Heme Oxygenase-1 Inhibition Sensitize Pancreatic Cancer to Gemcitabine Therapy
M.Y. Abdalla,1 S. Rachagani,2 N. Wasim,2 S. Batra,2 S. Kumar. 1Department of Pathology/Microbiology, University of Nebraska Medical Center, Omaha, NE; 2Department of Biochemistry and Molecular Biology, UNMC, Omaha, NE.
Background: Pancreatic cancer (PC) is a very lethal malignancy with five-year survival rate of 7.2 %, underscoring the need to develop novel therapeutic regimens. Heme Oxygenase-1 (HO-1) is a stress enzyme that is induced during chronic hypoxia, and has been demonstrated to facilitate PC progression. Extreme hypoxia is one of the characteristic feature of PC and is responsible for the resistance to therapy. The relationships between HO-1, hypoxia, and response to chemotherapy remain unclear. We hypothesize that HO-1 inhibition under hypoxic conditions suppress the growth of pancreatic tumor and sensitize to chemotherapy.
Method: PC cell lines (CD18/HPAF and BxPc3) were evaluated for proliferation, ROS production and apoptosis, under hypoxic and normoxic conditions in the presence of HO-1 inhibitors ZnPP and SnPP, and activator CoPP (20–100 μM). Further, CD-18/HPAF PC cells expressing luciferase were orthotopically implanted in nude mice, and treated with SnPP, or SnPP in combination with gemcitabine.
Results: Hypoxia induced the expression of HO-1 in PC cells and inhibition of HO-1 with ZnPP and SnPP significantly reduced the proliferation of PC cell line both under normoxic and hypoxic conditions. Further, HO-1 inhibitors induced apoptosis in PC cells. Mechanistically, inhibition of HO-1 increased the production of ROS (p<0.05), as demonstrated by increased DCFH, and Mitosox fluorescence. Treatment with SnPP, or SnPP in combination with gemcitabine significantly reduced the pancreatic tumor weight (p<0.05) and improved the efficacy of gemcitabine treatment (p<0.05) under in vivo conditions.
Conclusion: Our study demonstrate that HO-1 inhibition provides a novel therapeutic approach by reducing PC cell proliferation and enhance the efficacy of gemcitabine therapy to treat PC.
Novel Role of Peritoneal Mesothelial Cells That Lead to Pancreatic Cancer Peritoneal Dissemination Formation
T. Abe, K. Ohuchida, S. Kibe, Y. Ando, H. Nakayama, S. Takesue, S. Endo, K. Koikawa, T. Okumura, T. Moriyama, K. Nakata, Y. Miyasaka, T. Manabe, T. Ohtsuka, E. Nagai, K. Mizumoto, M. Nakamura. Department of Surgery and Oncology, Kyushu University, Fukuoka, Japan.
Background: Peritoneal mesothelial cells (PMCs) are located on the surface of the peritoneal cavity and generally work as a protective barrier for dissemination. But, previous study reported that the interactions between cancer cells and PMCs played an important role in peritoneal dissemination in gastric and ovarian cancer. However, the role of PMCs in the peritoneal dissemination of pancreatic cancer remains unclear.
Methods: In the present study, we investigated the interactions between pancreatic cancer cells and PMCs in the formation of peritoneal dissemination.
Results: PMC was activated by TGF-β administration. PMCs and activated PMCs (aPMCs) significantly promoted the migration and invasion of pancreatic cancer cells in indirect and direct co-culture (P<0.004). aPMCs significantly increased adhesion ability of pancreatic cancer cells compared with PMCs (P<0.001). Condition medium (CM) derived from PMCs significantly promoted proliferation of pancreatic cancer cells in adhered and suspended condition (P<0.001) and inhibited apoptosis of pancreatic cancer cells in suspended condition. CM derived from PMCs promoted mesothelial clearance by pancreatic cancer spheroids (P<0.001). Furthermore, we evaluated the invasion process of pancreatic cancer cells and PMCs with 3D collagen invasion assay. We found PMCs pre-invaded and the cancer cells followed them. We observed similar findings in the tissue of peritoneal dissemination from KPC (LSL-Kras G12D/+ ;LSL-Trp53 R172H/+;Pdx-1 -Cre) mouse.
Conclusions: The cancer-stromal interactions between pancreatic cancer cells and PMCs are important in the peritoneal dissemination of pancreatic cancer cells. Therapy targeting this interaction may improve the prognosis of patients with pancreatic cancer.
Acute Pancreatitis Admission Trends in Pediatrics: A National Estimate Through the Kids
M. Abu-El-Haija,1 S. El-Dika,2 A. Hinton,2 D.L. Conwell.2 1Cincinnati Children's Hospital Medical Center, Cincinnati, OH; 2Ohio State University-Wexner Medical Center, Columbus, OH.
Introduction: Despite the increased incidence, data is lacking on national health care utilization for pediatric Acute Pancreatitis (AP).
Hypotheses: Pediatric AP constitutes a significant healthcare burden that varies by patients’ age. Chronic Pancreatitis (CP) and obesity are associated conditions that affect AP trends.
Methods: The Kids' Inpatient Database (KID) for years 2006, 2009 and 2012 was queried for patients with a principal diagnosis of AP. Cases were grouped by age: young < 5 y, middle 5–14 y and older >14 y.
Results: A total of 27,983 AP discharges were identified. The number of admissions increased with age: young n=1,279, middle n=8,012, and older n=18,692. Females constituted 53-61% of cases in each age group. Length of stay (LOS) was highest in young children; [mean 7.18 days (95% CI 6.34, 8.02)], compared with middle [5.79 days (5.51, 6.07)] and older children [4.77 (4.65, 4.89)] (p<0.0001). Mean cost of the hospitalization decreased with age: $15,387 in young, $11,404 in middle and $9,306 in older children; (p<0.0001). Risks of AP varied by age, presence of gallstone pancreatitis, alcohol, hypertriglyceridemia, which were more common among older children compared to younger children (p<0.001). Viral infections were more common in young children, compared to middle and older children (5.3% vs. 3.5% and 1.2%, respectively). There was an increasing trend in AP, and associated CP, and obesity for the two older age groups, p<0.001.
Conclusion: AP admissions in pediatrics constitute a significant healthcare burden, with a rising incidence with age. However cost and LOS per admission are highest in young children. There is a positive association between AP, CP and obesity. Future studies are needed to measure the impact of AP on inpatient pediatric hospitalizations.
Regional Differences in Treatment Strategies of Pancreatic Cancer: A Finnish Register Study Covering the Entire Nation
R. Ahola,1 H. Hölsä,2 S. Kiskola,2 P. Ojala,2 A. Pirttilä,2 J. Sand,1 J. Laukkarinen.1 1Tampere University Hospital, Tampere, Finland; 2Tampere University, Tampere, Finland.
Background: Surgical resection is the only possibility for cure in pancreatic cancer (PC). Accessibility to health care facilities may affect the diagnostics and treatment outcome.
Aims: Our aim was to analyze whether PC treatment strategies show regional variation in Finland, a country with unrestricted, public health care.
Patients and Methods: PC patients diagnosed in 2003 or 2008 were selected from the nationwide Finnish Cancer register. The data regarding tumor, treatment and demographics were recorded from the patient archives. Other malignancies than PC were excluded. Time spans to the beginning of the treatments were calculated. Patients were grouped based on the health care region at the diagnosis and hospitals based on operative volumes.
Results: 1935 potential PC patients were identified from the register. After reviewing of patient records, 1589 PC patients in 23 health care regions were included. Median age was 72 years (range 34–97), and 45% were men. Age, sex, ASA distribution or the proportion of PC diagnosis set post mortem (2.7%) were similar, but proportion of stage IV varied (57–91 %; p=0.025) in the regions. In two days from the diagnosis 29-79% had a contact with the treatment hospital (p=0.000) and in 30 days 82-100% (NS). Proportion of radical resection varied regionally (5.1-26%, p=0.001), being 18% in the regions of high volume centers and 8-11% elsewhere (p<0.01). Use of chemotherapy varied from 22-56% (p<0.05). Palliative therapies (bile drainage 18%, other 28%) did not have regional differences. Median time span from the diagnosis to radical resection or to chemotherapy did not differ.
Conclusions: The accessibility to treatment of PC was region depended. Proportion of radical resections was larger in the health care regions including a hospital with high operative volumes.
A Case Series of Secondary Pancreatic Cancers
Y. Alazzawi, M. Mahmoud, S. Han, W. Wassef. Gastroenterology, UMass Memorial Medical Center, Worcester, MA.
Background: Metastatic lesions into the pancreas are rare and reported to represent only 2-5% of pancreatic neoplasm & commonly metastasized from the lung cancer & kidney.
Aim: Our Aim is to examine the characteristic demographics/imaging features of pancreatic metastases and the outcome to help accumulating more knowledge about this rate entity and guide future management protocols.
Method: A Retrospective review through pathology department database on pancreatic tumors FNAs between 1/2011 and 4/2016. The parameters were demographics, type of primary cancer, time from diagnosing the primary cancer to the detection of pancreatic metastasis, clinical presentation, endoscopy ultrasound findings, treatment received and outcome.
Result: A total of 10 patients were detected. The mean age of the group was 66.5 years. All of the patients were white with a female predominance (80%) and 20% male. Primary malignancies were; Large B cell lymphoma (30%), RCC (20%), CRC (20%), ovarian (10%), melanoma (10%) and Merkel cell (10%). Presentations were: on surveillance imaging (40%), abdominal pain (30%), jaundice (20%) and weight loss (10%). On EUS 83% of lesions were hypoechoic solid masses, 8% cystic and 8% mixed solid/cystic.50% were located in the head,33% body,8%tail and 8% body/tail. The mean time between primary diagnosis and development of Mets was 33 months. 90% of cases had mets to other organs including LNs, liver, spleen, stomach, esophagus, Lung and muscles. 60% of patients were treated with chemotherapy, 10% radiation, 10% resection and 20% had no intervention.
Conclusion: Our study showed that the characteristic features of secondary pancreatic cancers are undistinguishable from primary pancreatic adenocarcinoma on imaging. Since most secondary tumors underwent chemotherapy regimen based on their primary cancer, such a differentiation will have a significant impact on management & prognosis and will avoid unnecessary surgical intervention. We encourage that all the pancreatic lesions should be evaluated with EUS/FNA.
Predictive Factors for Long-Term Survival Following Hepatectomy for Liver Metastases From Pancreatic Cancer
A. Andreou, F. Klein, R.B. Schmuck, A.R. Noltsch, J. Pratschke, M. Bahra. Charité Universitätsmedizin Berlin, Campus Virchow Klinikum, Berlin, Germany.
Background: The role of hepatectomy for patients with liver metastases from ductal adenocarcinoma of the pancreas (PLM) remains controversial. The present study examined the morbidity, mortality and long-term survivals after liver resection for PLM.
Methods: Clinicopathological data of patients who underwent hepatectomy for PLM between 1993 and 2015 were assessed and predictors of overall survival (OS) were identified.
Results: During the study period 76 patients underwent resection for pancreatic cancer and concomitant hepatectomy for synchronous PLM. Pancreatoduodenectomy, distal pancreatectomy, and total pancratectomy were performed in 67%, 25% and 8% of the patients respectively. The median PLM size was 1 (1–13) cm and 36% of patients had multiple PLM. The majority of patients (97%) underwent a minor liver resection. After a median follow-up time of 130 months, 1-, 3-, and 5-year OS rates were 44%, 14% and 7%, respectively. Postoperative morbidity and mortality rates were 50% and 5%, respectively. Preoperative and postoperative chemotherapy was administered to 4% and 82% of patients, respectively. In univariate analysis, resection and reconstruction of the superior mesenteric artery (P = 0.016), T4 stage (P = 0.033), lymph node metastases (P = 0.016), poorly differentiated cancer (G3) (P = 0.037), and no postoperative chemotherapy (P= 0.005) were significantly associated with worse OS. In the multivariate analysis, poorly differentiated cancer (G3) (median OS 19 vs. 6 months; hazard ratio [HR] = 1.74; 95% confidence interval [CI] = 1.02-2.96; P = 0.042), and no postoperative chemotherapy (median OS 12 vs. 4 months; HR = 1.78; 95% CI = 1.01-3.14; P = 0.045) independently predicted worse OS.
Conclusions: Liver resection for PLM is feasible and safe and may be recommended within the framework of an individualized cancer therapy. A multimodal treatment strategy including hepatectomy and systemic therapy for selected patients with less unfavorable tumor biology may provide prolonged survival in patients with metastatic pancreatic cancer.
Comparison of Remnant Invagination and Duct-To-Mucosa Pancreaticojejunostomy Following Whipples Resection: Randomized Clinical Trial in Patients at High Risk of Postoperative Pancreatic Fistula
C. Ansorge,1 S. Sanjeevi,1 M. Del Chiaro,1 B. Björnsson,2 J. Sand,3 R. Segersvärd,1 L. Lundell,1 Å. Andrén-Sandberg.1 1Div. of Surgery, Dept. of Clinical Science, Intervention and Technology (CLINTEC), Karolinska Institute, Stockholm, Sweden; 2Dept. of Surgery, Linkoping University, Linkoping, Sweden; 3Dept. of Gastroenterology and Alimentary Tract Surgery, Tampere University Hospital, Tampere, Finland.
Introduction: Pancreatic fistula (POPF) following pancreaticoduodenectomy (PD) is a potentially lethal complication that may occur after surgical manipulation of viable pancreatic parenchyma. The aim of this randomized clinical trial was to evaluate the outcomes of a non-traumatic remnant-invaginating pancreaticojejunal reconstruction technique (RI) in a patient cohort at high risk of POPF, and to compare it to the traditional duct-to-mucosa technique (DM) regarding the risk of associated morbidity.
Methods: Prospective randomized clinical conducted between 2012 and 2015 in patients undergoing PD. Eligible candidates were identified by radiological criteria at the multidisciplinary pancreatic conference. The POPF risk was estimated intraoperatively according to a standardized assessment of pancreatic texture softness and main pancreatic duct diameter. Patients who were assessed to have a high risk of POPF were randomized to either RI or DM. Associated morbidity was classified according to ISGPF and Dindo-Clavién.
Results: 120 consecutive patients were included in the analysis (60 RI, 60 DM). Pancreatic duct diameter and gland circumference were median 2 and 80 mm, respectively. RI and DM groups were comparable regarding duration of surgery (336 min) and blood loss (400 mL). The RI reconstruction was completed faster (28 min) than DM (36 min; p = 0.001), but caused more technical problems (RI 14 pts, DM 7 pts). Severe postoperative complications were observed in 48% (57 pts; RI 25 pts, DM 32 pts; p = 0.273), overall mortality was 7.5% (9 pts; RI 2 pts, DM 7 pts; p = 0.163), POPF-associated mortality was 5% (6 pts; RI 1 pt, DM 5 pts; p = 0.207). Clinically-relevant POPF (B/C) was observed in both groups (RI 30 pts, DM 31 pts); however, RI had significantly fewer cases of severe POPF C (2 pts; 3.3%) than DM (12 pts; 20%; p = 0.008).
Conclusions: Remnant invagination is a suitable technique for safe pancreaticojejunal reconstruction following PD. In patients at high risk of POPF, it was associated with a lower risk of severe POPF than the traditional duct-to-mucosa technique.
MPTP-Independent Modulation of Bioenergetics by Oxidants Determines Pancreatic Acinar Cell Death Pathway Activation
J. Armstrong,1 N. Cash,2 J. Morton,2 Y. Ouyang,1 A. Tepikin,2 R. Sutton,1 D. Criddle.2 1NIHR Pancreas Biomedical Research Unit, University of Liverpool, Liverpool, United Kingdom; 2Cellular and Molecular Physiology, University of Liverpool, Liverpool, United Kingdom.
Introduction: Oxidative stress is elevated in acute pancreatitis, although mechanisms whereby ROS influence pancreatic acinar cell (PAC) death pathways are unclear. This study has examined effects of oxidants on mitochondrial bioenergetics and PAC death, including potential involvement of the mitochondrial permeability transition pore (MPTP).
Methods: The effects of oxidants (H2O2 and menadione) on murine PAC bioenergetics, mitochondrial dysfunction, ATP levels and cell death (apoptosis and necrosis) were evaluated using confocal microscopy, plate-reader and Seahorse XF24 respiratory analysis.
Results: H2O2 (1μM-1mM) concentration-dependently modulated cell death; 1-10μM preferentially promoted apoptosis, whilst high levels (500μM-1mM) elicited rapid necrosis. H2O2 decreased mitochondrial NADH/FAD+ redox ratio and [INCREMENT]Ψm, effects maximal at 500μM. Basal O2 consumption rate (OCR) of PACs was 462.2±28.8 pmol/min (n=10); H2O2 (<50μM) inhibited OCR with no decrease of ATP turnover. Higher levels (350μM) significantly diminished spare respiratory capacity and ATP turnover and increased proton leak; bioenergetic collapse, ATP depletion and cell death ensued. Menadione (5-30μM) produced similar detrimental effects on PAC bioenergetics to H2O2, with actions inhibited by the antioxidant N-acetylcysteine. H2O2-induced bioenergetic changes and [INCREMENT]Ψm falls were not significantly different between cyclophilin D knockout (PPIF−/−) and wild type controls.
Discussion: Oxidants altered bioenergetics and determined cell death independently of MPTP formation. A shift from apoptosis to necrosis was associated with decreased respiratory capacity and ATP production, possibly via enhanced proton leak in mitochondria; this may be important for drugs, such as Valproate, known to increase ROS and cause AP.
Elevated Plasma Soluble Urokinase Plasminogen Activator Receptor (P-suPAR) on Recovery After First Acute Alcohol-Induced Pancreatitis (AAP) Predicts 10-Year Mortality
A. Aronen,1 J. Aittoniemi,2 R. Huttunen,3 A. Nikkola,1 J. Nikkola,1 O. Limnell,4 I. Nordback,1 J. Sand,1 J. Laukkarinen.1 1Department of Gastroenterology and Alimentary Tract Surgery, Tampere University Hospital, Tampere, Finland; 2Fimlab Laboratories, Tampere, Finland; 3Department of Internal Medicine, Tampere University Hospital, Tampere, Finland; 4School of Medicine, University of Tampere, Tampere, Finland.
Objectives: SuPAR (soluble urokinase plasminogen activator receptor) is a biomarker associated with inflammatory and certain malign diseases. Previously we have shown that high plasma suPAR (P-suPAR) on admission of acute alcohol-induced pancreatitis (AAP) predicts a more severe disease. Our aim was to investigate P-suPAR levels during the follow-up after first AAP.
Methods: 83 patients (median age 47.5, range 25–71 years) suffering their first AAP during 2001–2005 were prospective followed up for 9 years. Median follow-up time was 7.0 (range 0.3-9.8) years. P-suPAR was measured by enzyme-linked immunosorbent assay (ELISA) on admission, after discharge, 5, 7 and 9 years. Survival was registered in November 2014. Hazard ratio and covariate effect on prognosis were established with COX regression analysis, the cut-off value for P-suPAR indicating a higher risk for 10-year mortality using ROC curve analysis. Survival curves were established with Kaplan-Meier method.
Results: 19/83 (23%) of the patients died during the study period, and median survival time was 8 (3.2-13.3) years after the first AAP. 32 (39%) patients had at least one recurrent AAP episode during follow-up. P-suPAR level on admission or on recovery did not predict the recurrence of AAP. Higher P-suPAR measured on recovery after the first AAP (3.6 vs. 2.9 ng/mL) predicted mortality during the follow-up period (hazard ratio 1.48, p = 0.008). The cut-off value for P-suPAR indicating a higher risk for 10-year mortality resulted a value of ≥ 3.4 ng/mL. When adjusted for other covariates, P-suPAR above cut-off level retained its statistical significance as an independent factor.
Conclusions: P-suPAR level on admission or on recovery does not predict the recurrence of AAP during the long-term follow-up. However, P-suPAR ≥ 3.4 mg/mL measured on recovery from the first AAP is associated with an increased risk of 10-year mortality as an independent factor.
Beneficial Effects of Berberine on Acute Necrotizing Pancreatitis and Associated Lung Injury
G.-S. Bae, S.-J. Park, D.-G. Kim, M.J. Kim, S. Choi, J.H. Jeong. Department of Herbology, WonKwang University, WonKwang, Korea.
AIM: We set out to examine whether BBR might affect severity of pancreatitis and pancreatitis-associated lung injury in choline-deficient ethionine supplement (CDE) diet-induced severe acute pancreatitis (SAP).
Background: Acute pancreatitis (AP) is an acute inflammatory process of pancreas that affects other regional tissues or remotes organ systems. Berberine (BBR) is well-kwon to exhibit an anti-inflammatory activity in different disease models. However, the effect of BBR on AP is not well studied.
Method: SAP was induced by feeding a CDE diet for 3 days. BBR was administrated intraperitoneally during CDE diet. Mice were sacrificed on day 1, 2, and 3 after the onset of CDE diet. The severity of pancreatitis was evaluated by changes of pancreas and lung and survival rate. Blood samples were obtained to determine serum amylase and lipase levels and inflammatory cytokine production. The pancreas and lung were rapidly removed for examination of histologic changes, myeloperoxidase (MPO) activity, and real-time reverse transcription-polymerase chain reaction. Furthermore, the regulating mechanisms of BBR were evaluated.
Results: Administration of BBR significantly inhibited histological damage in pancreas and lung, serum level of amylase and lipase, MPO activity, and cytokine production and showed a reduced mortality rate. Furthermore, Administration of BBR inhibited activation of nuclear factor kappaB (NF-kB) and p38, c-Jun N-terminal kinases (JNK) in the pancreas during CDE diet.
Discussion and Conclusion: In conclusion, these findings suggest that BBR attenuates the severity of pancreatitis by inhibiting activation of NF-kB and p38, JNK and BBR could be a new candidate for AP.
