h IP ×2) (FAEE-AP).Blood, pancreas, lung and liver were harvested 12h or 24h after AP induction. IFN-α-induced immune responses were examined by liver mRNA expression of interferon-induced protein with tetratricopeptide repeats (IFIT 1 and 2). AP was assessed by standard biomarkers and blinded histopathology.
Results: Administration of IFN-α resulted in higher liver inflammatory gene expression and standard AP parameters than without IFN-α (p<0.05). Application of IFN-α at 1or 3 MIU/kg caused marked increases of lung MPO activity inall AP models (80% in CER-AP, 280% in TLCS-AP and 40%in FAEE-AP vs AP without IFN-α, p<0.05). Lung histopathology showed an increase of alveolar membrane thickness score at 10 MIU/kg (p<0.05).
Conclusion: This study suggests that IFN-α exacerbates AP through immune responses that contribute to pancreatic and distant organ injury, confirming that distant organ injury is immune-mediated. The mechanism by which IFN-α initiates AP has not been addressed.
Trends in Pancreatic Surgery: Indications, Operative Techniques and Postoperative Outcomes of 1120 Pancreatic Resections
U.A. Wittel, F. Makowiec, T. Keck, U. Adam, H. Riediger, U. Wellner, U. T. Hopt. Department of Surgery, University of Freiburg, Freiburg, Germany.
Introduction: Hospital volume, surgeon volume and adequate management of complications are factors contributing to a better outcome from pancreatic resection. The aim of our study was to analyze trends in indications, operative techniques and postoperative outcome in 1120 PaRes performed since 1994.
Methods: 1120 PaRes were performed since 1994. The vast majority of the operations were performed by three surgeons. The perioperative data were documented in a database. For our analyses the study period was subclassified into three periods (A 1994-2001/n=363; B 2001-2006/n=305; C since 2007/n=452).
Results: 81% of the PaRes were performed by one of the 3 principal surgeons. The average annual number of PaRes increased from 52 (period A) to 80 (period C; n=107 in 2011). The median age increased from 51 (A) to 65 years (C; p<0.001). In the entire group (n=1120) indications for surgery were pancreatic/periampullary cancer (49%), chronic pancreatitis (CP; 33%) and various other lesions (18%). The frequency of IPMNs increased from below 1% (A) to 8% (C; p<0.05). About two thirds of the operations were pancreaticoduodenectomies. Total pancreatectomies increased due to a more aggressive approach with cancer from 1% (A) to 6% (C). The frequency of mesenterico-portal vein resections increased from 8% (A) to 20% (C; p<0.01). Distal resections were performed in 17%. Overall mortality was 2.4%. Overall complication rates increased from 42% (A) to 56% (C; p<0.01). The rate of pancreatic leak grade B/C also increased from 5% (A) to 12% (C; p<0.01) but the frequencies of relaparotomies were comparable (10-14%; n.s.)
Conclusions: Operative mortality in our institutional series of 1120 pancreatic resections was 2.4% and remained low despite a more aggressive surgical approach to pancreatic disease. An increased overall morbidity may be explained by more clinically relevant pancreatic fistulas and better documentation.
Statins are Associated With Reduced Risk of Acute Pancreatitis: Findings From a Population-Based Cohort Study
B.U. Wu,1 S.J. Pandol,2 I.L. Liu,3. 1Center for Pancreatic Care, Southern California Permanente Medical Group; 2Cedars-Sinai Medical Center, Los Angeles CA; 3Department of Research and Evaluation, Pasadena CA, Southern California Pancreas Study Group.
Background: Recent data suggest that statin use may be protective against acute pancreatitis (AP). The aim of this study was to further characterize the relationship between use of statins and risk of AP.
Methods: We conducted a retrospective cohort study (2001-2012) on data from an integrated care system in southern California. Inclusion criteria: age>18 and ≥6 months of continuous follow-up. Exposure to simvastatin or atorvastatin was ascertained by electronic pharmacy data. Medication exposure was calculated from time of initial dispensation until 6-months following prescription termination. AP cases were defined by diagnosis 577.0 and serum lipase>3 times normal. Patients were censored at death, last follow-up, and onset of AP or end-of-study. Cox proportional hazards regression was used to generate hazards ratio (HR) for statin use adjusted for age, gender, race/ethnicity, gallstone-related disorders, hypertriglyceridemia (triglycerides>1000mg/dL) and alcohol dependence. Separate models were constructed for use of either simvastatin or atorvastatin.