Deletion of Atrx in Adult Pancreatic Acinar Cells Leads to Increased Cell Stress, DNA Damage, and Sensitivity to Pancreatitis
R. Baker,1 C. Young,2 C. Howlett,3 C. Pin.4 1Biology, Children's Health Research Institute, University of Western Ontario, London, Canada; 2Physiology & Pharmacology, University of Western Ontario, Children's Health Research Institute, London, Canada; 3Pathology and Laboratory Medicine, Schulich School of Medicine & Dentistry, University of Western Ontario, London, Canada; 4Paediatrics, University of Western Ontario, London, Canada.
Introduction: Pancreatitis involves inflammation and fibrosis of the pancreas. Chronic and hereditary forms of pancreatitis are a main susceptibility factor for pancreatic adenocarcinoma. Recent studies have suggested that chromatin remodeling proteins, including EZH2, BMI1 and BRG1, may be involved in the regenerative stage of pancreatitis. In this study, we examined the effects of loss of ATRX, a chromatin remodelling protein involved in DNA damage repair and chromosome segregation, on the response to cerulein-induced pancreatitis (CIP). We hypothesized that an absence of ATRX would exacerbate the response to pancreatic injury in experimentally induced and genetically susceptible models of pancreatitis, and would be required for proper regeneration of the exocrine pancreas.
Methods: Mice containing an inducible cre-recombinase (CreERT) in place of the Mist1 gene allowed for acinar-specific Atrx ablation in the exocrine pancreas (Mist1 +/CreERT Atrx flx). 2–4 month old Mist1 +/CreERT Atrx flx or Mist1 CreERT/CreERT Atrx flx mice were orally administered tamoxifen then sacrificed 7, 35 or 60 days following the last gavage. Additional mice were exposed to CIP 7 days after treatment, or to recurrent cerulein 60 days after treatment.
Results: Long term absence of ATRX promoted increased DNA damage and apoptosis in acinar cells and Mist1 +/CreERT Atrx flx mice showed increased pancreatic injury based on elevated serum amylase, intracellular carboxypeptidase activity and tissue necrosis. Following CIP, Mist1 +/CreERT Atrx flx mice showed increased proliferation and delayed regeneration relative to WT, as well as increased tissue damage.
Conclusions: These results suggest that absence of ATRX leads to improper pancreatic regeneration following CIP. Future studies are aimed at understanding how ATRX affects the pathways involved in regeneration.
In Chronic Pancreatitis, a Predictive Model for Small Intestinal Bacterial Overgrowth Influences Decisions to Test or Just Treat
J. Baker,1 M.J. Dimagno,2 E. Wamsteker,3 A. Lee,3 R. Saad.3 1University of Michigan, University of Michigan, Ann Arbor, MI; 2Gastroenterology, University of Michigan, Ann Arbor, MI; 3University of Michigan, University of Michigan, Ann Arbor, MI.
Background: The prevalence of small intestinal bacterial overgrowth (SIBO) using glucose hydrogen breath testing (GHBT) in patients with chronic pancreatitis (CP) varies between 15-40%. In our retrospective study of 9517 patients referred for GHBT between 1989–2016 (reported here), 43% of 83 patients with chronic pancreatitis had SIBO which is significantly greater than published data (PMC3099351) for similar older age (>61 years) healthy controls (15.8%; P<0.001). We aimed to determine if CP patients have different characteristics that predispose to SIBO.
Methods: We collected data for 83 patients with CP and examined a priori variables (gastroparesis, diabetes (DM), UGI surgery) and investigational variables (age, gender, GI symptoms, BMI, co-morbidities, PPIs, opiates, alcohol/smoking use, severe CP features, cholecystectomy and diverticulosis). We defined CP by Mayo Score (0–16) ≥ 4 & equated SIBO (positive GHBT) to a rise of breath H2 or CH4 ≥ 12 ppm over baseline.
Results: In univariate analysis, 4 of 21 variables predicted SIBO: alcohol use (p=0.02), DM (p=0.002), bloating (p=0.03), and abdominal pain (p=0.02). Multivariate analysis (of a priori variables and others with p<0.1 in the univariate analysis) identified 4 independent variables for SIBO: DM (p=0.002), opiate use (p=0.04), vomiting (p=0.02), and abdominal pain (p=0.04). The regression model for SIBO in patients with CP has n=4 variables (DM, opiate use, vomiting, and abdominal pain) with a C-statistic=0.928 (p<0.001).
Conclusions: A large retrospective study confirmed the high prevalence of SIBO in CP using GHBT. We derived a predictive model for SIBO in patients with CP that could influence decisions to pursue GHBT for lower risk patients and empiric treatment for higher risk patients.
Cannabis Induced Acute Pancreatitis: A Systematic Review
J.A. Barkin,1 Z. Nemeth,2 A.K. Saluja,3 J.S. Barkin.1 Departments of 1Medicine, Division of Gastroenterology; 2Health Informatics; and 3Surgery, University of Miami, Leonard M. Miller School of Medicine, Miami, FL.
Introduction: Cannabis is the most frequently consumed illicit drug in the world, with higher prevalence under age 35. Cannabis was first reported as a possible cause of Acute Pancreatitis (AP) in 2004. The aim of this systematic review is to examine cannabis use as an etiology of AP.
Methods: A systematic review using PubMed/Medline, Embase, Scopus, and Cochrane was performed with a reference librarian, without language or year limitations. Search terms were “Cannabis” and “Acute Pancreatitis” with all permutations. AP was defined by meeting 2 of 3 Revised Atlanta Classification criteria (Pain consistent with AP; amylase or lipase > 3x upper limit of normal; imaging consistent with AP). Cannabis induced AP was defined by preceding use of cannabis, and exclusion of common causes of AP when reported (alcohol, biliary, hypertriglyceridemia, medications, hereditary). Two authors reviewed each study for eligibility. The search yielded 239 results. 16 met inclusion criteria (1 prospective series, 1 case series, 12 case reports, 2 abstracts of case reports) dating from 2004 to 2016.
Results: There were 26 cases of cannabis induced AP, 23/26 (88.5%) men, 22/26 under age 35. AP correlated with increased cannabis use in 22 patients. Two patients had concomitant alcohol use. Recurrent AP related temporally to cannabis use was reported in 12/26. There are 9 reports of no further AP episodes after cannabis cessation. Genetic studies were available in 13, which were negative.
Conclusion: Cannabis is a probable cause of AP and recurrent AP, though its mechanism remains unclear. It occurs primarily in younger patients under age 35. This trend is likely to increase as cannabis availability increases. Cannabis should be included as a probable cause of toxin induced AP. Toxicology screens should be considered in all patients with AP.
The Micro-Forceps for Pancreatic Cysts: A Game Changer?
O. Basar,1 O. Yuksel,1 D. Yang,2 J. Samarasena,3 C.J. DiMaio,4 M.S. Wagh,5 D.G. Forcione,1 A.N. Ronald,6 M.B. Pitman,6 W.R. Brugge.1 1Department of Gastroenterology, Massachusetts General Hospital, Boston, MA; 2Gastroenterology, University of Florida Health, Gainesville, FL; 3Gastroenterology, University of California, Irvine, Orange, CA; 4Gastroenterology, Mount Sinai Hospital, New York, NY; 5Gastroenterology, University of Colorado, Denver, CO; 6Pathology, Massachusetts General Hospital, Boston, MA.
Background: An ideal modality for evaluation of pancreatic cystic neoplasms (PCN) has not been established yet. The current device, disposable MorayTM micro-forceps, passes through a 19 G needle and allows tissue sampling from PCN.
Aim: To compare the diagnostic yield of micro-biopsy forceps with cyst fluid cytology results in a retrospective analysis.
Material – Methods: Thirty subjects were enrolled into the study. The cystic fluid was aspirated on EUS-FNA followed by obtaining biopsies with the MorayTM micro-forceps, without removing the 19G needle. Hospital reports were collected and analyzed.
Results: The mean age of 11 men and 19 women was 71.4 (55–81) and 66.9 (46–83), respectively. The mean pancreatic cyst size was 28.3 mm (15–60). Cysts were located in pancreatic head in 12 patients, 12 in the body and 6 in the tail.
The tissue acquisition yield was greater with forceps (28/30 93%) compared to FNA cytology (25/30 83.3%)
The diagnostic yield of FNA cytology (53%) was not statistically significantly different when compared with forceps (73.3%) (p: 0.179) (Table1). The specific diagnosis yield of forceps (36.66%) was significantly higher than FNA cytology (3.33%) (p: 0.002).
Discussion: The micro-forceps was able to provide tissue, but was not sufficient for histological evaluation in 13% of patients. When compared with FNA, the diagnostic yield of micro-forceps was greater but it was not statistically significant. However, the micro-forceps was superior to FNA for determining the subgroups of PCNs. Furthermore, it opens new horizons in the diagnosis of pancreatic cysts via allowing molecular analyses on cystic tissue.
Loss of EZH2 Does Not Enhance Oncogenic KRAS-promoted PDAC in Adult Tissue Unless Combined With Events That Affect Acinar Cell Maturation
K. Berger,1 C. Johnson,1 G. Lomberk,2 C. Howlett,3 R. Urrutia,4 C. Pin.1,5 1Paediatrics, University of Western Ontario, London, Canada; 2Medicine, Mayo Clinic College of Medicine, Rochester, MN; 3Pathology and Laboratory Medicine, Schulich School ofMedicine & Dentistry, University of Western Ontario, London, Canada; 4Biochemistry and Molecular Biology, Mayo Clinic College of Medicine, Rochester, MN; 5Physiology and Pharmacology, and Oncology, University of Western Ontario, London, Canada.
Background: Pancreatic ductal adenocarcinoma (PDAC) is the most common form of pancreatic cancer in North America. Genetic studies have identified KRAS mutations in 97% of the patient population; however, mice harbouring this same mutation rarely develop PDAC without additional causes. Recent studies suggest Enhancer of Zeste homolog 2 (EZH2) loss in mice increases sensitivity to oncogenic KRAS, yet in human patients, elevated levels of EZH2 expression correlate to a poorer prognosis. These contradictory results lead to our hypothesis that EZH2 plays temporally distinct roles in KRAS-mediated PDAC progression.
Methods: To address this hypothesis, we developed a mouse model allowing simultaneous deletion of EZH2, and activation of oncogenic KRAS (KRASG12D) via CreERT from the Mist1 gene (K+E-). Mice combining Mist1 and Ezh2 loss with KRASG12D activation (K+E-M-) were also generated. Mice were treated with tamoxifen at 2–4 months of age, and assessed for morphological and molecular differences 60 days after treatment.
Results: Ezh2 loss in adult pancreatic tissue had minimal morphological effects on its own, or in KRASG12D–expressing tissue. As previously reported, mice expressing KRASG12D in the absence of Mist1 showed PanIN lesions and fibrosis 60 days after tamoxifen treatment. K+E-M- mice showed a dramatically worse phenotype; 1/3 of K+E-M- mice died prior to the experimental endpoint, whilst the remaining K+E-M- mice showed an almost complete loss of acinar tissue, extensive fibrosis, inflammation, acinar to duct cell metaplasia, and focal PDAC.
Conclusions: Our data suggests that the effects of EZH2 loss on KRAS-initiated PDAC are dependent on the differentiation status of the acinar cells containing oncogenic KRAS. Future experiments are aimed at defining the targets of EZH2 that may affect the potential of oncogenic KRAS.
Corticotropin-Releasing Factor Receptor 2 (CRF2R) Deficiency Alters Metabolic and Pancreatic Function in a Sex-Specific Manner in Mice
A. Bhargava,2 S. Paruthiyil,1 E. Kaushal,1 B. Hasdemir.1 1Osher Center, UCSF, San Francisco, CA; 2ObGyn & Osher Center, UCSF, San Francisco, CA.
The CRF family mediates a myriad of physiological functions from stress responses to food intake and metabolic function via activation of two GPCRs CRF1R and CRF2R. The role of CRF2R in mediating pancreatic function or in metabolic syndrome remains unknown. Wild type (Wt) mice of both sexes showed ~34% increase in their body weight (BW) after 8 weeks on high fat diet (HFD, 60kCal%). CRF2R−/− (KO) male mice gained 41%, whereas female KO mice gained 28% BW despite similar HFD consumption. Mesenteric white adipose tissue (mWAT) weight per BW was significantly higher in WT mice on HFD in both sexes compared with KO mice on HFD or chow. Surprisingly, gonadal WAT weight in male mice was not different between HFD and chow groups. WT, but not KO female mice on HFD had significantly increased gonadal WAT compared with mice on chow. Glucose tolerance test showed that male WT and KO mice on HFD had significantly elevated blood glucose levels at 30 min compared to male mice on chow. In contrast, only Wt female mice on HFD showed elevated 30 min glucose levels; KO mice on HFD did not differ in glucose levels than females on chow diet. Glucose release from the islets and amylase secretion from the acinar cells after food intake are Ca2+-dependent processes. Redistribution of F-actin is key for Ca2+-mediated secretion from acinar cells. CRF resulted in F-actin redistribution in acinar cells from mice fed HFD, but not chow. Molecular mechanisms that prevent mWAT weight increases in KO mice of both sexes on HFD and signaling pathways that might contribute to sex-specific difference in pancreatic function will be discussed. To conclude, our study aims to identify novel mechanisms by which CRF2R mediates effect of diet on pancreatic function in a sex-specific manner.
Functional Role of 4F2hc in Pancreatic Ductal Adenocarcinoma
D. Bianconi,1 M. Herac,2 A. Gleiss,3 M. Unseld,1 R. Weigl,1 M. Schindl,4 W. Scheithauer,1 C. Zielinski,1 G. Prager.1 1Internal Medicine I, Oncology, Medical University of Vienna, Vienna, Austria; 2Clinical Institute of Pathology, Medical University of Vienna, Vienna, Austria; 3Section for Clinical Biometrics, Center for Medical Statistics, Informatics, and Intelligent Systems, Medical University of Vienna, Vienna, Austria; 4Department of Surgery, Medical University of Vienna, Vienna, Austria.
Introduction: PDAC represents one of the most aggressive cancers with limited prognosis. With less than 5 % five-year-overall survival, the characterization of new therapeutic targets is urgently needed. 4F2hc is a cell surface-antigen overexpressed in some tumor cells and therefore, it represents a promising novel therapeutic target. In the current study, we investigated the functional role of 4F2hc in PDAC.
Methods: 4F2hc protein expression level was analyzed in human pancreatic cancer tissue (222 cases) and matched adjacent tissue (141 cases) via immunohistochemistry (IHC). Additionally, pancreatic cancer cells were isolated from fresh human tumor tissue and 4F2hc+ cells were phenotypically analyzed via flow cytometry. To characterize the functional role of 4F2hc in PDAC, in vitro experiments were performed.
Results: Using IHC, we demonstrated that 4F2hc is aberrantly expressed in PDAC and matched adjacent tissue. Although Kaplan-Meier survival curves and Cox analyses did not revealed a significant association between 4F2hc expression and OS, we observed a trend that suggests an association between absence of 4F2hc expression and prolonged survival. Additionally, our results showed that 4F2hc+ cells isolated from fresh tumor tissue co-expressed other known markers of PDAC such as MUC4 and MUC1.To determine the role of 4F2hc in PDAC, cell behavior between 4F2hc+ cells and 4F2hc low expressing cells was compared. We found that 4F2hc downregulation significantly inhibited tumorsphere formation and cell proliferation by arresting cell cycle.
Conclusion: Herein, we demonstrated that 4F2hc is overexpressed in resected tumor tissue as well as in matched adjacent tissue of patients with pancreatic cancer. Moreover, our data suggests that 4F2hc expression increases tumorigenesis by enhancing cell proliferation and promoting anchorage-independent growth in vitro. Although further studies are needed, our results suggest that 4F2hc might be a novel therapeutic target of PDAC.
The Relationship of Nutritional Status With Pain Medication Use in Chronic Pancreatitis Patients
L. Bocelli,1 M. Min,2 B. Patel,3 S. Han,4 J. Kheder,1 W. Wassef.1 1Gastroenterology; 2Internal Medicine, University of Massachusetts Medical Center, Boston, MA; 3Internal Medicine Residency, UMass Medical School, Worcester, MA; 4Gastroenterology, University of Colorado School of Medicine, Denver, CO.
Background: Chronic pancreatitis is well-known to cause pancreatic insufficiency, resulting in fat malabsorption. While this malabsorption will result in steatorrhea and lead to malnutrition, it is unknown whether malnutrition is associated with pain in this population. The primary endpoint of this study was to examine pain medication use based on nutritional status.
Methods: 45 patients with chronic pancreatitis were administered the Malnutrition Universal Screening Tool (MUST) and had albumin and pre-albumin levels measured. Their daily pain medication regimen was then compared to their nutritional status.
Results: There was a negative correlation between albumin levels and pain medication (r=−0.29, p<0.02), but there was no significant correlation between MUST scores (r=−0.18, p<0.23) and pre-albumin levels (r=−0.32, p<0.09) and pain medication use. Comparing pain medication consumption between patients considered malnourished (MUST score > 2) and patients not considered to be malnourished (MUST score ≤ 2), there was also no difference (88.3 vs. 151.1 mg of oral morphine/day, p<0.12).
Discussion: As expected, there tended to be a negative correlation between nutritional status and pain medication use, suggesting that patients with a worse nutritional status require more pain medications, although only albumin levels were found to have a significant correlation. Albumin and pre-albumin appear to be more accurate in predicting opiate use, while the MUST score appears to have less utility in forecasting analgesic use. While emphasizing the importance of nutrition in this patient group, this study recognizes the need for further studies to assess whether nutritional improvement can improve pain levels, thereby providing another avenue to manage this disease.
Functional Studies Implicate an Imbalanced Activation of Dendritic Cells in the Pathogenesis of Murine Autoimmune Pancreatitis
L. Borufka,1 E. Volmer,1 S. Müller,1 R. Engelmann,2 H. Nizze,3 S. Ibrahim,4 R. Jaster.1 1Department of Medicine, Division of Gastroenterology; 2Institute of Immunology and Core Facility for Cell Sorting & Cell Analysis; and 3Institute of Pathology, Rostock University Medical Center, Rostock, Germany; 4Institute of Experimental Dermatology, University of Luebeck, Luebeck, Germany.
Aim: Aged MRL/MpJ mice spontaneously develop an autoimmune pancreatitis (AIP) and are frequently used as a model to study the immunological and molecular basis of the disease. Based on the results of previous genetic studies, we raised the hypothesis that distinctive features of their dendritic cells (DCs) may predispose MRL/MpJ mice to the chronic inflammation. Here, we aimed at an experimental validation of this concept.
Methods: Conventional DCs (cDCs) were derived from bone marrow (BM) cells of young (healthy) MRL/MpJ mice, adult (sick) individuals and AIP-resistant CAST/EiJ mice. The cells were subsequently characterized employing flow cytometry, cell proliferation assays, ELISA tests and real-time PCR measurements. Cocultures with syngeneic T-cells were performed to monitor DC-mediated T-cell activation.
Results: MRL/MpJ mice at an age of approximately 6 months developed pancreatic lesions that histologically resemble human AIP type 1. Within these inflammatory foci, cells expressing the murine DC marker CD11c and CD3+ cells were found in close spatial proximity to each other. In vitro, many cDCs from diseased MRL/MpJ mice displayed a mature phenotype (CD11c+/CD83+/MHCIIhigh/CD40high/CD80high/CD86high) already prior to incubation with lipopolysaccharide (LPS). After LPS-treatment, cDC cultures of both MRL/MpJ mouse cohorts contained more mature cells, proliferated at a higher rate and secreted less interleukin-10 than cultures of CAST/EiJ mice. Furthermore, LPS-free cultured cDCs from MRL/MpJ mice expressed less mRNA of the inhibitory receptor triggering receptor expressed on myeloid cells 2 (trem2) than cells of the control strain. Under co-culture conditions, DCs displayed the strongest pro-mitogenic effect on T-cells from mice with an advanced AIP.
Conclusions: The results of this study implicate an imbalanced DC activation into the pathogenesis of murine AIP in the MRL/MpJ model. As a possible mechanism, we suggest an enhanced DC maturation that triggers a dysregulated immune response.
New Potential Role for Transcription Factor EB in DNA Repair
M.-J. Boucher, M. Groleau, B. Marchand. Department of Medicine, University of Sherbrooke, Quebec, Canada.
We previously demonstrated that prolonged GSK3 inhibition triggers an apoptotic response specifically in pancreatic cancer cells leaving intact pancreatic normal cells. However, we recently observed that the apoptotic response is counterbalanced by pro-survival autophagic signals dependent on the transcription factor EB (TFEB). In order to identify potential mechanisms by which TFEB limits cell death, mass spectrometry analysis was performed to identify new TFEB interacting partners. Many proteins involved in DNA repair were found associated with TFEB upon GSK3 inhibition including PARP1. The aim of this study was to assess whether TFEB participates in DNA damage detection/repair in human pancreatic cancer cells. The experiments were performed using stable population of pancreatic cancer cells (PDAC-shCTL) with reduced expression levels of TFEB (PDAC-shTFEB).
Results: GSK3 inhibition and DNA damaging agents (doxorubicin, etoposide) induced DNA damages that were amplified in PDAC-shTFEB populations as compared to control PDAC-shCTL populations. TFEB depletion impaired DNA repair upon treatment with DNA damaging agents. PDAC-shTFEB cells were more sensitive to cell death upon GSK3 inhibition or treatment with DNA damaging agents.
Conclusion: Our results provide evidence that TFEB-depleted PDAC cells are less efficient at repairing DNA damage and are more prone to apoptosis upon treatment with DNA damaging agents. These results suggest that interfering with TFEB function may synergize with GSK3 inhibition and/or DNA damaging agents to promote death of PDAC cells.
Does Family History Predict Genetic Test Results for Chronic Pancreatitis?
R.E. Brand,1 N. Shah,2 D. Yadav,1 A. Slivka,1 J. Larusch,1 D.C. Whitcomb.1 1Department of Medicine, University of Pittsburgh Medical Center, Pittsburgh, PA; 2Dental Public Health, University of Pittsburgh Medical Center, Pittsburgh, PA.
Introduction: Genetic testing for known pathogenic variants (PVs) in PRSS1, CFTR, SPINK1 and CTRC, which contribute to chronic pancreatitis (CP) susceptibility, is evolving with more research required to better define appropriate candidates for whom testing is indicated. Presently a CP-associated genotype would include a single pathologic variant (PV) in PRSS1 or a combination of two PVs in CFTR, SPINK1 and CTRC. Our aim was to determine whether family history (FH) predicts the presence of PVs for CP.
Methods: Reported family histories for CP and genetic testing results for PRSS1, CFTR, SPINK1 and CTRC from 886 CP patients enrolled in NAPS2 Studies were analyzed. FH of CP was defined as a CP proband having at least1 first-degree relative (FDR) with CP.
Results: Seventy-seven patients with a CP-associated genotype were identified in the 886 CP cases (9%). Of the 83 patients with a FH of CP, 20 (24%) had a PRSS1 PV and 9 (11%) had two or more non-PRSS1 PVs. Forty-three PVs were found in the 803 CP patients with no FH, 14(2%) PRSS1 PVs and 34(4%) two or more non-PRSS1 PVs. Using FH to predict whether a proband has PRSS1 or two other mutations showed a sensitivity of 37.7%, specificity of 96.9%, PPV of 34.9% and NPV of 94.0%.
Conclusion: The findings that a FH of CP in at least one FDR has a yield of 35% in identifying CP-associated genotypes supports genetic testing based on FH. Since the majority of CP patients with a CP-associated genotype (62%) were not identified by FH, genetic testing should not be excluded based on FH alone. Further research is needed to identify additional genetic susceptibility factors responsible for the CP clustering in a subset of CP families and identifying additional populations with a high prevalence of CP-associated genotypes.
Association of High HLA Class I Antigen Expression With Poor Prognosis in Intraductal Papillary Mucinous Neoplasms (IPMN)
L. Cai,1 T. Michelakos,1 C. Fernández-Del Castillo,1 M. Mino-Kenudson,2 A.L. Warshaw,1 K.D. Lillemoe,1 S. Ferrone,1 C.R. Ferrone.1 1Department of Surgery, Massachusetts General Hospital, Boston, MA; 2Department of Pathology, Massachusetts General Hospital, Boston, MA.
Background: The potential role of immune surveillance in the control of tumor growth has prompted us to investigate the role of immunological events in IPMN. To this end, we have correlated the expression of HLA Class I Antigen Processing Machinery (APM) components with prognosis in IPMN since these molecules play a crucial role in the interactions of malignant cells with T cells and NK cells.
Methods: Clinicopathologic data was collected for 59 IPMN patients from a single academic center between 2006–2013. Formalin fixed, paraffin-embedded TMAs were stained with HLA class I APM component, CD4- and CD8-specific mAbs. Results were correlated with clinicopathologic parameters.
Results: This study included 59 patients whose tumors were available; 56% were male, 90% Caucasian. The median age was 67y. HLA class I antigen expression was within normal ranges in 4 of the 34 gastric type, 5 of the 18 intestinal type, 4 of the pancreatobiliary type and the 2 oncocytic type tested. Within each subgroup, HLA class I expression was correlated with tumor grade, being lower in low grade than in high grade tumors. But it was not correlated with intratumoral CD8+ T cell infiltration. Infiltrating CD4+ T cells were scarce. A univariate analysis showed an association of high HLA-A (p=0.037) and high Tapasin (P=0.047) expression with a worse OS. Lastly, a multivariate analysis showed that high Tapasin expression (HR=1.02; P=0.037) and lymphovascular invasion (HR=8.9; P=0.002) are independent predictors of OS.
Conclusion: The association of high HLA class I antigen expression with poor prognosis suggests a role of NK cells in the immune surveillance of IPMN. Consistent with this possibility is the lack of evidence of a role of T cells in the immune surveillance of this malignancy.
Genetic Deletion of the Adaptor Protein, AP3, Results in Secretory and Processing Defects in Acinar Cells
A.J. Ceplenski,1 C.A. Shugrue,1 T. Kolodecik,1 G. Groblewski,2 S. Messenger,2 D.D. Thomas,2 F. Gorelick.1,3 1Internal Medicine, Digestive Diseases, Yale University, New Haven, CT; 2University of Wisconsin, Madison, WI; 3Veterans Administration CT Healthcare, West Haven, CT.
AP3 is a clathrin adaptor protein complex involved in trans-Golgi and endocytic protein and vesicular trafficking. We find that an acinar cell minor secretory compartment (MSC) mediates zymogen granule maturation, secretion and lysosomal biogenesis. We hypothesize that MSC function is important in acute pancreatitis and may be AP3-dependent. We have compared pancreatic acini and lobules from a AP3 knockout (KO) mice to wildtype (WT) to examine AP3’s acinar cell role. KO acini had increased (~100%) basal amylase release compared to WT, but relatively (to basal) reduced cerulein (CER) or carbachol (CARB induced secretion. AP3 showed considerable overlap using immunolocalization with another regulator of the MSC, D52, but D52 overexpression by viral vector did not enhance basal or stimulated secretion in KO acinar cells as it does in WT. Basal and stimulated (CER hyperstimulation) protease (trypsin, chymotrypsin) levels were significantly lower in KO acini. However, basal levels of TAP were dramatically increased in KO acini, indicating increasing trypsinogen processing. Evidence for enhanced processing of carboxypeptidase A1 was also observed. LDH release, a marker of injury, was significantly increased under basal or hyperstimulation (CER, CARB) conditions. In KO acini, lysosomal hydrolase activity (Cathepsin B & L) was 2–3 times lower than in WT. Electron microscopy of KO mice appeared to show enlarged less dense zymogen granules compared to WT, consistent with a maturation defect. In summary, genetic AP3 deletion results in a complex acinar cell phenotype including increased basal secretion and baseline injury, which may be due to defects in the minor secretory pathway and lysosomal function.
Genome-Wide RNAi Screening Identified Metastasis Suppressor Genes in an Orthotopic Pancreatic Cancer Mouse Model
Y. Chen, T. Xia. School of Biomedical Sciences, Faculty of Medicine, The Chinese University of Hong Kong, Hong Kong, China.
Objectives: Pancreatic cancer is an aggressive malignancy with extremely poor prognosis. It is usually diagnosed when metastases are already present. To identify genes that play critical roles in the processes of pancreatic cancer metastasis, a whole genome RNAi screening was performed.
Methods: An shRNA library targeting all human genes was introduced into a human pancreatic cancer cell line capan-2. The infected cells were then transplanted into the pancreas of nude mice. Because capan-2 is of low metastatic potential, we hypothesized that knocking down of metastasis suppressor genes would facilitate capan-2 cells to spread to the liver. By retrieving shRNA templates from the liver metastatic nodules, several candidate genes were found. One of them, SOX9, has been validated as metastasis suppressor gene in vivo, implying that loss of expression of SOX9 promotes pancreatic cancer metastasis.
Results: Our genome wide RNAi screening identified SOX9 as a metastasis suppressor gene in pancreatic cancer. In the validation experiments, 10 and 11 nude mice were used for control shCtrl group and shSOX9 group respectively. All of them had primary tumors established. 7 out of 11 mice in shSOX9 group developed liver metastasis, whereas only 2 out of 10 metastasized in control group. Knocking-down of SOX9 promoted the orthotopically transplanted capan-2 cells to metastasize to the liver.
Laparoscopic-Assisted Versus Open Total Pancreatectomy and Islet Autotransplantation: A Case-Matched Study of Pediatric Patients
S. Chinnakotla,1 M. Berger,1 T.B. Dunn,1 G.J. Beilman,1 M. Freeman,2 M. Bellin,3 S.J. Schwarzenberg.3 Departments of 1Surgery; 2Medicine; and 3Pediatrics, University of Minnesota, Minneapolis, MN.
Total Pancreatectomy and Islet Autotransplantation (TPIAT) is a potential treatment for patients who are refractory to medical endoscopic or surgical drainage procedures. A minimally-invasive approach has proven advantageous for other pediatric procedures, but its value is not clear for this rare operation.
Children (n=21) who underwent hand-assisted laparoscopic TPIAT were compared with case-matched open TP-IAT procedures (n=21) Data reviewed included post operative complications, operative time, estimated blood loss, intraoperative blood transfusions, number of islet equivalents (IEQ)/kg body weight transfused, hospital length-of-stay, graft function, narcotic use, and PSAQ scores.
There was no perioperative mortality. Surgical complications were similar between the two groups (p=0.51) and included wound complications (n=11), chyle leak (n=7), bowel obstruction (n=5), bile leak (n=3), GI bleed (n=2), and pneumonia (n=1). Operative times were not significantly different (p=0.18). EBL was nearly identical between groups (p=0.96). The mean number of islet equivalents (IEQ)/kg body weight did not differ significantly between open (4352±651 IEQ/kg) and laparoscopic cases (5751±679 IEQ/kg), p=0.15. LOS did not differ between groups 20.67±2.40 days for open vs. 22.10±2.22 days for laparoscopic-assisted, p=0.66). The overall rate of insulin independence at 1 year was 28.6% with an additional 37.1% of patients receiving only once-daily long-acting insulin. There was no difference in rates of insulin independence between the two surgical groups (p=1.0). There was no difference in prevalence of narcotic use at 3 months (p=1.0), 6 months (p=0.06), and 1 year (p=0.42) post-operatively. Patient satisfaction with the surgical scar was similar between the groups (p=0.26).
Preliminary data indicates that outcomes for hand-assisted laparoscopic TPIAT are comparable to open TPIAT in all measures. In young patients, a minimally-invasive approach does not compromise safety, effectiveness, or operative efficiency and may be used based on surgeon and patient preference.
Two Gaseous Transmitters in L-ornithine-induced Acute Pancreatitis in Rats
S. Chooklin, S. Chuklin, B. Pidhirnyy. Regional Clinical Hospital, Lviv, Ukraine.
Introduction: In recent years, interest has been directed towards other naturally occurring gases such as hydrogen sulfide (H2S) and nitric oxide (NO). It is now becoming increasingly apparent that H2S and NO are exert complex effects on inflammation. Recent research has highlighted the mechanism by which H2S and NO may contribute to inflammation in acute pancreatitis. We investigated the roles and interrelations of NO and H2S in the development of experimental acute necrotizing pancreatitis.
Materials and Methods: For the experiment were used twelve Wistar albino rats. Acute necrotizing pancreatitis was induced by intraperitoneal injection with 3 g/kg L-ornithine-HCl in 6 rats. 6 rats served as control. To determine the activity of NO-synthase in pancreas (Ca2+-dependent and Ca2+-independent) used a combination of classic method and present its modification, adapted to the measurement of one of the reaction products – L-citrulline. The concentration of H2S in serum was determined in the blood serum using N,N-dimethyl-p-phenylenediamine. The level of NO was expressed as NO2−+NO3− and it was measured in reaction with Griess reagent.
Results: A significant decrease in the H2S level was observed in rats with acute pancreatitis (the level was 13.92% less than in control group) (p<0.01). In this time, level of NO was 12.15% higher than in the control group (p<0.05). Furthermore, the activity of iNOS and cNOS was 122.80% (p<0.05) and 63.48% (p<0.01) higher than that of the control group, respectively. However, the activity of physiologic NOS was lower by 22.63% in acute pancreatitis. Levels of H2S negative correlate with NO levels (R=−0.823964, p<0.001) Changes of H2S levels negative correlate with changes of iNOS (R=−0.748252, p<0.01) and cNOS (R=−0.762238, p<0.01) activity. Concentrations of H2S negative correlated with levels of P-amylase (R=−0.734266, p<0.01), in this time, P-amylase levels positive correlate with NO levels (R=0.612691, p<0.05).
Conclusion: These data can be an evidence of different roles and interaction of H2S and NO in acute necrotizing pancreatitis.
Endosomal Regulatory Protein D52 Interacts With ATG16L1 to Coordinate Secretion and Autophagy in Acinar Cells
M. Cooley, D.D. Thomas, S. Messenger, G. Groblewski. University of Wisconsin, Madison, WI.
Tumor protein D52 is an endosomal regulatory protein known to interact with Rab5 and acutely control acinar cell secretion in a phosphorylation-dependent manner. D52 is highly expressed in secretory cells containing large granules including acinar, Paneth, goblet, and chief cells. Computer-based analysis of potential D52 binding proteins using Phyre2 software indicated a high probability of interaction with ATG16L1 based on periodicity of hydrophobic and charged amino acids in their coiled-coil domains. ATG16L1 is an autophagy regulatory protein that is required for LC3 lipidation and expansion of the phagophore. Endogenous D52 and ATG16L1 β isoform strongly co-immunoprecipitated from acinar lysates. Colocalization of D52 and ATG16L1 in acinar cells is present in both basal and CCK-8 stimulated states, where they accumulate in a subapical compartment. Ectopic expression of WT D52 or phospho-null S136A mutant in CHO-CAR cells showed a marked colocalization with ATG16L1 in a perinuclear compartment, while expression of D52 phosphomimetic S136E mutant revealed an expanded colocalization of both proteins in punctate structures throughout the cytoplasm. Secretory and supraphysiological stimulation of acinar cells with CCK-8 revealed enhanced autophagic flux with accumulation of LC3 puncta that colocalize with ATG16L1 and Rab11a in a subapical compartment. Moreover, WT D52 expression in HeLa cells significantly increased autophagic flux as measured by LC3-II accumulation; conversely, knockdown of D52 using shRNA-expressing lentivirus in A549 cells strongly reduced autophagic flux. Finally, HCT 116 cultured cells that do not express ATG16L1 have increased D52 expression and do not accumulate LC3-II. These data suggest a pivotal role for D52 in acinar secretion and autophagy.
Endoscopic Minor Papilla Sphincterotomy is Effective for the Treatment of Symptomatic Santorinicele: Long-Term Results in a Large Series
S.F. Crinò,1 L. Bernardoni,1 M.C. Conti Bellocchi,1 G. Malleo,2 R. Manfredi,3 I. Breoni,1 A. Amodio,1 L. Frulloni,1 A. Gabbrielli.1 1Gastroenterology and Digestive Endoscopy Unit; 2Unit of General and Pancreatic Surgery; and 3Department of Radiology, Pancreas Institute, University of Verona, G. B. Rossi University Hospital, Verona, Italy.
Background & Aims: Santorinicele (SC) is a cystic dilatation of the intramural portion of the dorsal pancreatic duct associated with pancreas divisum and recurrent pancreatitis (RP). Endoscopic minor papilla sphincterotomy (EPSm) has been proposed as treatment in case reports or small series with encouraging results, but EPSm treatment was never systematically investigated. The aim of this study is to assess the outcome of endoscopic EPSm treatment in a large series with long-term follow-up.
Methods: From 2009 to 2015, 33 patients with SC and RP underwent EPSm at the pancreas institute of Verona. The average numbers of pancreatitis per year and the number of pancreatitis during a comparable time period before and after treatment were evaluated. Outcomes were related to pre-treatment secretin-enhanced magnetic resonance cholangiopancreatography (s-MRCP) findings.
Results: The average number of pancreatitis/year and during a comparable time period before and after EPSm was respectively 1.586 vs 0.18 and 2.63 vs 0.67 (p<0.0001). The mean follow-up was 43.8 months (range 12–82). Only 1 severe EPSm-related adverse event was observed (retroperitoneal perforation). Complete response was obtained in 80% of patients. Six patients (20%) relapsed after a mean time of 16 months after EPSm. In 5 cases, we found another potential cause of RP (2 post-sphincterotomy stenosis, 1 alcohol, 1 CFTR gene mutation, or 1 side-branch IPMN). No relation between pre-treatment s-MRCP findings and clinical outcomes was observed.
Conclusions: EPSm is highy effective in reducing the number of RP in patients with SC. Therefore, SC in RP patients may be an indication for EPSm.
Lipocalin-2 Promotes Obesity-Induced Pancreatic Ductal Adenocarcinoma by Regulating Inflammation in the Tumor Microenvironment
Z. Cruz-Monserrate,2 S.B. Gomez,1 A.K. Swidnicka-Siergiejko,1 N. Badi,2 M. Chavez-Tomar,2 G. Lesinski,2 T. Bekaii-Saab,3 M.R. Farren,2 T. Mace,2 C. Schmidt,4 Y. Liu,1 D. Deng,1 R. Hwang,5 L. Zhou,5 T. Moore,5 D. Chatterjee,6 H. Wang,6 X. Leng,7 R. Arlinghaus,7 C.D. Logsdon.1 1Cancer Biology, University of Texas, M. D. Anderson Cancer Center, Houston, TX; 2Department of Internal Medicine, The Ohio State University Wexner Medical Center, Columbus, OH; 3Hematology and Medical Oncology, Mayo Clinic, Scottsdale, AZ; 4Surgery, The Ohio State University Wexner Medical Center, Columbus, OH; Departments of 5Surgery; 6Pathology; and 7Translational Molecular Pathology, University of Texas, M. D. Anderson Cancer Center, Houston, TX.
Background: Lipocalin-2 (LCN2) is a molecule known to have a pro-tumorigenic role in many cancer types and is significantly upregulated in patients with pancreatic ductal adenocarcinoma (PDAC) and in obese individuals. Obesity is a risk factor for PDAC and is associated with poor survival. We investigated whether Lcn2 depletion affected obesity-associated PDAC development and mouse survival as well as its role in the tumor microenvironment.
Methods: We studied mice with acinar cell-specific expression of KRasG12D (KRasG12D/CRE) or crossed with Lcn2 knockout (Lcn2−/−) animals (Lcn2−/−/KRasG12D/CRE). Animals were fed isocaloric diets with varying amounts of saturated fat content. Moreover, the role of Lcn2 in tumor growth was tested in a syngeneic orthotopic PDAC mouse model. The pancreas of these animals were collected and analyzed for inflammation, pancreatic intraepithelial neoplasia (PanIN) and PDAC. Tumor growth in syngeneic model was measure via IVIS imaging. In addition, we studied the role of LCN2 and its specific receptor solute carrier family 22, member 17 (SLC22A17) in pancreatic stellate cells.
Results: In this study, depletion of Lcn2 diminished extracellular matrix deposition, immune cell infiltration, PanIN formation, tumor growth and significantly increased survival in both an obesity-driven PDAC genetic mouse model and in a syngeneic orthotopic PDAC mouse model. In addition, we found that LCN2 modulated the secretion of pro-inflammatory cytokines in human pancreatic stellate cells, key regulators of the PDAC tumor microenvironment, via the LCN2-specific receptor, SLC22A17.
Conclusions: Our results reveal a previously unknown role of LCN2 in the establishment and regulation of the PDAC tumor microenvironment, suggesting a link between LCN2, obesity, inflammation and PDAC.
Renalase Forms High Molecular Weight Complex in Plasma for Tissue Translocation in Acute Pancreatitis
K. Date,1,2 T. Kolodecik,1 F. Gorelick.1,3 1Internal Medicine, Digestive Diseases, Yale University, New Haven, CT; 2Graduate School of Humanities and Science, Ochanomizu University, Tokyo, Japan; 3Veterans Administration CT Healthcare, West Haven, CT.
Renalase (RNLS) is a protein secreted by the kidney and circulates in the blood. Levels of circulating RNLS drop dramatically in patients with renal disease. Our lab has previously presented data showing that RNLS can protect pancreatic acinar cells from secretagogue induced pancreatitis. In our current study, we examined plasma and tissue levels of RNLS in cerulein (CER) induced pancreatitis. We also explored whether RNLS binds to other serum proteins in circulation. We found that like in kidney disease, plasma RNLS levels were significantly decreased in a time-dependent manner in CER treated animals. In contrast, RNLS levels in both kidney and pancreas were increased at 2 hours after CER treatment, a time when serum RNLS levels are reduced. This increase in tissue RNLS levels may be due to its sequestration from the blood and could be mediated by RNLS binding to other circulating proteins. To detect RNLS binding proteins, plasma from C57/BL6 mice was separated by gel-filtration chromatography and RNLS was detected by immunoblot in high molecular weight fractions (>200kDa) but not the 37kDa fraction, the predicted size of the RNLS monomer. Neither NaCl (0–300 mM) nor calcium affected which fraction contained RNLS. Plasma was also subjected to anion-exchange chromatography with RNLS eluting at 200 mM NaCl a concentration which would keep RNLS complexes intact. RNLS complexes were separated by anion-exchange chromatography followed by gel-filtration. The RNLS-containing fraction was separated by SDS-PAGE and demonstrated a discrete set of plasma proteins that co-eluted with RNLS. These results suggest that RNLS forms high molecular weight complexes in plasma that may be sequestered early.
Incidence and Timing of the Development of Concomitant Pancreatic Ductal Adenocarcinoma During Surveillance for Resected and Unresected Intraductal Papillary Mucinous Neoplasms
K. Date,1 T. Ohtsuka,1 S. Nakamura,1 Y. Gotoh,1 Y. Nakashima,1 T. Fujimoto,1 K. Saeki,2 N. Mochidome,2 Y. Mori,1 Y. Sadakari,1 K. Nakata,1 Y. Miyasaka,1 K. Ohuchida,1 T. Manabe,1 E. Nagai,1 Y. Oda,2 M. Nakamura.1 Departments of 1Surgery and Oncology; and 2Anatomic Pathology Kyushu University, Fukuoka, Japan.
Background: Intraductal papillary mucinous neoplasm (IPMN) of the pancreas has been believed to be an important clue to detect pancreatic ductal adenocarcinoma (PDAC), and the aim of this study is to elucidate the incidence and timing of development of concomitant PDAC.
Methods: Medical records of 481 patients having IPMN were retrospectively reviewed. There were 26 patients who had undergone pancreatectomy for concomitant PDAC with and without combined resection of IPMN (PDAC-resection group), 198 with pancreatectomy for IPMN (IPMN-resection group), and 257 who were conservatively surveyed without pancreatectomy for IPMNs (CS group). Patients with surveillance period of less than 6 months and those who had undergone total pancreatectomy were excluded.