Results: 6399 patients developed AP in a study population of 3.9 million (mean age 42.1, SD 16.2; 51.2% women). Median duration of follow-up was 3.4 years (IQR 1.3,7.0). 707,236 patients received simvastatin and 172,979 atorvastatin during the study period: 1384 (0.16%) patients developed AP while on medication. Overall risk of AP was significantly reduced with statin use in multivariate analysis, simvastatin HR 0.29 [95% CL 0.27,0.31] after adjusting for age, gender, race/ethnicity, gallstone disorders HR 23.2[22.1,21.4], alcohol dependence HR 5.2[4.8,5.5] and hypertriglyceridemia HR 12.7[11.3,14.2]. Similar results were noted with atorvastatin, adjusted HR 0.33[0.29,0.37].
Conclusion: Use of statins was independently associated with reduced risk of AP in this large population-based setting. Further investigation into potential mechanisms and prospective validation of the present study findings are warranted.
GSK-3β Regulation of Survival and EMT in Pancreatic Cancer
S. Xu, S. J. Pandol, M. Edderkaoui. University of California Los Angeles, Department of Veterans Affairs & Cedars-Sinai Medical Center, Los Angeles, CA.
Background: Strong correlation exists between high expression level of glycogen synthase kinase 3 beta (GSK-3β) and pancreatic cancer (PaCa) progression in humans. GSK-3β inhibition in a mouse orthotopic model of PaCa induced tumor shrinkage and inhibition of NF-κB activity. However, GSK-3β inhibition stimulates pro-metastasis epithelial to mesenchymal transition (EMT). We hypothesize that combination of GSK-3β and EMT inhibitions will prevent both cancer cell survival and EMT.
Methods: Pdx-Cre;LSL-Kras mice were treated with GSK-3β inhibitor TDZD-8 (4mg/Kg). Pancreas levels of proliferation and EMT were measured by immunohistochemistry and Western. Inhibition of GSK-3β in MIA PaCa-2 and AsPC-1 cell lines was achieved by applying GSK-3β inhibitors tideglusib or TDZD-8, or by GSK-3β siRNA. Inhibition of Zeb1 was achieved by using Zeb1 siRNA. EMT markers vimentin and E-cadherin, as well as EMT transcription factor levels were measured by Western. Cell proliferation and apoptosis were measured by MTT assay and DNA fragmentation, respectively.
Results: Inhibition of GSK-3β in mice stimulated EMT markers in pancreas. Pharmacological and molecular inhibitions of GSK-3β significantly and dose dependently decreased proliferation and at a lesser extent stimulated apoptosis in PaCa cells. GSK-3β inhibition stimulated EMT by increasing the level of vimentin in MIA PaCa-2 and decreasing the level of E-cadherin in AsPC-1. EMT transcription factor Zeb1 level was increased in cells treated by GSK-3β inhibitors. Zeb1 siRNA reversed the effect of GSK-3β inhibitors on EMT.
Conclusion: Combination of Zeb1 inhibition and GSK-3β inhibitor induced a triple beneficial effect by decreasing proliferation, stimulating apoptosis and inhibiting EMT. Tideglusib applied here is in clinical trials for a different disease, which makes it promising to translate our finding to humans.
Alternative Activated Macrophages Activate Pancreatic Stellate Cells and Promote Chronic Pancreatitis
J. Xue, A. Habtezion. Department of Medicine, Division of Gastroenterology & Hepatology, Stanford University School of Medicine, Stanford, CA.
Chronic pancreatitis (CP), is a progressive inflammatory and fibrotic disease, which eventually leads to exocrine and endocrine deficiency. The management of CP is challenging, and most patients remain symptomatic despite limited available therapy. Current therapy mainly focuses in managing complications associated with the disease. Activated pancreatic stellate cells (PSCs) contribute to fibrogenesis and promote CP progression. Alternatively activated macrophages (M2), which are driven by Th2 cytokines (IL-4/IL-13), facilitate wound repair and suppress inflammation. However, the role of macrophage, especially M2, in CP development remains poorly understood. Using caerulein mouse model of CP, we show that M2-like macrophages (CD206hiIL-10+) are dominant in CP whereas acute pancreatitis is dominated by classically activated macrophages (M1). Interestingly, transgenic mice that lack the ability to polarize macrophages to M2 (e.g. IL-4R1-/-) and produce IL-10 (IL-10-/-) are less susceptible to caerulein-induce CP, suggesting that M2 macrophages play an important role in CP progression. To understand mechanisms involved, we set up ex-vivo cultures of bone marrow derived macrophages (BMDM) with primary PSCs, and assess macrophage and PSC activation as well as function. Results from these studies show that PSCs promote BMDM polarization towards M2, and in turn M2 macrophages, but not IL-10 deficient macrophages, have the ability to activate PSCs. Taken together, our data demonstrate an important crosstalk between macrophages and PSCs in the development of CP. Further understanding into the interactions involved between macrophages and PSCs offers potential future novel therapeutic approaches that may either halt or reverse CP progression.