Results: During the median surveillance period of 39 months (range 6 to 262), a total of 20 patients developed concomitant PDAC. Among them, 5 (19%) of PDAC-resection patients developed secondary PDAC during the median surveillance period of 65 months (range 33 to 160). The estimated 5- (17%) and 10-year (48%) cumulative incidence of secondary PDAC in PDAC-resection patients was significantly higher than those of the other 2 groups (all, p<0.01). On the other hand, 6 (3.0%) of IPMN-resection patients and 9 (3.5%) of CS patients developed concomitant PDAC. There was no significant difference between IPMN-resection (2.7% and 9.5%) and CS patients (4.7% and 4.7%) in the estimated 5- and 10-year cumulative incidence (p=0.55), and the timing of the development of concomitant PDAC (median 34 [range 18 to 40] vs. 19 months [range 7 to 139], p=0.22).
Conclusions: Patients with a history of pancreatectomy for concomitant PDAC are at high risk of developing secondary PDAC in the remnant pancreas. In addition, the incidence and the timing of the development of concomitant PDAC are equal between the patients with and without pancreatectomy for IPMN.
Sp1 Downregulation Leads to Disruption of Endoplasmic Reticulum Homeostasis and Cell Death
P. Dauer,1 A. Nomura,1 V.K. Gupta,1 V. Dudeja,1 S. Banerjee,1 A.K. Saluja.2 1University of Miami, Miami, FL; 2Dept of Surgery, University of Miami, Leonard M. Miller School of Medicine, Miami, FL.
Background: Transcription factor, Sp1, is overexpressed in pancreatic cancer. Sp1 is essential to tumor cell survival, and downregulation results in cell death. Preliminary data suggests that downregulation of Sp1 results initiates endoplasmic reticulum (ER) stress. We have previously published that triptolide, a novel compound in preclinical studies for pancreatic cancer, causes Sp1 downregulation and cell death, as well as chronic ER stress and cell death.
Aim: To study how Sp1 downregulation prevents ER homeostasis and causes pancreatic cancer cell death.
Methods: MIA PaCa-2 cells were treated with mithramycin and transfected with siSp1. Protein and RNA were collected over 48 hours for gene expression studies. ChIP was performed for Grp78, using Sp1 for immunoprecipiation. Lysosome membrane permeabilization (LMP) was detected by confocal microscopy. Cytosolic calcium was measured using Calcium Orange with mithramycin treated and siSp1 transfected cells.
Results: Sp1 downregulation results in increased gene expression of downstream ER stress markers. However, since Sp1 has bindings sites on the Grp78 promoter region, Sp1 downregulation does not result in a robust increase in Grp78. ER stress induced by mithramycin results in LMP, a sustained release of calcium at 24 hours, and eventually cell death in MIA PaCa-2 cells.
Conclusions: Our data suggests that Sp1 downregulation leads to ER stress, and the activation of the unfolded protein response (UPR). Grp78 is the master regulator of UPR, and is an essential component to regulating cell homeostasis. Importantly, Sp1 has binding sites on the Grp78 promoter region; without proper regulation of Grp78, the cell experiences an overwhelming amount of stress, which leads to cell death. Further, as a result of Sp1 downregulation, a sustained release of cytosolic calcium and LMP are observed, which lead to cell death.
Predictive Biomarkers for Short-Term Survival After Curative Intent Pancreatic Cancer Surgery: Identifying the Biologically Ill-Fated
S.W.L. De Geus, J.S.D. Mieog, R.-J. Swijnenburg, H.A.J.M. Prevoo, C.F.M. Sier, H. Morreau, C.J.H. Van De Velde, B. Bonsing, A.L. Vahrmeijer, P.J.K. Kuppen. Leiden University Medical Center, Leiden, Netherlands.
Background: Sedulous patient selection of upfront surgical candidates is pivotal to pancreatic cancer care. Accurate selection protects patients with rapidly progressive disease from the unnecessary harm of surgery and facilitates early systemic treatment. The purpose of this study was to identify predictive biomarkers for short-term, overall (OS) and disease-free (DFS) survival after curative-indent surgery for pancreatic adenocarcinoma.
Methods: A tissue microarray was constructed including 137 patients who underwent resection for pancreatic adenocarcinoma with curative intent. CEA, cMET, EGFR, EpCAM, HC10, HCA2, HER2, HLA-E, HLA-G, integrin avb6 and VEGFR expression was evaluated by immunohistochemistry. Multivariate logistic regression for short-term (<12 months) survival and Cox proportional hazard survival analyses were performed.
Results: 29.9% (n=41) of patients died within 1 year of surgery. Median OS and DFS were 17 and 13 months, respectively. HLA-G (OS:HR=1.780, p=0.049; DFS:HR=2.083, p=0.010), integrin avb6 (OS:HR=2.872, p=0.005; DFS:HR=1.867, p=0.045), cMET (OS:HR=2.664, p=0.005; DFS:HR=2.253, p=0.009) and loss of EpCAM (OS:HR=1.880, p=0.002; DFS:HR=1.709, p=0.008) expression were significant independent risk factors for OS and DFS. Additionally, HLA-G (OR=3.206, p=0.043), integrin avb6 (OR=8.935, p=0.038), cMET (OR=8.247, p=0.046) and loss of EpCAM (OR=3.034, p=0.007) were also predictive for short-term survival on multivariate analysis.
Conclusions: The results of this study show that HLA-G, integrin avb6, cMET and EpCAM expression are predictive for short-term survival, OS and DFS in patients with pancreatic adenocarcinoma. These biomarkers may promote identification of biologically ill-faded patients that benefit less from upfront surgery.
Transgastric Therapeutic Pancreatic Hypothermia as a Novel Therapy for Acute Pancreatitis (AP)
C. De Oliveira, K. Patel, V. Mishra, R.N. Trivedi, J. Bradley, J.R. Yaron, V.P. Singh. Department of Medicine, Mayo Clinic, Scottsdale, AZ.
Background: Multiple deleterious signaling cascades are simultaneously activated in AP, which may limit the success of targeted pharmacologic approaches. Hypothermia slows biologic processes, decreasing oxygen requirements and metabolic demand, but generalized hypothermia can compromise vital functions. Here we tested the efficacy of a novel transgastric local pancreatic hypothermia approach for reducing AP severity while avoiding generalized hypothermia.
Methods: In vitro acinar studies helped determine the temperatures to be used in vivo. A collapsible gastric balloon with a dual lumen perfusion system was placed in anesthetized rats. Glyceryl trilinoleate (GTL) was used in vitro (300mM) or injected intraductally (50μL/100g) as described (PMID: 24854864), followed by perfusing the balloon with cold water. Vitals signs, pancreatic necrosis, serum amylase, lipase, cytokines (pg/ml) and fatty acids (mM) were monitored. Animals (8rats/group) were followed till mortality or duration of anesthesia (6.5h post AP induction). Values are mean ± SEM with p<0.05 being significant.
Results: Acinar cooling to 25°C reduced necrosis by >80% vs. 37°C. In vivo, after 1 hour the pancreatic temperature was 25.5±0.8°C with rectal being 38°C. Cooling improved pulse distention (396 ± 26 μm vs 266 ± 35 μm), reduced the increase in necrosis (from 47% to 30%) and improved survival from 0% to 85%, with median mortality in the GTL (± uncooled balloon) groups occurring at 3.5 hours. Cooling significantly reduced Serum IL-1b (26±6.5 vs.152±27), IL-6 (452±211 vs 3294±768), TNF-a (8±1 vs. 37±5.4) and the linoleic acid metabolite arachidonate (7.5±1.5 vs. 128±15 mM) to control levels at 3.5 hours vs. the GTL group.
Conclusions: Therapeutic transgastric pancreatic cooling is rapidly inducible without causing generalized hypothermia and reduces local injury, lipotoxic mediator generation, and systemic inflammation in severe acute pancreatitis. Thus local pancreatic hypothermia may be a valid therapeutic option for AP.
Glycogen Synthase Kinase-3beta Ablation Limits Pancreatitis Induced Acinar-To-Ductal Metaplasia
L. Ding,1 G.-Y. Liou,2 J.-S. Zhang,1 P. Storz,2 D.D. Billadeau.1 1Division of Oncology Research, Schulze Center for Novel Therapeutics, Mayo Clinic, Rochester, MN; 2Department of Cancer Biology, Mayo Clinic, Jacksonville, FL.
Acinar-to-ductal metaplasia (ADM) is a reversible process involving trans-differentiation of pancreatic acinar cells into ductal-like structures. These ADM lesions are thought to be precursor lesions that can progress toward pre-malignant PanIN lesions and ultimately develop into pancreatic adenocarcinoma (PDAC). Recently we showed that glycogen synthase kinase-3beta (GSK-3β) is overexpressed in PDAC and accumulates in the nucleus of moderate and poorly differentiated PDAC. In addition, pharmacologic inhibition of GSK-3 kinase activity using small molecule inhibitors or genetic depletion of GSK-3β by RNA interference leads to decreased cancer cell proliferation and survival. However, the role of GSK3β in early ADM is still unknown. Herein, using a combination of genetic, biochemical and pharmacological approaches, we show that GSK3β promotes TGFα-induced ADM in 3D cultured primary acinar cells and deletion of GSK3β attenuates caerulein-induced ADM formation and PanIN progression in KRasG12D transgenic mice. Furthermore, we demonstrate that the inhibition effects of GSK3β ablation on ADM formation and PanIN progression in KRasG12D mice mainly act by suppressing the proliferation of cells within the ADM and PanIN lesions. Mechanistically, we show that GSK3β regulates proliferation by increasing the phosphorylation of mTOR signaling pathway substrate, ribosomal protein S6. Taken together, these results indicate that GSK3β plays an important role in early pancreatitis induced ADM and could be a target for the treatment of chronic pancreatitis.
Blocking P2 Receptor by Suramin Reduces the Severity of Acute Pancreatitis
A. Dixit,1 J. George,2 Y. Ryu,1 H. Cheema,1 V. Dudeja,3 R. Dawra,1 A.K. Saluja.1 1Department of Surgery, University of Miami, Miami, FL; 2Surgery, University of Miami, Miami, FL; 3University of Miami, Miami, FL.
Background: Pancreatitis in its severe form causes multiple organ dysfunction syndrome (MODS), which is the main cause of mortality associated with severe acute pancreatitis (SAP). The mechanism of local injury leading to MODS in SAP, is not well understood. Earlier, we have reported that increased levels of extracellular ATP (eATP) in different mouse models of acute pancreatitis. eATP can induce cellular signaling, by interacting with P2 receptors. Presence of different P2 receptors on pancreatic acinar cells and their role in pancreatitis not known.
Aim: of the current study was to evaluate expression of P2 receptors on pancreatic acinar cells in acute pancreatitis and effect of pharmacologically blocking the P2 receptors on severity of acute pancreatitis.
Method: Expression levels of different P2 receptors in freshly prepared acinar cells and in pancreas after induction of acute pancreatitis was measured by qPCR. Effect of suramin (10 mg/kg, i.p x2), a P2 receptor antagonist was evaluated on cerulein-induced pancreatitis. Serum cytokines were measured using ELISA.
Results: Pancreatic acinar cells have P2X1, P2X2, P2X3, P2X6, P2Y2, P2Y6, and P2Y12 but P2X3, P2Y2 and P2Y12 expression was significantly lower level than others. In acute pancreatitis P2X2 and P2X6 were most over expressed receptors. Blocking of P2 receptors using suramin resulted in significantly reduced level of serum IL-6 and TNF-α. Suramin treatment also, significantly reduced pancreatic and lung injury as measured by MPO activity.
Conclusions: P2 receptors are present on acinar cells. Activation of P2 receptors contributes to severity of acute pancreatitis and associated lung injury. Blocking these receptors can reduce the MODS associated with severe acute pancreatitis.
Diet-Independent Visceral Adipose Tissue Inflammation Accelerates Pancreatic Cancer Development in the Conditional KrasG12D Mouse Model
G. Eibl, M. Xu, X. Jung, A. Moro, C. Chou, A. Schmidt, H.-H. Chang, O.J. Hines. Department of Surgery, David Geffen School of Medicine at UCLA, Los Angeles, CA.
There is strong evidence that obesity increases the risk of pancreatic cancer (PDAC). We have previously demonstrated that a diet high in fats and calories accelerates pancreatic neoplasia in the conditional KrasG12D mouse model (KC). This was associated with a substantial expansion of the visceral adipose tissue (VAT) and a strong VAT inflammation. The aim of the current study was to delineate the role of adipose tissue inflammation alone in PDAC development in the KC model. Hormone sensitive lipase (hsl) is an enzyme important for triglyceride hydrolysis in adipose tissue. Hsl-null mice are characterized by impaired fatty acid release with subsequent inflammation of the adipose tissue. KC mice were crossed with hsl-null mice to create KC;hsl+/+ and KC;hsl−/− mice. Mice were fed a control diet for 6 months. Both genotypes gained weight similarly. At sacrifice, there was a significant difference in PDAC between KC;hsl−/− and KC;hsl+/+ mice. While there were no cancers in KC;hsl+/+ mice at 6 months, 20% of KC;hsl−/− mice developed PDAC. This was associated with a significant difference in adipose tissue inflammation. Compared to KC;hsl+/+ mice, KC;hsl−/− mice had about 12-fold more “crown-like structures”, known histologic features of adipose tissue inflammation, and significantly more F4/80 positive macrophages (by immunofluorescence) in the mesenteric adipose tissue. The increased adipose tissue inflammation in the KC;hsl−/− mice was associated with enhanced proliferation of PanIN and pancreatic stromal cells (by Ki67 immunohistochemistry). In conclusion, our data demonstrated that VAT inflammation independent of diet and diet-induced obesity increased the incidence of PDAC in the KC mouse model, thereby supporting the importance of VAT and VAT inflammation in PDAC development.
Training for Endoscopic Retrograde Cholangiopancreatography (ERCP) in Children: Insights From the Kids
S. El-Dika,1 K. Williams,2 A. Hinton,1 S. McCarthy,1 J.R. Groce,1 P. Hart,1 S.G. Krishna,1 D.L. Conwell.1 1Ohio State University-Wexner Medical Center, Columbus, OH; 2Nationwide Childrens Hospital, Columbus, OH.
Background: ERCP is less commonly performed in children when compared to adults. Training and credentialing endoscopists to perform ERCP in children poses a challenge, and no existing guidelines address this important issue.
Aim: We used the (kids’ inpatient database) KID to analyze national inpatient ERCP practice patterns in order to better understand the requirements and need for optimal training and credentialing for pediatric ERCP.
Methods: We analyzed the KID from 2006, 2009, and 2012. The KID is an Agency for Health Care Research and Quality (AHRQ) United States inpatient database exclusively dealing with children (ages 0–20 years) released every 3 years. The ICD-9-CM procedural and diagnostic codes were utilized for identification of diagnoses and procedures.
Results: A total of 16,661 inpatient ERCPs were performed during the 3 study periods. Although a majority of the ERCPs (n= 15,021) were performed in children > 10 years of age (76% for 16–20 years, 14% for 11–15 years of age), a notable subset (10%, n=1,580) of ERCPs were performed in children ≤ 10 years of age. The majority of procedures (60% of all ERCPs) were performed at urban teaching hospitals. A majority (87%; n=14,548) of these procedures had an associated diagnosis of gallbladder or biliary disease. A smaller group 4% (n= 722) had an associated diagnosis of chronic pancreatitis, while 3% (n= 539) had an associated diagnosis of anomalies of gallbladder, bile ducts, and liver. While the demand for ERCP has increased from 2006 to 2012, the proportion of diagnostic ERCPs (compared to therapeutic) significantly declined (17% in 2006 to 11% in 2012; P<0.001).
Conclusion: Demand for therapeutic ERCPs in pediatric population is increasing. Endoscopists performing pediatric ERCPs should be familiar with general endoscopy in very young children, aware of pancreatico-biliary anomalies observed in childhood, and at the same time skillful in pancreatic endotherapy. Reaching this level of training is best achieved with collaborative efforts of both pediatric and adult gastroenterology training programs.
Autophagy Drives Pancreatic Stellate Cells Activation and Promotes Pancreatic Cancer
S. Endo,1 K. Nakata,1 K. Ohuchida,1 Y. Ando,1 S. Kibe,1 S. Takesue,1 H. Nakayama,1 T. Abe,1 K. Koikawa,1 T. Okumura,1 Y. Mizuuchi,2 T. Moriyama,1 Y. Miyasaka,1 T. Manabe,1 T. Ohtsuka,1 E. Nagai,1 K. Mizumoto,1 Y. Oda,2 M. Nakamura.1 Departments of 1Surgery and Oncology; and 2Anatomical Pathology, Kyushu University, Fukuoka City, Japan.
Introduction: Pancreatic stellate cells (PSCs) secrete extracellular matrix and cytokines, and can increase the aggressiveness of pancreatic cancer through tumour stromal interactions. However, little is known about the mechanisms of PSCs activation, or how to transform activated PSCs back to a quiescent state. In this study, we tested the hypothesis that autophagy is related to activation of PSCs and that autophagy-induced activation of PSCs promotes the growth and metastasis of pancreatic cancer.
Methods: We investigated the relationships between autophagy and PSCs activation using pancreatic cancer specimens and PSCs isolated from pancreatic cancer tissues.
Results: In immunohistochemical staining, autophagy activity had significant associations with pathologic T category (P=.0179) histological grade (P=.0005), lymph node metastases (P<.0001), stage (P=.0094), perilymphatic invasion (P=.0006), and perivascular invasion (P=.0028). Autophagy activation in PSCs is related to poor survival of patients with pancreatic cancer and microtubule associated protein 1 light chain 3 (LC3) expression in PSCs was an independent poor prognostic marker in pancreatic cancer patients, with a relative risk of 1.564. Autophagy activity was higher in PSCs derived from pancreatic cancer compared with PSCs derived from chronic pancreatitis. Autophagy inhibition caused PSCs to become quiescent, which reduced the production of extracellular matrix and attenuated the aggressiveness of pancreatic cancer cells.
Conclusions: Repressing autophagy in PSCs can be a promising therapy to inhibit the invasiveness of pancreatic cancer by decreasing tumour stromal interactions with PSCs.
The Routine Clinical Yield of Molecular Analysis for Precision Medicine in Pancreatic Cancer Using Pancreatic FNA Biopsy Material
J.J. Farrell,1 J. Wong,2 K. Burnett,3 M. Baker,3 T. Maney.3 1Yale University, New Haven, CT; 2University of Hawaii, Honolulu, HI; 3Caris Life Science, Phoenix, AZ.
Background: The value of EUS-FNA (FNAspiration and FNCore Biopsy) acquired tissue for molecular analysis to guide pancreatic cancer treatment is limited due to the amounts of tissue obtained. We hypothesize that commericial based molecular analysis performed using FNA acquired material is inferior compared with using NonFNA (surgical resection/excision) material.
Methods: We studied a large commercial dataset of patient pancreatic ductal adenocarcinoma tissue samples submitted for molecular analysis for precision medicine, (CarisLS, AZ). We classified tissue samples based on location (primary vs. metastatic), method of tissue acquisition ("Non-FNA" vs. "FNA"), type of tissue submitted (tissue vs. cell block) and their associated molecular analysis results (“Complete Results”, defined as DNA sequencing (44+ gene mutation panel) and/or Protein IHC analysis (15+ protein IHC panel), vs. “No results”). The primary outcome was defined as any DNA or IHC results.
Results: 970 pancreatic ductal adenocarcinoma samples (Primary 472/Metastatic 498) were analyzed. For primary samples, the majority, 402, were by “Non FNA” compared with 70 using “FNA” methodology, with a statistically significant greater yield of molecular analysis using the “Non FNA” samples (94.7 % vs 57.1%, p<0.001). Of the “Metastatic” Tissue samples, 459 were from “Non FNA” sources compared with 39 from “FNA” sources, again with a statistically significant greater yield of molecular analysis for the “Non FNA” samples (91.7 % vs 78.1%, p=0.006). For the ‘FNA” samples alone, location of biopsy, method of biopsy (FNAspiration vs FNCore Biopsy) or type of tissue submitted does not influence the success of molecular profiling.
Conclusions: A minority of samples (9%) undergoing commercial molecular analysis are ‘FNA” samples with an inferior yield compared with “NonFNA” for both primary and metastatic pancreatic sources. Location, biopsy technique or tissue submitted has no effect on outcome. Further attention to improving tissue yields using EUS- FNA may improve its role in pancreatic cancer precision medicine.
DNA Analysis of Pancreatic Cystic Fluid has Incremental Predictive Value in Assessing Future Risk of Malignant Outcomes
J.J. Farrell,1 S. Jackson,2 N. Toney,2 T. Gonda.3 1Yale Center for Pancreatic Disease, Yale University, New Haven, CT; 2Clinical Development, Interpace Diagnostics Corporation, Pittsburgh, PA; 3Division of Digestive and Liver Disease, Columbia University, New York, NY.
Background and Aim: To determine the incremental utility of DNA analysis of pancreatic cyst fluid in assessing risk of malignancy over long term follow-up, given the predictive value clinical features used to routinely evaluate such lesions.
Methods: Patient outcomes (benign or malignant) were used to determine the positive predictive value (% PPV) of individual and cumulative DNA abnormalities (DNAB) in various clinical scenarios. DNAB included tumor suppressor gene (TSG) loss of heterozygosity mutations, KRAS mutations, and/or elevated DNA quantity. The PPV for malignancy of 0–3 DNAB in cysts lacking or having concomitant high risk stigmata (HRS) or clinical worrisome features (WF) is reported.
Results: 492 cases (66 malignant) with surgical pathology (n=209) or long-term follow-up (n=283) were analyzed. Malignancy was found in 58% initially having HRS. Malignancy was found in only 10% initially lacking HRS but in 30% when 2–3 DNAB were also initially present and only 3% when all DNAB were initially absent. Malignancy was found in 4% initially lacking HRS and all WF but in 9% when 2–3 DNAB were also initially present. Malignancy was found in 17% initially lacking HRS and having one WF but in 50% when 2–3 DNAB were also initially present and in only 2% when all DNAB were initially absent. Malignancy was found in 30% initially lacking HRS and having two or more WF but in 80% when 2–3 DNAB were also initially present and in only 8% when all DNAB were initially absent.
Discussion: In the absence of HRS, pancreatic cyst fluid analysis for TSG mutations, KRAS and DNA quantity provides predictive value for risk of future malignancy beyond that achieved by examining WF alone. This may have clinical implications for personalized surveillance planning.
The Pancreas-Specific Isoform of SPCA2 Affects Store Operated Ca2+ Entry
M. Fenech,1 S. Brar,1 P. Stathopulous,2 C. Pin.3 Departments of 1Physiology and Pharmacology; 2Physiology and Pharmacology; and 3Paediatrics, University of Western Ontario, London, Canada.
Regulated exocytosis in pancreatic acinar cells involves spatial and temporal accumulation of cytosolic Ca2+. Disruptions in Ca2+ signalling have been linked to perturbed exocytosis, premature enzyme activation, and initiation of pancreatitis. Our laboratory has identified a pancreas-specific isoform of Secretory Pathway Ca2+ -ATPase 2 (SPCA2C) that lacks domains required for ATPase function. SPCA2C elevates cytosolic Ca2+ when expressed in cultured cells, which is uncharacteristic of other Ca2+ ATPases. In this study we investigate SPCA2C’s involvement in Store-Operated Ca2+ entry (SOCE). SOCE is regulated by ORAI1 and STIM1 proteins, and recent studies show that inhibiting SOCE reduced the severity of pancreatitis. We hypothesized that SPCA2C regulates cytosolic Ca2+ levels through a SOCE-mediated pathway.
HEK293 cells +/− ORAI1 were transfected with tagged SPCA2C, and co-IF performed showing cell localization and association with ORAI1. Cytosolic [Ca2+] changes were examined in the same cells during carbachol stimulation, or thapsigargin treatment +/− inhibition of SOC channels. Finally, we generated mice over-expressing SPCA2CMYC (Elas1-SPCA2C MYC) and characterized pancreatic morphology and the response to cerulein-induced pancreatitis (CIP).
Co-IF revealed SPCA2C localization to the endoplasmic reticulum and Golgi following transfection, which was altered in the presence of ORAI1. SPCA2C over-expression affected cytosolic [Ca2+] in HEK293 cells before and after stimulation with carbachol, and Elas1-SPCA2C MYC mice showed an altered response to CIP.
Our findings suggest SPCA2C plays a unique role in regulating cytosolic Ca2+ entry potentially through a SOCE pathway. Future studies will focus on understanding the mechanisms by which SPCA2C influences cytosolic Ca2+.
Loss of Necroptotic Rip3 Can Not Attenuate Impaired Autophagy-Induced Pancreatitis
F. Fortunato,2 X. Zhou,1 L. Xie,2 F. Bergmann,3 O. Strobel,4 M.W. Büchler,4 T. Hackert.4 1Section Surgical Research, University Clinic Heidleberg, Heidleberg, Germany; 2Section of Surgical Research, University Clinic Heidelberg, Heidelberg, Germany; 3Institute of Pathology, University Clinic Heidleberg, Heidleberg, Germany, 4Department of General Surgery, University Hospital Heidelberg, Heidelberg, Germany.
Background & Aims: Autophagy has been implicated in various diseases, including pancreatitis. We investigated whether Atg7, an essential autophagy protein, plays a role in pancreatitis and whether acinar cell necroptosis is the predominant cell death in pancreatitis.
Methods: Mice were generated with autophagic Atg7-specific deletion in the pancreas and crossed with necroptotic Rip3-deleted mice, followed by an evaluation of tissue morphology, acinar cell autophagy, necroptosis and apoptosis, as well as inflammation and fibrosis, using immunofluorescence, WB and qPCR methods.
Results: Mice with conditional knockout of Atg7 developed pancreatitis spontaneously, with evidence of a progression from acute to chronic pancreatitis (CP). The mice exhibited reduced autophagic activity as well as increased inflammation and fibrosis, leading to severe pancreatic exocrine and endocrine damage with a reduced survival rate. The remarkable damage to pancreatic acinar and islets cells was induced by apoptotic and necroptotic cell death and age-dependent acinar-to-ductal cell metaplasia (ADM). However, double Rip3-Atg7 mouse was unable to reduce loss of autophagy-induced acinar and islets cell damage, indicating that necroptosis seem not to play a critical role Pancreatitis and diabetes.
Conclusion: Necroptosis inhibition is unable to prevent loss of autophagy-induced pancreatitis and should be considered for studies using necrostatin for the treatment of necroptosis in pancreatitis and diabetes.
Participation of Bile Acid Receptor FXR in Suppression of Acinar Cell Autophagy in Human Chronic Pancreatitis
F. Fortunato,2 X. Zhou,1 L. Xie,2 F. Bergmann,3 V. Endris,3 O. Strobel,4 M.W. Büchler,5 T. Hackert.4 1Section Surgical Research, University Clinic Heidleberg, Heidleberg, Germany; 2Section of Surgical Research, University Clinic Heidelberg, Heidelberg, Germany; 3Institute of Pathology, University Clinic Heidelberg, Heidleberg, Germany; 4Department of General Surgery, University Hospital Heidelberg, Heidelberg, Germany; 5University Hospital Heidelberg, Heidelberg, Germany.
Background & Aims: Autophagy has been implicated in various human diseases, including pancreatitis. The aim of this study was to investigate whether the bile acid receptor FXR and autophagy play a critical role in human pancreatitis.
Methods: We investigated human chronic pancreatitis (CP) patients and evaluated tissue morphology, acinar cell autophagy, apoptosis and necroptosis, inflammation and fibrosis. Since depletion of pancreatic Atg7 induced pancreatitis in mice, we also determined bile acid nuclear farnesoid X receptor (FXR) and other transcription factors as possible repressors of autophagy, using immunofluorescence, WB and qPCR methods.
Results: Tissue from chronic pancreatitis patients displayed a reduced acinar cell ATG7 and ATG5 levels and reduced autophagy activity, increased inflammation and fibrosis. Apoptosis and necroptosis were also increased in acinar cells from CP tissue. Investigation into the regulation of reduced acinar cell autophagy showed that increased nuclear FXR is the most prominent factor, along with increased cytoplasmic FOXO3 and nuclear NFкB, reducing acinar cell ATG7 and autophagy activity, promoting the development of chronic pancreatitis.
Conclusion: Based on existing knowledge, this is the first study that correlates not only FXR to pancreatitis but also demonstrated that FXR diminished acinar cell autophagy activity that facilitated the development of CP in humans. Since animal models with deleted acinar cell autophagy promote CP, the bile acid nuclear receptor FXR and the autophagy activity may be a potential new target to suppress the progression of CP.
Successful Ablation of Lymph Nodes Using Irreversible Electroporation (IRE) in a Porcine Survival Model
S. Fritz,1, 2 C.M. Sommer,3 T. Longerich,4 C. Kuhn-Neureuther,5 B. Radeleff,2 J. Werner,6 J. Köninger,3 M.W. Büchler,2 T. Hackert.2, 1 General, Visceral and Transplantation Surgery, Katharinenhospital, Stuttgart, Germany; 2University of Heidelberg, Heidelberg, Germany; 3Katharinenhospital Stuttgart, Germany; 4University Hospital RWTH Aachen, Germany; 5AngioDynamics Heidelberg, Germany, 6University of Munich, Munich, Germany.
Background: Irreversible electroporation (IRE), a new non-thermal ablation technique, has been shown to be effective and safe in various organs, such as in kidney, liver, prostate, or in the pancreas. In contrast to radiofrequency or microwave ablation, IRE can be performed in the neighborhood of major vessels. Many human cancers reveal lymphatic spread. To our knowledge, this is the first report showing successful IRE of lymph nodes in a standardized porcine survival model.
Methods: A total of ten pigs (German landrace) were divided into two study groups. In the acute phase experiment, five animals received IRE of mesenteric lymph nodes of the small bowel and were sacrificed two hours after ablation. In a second study cohort, IRE was performed and the animals followed up for seven days. Clinical parameters, laboratory results, abdominal imaging by computed tomography, as well as histology were obtained from all animals at different time points.
Results: During and after IRE ablation, no cardiac side effects were noted in any of the animals. Computed tomography revealed a hypodense lesion following IRE already at two hours. Histopathology showed coagulation necrosis of the treated lymph nodes.
Conclusions: This porcine survival model shows that IRE can safely and effectively be performed in lymph nodes. Thus, IRE might display a novel approach for therapy of lymph node metastasis. Further clinical studies are needed to evaluate the oncologic outcome of IRE ablation in lymph node metastasis.
Combination of L1156F and M470V in CFTR Gene Associated With Alcoholic Chronic Pancreatitis in Japanese
K. Fujiki,2 S. Kondo,1 S.B.H. Ko,3 A. Yamamoto,1 M. Nakakuki,1 Y. Ito,1 M. Kitagawa,2 S. Naruse,4 H. Ishiguro.1 1Nagoya University Graduate School of Medicine, Nagoya, Japan; 2Department of Nutrition, Nagoya University of Arts and Sciences, Nisshin, Aichi, Japan; 3Keio University School of Medicine, Shinjuku-ku, Japan; 4Miyoshi Municipal Hospital, Miyoshi-Shi, Japan.
Introduction: Although cystic fibrosis is rare in Japanese, measurement of sweat Cl− suggested mild dysfunction of CFTR in some patients with chronic pancreatitis. In the present study, we have investigated the functional characteristics of the Japanese-, and pancreatitis-specific CFTR variant, L1156F.
Materials and Methods: Seventy patients with alcoholic chronic pancreatitis, 18 patients with idiopathic chronic pancreatitis, and 180 normal subjects participated. All exons and their boundaries of the CFTR gene were sequenced. HEK 293 cells and CFPAC-1 cells were transfected with 3 CFTR variants (M470V, L1156F, and M470V+L1156F). Effects of ethanol (EtOH), acetaldehyde (ALD), palmitoleic acid (POA), and palmitoleic acid ethyl ester (POAEE) on the CFTR protein expression were examined in HEK 293 cells. Cl−-HCO3 − exchange activity was examined in CFPAC-1 cells. An atomic model of CFTR protein was constructed by homology modeling using Discovery studio.
Results: The allele frequencies of L1156F in alcoholic chronic pancreatitis were significantly higher than those in normal subjects (5.0 v.s. 0.6 %, p<0.01). L1156F was linked with the world-wide CFTR variant M470V. Although the expression of M470V-CFTR and L1156F-CFTR was not significantly different from that of wild-type CFTR, the expression of M470V+L1156F-CFTR was reduced to ~60% of wild-type CFTR. The reduction was not affected by EtOH, ALD, POA, and POAEE. The Cl−-HCO3 − exchange activity of M470V+L1156F-CFTR was reduced to 50~60% of wild-type CFTR. A homology model of CFTR in the inward-facing conformation showed M470V and L1156F are not close to each other.
Conclusion: Combination of L1156F and M470V causes mild dysfunction of CFTR and increases the risk of alcoholic chronic pancreatitis in Japanese.
Serum Level of Wisteria Floribunda Agglutinin-Positive Mac-2-Binding Protein Reflects the Severity of Chronic Pancreatitis
T. Fujiyama, K. Ueda, Y. Tachibana, M. Miki, K. Yasunaga, T. Takaoka, K. Kawabe, T. Ito. Department of Medicine and Bioregulatory Science, Graduate School of Medical Sciences, Kyushu University, Fukuoka City, Japan.
Background: Chronic pancreatitis (CP) is diagnosed by imaging, function test, and clinical symptoms. Endoscopic retrograde cholangiopancreatography (ERCP), which reveals the main pancreatic duct and its branches, is the standard technique for grading chronic pancreatitis. However, ERCP is invasive and post-ERCP pancreatitis is a known complication. Therefore, non-invasive markers for diagnosis and grading for CP are needed. Recently, Wisteria floribunda agglutinin-positive Mac-2-binding protein (WFA+-M2BP) was developed as a liver fibrosis marker.
Aim: To evaluate the utility of serum WFA+-M2BP level as a marker for CP.
Methods: We measured serum WFA+-M2BP level of 49 patients with CP who had undergone ERCP and 20 normal controls (NC) using the glycan sugar chain-based immunoassay and examined the relationship between serum WFA+-M2BP level and Cambridge classification.
Results: Serum WFA+-M2BP level was significantly higher in patients with CP (0.63 ± 0.26) than in NC (0.35 ± 0.28; P < 0.001). CP was classified as Mild, Moderate, and Marked and the serum WFA+-M2BP levels for each category were 0.45, 0.66, and 0.87, respectively. Thus, serum WFA+-M2BP levels increased with increasing CP severity. The data for receiver operating characteristic curve analyses, area under the curve, sensitivity, and specificity were 0.34, 0.834, 91.8%, 65.0% for Mild; 0.60, 0.902, 88.5%, 90.0% for Moderate; and 0.62, 0.919, 93.8%, 90.0% for Marked, respectively. Multivariate regression analysis revealed that elevated serum WFA+-M2BP was associated with Moderate CP, whereas elevated serum WFA+-M2BP, the presence of diabetes mellitus, and the presence of abdominal or back pain were associated with Marked CP.
Conclusions: Serum WFA+-M2BP level is a useful marker for grading CP severity.
Investigating the Novel Function of Hippo Signaling in Pancreatic Acinar Cells
M. Gao,1 J. Liu,1 J. Leighton,1 X. Yin,1 R.L. Johnson,2 P. Wang.1 1Department of Cellular and Structural Biology, UT Health Science Center at SA, San Antonio, TX, 2Department of Cancer Biology, Division of Basic Science Research, The University of Texas MD Anderson Cancer Center, Houston, TX.
Background: The Hippo signaling pathway regulates diverse cellular processes, including proliferation, differentiation, cell survival, and organ size. We found that the core components of the pathway, large tumor suppressor kinase 1 & 2 (Lats1&2) and their targets Yap1&Taz, are dynamically regulated during cerulein induced acute pancreatitis. However, the function of Lats1&2 and YAP1&TAZ in pancreas and pancreatitis is unknown.
Methods and results: To investigate the function of Lats1&2 and Yap1&Taz in pancreas, we generated mice with adult pancreatic acinar cell–specific deletion of Lats1&2 genes and Yap1&Taz genes respectively. Interestingly, the deletion of Lats1&2 genes in pancreatic acinar cells resulted in severe pancreatitis phenotype, while Yap1&Taz are dispensable for normal pancreas function. To further determine the mechanisms, we took advantage of Rosa26 reporter to trace individual Lats1&2 null cells in vivo. We found that the loss of Lats1&2 did not affect cell proliferation and cell apoptosis. We then deleted YAP1&TAZ in Lats1&2 null acinar cells to investigate whether this phenotype is YAP1&TAZ dependent. Interestingly, we found deletion of YAP1&TAZ rescued the pancreatitis phenotype of Lats 1&2 null mice.
Conclusion and significance: We found deletion of Lats1&2 genes in acinar cells caused inflamed pancreas. More importantly, our data suggested YAP1&TAZ played a potential role in inflammation initiation in pancreas, and anti-YAP1&TAZ could provide new strategies for controlling pancreatic inflammation.
NFkB in Tumor Stroma Modulates Cancer Growth in Mouse Models of Pancreatic Cancer
B. Garg,1 B. Giri,2 S. Modi,1 V. Sethi,3 S. Banerjee,3 A.K. Saluja,4 V. Dudeja.3 1University of Miami, Miami, FL; 2University of Miami, Miami, FL; 3University of Miami, Miami, FL; and 4University of Miami, Miami, FL.
Background: Interaction between cancer and tumor stroma contributes to aggressiveness of pancreatic cancer. Cancer associated fibroblasts, which in pancreatic cancer primarily arise from pancreatic-stellate-cells (PSCs), promote tumor growth. While NFkB signaling in cancer cells is known to promote tumor growth, impact of NFkB signaling in stroma on pancreatic cancer growth is not known. We have evaluated the role of NFkB signaling in tumor stroma on the growth of pancreatic cancer.
Methods: Cancer cells from tumor developing in KrasG12D TP53 PDX-1 cre (KPC) were co-injected with PSC from (WT) or p50−/− (from mice lacking p50 subunit of NFkB) in WT or athymic nude mice and tumor growth were evaluated at 30 days. To confirm our findings in other cancers, LLC (Lewis Lung Carcinoma) and B16 melanoma cells were co-injected with lung and dermal fibroblasts (extracted from WT and p50−/−) in WT mouse respectively. Viability, proliferation, invasion and migration of KPC cancer cells were measured after co-culturing them with WT and p50−/− PSC.
Results: Injection of p50−/− PSC with KPC cells led to decreased pancreatic cancer growth as compared to co-injection with WT PSC. Similarly, stromal NFkB inhibition decreased growth of lung cancer and melanoma. In the absence of T cells (KPC with PSC injected in athymic nude mice) lack of NFkB in tumor stroma did not inhibit tumor growth. In-vitro data showed that loss of P50−/− in stromal cells were unable to augment viability, proliferation, migration and invasion of pancreatic cancer cells.
Conclusion: NFkB signaling in stroma promotes pancreatic cancer growth by protecting cancer cells from immune mediated-cytotoxicity. NFkB inhibition in stroma could emerge as novel strategy against pancreatic cancer, either alone or in combination with immunotherapy.
Optimization of Targeted Radionuclide Therapy (TRT) for Pancreatic Cancer
S.K. Gautam,1 M.W. Nasser,1 S. Gupta,1 S.K. Batra,1, 2 M. Jain.1, 2 1Biochemistry and Molecular Biology, University of Nebraska Medical Center, Omaha, NE; 2Fred and Pamela Buffett Cancer Center, Omaha, NE.
Background: TRT using monoclonal antibodies (MAbs) recognizing tumor cell surface antigens is a promising approach to target overt and occult metastatic lesions. However, obstructive stroma, high interstitial fluid pressure, and heterogeneous blood flow contribute to limited success of TRT in pancreatic cancer (PC). Here we describe the impact of selective modulators of tumor blood flow on the uptake of radiolabeled MAbs recognizing TAG72 (CC49) and MUC4 (8G7) that are highly expressed in PC using xenograft mouse model.
Methods: Pair-labeled biodistribution was performed at various time points using radioiodinated mAbCC49 (131I) and mAb8G7 (125I) in athymic mice bearing xenografts derived from antigen positive (Capan1) and negative (ASPC1) human PC cell lines. Radioimmunoconjugates were administered i.v., alone or in combination with angiotensin II (AT II) or BQ123 (ETAR antagonist). Impact of BQ123 on tumor perfusion was studied using MRI while the changes in hypoxia were studied using IHC.
Results: Both CC49 and 8G7 exhibited specific uptake in Capan1 tumors at 48 h with peak %ID of 20.98 and 6.44 respectively. ATII treatment resulted in 6.3-fold increase in CC49 and 2.53 fold increase in 8G7 in Capan1 tumors while the BQ123 increased tumor uptake by 3.64 and 1.52 fold, respectively. ATII and BQ123 did not alter the tumor uptake in antigen negative ASPC1 tumors. Combined treatment with ATII and BQ123 resulted in additive increase in MAbs in the tumor tissues without impacting the uptake in non-target tissues. BQ123 treatment resulted in selective enhancement in the perfusion and decrease in hypoxia in xenograft tumors.
Conclusions: TAG-72 and MUC4 are promising candidates for TRT of PC and selective modulators of tumor blood flow can improve the uptake and possibly efficacy of antibody-based drugs.
The Met Receptor Tyrosine Kinase is Indispensable for Acinar Regeneration Following Recurrent Injury
I. Gaziova,1 C. Elferink,2 L. Elferink.1 Departments of 1Neuroscience and Cell Biology; and 2Pharmacology, University of Texas Medical Branch, Galveston, TX.
Introduction: The pathophysiology of pancreatitis involves acinar cell dysfunction incurred during repeated bouts of injury. Here we examined the importance of Hepatocyte Growth Factor Receptor/MET signaling as an intrinsic repair mechanism for acinar cells following repetitive (recurrent) injury.
Methods: Mice with acinar cell-specific targeted disruption of MET (MET−/−) were generated to assess the role of MET signaling for acinar survival and regeneration. Using the repetitive cerulein administration model, we assessed the role of acinar MET for proinflammatory and profibrotic responses associated with pancreatic damage using morphometric, proteomic and histological analyses.
Results: MET exerted protective and regenerative effects in cerulein treated animals, evident as decreased edema, histological damage, macrophage inflammation, amylase activity and extracellular collagen deposition, relative to MET−/− mice. Moreover, loss of acinar MET resulted in increased acinar-ductal metaplasia (ADM), a process by which acinar cells transdifferentiate into a transient, ductal-like epithelia to enable pancreatic homeostasis.
Conclusion: Our data support a pivotal role for MET in this model of pancreatic injury, in that loss of acinar MET increased changes in pancreatic architecture, inflammation and ECM deposition. Thus MET signaling appears indispensable for acinar homeostasis and regeneration, a novel finding that may form the basis for targeted therapeutics designed to attenuate the symptomology of pancreatitis.
Evaluation of Secretin-Enhanced MRCP in Chronic Pancreatitis
P. Gecov,2 S. Siminkovitch,1 B. Vladimirov,1 G. Nedelkov,2 M. Kovacheva-Slavova,1 B. Golemanov.1 1Gastroenterology, University Hospital Tsaritsa Ioanna-ISUL, Sofia, Bulgaria; 2Medical Imaging, University Hospital Tsaritsa Ioanna-ISUL, Sofia, Bulgaria.