Loss of TFF2 in Pancreatic Duct Glands (PDG) Results in the Formation of IPMN Suggesting TFF2 May Function as a Tumor Suppressor
J. Yamaguchi,1 M. Mino-Kenudson,2 A.S. Liss,1 K.D. Lillemoe,1 C. Fernández-del Castillo,1 A.L. Warshaw,1 S.P. Thayer,1. Warshaw Institute and Departments of 1Surgery and 2Pathology, Massachusetts General Hospital and Harvard Medical School, Boston, MA.
Background: PDG are a novel epithelial progenitor compartment important for regeneration of the ductal epithelium. Our early correlative studies suggest that dysregulation of this compartment may result in formation of IPMN. This compartment uniquely expresses trefoil family factor 2 (TFF2). PDG functionally and molecularly resemble gastric glands. In the stomach TFF2 has been reported to be the tumor suppressor of gastric cancer, suggesting that TFF2 also act as the tumor suppressor to prevent the initiation of pancreatic mucinous neoplasms.
Methods: In vivo TFF2-knockout mice (TFF2-KO) were bred to generate TFF2KO;Pdx-Cre;LSL-KRASG12D, which were harvested at 2, 4 and 6 months then histologically evaluated. In vitro a pancreatic cancer cell line (Panc-1) was transfected with a TFF2-overexpressing vector to evaluate the impact of TFF2 on proliferation.
Results: In the pancreas of KRASG12D and TFF2-KO mice, no neoplastic epithelium was identified at 2, 4 and 6 months old. The absence of TFF2 in combination with the background of activated KRASG12D mutation results not only in expansion of PDG compartment, but also in the formation of intraductal papillary mucinous neoplasms (IPMN) predominantly of the main pancreatic duct by 2 month. High to moderate grade IPMN were identified by 4-6 months. In vitro the induction of TFF2-expression in cancer cell line resulted in a decrease in proliferation. TFF2 over expression and its effect on proliferation was associated with an increase in nuclear SMAD4.
Conclusion: TFF2 act as the tumor suppressor in PDG. This effect may be mediated by its effect on SMAD 4.
Development of a Novel Next Generation Sequencing (NGS) Method for Detection of Low Concentration Sequence Variants
L. Yan, J. A. Nicholson, L. Bennet, K. Bullock, M. Lombard, H. Smart, J. Evans, C. Halloran, P. Ghaneh, E. Costello, R. Sutton, J. P. Neoptolemos, W. Greenhalf. NIRH Pancreas Biomedical Research Unit, University of Liverpool, UK.
Background: The detection of low frequency somatic mutations is used to screen for pancreatic cancer. Conventional sequencing methods do not offer sufficient sensitivity and mutation specific PCR requires the specific sequence to be known. Deep sequencing has low specificity.
Aim: To evaluate a next generation sequencing workflow for detection of p53 mutations in pancreatic juice.
Methods: Ion Torrent sequencing was used with samples of wild type and mutant p53 using different ratios. These were combined with serially diluted DNA from CFPAC2 cells. Limiting dilution was used to improve specificity.
Results: PCR error gave a false positive result in 2% of sequences. Ten genome equivalents of juice DNA eliminated non-specific detection and allowed identification mutant sequences. Mutations were confirmed by Sanger sequencing following functional enrichment of mutations in yeast.
Conclusion: The strategy can be used in screening of clinical samples for low concentration mutations.
Trends and Regional Variation for Inpatient Costs in Acute Pancreatitis: An Opportunity for Savings?
J. Yeh, B. Wu. Center for Pancreatic Care, Department of Gastroenterology, Kaiser Permanente Los Angeles Medical Center, Los Angeles, CA.
Introduction: Health care reform has placed a strong emphasis on containment of the nation’s rising health care costs. Acute pancreatitis (AP) is the most common diagnosis for gastrointestinal hospitalizations. This study aims to highlight trends in hospital costs for AP and evaluate cost variation on a state-by-state level.
Methods: The mean cost per hospitalization for AP [ICD-9 577.0] was calculated using the 2006 to 2010 Nationwide Inpatient Sample (NIS), the largest all-payer inpatient care database covering 1051 hospitals across 45 States. The impact of patient age, sex, race, AHRQ comorbidities and hospital bed size, teaching status, location and ownership were analyzed using linear regression and ANOVA testing. Log-transformed linear regression model with smearing retransformation was performed with total cost as the dependent variable.
Results: In 2010, there were a total of 288,597 (S.E 6,518) admissions with a principle diagnosis of AP. The mean total cost per hospitalization increased from $9,348 (+/- $139) in 2006 to $10,069 (+/- $192) in 2010, Wilcoxon p<0.001. After adjusting for both patient level and hospital level confounders, the mean cost per hospitalization was highest in Vermont ($18,795) and lowest in Kansas ($4,873), representing a 3.8 fold degree of variation after adjusting for local price indices and mortality. Compared to the median represented by Missouri ($12,717), the states at both ends of spectrum are statistically significant (Wilcoxon p <0.0001).
Conclusions: Inpatient costs for acute pancreatitis in the United States have increased from 2006 to 2010. Significant variations in cost were noted among states even after adjusting for patient factors, hospital level covariates, price indices and outcomes, in particular in-hospital mortality. Further studies are needed to understand the sources of this variation and may identify opportunities for more cost effective care.
Atrophy, Calcifications, and Small Volume Pancreata by CT/MRI Predict Lower Islet Mass and Insulin Dependence in Total Pancreatectomy-Islet Autotransplantation (TPIAT)
M. Young, J. Theis, G. Beilman, S. Walker, T. Pruett, S. Chinnakotla, T. Dunn, S. Schwarzenberg, M. Freeman, M. Bellin. University of Minnesota, Minneapolis, MN.
Background: Patients with chronic pancreatitis undergoing TP-IAT have varying extents of pancreatic damage which may affect IAT results. We investigated the relationship of preoperative imaging with islet isolation and patient outcomes.
Methods: Pre-operative CT (n=26) and MRI (n=31) were reviewed from 55 patients (mean age 31y; 10 male) who underwent TP-IAT. Presence of atrophy and calcifications were recorded. Pancreatic volume was calculated using Vitrea imaging software and was correlated with trimmed pancreas weight, total islet equivalents (IEQ) isolated, and digest tissue volume (TV) using linear regression modeling. Insulin requirements were assessed at 1 year or most recent for patients <1 year post-IAT.
Results: Volume by CT and MRI each correlated with IEQ, pancreas weight, TV, and number of purifications (P<0.01). Volume by CT was a slightly better predictor than MRI. Mean pancreatic volume by CT was lower in insulin dependent patients (p=0.05), while differences in MRI- volume were not significant (p=0.4). Patients with calcifications (n=10) had lower islet mass (97,000±91,000 IEQ vs 372,000±201,000 IEQ, p=0.0001), lower digest tissue volume (4.6±3.7 mL vs 20.0±10.4 mL, p<0.0001), fewer purifications, and none were insulin independent (vs 50% insulin independent in non-calcific, p=0.01). Patients with atrophy (n=21) similarly had a lower islet mass (p<0.0001), lower digest tissue volume (p<0.0001), fewer purifications, and lower likelihood of insulin independence (p=0.007).
Conclusions: Radiographic pancreatic volume was closely associated with islet mass, and in the case of CT volume, later insulin dependence. Patients with calcifications or atrophy were at high risk for low islet mass and insulin dependence. CT volume may assist in prediction of need for islet purification.
Alcohol-induced Experimental Pancreatitis: Role of Protein Kinase D
J. Yuan, Y. Liu, T. Tan, L. Li, S.J. Pandol. Veterans Affairs Greater Los Angeles Healthcare System, and Cedars-Sinai Medical Center, Los Angeles, CA.
Acute pancreatitis is a common and potentially lethal acute inflammatory disease and alcohol abuse is a major cause of pancreatitis. Our previous studies have demonstrated that ethanol augments NF-κB activation and cell necrosis in pancreatic acinar cells resulting in a more severe form of pancreatitis. However the mechanisms mediating the modulatory effects of ethanol on the pathologic responses of pancreatitis have not been completely revealed.
We recently reported that protein kinase D (PKD/PKD1) signaling is required for inflammation and cell necrosis in pancreatitis. Downregulation or inhibition of PKD1 with two novel small molecule PKD inhibitors CID755673 and CRT0066101 ameliorates these pathologic responses and the severity of pancreatitis. In current study, we explored whether PKD is involved in the mechanisms mediating the modulatory effects of ethanol on pancreatitis.