Introduction: Secretin-Enhanced Magnetic Resonance Cholangiopancreatography (sMRCP) is one of the most accurate methods for diagnosis and classification of chronic pancreatitis (CP) simultaneously with exocrine function and pancreatic anomalies assessment.
Aims & Methods: To investigate by sMRCP morphological changes, functional data, and presence of pancreatic anomalies, the relation between them and clinical and laboratory data in patients (pts) with suspected CP. 53 pts were enrolled (33 male), mean age 48.5±13.3 yrs. Morphological data were assessed by Cambridge classification for CT/MRCP- grade I-IV. sMRCP was performed with Secrelux on MRI scanner-GE Signa HDe 1.5 Tesla. After secretin administration were evaluated pancreatic excretory capacity via duodenal filling (DF-grade 0–3), low main pancreatic duct compliance (LPDC), visible side-branches (SB) and pancreatic anomalies. Statistical analysis was performed by SPSSv22.
Results: Pre-secretin data: 10 pts had normal pancreas morphology, 8- grade (gr.) I by Cambridge, 13-gr.II, 8-gr.III and 14-gr.IV. Post-secretin data: normal DF in 17 pts (gr.3), gr.2- in 28 and gr.1- in 8; with LPDC below 1 mm dilatation were 34 pts; visible SB were observed in 26, and Santorini duct- in 22 pts. Cambridge grades correlated with DF grades (r=−0.52), LPDC (r=0.51) and visible SB (r=0.55), p<0.05. DF grades correlated positively with LPDC, visible SB and pain presence. Pancreas divisum was single aetiology factor for CP in 3 pts, and Santorini duct-in 5. Cambridge grades related positively with smoking, HDL, albumin, p<0.05, and DF grades-with albumin.
Conclusion: sMRCP is a reliable method for evaluation of early morphological changes, exocrine dysfunction and CP development based on pancreatic anomalies.
Therapeutic ADAM 10 and 17 Inhibition Reduces Local and Systemic Inflammation in Acute Pancreatitis
J. George,1 A. Dixit,2 A. Sareen,3 H. Cheema,2 B. Giri,4 V. Dudeja,5 R. Dawra,2 A.K. Saluja.2 1Department of Surgery, University of Miami, Miami, FL; 2Department of Surgery, University of Miami, Miami, FL; 3Department of Surgery, University of Minnesota, Minneapolis, MN; 4University of Miami, Miami, FL; 5University of Miami, Miami, FL.
Background: Despite decades of research there is no specific therapy for acute pancreatitis (AP). While the inflammation during acute pancreatitis (AP) is initiated in acinar cells, over disease course the inflammation spills into systemic circulation and leads to acute lung injury (ALI). ‘A Disintegrin & Metalloproteinases’ (ADAM) are enzymes that can modulate activity of cytokines and their receptors by cleavage and release of soluble ecto-domains.
AIMS: To evaluate the effect of therapeutic ADAM-10/17 inhibition in AP.
Methods: ADAM 10&17 was inhibited in a therapeutic fashion after initiation of caerulein AP or L-Arginine AP. The effect of treatment on pancreatic injury and inflammation was evaluated by histology (necrosis, edema and infiltrates were measured on a scale of 1–4, averaged from 10 fields), pancreatic MPO activity, tissue cytokines and leukocytic infiltration by flow cytometry and IHC. Systemic injury was evaluated using lung histology and neutrophilic infiltration.
Results: ADAM-10/17 inhibition in a therapeutic setting (8 hrs after induction of caerulein AP) significantly reduced pancreatic necrosis (1.3±0.1 vs 3.2±0.6), inflammatory infiltrate (1.4±0.1 vs 2.9±0.1) and edema (2.0±0.1 vs 3.3±0.1) compared to AP alone. Pancreatic and Lung neutrophil recruitment, as measured by MPO assay, FACS and leukocyte staining on IHC, were significantly reduced after ADAM-10/17 inhibition. ADAM inhibition led to significant reduction in the pro inflammatory cytokines in both pancreas and lung tissue ELISA. There was a significant reduction in serum amylase, CRP & HMGB1 also.
Conclusions: This is the first study to evaluate the therapeutic potential of ADAM-10/17inhibition in AP. Our data clearly shows that ADAM-10/17 inhibition reduces local and systemic injury in acute pancreatitis.
Naltrexone Reduces and Morphine Worsens Chronic Pancreatitis Progression in Mouse Models of the Disease
J. George,1 H. Cheema,1 A. Dixit,2 U. Barlass,1 B. Giri,3 Y. Ryu,2 S. Banerjee,1 S. Roy,4 R. Dawra,2 A.K. Saluja,2 V. Dudeja.5 1Department of Surgery, University of Miami, Miami, FL; 2Department of Surgery, University of Miami, Miami, FL; 3University of Miami, Miami, FL; 4Department of Surgery, University of Minnesota, Minneapolis, MN; 5University of Miami, Miami, FL.
Background: Chronic pancreatitis (CP) is a progressive fibro-inflammatory disease of the pancreas, characterized clinically by associated severe abdominal pain, exocrine insufficiency and diabetes mellitus. Recurrent Acute Pancreatitis (RAP) is a known risk factor of CP. Narcotics such as morphine and its derivatives are the primary pain management modality in chronic pancreatitis.
AIMS: To study the effects of naltrexone and morphine adminstration in L Arginine and Caerulein induced mouse models of recurrent acute pancreatitis.
Methods: RAP was induced by administration of caerulein (50ug/kg, i.p., hourly x6x 2 days/week x 6) to mice (C57BL/6). For the L Arginine model, RAP was induced by adminstration of L- Arginine (4.5g/kg x 2 x 2weeks).The effect of opioid signaling on severity of RAP and progression to CP was evaluated by treating with morphine (25 mg/kg i.p, bid) an opioid agonist and naltrexone (25 mg/kg, i.p, bid) an opioid antagonist. The effect of treatment on pancreatitis was evaluated by measuring changes in pancreas to mouse weight ratio, pancreatic histology and Sirius Red staining for collagen.
Results: Pancreatic histology of morphine treated mice had significantly increased chronic inflammatory infiltrate and fibrotic changes compared to RAP alone group in both mouse models. Morphine treated mice showed increased collagen staining compared to caerulein alone group, suggestive of increased fibrosis after morphine treatment. Interestingly naltrexone treatment after RAP significantly reduced pancreatic atrophy, inflammatory infiltrate and fibrotic changes.
Conclusions: Our results suggest that opioid analgesics should be used with caution in the management of RAP & CP as they can worsen the disease progression. This is the first study to show that an opioid antagonist, naltrexone reduces pancreatic fibrosis of chronic pancreatitis. Our study suggests that opioid signaling pathways play an important role in modulating inflammation and fibrosis in pancreatitis.
Targeting Pancreatic Cancer by EGCG in Gemcitabine Resistance
C. Ghosh,1 S. Paul,1 S. Anant,2 A. Dhar.1 1Cancer Biology, The University of Kansas Medical Center, Kansas City, KS; 2Surgery, The University of Kansas Medical Center, Kansas City, KS.
Introduction: Pancreatic ductal adenocarcinoma (PDAC) is a lethal malignancy with poor prognosis and high mortality rate in USA. Currently, there is no effective treatment available for PDAC. Gemcitabine is the firstline drug for the treatment of PDAC but improves survival rate upto 6 months due to the incidence of resistance. Hence, there is need for novel drugs for the treatment of PDAC. Recently natural compounds gained more attention due to their better safety profile and ability to target multiple pathways. Green tea is one of the most popular beverages consumed around the world. EGCG (epigallocatechin gallate) is the most abundant polyphenol in green tea and known to possess antioxidant and anticancer activity. It is also reported to sensitize certain chemotherapeutic drugs in cancer cells. However, the exact mechanism of its action in PDAC remains unclear.
Aim: We investigated the role of EGCG in gemcitabine resistant PDAC.
Method: We studied the effect of EGCG on proliferation, formation of colony, spheroids and invasion of human PDAC cells (MiaPaCa-2 and S2-007). We also observed the cytotoxic effect of EGCG against gemcitabine resistant in vitro and in vivo models.
Result: EGCG inhibited pancreatic cancer cell proliferation, colony formation and spheroid formation in dose dependent manner. EGCG induced apoptosis by arresting the cell cycle in PDAC cells. EGCG induces cytotoxic effect in gemcitabine resistant cells.
Conclusion: EGCG impairs stemness that could be potent targets for new therapeutic strategies for inhibition of PDAC tumorigenesis and have cytotoxic effect on gemcitabine resistant cells.
Inhibition of Histone Acetylation Results in Pancreatic Cancer Cell Death and Apoptosis
B. Giri, S. Modi, V. Sethi, J. George, B. Garg, S. Banerjee, A.K. Saluja, V. Dudeja. Department of Surgery, University of Miami, Miami, FL.
Background: Histone based epigenetic mechanisms have been shown to be important for pancreatic tumorigenesis. Histone acetyltransferases (HAT) acetylate histone residues resulting in transcriptional activation. Potential of histone acetyltransferase inhibitors as a novel therapeutic strategy against pancreatic cancer has not been evaluated. The aim of the current study was to evaluate HAT inhibitors as novel therapy against pancreatic cancer.
Methods: HAT was inhibited in pancreatic cancer cell line MIA PaCa-2 and S2-VP10 cells by C646; a selective Histone Acetyl transferase inhibitor and cell viability was measured using WST-8 assay. Impact of HAT inhibition on apoptosis and cell cycle was evaluated by measuring caspase-3 cleavage and by performing cell cycle analysis using flow cytometry
Results: Treatment with C646 (1–100 μM) resulted in a dose and time dependent cytotoxicity in MIA PaCa-2 and S2-VP10 cells at 24, 48 and 72 hours with corresponding increase in caspase 3 cleavage as measured by flow cytometry. We also observed a G2/M phase arrest on S2VP10 and Mia PaCa-2 cells on being treated with C646 at a dose of 25 μM for 48 hours.
Conclusion: Histone acetyl transferase inhibition leads to pancreatic cancer cell apoptosis and cell death. HAT inhibitors could emerge as novel therapeutic strategy in future, either alone or in combination with other chemotherapeutics.
HSP70 Deficient Immune Cells Lead to Greater Immune Mediated Killing in Pancreatic Cancer
B. Giri, B. Garg, S. Modi, V. Sethi, J. George, S. Ramakrishnan, S. Banerjee, A.K. Saluja, V. Dudeja. Department of Surgery, University of Miami, Miami, FL.
Background: Although cancer cells have been shown to be dependent on HSP70 for their growth, the role of HSP70 in tumor microenvironment is not clearly elucidated. We aim to understand how HSP70 in the stroma and immune cells affects growth of pancreatic cancer cells.
Methods: Tumors from Pdx1-Cre; K-Ras+/LSLG12D; p53R172H/+ (KPC) mice were surgically implanted into pancreas of WT (C57BL/6) or Hsp70 −/− (mice lacking inducible form of HSP70) mice. The results were confirmed by injecting cancer cells isolated from tumors developing in KPC model orthotopically in the pancreas of WT or HSP70 −/−mice. Pancreatic Stellate cells (PSCs) from HSP70−/− or WT mice were co-injected with KPC cells (ratio=9:1) in the pancreas of WT mice and tumor size was measured. Splenocytes harvested after immunizing HSP70 −/− or WT mice with KPC cells were subsequently co-incubated with cancer cells to assess cell mediated cytotoxicity.
Results: Lack of HSP70 in tumor microenvironment led to smaller tumors in both surgical implantation (HSP70−/−: 0.49±0.1 vs WT: 1.41± 0.1 g, p<0.05) as well as in orthotopic injection (HSP70−/−: 0.14± 0.1 vs WT: 1.23 ± 0.7 g, p<0.05). Co-injection of KPC cells with WT PSC or HSP70 −/−PSC did not show any difference in tumor size suggesting that inhibition of tumor growth observed in absence of HSP70 in tumor microenvironment is not mediated through lack of HSP70 in stroma. Surprisingly co-incubation of HSP70 −/− splenocytes with KPC cells led to greater cell cytotoxicity when compared to WT splenocytes suggesting enhanced immune mediated cancer killing in the absence of HSP70.
Conclusion: Absence of HSP70 in the stroma of pancreatic cancer inhibits its growth possibly by enhancing immune mediated cytotoxicity and not mediated by absence of HSP70 in stellate cells.
Visceral Arterial Calcium Burden and Likelihood of Pancreatic Fistula After Pancreatic Resection
A. Gomes,1 I. Santiago,2 R. Rocha,1 R. Marinho,1 M. Sousa,1 M. Fragoso,1 D. Aparício,1 A. João,1 A. Soares,1 V. Nunes.1 1Department of Surgery, Hospital Prof. Dr. Fernando Fonseca, Amadora, Portugal; 2Champalimaud Foundation, Lisbon, Portugal.
Introduction: Pancreatic fistula (PF) is one of the most frequent causes of morbidity in pancreatic resection. High abdominal arterial calcium burdens have been associated with risk of anastomotic leakage in esophageal and colorectal surgery, likely surrogating chronic reduction in blood supply and consequent ischemia. Our aim is to evaluate the relation between aortic/splancnic calcium burden and the occurrence of PF, delayed hospital leave and surgical mortality after pancreatic resection for oncologic disease.
Methods: Calcium burden was retrospectively quantified based on preoperative non-enhanced abdominal CT using Phillips CT calcium scoring software. Agatson score, n. ROIs and surface in sqmm were recorded for abdominal aorta, celiac trunk, superior and inferior mesenteric arteries. Patients with R0 pancreatoduodenectomy or distal pancreatectomy for oncological disease were analysed. Outcome variables were PC (presence/absence) diagnosed and graded according to ISGPD, length of hospitalization, 30-day surgical mortality and morbidity. Univariate and multivariate analysis with logistic regression were performed, controlling for comorbidities and surgical technique. Non-parametric statistics were used.
Results: 91 patients included, 60 males, median age 68y (25–85), 74 duodenopancreatectomies and 17 distal resections. 31 patients presented with PC(34,1%): 22,6% type B and 1,6% type C. In univariate analysis, no significant assotiations were found between calcium burden parameters and outcomes. In multivariate analysis, none of the logistic regression models found calcium score as a risk factor for PC, death or other complications.
Conclusions: PC, PC grade, other surgical complications and surgical death were independent of calcium burden for aorta and splancnic circulation.
Pigment Epithelium-Derived Factor (PEDF) Inhibits Notch Signaling in PanIN Cells Author Block
J. Gong,1 G. Belinsky,1,2 C. Chung.1,2 1Yale University School of Medicine, New Haven, CT; 2VA CT Healthcare, West Haven, CT.
Notch signaling is an evolutionarily conserved pathway that has been implicated in pancreatic carcinogenesis. Multiple pathway components are increased in pancreatic premalignant samples compared with the normal pancreatic epithelium. Moreover, pancreatic intraepithelial neoplasms (PanIN) lesions found in the Pdx1-Cre;LSL-Kras G12D mice reveal increased expression of the Notch target Hes1 compared with normal ducts. We recently crossed this model of PanIN formation with a murine model of pigment epithelium-derived factor (PEDF) deficiency and found accentuation of the malignant phenotype with increased stromal and inflammatory responses (Oncotarget 2016). We therefore asked whether PEDF influences Notch signaling in PanIN cells. We evaluated PEDF’s effects on various components of the Notch signaling pathway using two PanIN cells lines derived from a model of mutant Kras G12D, PI5505 (PanIN-2) and PI34 (PanIN-3). With PEDF treatment, expression of Notch1, Notch 2 and Jagged-1 were inhibited 10-20% in both murine PanIN cells. In contrast, expression and protein levels of transcriptional factor Hes1 were inhibited nearly 50% by PEDF. Similarly, the protein levels of Jagged-1 were also significantly reduced by PEDF, suggesting that inhibition of Notch signaling is a target of PEDF. Our study demonstrates that the tumor-inhibitory role of PEDF extends to Notch signaling in PanIN cells and provides an explanation for the acceleration of tumorigenesis in the Pdx-Cre:LSL-Kras G12D model.
Pancreatic Microtumors: A Novel Platform for Screening Chemotherapeutic Agents
M. Goodwin,1,2 S. Urs,1,2 Z. Sila,1,2 D.M. Simeone.1,2,3 Departments of 1Surgery; 2Translational Oncology Program; and 3Molecular and Integrative Physiology, University of Michigan, Ann Arbor, MI.
Introduction: Pancreatic tumors have an abundance of cancer associated fibroblasts (CAFs) and immune cells. We propose that 3D pancreatic microtumors composed of pancreatic tumor cells, CAFs and tumor associated macrophages (TAMs) provide a platform for screening of chemotherapeutic agents that more accurately represents the tumor microenvironment.
Methods: 3D pancreatic microtumors were generated by combining primary pancreatic cancer cells, CAFs and TAMs with collagen. TAMs were isolated from peripheral blood mononuclear cells (PBMCs) by density centrifugation and enriched for CD14+ using a magnetic column. TAMs were then generated by growing them in the presence of IL-6, M-CSF and apoptotic tumor cells and then characterized by flow cytometry. Microtumor viability following treatment with chemotherapeutic agents was assessed for viability using a 3D luminescence assay.
Results: 3D pancreatic microtumors develop histological similarities to pancreatic adenocarcinoma. We were able to successfully generate TAMs from CD14+ monocytes that were characterized by the presence of CD 206 and CD 163. Using our primary pancreatic cancer cells we demonstrate that 3D cultures demonstrate increased resistance to gemcitabine compared to 2D monolayer culture (6-fold increase, p< 0.05) and that addition of TAMs and CAFs to the 3D cultures to create microtumors that are more resistant to gemcitabine then both 2D and 3D tumor cells alone (2-fold increase, p<0.05).
Conclusion: 3D pancreatic microtumors provide a novel platform for screening of chemotherapeutic agents. This model may allow a more cost effective screening platform for chemotherapeutic agents, and potentially immunomodulators prior to in vivo animal models.
Serum Exosomal MicroRNA-191, −21, −451a are Considered to be Efficient Diagnostic Markers of Pancreatic Neoplasm
T. Goto, H. Konishi, J. Sasajima, S. Fujibayashi, T. Utsumi, H. Sato, Y. Sugiyama, T. Iwama, M. Ijiri, K. Takahashi, K. Tanaka, A. Sakatani, Y. Nomura, N. Ueno, S. Kashima, S. Takauji, K. Moriichi, M. Fujiya, T. Okumura. Asahikawa Medical University, Asahikawa, Japan.
Background: Pancreatic cancer (PC) is associated with an extremely poor prognosis due to difficulty in its early detection, indicating the need of the diagnostic markers detecting in the early stage. While microRNAs (miRs) are known to be dysregulated in various cancers, the significance of serum miR levels in pancreatic neoplasm diagnosis remains unclear. We herein evaluated the usefulness of serum miR levels, particularly in exosomes, as diagnostic markers in patients with PC and intraductal papillary mucinous neoplasm (IPMN).
Methods: The miRs enclosed in exosomes (ExmiRs) from patients with PC or IPMN, and patients without malignant or neoplastic lesions (Controls) were enriched using ExoQuick-TC™. The expression of ExmiRs was evaluated using a next-generation sequencing analysis, and the expression of the miRs selected through this analysis was confirmed by a quantitative real-time polymerase chain reaction.
Results: Thirty-two patients with newly diagnosed PC and 29 with IPMN were enrolled in this study. The median age in PC and IPMN group was 64.0 ± 10.1 and 73.8 ± 7.8 years old, respectively. Among the PC patients, 12 underwent surgical resection, and 28 received chemotherapy. All of the patients in the IPMN group were diagnosed with branched type (BD)-IPMN, and four underwent surgical treatment. The expressions of ExmiR-191, ExmiR-21 and ExmiR-451a were up-regulated in patients with PC and IPMN compared to the Controls. No abnormal expression of miR-191, miR-21, or miR-451a was detected in the circulating miR (CmiR) extracted from whole serum.
Conclusions: The level of ExmiR-191, ExmiR-21 and ExmiR-451a can thus serve as early diagnostic markers of pancreatic neoplasm. The ExmiRs were also considered a useful marker than the CmiRs in pancreatic neoplasm.
Metachronous Secondary Lesions and Recurrence in the Remnant Pancreas After Pancreatectomy for Pancreatic Ductal Adenocarcinoma
Y. Gotoh,1 T. Ohtsuka,1 S. Nakamura,1 Y. Nakashima,1 K. Date,1 T. Fujimoto,1 N. Mochidome,2 Y. Mori,1 Y. Sadakari,1 K. Nakata,1 Y. Miyasaka,1 K. Ohuchida,1 T. Manabe,1 E. Nagai,1 Y. Oda,2 M. Nakamura.1 Departments of 1Surgery and Oncology; and 2Anatomic Pathology, Kyushu University, Fukuoka, Japan.
Background: We sometimes experience the patients who have metachronous secondary or recurrent lesions in the remnant pancreas after pancreatectomy for pancreatic ductal adenocarcinoma (PDAC). The aim of this study was to clarify the beneficial effect of resection for these lesions.
Methods: The medical records of 411 patients who underwent pancreatectomy for PDACs between 2000 and 2015 were retrospectively reviewed. To discriminate the metachronous secondary lesion to recurrent disease, pathological assessment as well as KRAS mutation analysis and immunohistochemical analysis for p16, p53 and SMAD4 were investigated.
Results: There were 12 patients having PDAC in the remnant pancreas including 8 with metachronous secondary lesion and 4 with recurrent lesion. The duration from initial operation to the diagnosis of PDAC in the remnant pancreas was significantly longer in patients with metachronous secondary lesion than those with recurrent lesion (median 43 [range 24–72] vs. 21 months [range 9–35]; p < 0.01). There was no significant difference in disease specific survival (DSS) between the patients after repeat pancreatectomy for metachronous secondary lesion and those after initial pancreatectomy for PDACs in our facilities, and also in median survival time (12 [range 3–72] vs. 22 months [range 1–183]; p = 0.201). The MST after repeat pancreatectomy in patients having the recurrent lesions (17monsths) seemed to be longer than previously reported MST in patients with unresectable PDACs (12months).