Methods: Rats were pair-fed control and ethanol-containing Lieber-DeCarli diets for 6 weeks followed by administration of 4 hourly intraperitoneal (IP) injections of the cholecystokinin (CCK) analog, cerulein at 0.5 μg / kg. PKD inhibitor CID755673 (15mg/kg) dissolved in DMSO was applied in rats as a single IP injection 60 min before cerulein treatment. Measurement of pancreatic pathology included serum amylase and lipase activity, pancreatic necrosis, edema, vacuoles, and inflammatory cell filtration.
Results: Ethanol feeding promoted PKD1 expression and potently enhanced cerulein-induced PKD1 phophosphorylation and activation that were associated with ethanol-sensitized NF-κB activation and cell necrosis in pancreatitis. PKD/PKD1 inhibition by CID755673 significantly attenuated inflammation, necrosis and the severity of pancreatitis caused by cerulein administration in ethanol-fed rats.
Conclusions: Alcohol treatment worsens experimental pancreatitis at least in part by augmenting PKD1 expression and activation. Inhibition of PKD markedly improves pancreatitis severity.
Inhibition of Nuclear Factor kappa B Essential Modifier Ameliorated Severity of Acute Pancreatitis
Z. Yuan, S.K. Garg, U. Barlass, R. Dawra, A. Saluja. Division of Basic and Translational Research, Department of Surgery, University of Minnesota Medical School, Minneapolis, MN.
Background: The role of nuclear factor kappa B (NFκB) activation in the pathogenesis of acute pancreatitis (AP) remains controversial. Nuclear factor kappa B essential modifier (NEMO) is required for activation of NFκB.
Aim: To study the effect of NEMO inhibition on severity of acute pancreatitis.
Methods: Two different inhibitors, a specific inhibitory peptide, and a chemical inhibitor, withaferin A (WA) were used to treat acini for inhibiting NEMO. NFκB activation was compared in response to pathological caerulein stimulation with untreated acini. WA was also given intraperitoneally along with supramaximal caerulein for in vivo studies. Acinar cell death, tissue damage, and local and systemic inflammation were compared among saline, caerulein and treatment group.
Results: Both inhibitors impaired acini p65 NFκB nuclear translocation stimulated by pathological concentration of caerulein. High concentration of WA (5μM) even completed blocked p65 nuclear translocation and the degradation of IκBα, but did not affect cell viability. For in vivo study, compared to the caerulein control, optimal concentration of WA (4mg/kg) given intraperitoneally significantly decreased plasma amylase level and pancreatic myeloperoxidase activity but not edema. The pancreas necrosis was reduced in the treatment group.
Conclusions: Inhibition of NEMO, which block NF-κB activation, is protective against acute pancreatitis. This is the first report of using NEMO chemical compound inhibitor to attenuate acute pancreatitis.
Effect of Noninvasive Positive Pressure Ventilation on Acute Respiratory Failure in Patients with Predicted Severe Acute Pancreatitis
X.L. Zhao, J. Li, Y.L. Liu, M.H. Wan, S.F. Zhu, X.H. Peng, Q. Xia, W.F. Tang. Sichuan Provincial Pancreatitis Centre, Department of Integrative Medicine, West China Hospital, Sichuan University, Chengdu, China.
Background: The value of noninvasive positive pressure ventilation (NPPV) in severe acute pancreatitis (SAP) complicated with acute respiratory failure (ARF) remains unclear.
Methods: Predicted SAP patients admitted to our unit fulfilling inclusion and exclusion criteria between Jan 1, 2011 and Dec 31, 2012 were included. All patients with ARF were initially treated with NPPV for more than 24 hours. Intubation was implemented if there was no significant improvement of gasometric findings and clinical situation in at any time after NPPV treatment. Demographic and clinical data were collected for comparisons between designated groups.
Results: A total of 127 patients were included. There were 48 patients in the acute lung injury (ALI) group with 4 developed acute respiratory distress syndrome (ARDS) and intubated, but no patients died. There were 79 patients in the ARDS group with 20 intubated. All 9 patients died were from the intubated subgroup. Patients responded to NPPV had significantly higher proportion of ALI patients, lower CT severity index, decreased heart rate, improved respiratory parameters, less local and systemic complications, earlier oral refeeding time, shorter hospitalization, lower operation rate and mortality. Compared to ALI group, the ARDS group was associated with more pulmonary infection, altered consciousness and mortality. In NPPV successful patients, ARDS group had significantly delayed time of admission and NPPV application, lower oxygenation index, higher FiO2% initial and mean value, postponed oral refeeding time and prolonged hospitalization compared to those in ALI group.
Conclusion: NPPV is effective and safe for most of the ALI and part of ARDS for the predicted SAP. Well-designed prospective RCTs are warranted to confirm these findings.