Conclusion: Curative repeat pancreatectomy may bring beneficial effects on prognosis for patients with both metachronous secondary and recurrent PDACs.
Initial Pain Management for Children Presenting to a Pediatric Emergency Department With Acute Pancreatitis
A.S. Grover,1 V. Kadiyala,2 S.F. Manzi,3, 4 V.L. Fox.1 1Division of Gastroenterology, Hepatology and Nutrition, Boston Children's Hospital, Boston, MA; 2Center for Pancreatic Disease, Division of Gastroenterology, Hepatology, and Endosocopy, Brigham and Women's Hospital, Boston, MA; 3Division of Genetics and Genomics; and 4Department of Pharmacy, Boston Children's Hospital, Boston, MA.
Background: Currently, there is no published data examining the management of pain due to acute pancreatitis (AP) in children at the time of initial presentation. Management approaches are often extrapolated from adult practice and choices in therapy often are based on anecdotal experience in the absence of objective data.
Aim: The aim of our study was to examine the initial provision of analgesia to children who present to a pediatric emergency department (ED) with acute pancreatitis.
Methods: Medical records were reviewed retrospectively for all children presenting with AP to Boston Children’s Hospital from January 1st, 2013 to December 31st, 2015. The diagnosis was confirmed based on clinical presentation and elevated amylase and/or lipase levels >3X upper limit of normal. Data extraction included age, gender, type of analgesia provided, and time to first provision of analgesia in the ED.
Results: A total of 251 patients (89 (36%) male, median age 14 years (IQR 10–17)) were evaluated. Analgesia (opioid or non-opioid) was provided in 61% of encounters in the ED. Of these 71% were treated with either opioids alone or in combination with non-opioid analgesics, while 29% were treated exclusively with a non-opioid analgesic (p<0.001). In 2% of cases a non-opioid was given first followed by subsequent opioid analgesia. The median time to administration of analgesia was significantly shorter (p<0.001) for those who received an opioid first, 1.6 hours (IQR 1.1 – 2.5), compared to non-opioid, 2.6 hours (IQR 1.5 – 4.1).
Conclusion: Significantly more encounters of acute pancreatitis were treated with opioid analgesia, compared to non-opioids, in the ED. Given the increasing incidence of pediatric opioid abuse and dependence, future prospective studies are needed to establish best practice for opioid and non-opioid analgesia in acute pancreatitis.
Polymorphism of the Heme Oxygenase-1 (HO-1) Promoter and Cytokines Expression in Acute Pancreatitis
A.K. Gulla,1 A. Gulbinas,2 G. Barauskas,2 Z. Dambrauskas.2 1Department of Surgery, Georgetown University Hospital, Washington, DC; 2Department of Surgery, Lithuanian University of Health Sciences, Kaunas, Lithuania.
Introduction: Heme oxygenase-1 (HO-1) is an anti-oxidative, anti-inflammatory, and cytoprotective enzyme that is induced in response to cellular stress. The HO-1 promoter contains (GT)n dinucleotide repeats and is highly polymorphic. In this study we hypothesized that different expression of HO-1 determined by the number of GT repeats is associated with different expression of cytokines thus may predict severe onset and course of acute pancreatitis (AP).
Methods: AP patients (n= 40) and matched healthy controls (n= 28) were studied. Genomic DNA and mRNA were extracted from peripheral blood samples of patients and control groups. The HO-1 promoter region with the GT repeats was PCR amplified with fluorescent tagged primers, while mRNA expression by QRT-PCR. A short allele was defined as ≤ 27 GT repeats, whereas a long allele ≥ 27 repeats. Clinical data on study cohort was incorporated for the statistical analysis.
Results: The subjects were categorized into 3 groups based on the genotype results: one short and one long allele (S/L), two short alleles (S/S) and two long alleles (L/L). The presence of: S/L −42.3%, L/L- 47.2%, S/S- 10.5% was recorded in AP group. The levels of 15 different cytokines were measured and ninety percent of patients who were carriers of L/L genotype had increased levels of CD14 (p<0.003) levels compared to controls. These subjects were also at higher risk for developing severe acute pancreatitis.
Conclusion: The polymorphism of the GT repeats in the HO-1 promoter region, L/L genotype, may be a risk factor for altering cytokines and associated with severe course and outcome of acute pancreatitis.
Increased Caveolin-1 and Cholesterol Metabolism in CD133+ Tumor Initiating Cells Regulate Invasion and Chemoresistance in Pancreatic Cancer
V.K. Gupta, A. Nomura, P. Dauer, N.S. Sharma, V. Dudeja, A.K. Saluja, S. Banerjee. Department of Surgery, University of Miami, Miami, FL.
Introduction: Caveolins are integral membrane proteins of caveolae which play a major role in many biological processes that include cholesterol binding and cell signaling. Many of these processes and thus caveolin-1 contributes to cell transformation, tumorigenesis, and metastasis. In pancreatic cancer, Caveolin-1 has been associated with chemoresistance as well as aggressive biology. Interestingly, our results show that Caveolin-1 co-localizes with CD133+ tumor initiating cells in pancreatic cancer, and in concurrence with it had increased cholesterol content.
Aim: The present study investigates how association of CD133 with Caveolin-1 mediate chemo-resistance and aggression in Pancreatic Cancer.
Methods: Caveolin proteins expression were analysed in different cell lines by western blotting and QPCR. Detergent-free Purification of Caveolin-rich fractions was done by density gradient ultracentrifugation. CD133+ and CD133- cells were isolated by MACS sorting and a transcriptome analysis was done to identify differentially expressed genes.
Results: Analysis of expression of caveolin in pancreatic cancer cell lines showed a positive correlation with CD133 expression. Further, transcriptome analysis showed that CD133+ cells have around 1.5-fold higher expression of Caveolin genes compared to CD133- cells. Isolation of caveolae rich fraction by ultracentrifugation showed CD133 localizes to Caveolin-rich membrane fraction. In concurrence with increased expression of caveolin-1, CD133+ cells showed increased cholesterol cell content. In vitro treatment showed S2VP10 cell line more sensitive to Mevinolin, a Cholesterol synthesis inhibiting drug, compared to MiaPaCa-2 which have low CD133+ cell population.
Conclusion: Our results indicated that CD133+ cells have increased Caveolin-1 expression and accumulation of cholesterol which might play crucial role in their aggressive and chemoresistant phenotype.
A Systematic Review and Quantitative Analysis of Different Therapies for Pancreas Divisum
T. Hackert, M. Hafezi, B. Mayschak, P. Probst, M.W. Büchler, A. Mehrabi. Department of General, Visceral, and Transplantation Surgery, University of Heidelberg, Heidelberg, Germany.
Background: Pancreas divisum (PD) is the most common anatomical variant and the most frequent congenital anomaly of the pancreatic duct. Therapy of symptomatic PD depends on several parameters and includes different endoscopic intervention and various surgical procedures. The aim of this study was to summarize actual evidence of different treatment modalities.
Methods: A systematic literature search in MEDLINE and EMBASE was performed to identify all types of studies, investigating an endoscopic or surgical therapy for PD. An individual data simulation model was applied to compare endoscopic with surgical studies.
Result: A total of 56 observational studies were included in our analyses. Of these, 31 studies (1289 patients) investigated endoscopic and 25 studies (598 patients) surgical treatment. The patients were significantly younger in surgical group (p= < 0.0001) and most of them were female, especially in surgical group. Surgery was superior to endoscopic treatment in terms of success rate (72.5% vs. 62.3%; p<0.0001) and complication rate (23.8% vs. 31.3%; p<0.0001) as well as the re-intervention rate (10% vs. 28%, p<0.0001).
Conclusion: Based on this systematic review, surgical treatment may be superior to endoscopic treatment in terms of treatment success and complications. However, there is no study comparing surgical with endotherapy. A large, randomized trial with focus on comparing the endoscopic and surgical approach is urgently needed.
Extinction of ABCB5 Expression in Pancreatic Cancer Cells
T. Hank,1 M.W. Herbst,1 K. Hu,1 D. Maennle,1 A.S. Bauer,2 K. Felix,1 T. Hackert,1 N. Giese,1 O. Strobel.1 1Department of General, Visceral and Transplantation Surgery, University Hospital Heidelberg, Heidelberg, Germany; 2Department of Functional Genomics, German Cancer Research Centre (DKFZ), Heidelberg, Germany.
Background: Pancreatic ductal adenocarcinoma (PDAC) is an extremely aggressive, chemotherapy-resistant malignancy. The expression of a chemotherapeutics/xenobiotics exporter ABCB5 was found to mark therapy-refractory subpopulation of tumor cells conveying chemo-resistance in different cancer types.
Aim: To investigate relevance of ABCB5 in PDAC.
Methods: ABCB5 mRNA expression in pancreatic samples and tumor cell lines was studied using public databases, microarrays and qRT-PCR; protein expression - with 5 antibodies in Western blot, FACS and immunohistochemistry assays. Growth tests and FACS quantified responses to gemcitabine, 5-FU and doxorubicin in PDAC cells transfected with ABCB5 siRNA or plasmid.
Results: ABCB5 mRNA was nearly missing in all pancreatic biopsies and cell lines. Nevertheless, several antibodies detected abundant ABCB5-positivity of tumor cells in PDAC tissues, and fractional – in cultures. This immunopositivity, however, lacked sensitivity to siRNA- and plasmid-based deregulation, despite excessively overexpressed mRNA and its efficient reduction by siRNA. Neither down- nor over-expression of ABCB5 altered cellular growth, uptake or sensitivity to chemotherapeutics.
Conclusion: Together with dominant ABCB5-negativity of PDAC tissues and cell lines, missing protein expression in RNA-overexpressing cells indicated that ABCB5 presence might be detrimental for PDAC cells. Then, either cells could extinct ABCB5 at the level of translation or ABCB5 protein could instantly extinct the cells. Apparent immunopositivity, most probably, was non-specific, with available antibodies cross-reacting with other ABCB-transporters and not be trusted for ABCB5 characterization.
Nutritional Assessment of Dietary Intake in Chronic Pancreatitis Using a Web-Based Food Frequency Questionnaire
P. Hart, P. Madril, K. Roberts, D.L. Conwell, K. Crockett, M. Ramsey, M. Nahikian-Nelms. Gastroenterology, Hepatology and Nutrition, The Ohio State University Wexner Medical Center, Columbus, OH.
Background: Chronic pancreatitis (CP) is commonly associated with malnutrition, which is due to disease-related factors, such as coexistent diabetes mellitus, exocrine pancreatic insufficiency, and altered anatomy, and patient-related factors, including dietary intake. We performed a cross-sectional study to examine the dietary patterns of patients with CP using a web-based tool.
Methods: We recruited consecutive patients seen in our Pancreas clinic with a definitive diagnosis of CP from January-March 2016. The study used a web-based food frequency tool, VioScreen™, to assess dietary intake. This tool analyzes subjects’ responses to a series of 1,200 food pictures and portion sizes to estimate nutrient intake and food patterns for the preceding 90 days.
Results: A total of 27 subjects with CP completed the nutritional assessment during the study period. The median age in the cohort was 50 years (IQR, 38–59) and 19 (70.4%) subjects were male. One-third (n=9) of subjects had a history of pancreatic surgery. The median daily caloric intake compared to the recommended intake (based on ideal body weight (IBW)) was 72.6% (IQR, 45-121%), even though more than half of subjects (55.5%) weighed less than their IBW. Importantly, excessive intake of calories from high sugar foods occurred in 33.3% of subjects. The recommended consumption of fruits/vegetables, whole grains, fish, and low-fat dairy products were only achieved in 33.3%, 14.8%, 11.1%, 3.7%, respectively. The food intake of vitamins A, D, E, and K were inadequate (<100% of recommended dietary allowance) in 22.2%, 77.8%, 66.7%, and 70.4% of subjects respectively.
Conclusions: We demonstrate the feasibility of using a web-based tool to assess the dietary intake in patients with CP. These preliminary data identify multiple patterns of poor dietary intake including inadequate intake of calories, servings from healthy food groups, and fat-soluble vitamins. Suboptimal dietary intake is an important contributing factor to malnutrition, and requires further study to understand opportunities for nutritional interventions.
Neoadjuvant Chemotherapy for Pancreatic Cancer
E. Hashimoto, H. Shimamura, K. Takeda. Department of Surgery, Sendai Medical Center, Sendai, Japan.
Introduction: Development of chemotherapeutic drugs is gradually improving the prognosis of pancreatic cancer, though still on the way. While chemotherapy as the adjuvant setting seems to be established, neoadjuvant chemotherapy (NAC) remains to be exploited. Here we review our NAC cases, and discuss on the role of NAC.
Patients and Methods: Between October 2011 and May 2016, patients with pancreatic cancer (resectable or borderline resectable) who underwent NAC were recruited to this study. Transition of tumor markers after NAC, operability or clinical outcomes of these patients were retrospectively reviewed.
Results: Ten patients (5 males and 5 females, average age: 69.2) were enrolled. Before NAC, tumors of 7 patients were radiologically classified into borderline resectable cases, whereas the others resectable cases. Gemcitabine plus S-1 were intended to administer to all patients. But, 2 patients chose S-1 alone administration. After NAC, 6 patients could achieve curative resection, whereas 3 patients underwent palliative operation. We still continue NAC for one patient with higher tumor markers, which are gradually decreasing. Decreasing rate of CA19-9 in resected cases was much higher than that in palliative cases. Histological examinations of the resected specimens revealed pathological complete response in 2 cases.
Conclusion: NAC using gemcitabine plus S-1 or S-1 alone may be effective for patients with pancreatic cancer, especially in cases which tumor markers promptly decrease after NAC.
Minimally Invasive Versus Open Necrosectomy for Necrotizing Pancreatitis
R.A. Hollemans,1 S. Van Brunschot,2 O. Bakker,3 M.G. Besselink,1 T.H. Baron,4 H.G. Beger,5 M.A. Boermeester,1 T.L. Bollen,6 M.J. Bruno,7 R. Carter,8 R. Charnley,9 D. Coelho,10 B. Dahl,11 M.G. Dijkgraaf,12 N. Doctor,13 G. Farkas,14 P.J. Fagenholz,15 C. Fernández-Del Castillo,16 P. Fockens,17 M.L. Freeman,18 T.B. Gardner,19 H. Van Goor,20 H.G. Gooszen,21 G. Hannink,22 R. Lochan,23 C.J. McKay,24 M.P. Peev,25 J.P. Neoptolemos,26 A. Oláh,27 R.W. Parks,28 M. Raraty,29 B. Rau,30 T. Rösch,31 M. Rovers,22 H. Seifert,32 A.K. Siriwardena,33 K.D. Horvath,34 H.C. Van Santvoort.35 1Department of Surgery, Academic Medical Center, Amsterdam, Netherlands; 2Gastroenterology, Academic Medical Center, Amsterdam, Netherlands; 3Surgery, University Medical Center Utrecht, Utrecht, Netherlands; 4Gastroenterology and Hepatology, University of North Carolina, Chapel Hill, NC; 5Surgery, University of Ulm, Ulm, Germany; 6Radiology, St. Antonius Hospital, Utrecht, Netherlands; 7Gastroenterology and Hepatology, Erasmus University Medical Center, Rotterdam, Netherlands; 8Glasgow Royal Infirmary, Glasgow, United Kingdom; 9Department of HPB Surgery, Newcastle upon Tyne Hospitals, Newcastle Upon Tyne, United Kingdom; 10Hospital Clementino Fraga Filho, Rio De Janeiro, Brazil; 11Department of Internal Medicine, Oldenburg Municipal Hospital, Oldenburg, Germany; 12Clinical Research Unit, Academic Medical Center, Amsterdam, Netherlands; 13Surgery, Jaslok Hospital and Research Center, Mumbai, India; 14University of Szeged, Szeged, Hungary; 15Massachusetts General Hospital, Boston, MA; 16Department of Surgery, Massachusetts General Hospital, Boston, MA; 17Academic Medical Center Amsterdam, Netherlands; 18Gastroenterology, University of Minnesota, Minneapolis, MN; 19Gastroenterology and Hepatology, Dartmouth-Hitchcock Medical Center, Lebanon, NH; 20Surgery, Radboud University Medical Center, Nijmegen, Netherlands; 21Operating Rooms - Evidence Based Surgery, Radboud University Medical Center, Nijmegen, Netherlands; 22Radboud Institute for Health Sciences, Radboud University Medical Center, Nijmegen, Netherlands; 23Surgery, Freeman Hospital, Newcastle Upon Tyne, United Kingdom; 24Surgery, Glasgow Royal Infirmary, Glasgow, United Kingdom; 25Surgery, Massachusetts General Hospital, Boston, MA; 26National Institutes of Health Research Liverpool Pancreas Biomedical Research Unit, Royal Liverpool and Broadgreen University Hospitals, Liverpool, United Kingdom; 27Surgery, Petz-Aladár Teaching Hospital, Györ, Hungary; 28Surgery, University of Edinburgh, Edinburgh, United Kingdom; 29Surgery, Royal Liverpool and Broadgreen University Hospitals, Liverpool, United Kingdom; 30Surgery, University of Rostock, Rostock, Germany; 31Interdisciplinary Endoscopy, University Hospital Hamburg-Eppendorf, Hamburg, Germany; 32Internal Medicine, Oldenburg Municipal Hospital, Oldenburg, Germany; 33Surgery, Manchester Royal Infirmary, Manchester, United Kingdom; 34Surgery, University of Washington, Seattle, WA; 35Surgery, St. Antonius Hospital, Nieuwegein, Netherlands.
Background: Minimally invasive necrosectomy compared with open necrosectomy might improve outcomes in patients with necrotizing pancreatitis, especially in critically ill patients. Evidence from comparative studies with large sample sizes to allow evaluation of death as primary outcome and to adjust for potential confounding factors is lacking.
Methods: We combined individual patient data from 15 published and unpublished cohorts on pancreatic necrosectomy for necrotizing pancreatitis in 65 hospitals in 8 countries. Death rates were compared in patients undergoing open necrosectomy or minimally invasive necrosectomy (i.e. minimally invasive surgical or endoscopic necrosectomy). We adjusted for confounding by three types of analyses: logistic regression, stratification according to the predicted risk of death at baseline (low: <5%, intermediate: ≥5% to <15%, high: ≥15% to <35%, and very-high: ≥35%), and propensity-score matching.
Results: Among 1980 patients with necrotizing pancreatitis, 1167 underwent open necrosectomy, 467 underwent minimally invasive surgical necrosectomy, and 346 underwent endoscopic necrosectomy. There was a lower risk of death for minimally invasive surgical necrosectomy (odds ratio, 0.53; 95%-CI, 0.34 to 0.84; P=0.006) and endoscopic necrosectomy (odds ratio, 0.19; 95%-CI, 0.06 to 0.61; P=0.005). After risk stratification and propensity-score matching, minimally invasive surgical necrosectomy remained associated with a lower risk of death than open necrosectomy in the very-high-risk group (risk ratio, 0.70; 95%-confidence interval, 0.52 to 0.95; P=0.02). Endoscopic necrosectomy was associated with a lower risk of death than open necrosectomy in the high-risk group (risk ratio, 0.27; 95%-CI, 0.08 to 0.88; P=0.03) and the very-high-risk group (risk ratio, 0.43; 95%-CI, 0.24 to 0.77; P=0.005).
Conclusion: In high-risk patients with necrotizing pancreatitis, minimally invasive surgical and endoscopic necrosectomy reduced death rates compared with open necrosectomy.
Node-Negative Disease in Pancreatic Cancer Might Not Present Different Tumor Biology
K.C. Honselmann, I. Pergolini, C. Fernández-Del Castillo, A.L. Warshaw, K.D. Lillemoe, C.R. Ferrone. Department of Surgery, Massachusetts General Hospital, Boston, MA.
Background: Patterns of recurrence for pancreatic adenocarcinoma (PDAC) have been poorly characterized. Our aim was to evaluate the recurrence patterns of patients with negative lymph node status (pN0) after resection, as compared to patients with positive lymph nodes (N1). We hypothesized that patients with N0 disease would be more likely to first recur locally than patients with positive lymph nodes, possibly indicating differing tumor biology and offering a therapeutic window.
Methods: We reviewed 622 patients with resected exocrine pancreatic cancer without neoadjuvant treatment between 1985 and 2011. Follow up, thus far, has been confirmed for 266 patients.
Results: The 266 patients had a median age of 67 years. The median follow–up was 20 (10–43) months. Almost two thirds of patients (63%) were lymph node positive (pN1) on pathology. Tumor recurrence and time to recurrence were significantly more common and shorter in N1 patients as compared to N0 patients, (75% vs. 56%, p=0.001 and 10 months vs. 34 months, p<0.001). As first site of recurrence distant metastasis was found in 49% of patients (40% of pN0 and 54% of pN1), local recurrence in a total of 19% (17% of pN0 and 21% of pN1, p=0.8). Thus, location of recurrence did not differ between the two groups.
Conclusion: Patterns of recurrence for N0 vs. N1 patients did not differ. Time to recurrence was significantly longer for N0 patients. This may indicate that tumor biology is similar, but that N0 disease is identified earlier on the curve.
Pancreatic Enzyme Replacement Therapy in Chronic Pancreatitis: Systematic Review and Meta-Analysis
W. Huang,1, 2 D. Iglesia-Garcia,1, 3 P. Szatmary,1 I. Baston-Rey,3 J. Gonzalez-Lopez,4 G. Prada-Ramallal,5 A. Sud,1 R. Mukherjee,1 Q.M. Nunes,1 J.E. Domínguez-Muñoz,3 R. Sutton.1 1NIHR Liverpool Pancreas Biomedical Research Unit, Royal Liverpool University Hospital NHS Trust, University of Liverpool, Liverpool, United Kingdom; 2Sichuan Provincial Pancreatitis Centre, Department of Integrated Traditional Chinese and Western Medicine, West China Hospital, Sichuan University, Chengdu, China; Departments of 3Gastroenterology and Hepatology; and 4Pharmacy, University Hospital of Santiago de Compostela, Santiago de Compostela, Spain; 5Department of Preventive Medicine and Public Health, University of Santiago de Compostela, Santiago de Compostela, Spain.
Background & Aims: The magnitude of benefit from pancreatic enzyme replacement therapy (PERT) in chronic pancreatitis (CP) is unclear. We sought to conduct a meta-analysis of randomised controlled trials (RCTs) of PERT in exocrine pancreatic insufficiency (EPI) from CP.
Methods: We searched databases of studies from 1996–2015 for RCTs of PERT for EPI in CP. Seventeen studies were assessed using coefficient of fat absorption (CFA) and other parameters to determine the effects of PERT vs baseline or placebo, different doses, formulations and administration schedules.
Results: PERT improved CFA vs baseline (83.7±6.0 vs 63.1±15.0, P<0.00001; I 2=89%) and placebo (83.2±5.5 vs 67.4 ± 7.0, P=0.0001; I 2=86%). PERT improved the coefficient of nitrogen absorption vs baseline (P=0.001) and significantly reduced fecal fat excretion, fecal nitrogen excretion, fecal weight and abdominal pain vs baseline and placebo (all P<0.05). Follow up studies demonstrated PERT increased serum nutritional parameters, improved gastrointestinal symptoms and quality of life without significant adverse events. High dose or enteric-coated enzymes showed a trend to be more effective. Subgroup, sensitive and meta-regression analyses revealed that sample size, CP diagnostic criteria, study design and enzyme dose contributed to study heterogeneity. Although health inequalities have major impacts on CP, there were insufficient data to determine their magnitude.
Conclusion: PERT is indicated to improve nutrition in CP and may be optimised by higher doses, enteric coating, administration during food and acid suppression. Further studies are required to address the impact of PERT on complications and mortality from CP, and of health inequalities.
Early Drain Fluid Lipase as Predictor of Postoperative Pancreatic Fistula After Pancreatic Resection: A Prospective Pilot Study
T. Ingkakul, C. Pumpuang, A. Thienhiran, S. Hongjinda. Department of Surgery, Phramongkutklao Hospital, Bangkok, Thailand.
Background: Postoperative pancreatic fistula (POPF) is a common problem after pancreatic resection. Recent reports indicate early drain fluid amylase levels can predict POPF which benefit early drain removal but drain fluid lipase level is infrequently reported.
Aim: To determine drain fluid lipase level on postoperative day 1 (DFL1) as predictor of POPF.
Materials and methods: We prospectively analyzed 31 consecutive pancreatic resections performed in Phramongkutklao Hospital between June 2014 and June 2016. Twenty-six patients were included in this study and 5 patients were excluded due to incomplete data. The postoperative amylase and lipase levels in both serum and drain fluid were recorded. The diagnosis and grades of POPF were defined according to the International Study Group of Pancreatic Fistula (ISGPF). The correlation of drain fluid lipase and amylase levels were analyzed to predict POPF.
Results: Of 26 patients with pancreatic resection, 22 (84.6%) presented with postoperative pancreatic fistula. Majority of patients with POPF were grade A (18, 69.2%) followed by grade B (3, 11.5%) and grade C (1, 3.8%). A receiver operating characteristic (ROC) analysis was conducted to determine the threshold value of DFL1 associated with clinically significant POPF [AUC (95%CI)=0.981 (95%CI: 0.923 - 1.000), P=0.005]. A DFL1 cutoff value of 4356 U/L showed the best accuracy (94.1%), specificity (100%), sensitivity (92.3%), positive predictive value (100%) and negative predictive value (80%) which is similar to drain fluid amylase on postoperative day 1 (DFA1) cutoff value of 798 U/L. Both serum lipase and amylase levels on postoperative day 1 were not significantly associated with POPF.
Conclusion: Early DFL1 is useful to predict POPF with the same efficacy of DFA1.
Correlation of a Long Non-Coding RNA, H19, With Metastasis of Pancreatic Cancer
T. Ishiwata,1 H. Yoshimura,2 Y. Matsuda,3 N. Ishikawa,1 K. Takubo,1 T. Arai,3 J. Aida.1 1Department of Aging and Carcinogenesis, Research Team for Geriatric Pathology, Tokyo Metropolitan Institute of Gerontology, Tokyo, Japan; 2Division of Physiological Pathology, Department of Applied Science, School of Veterinary Nursing and Technology, Nippon Veterinary and Life Science University, Tokyo, Japan; 3Department of Pathology, Tokyo Metropolitan Geriatric Hospital, Tokyo, Japan.
Long non-coding RNAs (lncRNAs) comprise mRNA-like transcripts longer than 200 ribonucleotides and lack significant open reading frames. H19 is an imprinted lncRNA transcribed exclusively from the maternal allele and is an oncofetal RNA expressed in developing embryos and in tumors including bladder, breast, gastric, pancreatic, hepatocellular, and prostate cancers. Studies have shown that H19 enhances cancer invasion and metastasis; however, its role in cancer remains debatable. In this study, using a mouse implantation model, we found that 11 genes exhibited >10-fold increase in expression levels in lung metastasis-derived human pancreatic cancer cells than in parental cells. H19, the only non-protein coding gene, showed an 82.4-fold increase in expression levels —the second highest among the 11 genes—in metastasis-derived cancer cells. We determined the functions of H19 in pancreatic cancer development and metastasis by using in vitro and in vivo techniques. In situ hybridization analysis using tissue microarrays showed that H19 was detected in 17% (23/139) of invasive ductal carcinomas, and its expression positively correlated with higher histological tumor grades (P < 0.0001). Overexpression of H19 in PANC-1 pancreatic cancer cells induced higher motilities, whereas H19 inhibition using shRNA and siRNA showed opposite results; however, cell growth rates were unaffected. Intravenous injection of H19 shRNA vector-transfected PANC-1 cells markedly inhibited metastasis in the liver and lungs of immunodeficient mice. These findings suggest that H19 is correlated with pancreatic cancer metastasis and inhibition of H19 may have novel therapeutic potential for treating pancreatic cancer.
Treatment Strategy for Neuroendocrine Tumor of the Pancreas
J. Itakura, M. Watanabe, N. Hosomura, H. Amemiya, H. Kawaida, H. Okamoto, H. Kohno. Department of Surgery, University of Yamanashi, Yamanashi, Japan.
Background: According to progress in diagnostic imaging and EUS-FNA, patients of p-NET have increased in recent years. Moreover, the comparison of pathological classification and clinical course is important to establish the surgical adaptation and methods. In this study we analyze this point by referring p-NET cases resected in our institution.
Method: A WHO classification of 2010 and clinical findings were examined for 53 cases which underwent resection in our department from January, 2000 to May, 2016.
[Result] These 53 cases consist of 27 males and 26 female. The average age was 60.3 years old (33–81). The location of tumor was 18 on heads and 34 on body/tail and one had multiple lesions. 39 cases were G1 grade and 14 cases were G2 grade as WHO classification. The average diameter of the G1 cases was 13.7 mm, and that of the G2 cases was 27.5 mm. The average MIB-1 index of the G1 cases was 1.6%, and that of the G2 cases was 5.6%. Of these 53 cases 4 cases indicated concomitant metastasis and 2 cases had recurrence. Two of these six cases were diagnosed as MEN-1 of G1 grade and the other 4 cases were G2 grade. Two of the four G2 cases had liver metastasis concomitantly, and the other one had local recurrence two years after surgery, and the last one had lymph node metastasis and liver metastasis three years after surgery. The average diameter and MIB-1 index of the G2 cases which had metastasis or recurrence was 56.3mm and 7.5%, while that of the G2 cases which had no metastasis or recurrence was 16mm and 4.9%. ROC analysis indicated that the best cut-off size to which divides metastasis and non-metastasis is 19 mm. Two cases of concomitant liver metastasis died of hepatic failure, and 2 cases of MEN-1 and 2 cases of recurrence survived.
Conclusion: The standard operation with lymph node dissection is necessary for cases of over 20mm in diameter. Sufficient observation is necessary for cases of G2 grade. Minimal operation might be in consideration for cases of G1 grade with less than 20 mm in diameter.
Risk Factors for Pancreatic Stone Formation in Type 1 Autoimmune Pancreatitis: A Nationwide Survey by the Japan Pancreas Society
T. Ito,1 S. Kawa,2 K. Kubota,3 T. Kamisawa,4 K. Okazaki,5 T. Shimosegawa.6 1Gastroenterology, Shinshu University School of Medicine, Matsumoto, Japan; 2Center for Health, Safety, and Environmental Management, Shinshu University, Matsumoto, Japan; 3Gastroenterology, Yokohama City University Graduate School of Medicine, Yokohama, Japan; 4Gastroenterology, Tokyo Metropolitan Komagome Hospital, Tokyo, Japan; 5Gastroenterology, Kansai Medical University, Osaka, Japan; 6Gastroenterology, Tohoku University Graduate School of Medicine, Sendai, Japan.
Background and Aim: As autoimmune pancreatitis (AIP) has the potential to cause pancreatic stone formation, this investigation sought to clarify its risk factors.
Methods: Questionnaires on patients with type 1 AIP were sent to 21 high-volume medical centers across Japan to compare the clinical features of patients with pancreatic stone formation to those without.
Results: Of the completed records of 624 AIP patients (477 male median age [range]: 67 [38–76] years), 31 (5%) had experienced pancreatic stone formation (24 male) and 593 had not (453 male). Although there were no remarkable differences in serology results between the groups, BT-PABA findings were significantly lower and HbA1c was significantly higher in patients with pancreatic stone formation (p=0.04 and p=0.006, respectively). Moreover, imaging results disclosed pancreatic atrophy and stenosis of the hilar or intrahepatic bile ducts significantly more frequently in patients with pancreatic stones (p=0.001 and p=0.002, respectively). Pancreatic head swelling tended to be more frequent in this group as well (p=0.07). Neither prednisolone discontinuation nor recurrence was associated with stone formation. In multivariate analysis, follow-up period (OR: 2.62, p=0.04), pancreatic atrophy (OR: 2.44, p=0.04), stenosis of the hilar or intrahepatic bile ducts (OR: 2.65, p=0.03), and pancreatic head swelling (OR: 2.65, p=0.03) were independently associated with pancreatic stone formation.
Conclusion: The increased frequency of pancreatic head swelling in AIP patients experiencing pancreatic stones indicated a propensity for pancreatic juice stasis and subsequent stone development. The association of hilar or intrahepatic bile duct stenosis with stone formation in AIP requires further investigation.
Susceptibility Genes of Murine Autoimmune Pancreatitis
R. Jaster,1 S. Müller,1 L. Borufka,1 J. Bischof,2 Y. Gupta,2 F. Asghari,1, 2 S. Möller,2 H. Nizze,3 S. Ibrahim.2 1Department of Medicine, Division of Gastroenterology, Rostock University Medical Center, Rostock, Germany; 2Institute of Experimental Dermatology, University of Luebeck, Lubeck, Germany; 3Institute of Pathology, Rostock University Medical Center, Rostock, Germany.
Introduction: The specific genetic risk factors for autoimmune pancreatitis (AIP) are largely unknown. To study them in a mouse model, we have established a four-way autoimmunity advanced intercross line that originated from AIP-susceptible MRL/MpJ mice, the AIP-resistant strain CAST/EiJ as well as two further strains, BXD2/TyJ (susceptible to collagen- induced arthritis) and NZM2410/J (a model of lupus erythematosus). This concept was chosen to identify both general autoimmune disease associated genes and AIP-specific risk factors in parallel.
Aims: The goal of this study was to decipher quantitative trait loci (QTLs) of murine AIP and to fine-map them for the identification of susceptibility genes of the disease.
Methods: Outbred intercross mice of the generations G4 and ≥ G15 were phenotypically characterized by scoring histopathological changes of the pancreas and by flow cytometric analysis of splenocytes. Genotyping was performed employing single nucleotide polymorphism (SNP) arrays. Data were analysed with the R implementation of HAPPY and the DOQTL R package.
Results: At the stage of G4, five AIP-associated QTLs that partially overlapped with QTLs of other autoimmune diseases were mapped on chromosomes 4 (n=2), 2, 5, and 6. A fine-mapping of these QTLs was initiated at the stage of G15, when the reassortment of the parental genomes was completed. Using a SNP array with more than 77,000 SNPs, we could narrow down the confidence intervals of four QTLs (including a new QTL on chromosome 8) to less than 1 Mbp and the number of promising candidate genes to less than 20. The susceptibility genes mainly include regulators of immune cell activity, cell adhesion and growth factor signaling. Flow cytometric and genetic studies showed that the relative frequency of CD4+/CD44high memory T cells not only correlated with the severity of AIP, but was also controlled by overlapping QTLs.
Conclusion: Murine AIP is influenced by multiple genetic traits. To gain mechanistic insights into the pathogenesis of AIP, we suggest studies on the function of the newly identified murine candidate genes.
Da-Cheng-Qi Extract Identification and Therapeutic Potential in Experimental Acute Pancreatitis
T. Jin,1 D. Du,2 N. Shi,1 R. Zhang,3 Q.M. Nunes,4 M. Chvanov,5 D.N. Criddle,5 W. Huang,4 R. Sutton,4 Q. Xia.1 1Department of Integrated Traditional Chinese and Western Medicine, Sichuan Provincial Pancreatitis Center; 2West China-Washington Mitochondria and Metabolism Center; and 3Laboratory of Ethnopharmacology; West China Hospital, Sichuan University, Chengdu, China; 4NIHR Liverpool Pancreas Biomedical Research Unit, Royal Liverpool University Hospital NHS Trust; and 5Department of Cellular and Molecular Physiology; University of Liverpool, Liverpool, United Kingdom.
Background & Aims: Da-Cheng-Qi decoction (DCQD) ameliorates experimental acute pancreatitis (AP). We sought to determine the active ingredients.
Methods: DCQD aqueous (AE) and chloroform extracts (CE) were separated using chloroform after acetone precipitation and centrifugation. Isolated murine pancreatic acinar cells were exposed to taurolithocholic acid 3-sulfate (TLCS, 500μM) or caerulein (10nM) with or without AE or CE (5-500μg/ml) and cell fate determined with propidium iodide or caspase 3/7 substrate fluorescence over 12h. Mice received retrograde ductal TLCS (3mM) or seven hourly ip injections of caerulein (50μg/kg) to induce AP, with or without gavage of AE or CE (equivalent to 20g/kg DCQD, 2 hourly for 3 times) after AP induction. Severity of AP was assessed by biochemistry, immunology and histopathology. Ingredients in DQCD, AE and CE were analysed by UPLC-qTOF/MS/MS.
Results: AE, but not CE, reduced TLCS- and caerulein-induced necrotic (maximally at 50μg/ml) and TLCS-induced apoptotic cell death pathway activation, and consistently reduced pancreatic myeloperoxidase and histopathology scores in both EAP models (P<0.05). UNIFY analysis found nearly 3796, 1266 and 501 peaks in DCQD, AE and CE, respectively. The representative peaks in the mass spectrum of DCQD and deprotonated molecular ions suggested gallic acid, catechin, naringin, hesperidin, emodin-8-O-β-D-glucopyranoside, chrysophanol, emodin, and magnolol, respectively. The representative components in AE were gallic acid, catechin and naringin (tR=1.62, 3.21 and 5.15), and in CE this was emodin glycosides (tR=10.14).
Conclusion: AE of DCQD significantly ameliorates EAP, which may be due to gallic acid, catechin and/or naringin. Further work is required to identify the underlying mechanism of action of DCQD.
Enhancer of Zeste Homologue 2 is a Key Regulator of Acinar to Duct Cell Metaplasia
C. Johnson,1 L. Yu,2 L. Luyt,2,3 R. Urrutia,4 G. Lomberk,5 C. Pin.1,2,6 1Paediatrics, University of Western Ontario, London, Canada; 2Oncology, University of Western Ontario, London, Canada; 3Chemistry, University of Western Ontario, London, Canada; 4Biochemistry and Molecular Biology, Mayo Clinic College of Medicine, Rochester, MN; 5Medicine, Mayo Clinic College of Medicine, Rochester, MN; 6Physiology and Pharmacology, University of Western Ontario, London, Canada.
Enhancer of zeste homologue 2 (EZH2) is a component of the Polycomb Repressor Complex 2, responsible for trimethylation of H3K27. EZH2 is often expressed to high levels in pancreatic cancer cell lines and patient samples, correlating to poor outcome survival. However, targeted deletion of Ezh2 in pancreatic development promotes increased sensitivity to oncogenic KRAS suggesting a protective role for EZH2 in early pancreatic ductal adenocarcinoma (PDAC) progression. The objective of this study was to determine if EZH2 affects acinar to ductal metaplasia (ADM), and early events in PDAC.
Acinar cells were isolated from wild type (WT) mice, mice susceptible to ADM (Mist1 −/− and Mist1 creERT -LSLKRAS G12D), or mice with an inducible deletion of exons 14–16 in the Ezh2 gene (Ezh2flx/flx), and cultured in a collagen matrix for up to 9 days. In some instances, cultures were treated with EZH2 inhibitors UNC1999 or EPZ005687. The formation of ADM and gene expression were assessed over the course of the culture.
As previously reported, acinar cells from Mist1 −/− and Mist1 creERT -LSLKRAS G12D mice showed increased ADM in culture. Similar increases in ADM were also observed in Ezh2 flx/flx cultures. The combined loss of Ezh2 and Mist1 with oncogenic KRAS led to significantly faster progression through ADM than any individual combination. Surprisingly, treatment with the two EZH2 inhibitors produced different outcomes. EPZ005687 treatment increased ADM from WT cultures while UNC1999 treatment decreased ADM formed in the Mist1 −/− cultures.
The results suggest that EZH2 reduces ADM both in vivo and ex vivo, and may be protective against oncogenic KRAS early in the progression to PDAC. Future directions will be directed towards identifying the targets and role of EZH2 in this process.
Downsizing Chemotherapy for Locally Advanced Pancreatic Cancer Treated With Nab-Paclitaxel Plus Gemcitabine Followed by Radical Surgery: Toxicity and Clinical Outcome
S. Kagawa, H. Yoshitomi, H. Shimizu, M. Ohtsuka, K. Furukawa, T. Takayashiki, S. Takano, S. Kuboki, D. Suzuki, N. Sakai, H. Nojima, M. Miyazaki. Department of General Surgery, Chiba University, Chiba, Japan.
Background: In recent years, advantages of neoadjuvant chemotherapy for pancreatic cancer are reported. Furthermore, for initially unresectable cases, downsizing chemotherapy and subsequent surgical resection has been reported as an effective new approach compared with non-surgical therapy. We now report the safety and efficacy of the downsizing chemotherapy with nab-paclitaxel (nab-PTX) plus gemcitabine (GEM) combination and subsequent radical operation in cases of the initially unresectable (UR) or borderline resectable (BR) locally advanced pancreatic cancer with no distant metastasis.
Methods: 17 UR or BR locally advanced pancreatic cancer patients were treated with nab-PTX plus GEM. The resectability of the tumor was evaluated after two courses of treatment. Surgical resection was carried out for those determined to be curative after restaging. If it still unresectable, continued treatment.
Results: Tumor was significantly downsized in 11 patients. In these patients, tumor markers were decreased in 10 cases and SUV max levels of PET-CT were down in 8 of all examined cases. RECIST criteria showed PR in 5 and SD in 6 cases. Radical surgery was performed for 10 patients and palliative surgery was done for one patient, for liver metastasis. The median duration of treatment and frequency were 8 weeks and 6 times. Relative dose intensities of nab-PTX and GEM were 72% and 73% respectively. Grade 3/4 adverse events were occurred by 4 leukopenia (36%), 4 neutropenia (36%). Of these, R0 resection was performed in 9 cases and R1 in one case.
Conclusion: Preoperative nab-PTX plus GEM was safely carried out by appropriate management with satisfactory resection rate. A longer observation period should be required to accept the availability of this strategy.
Wrapping of Pancreaticojejunostomy With PGA Mesh Could Prevent the Pancreatic Fistula After Pancreatoduodenectomy
J.S. Kang, Y. Han, J.-Y. Jang, H. Kim, J.R. Kim, W. Kwon, S.-W. Kim. Department of Surgery and Cancer Research Institute, Seoul National University College of Medicine, Seoul, Korea.
Background & aim: The postoperative pancreatic fistula (POPF) after pancreatic surgery is still most serious complication, occuring in 5~40%.[JJ1] Recently, wrapping the polyethylene glycolic acid (PGA) mesh around the anastomotic site was developed to reinforce the pancreaticojejunostomy in pancreatoduodenectomy (PD) with favorable outcome in some small scaled study[JJ2]. The purpose of this study was to investigate the efficacy of PGA mesh for reducing POPF and to evaluate the other risk factors for POPF.
Methods: Between 2006 and 2015, 464 consecutive patients who underwent pancreatoduodenectomy with the identical surgical procedure by single surgeon were enrolled in this study. All pancreatico-enteric anastomosis was performed by double-layered, duct-to-mucosa, end-to-side pancreaticojejunostomy. PGA group consisted of 281 patients (60.6 %), and control group consisted of 183 patients (39.4 %). The effects of the PGA mesh for preventing the POPF and the risk factors for POPF were investigated. To reduce the effect of selection bias, propensity score analysis was performed.
Results: Mean age was 63.1 years and male to female ratio was 1.7:1. The overall POPF occurred in 144 (31.0%) patients and the clinically relevant POPF occurred in 83 (17.9 %) patients. The incidence of overall POPF in the PGA group was statistically significantly lower than that in the control group (27.0 vs. 37.2 %, p=0.024) and the incidence of clinically relevant POPF (Grade B, C) in the PGA group was lower than that in the control group (13.9 vs. 24.0 %, p=0.006). The incidence of clinically relevant POPF (12.6 vs. 22.4 %, p=0.024) and complications (40.2 vs. 63.8 %, p<0.001) were significantly lower in the PGA group than in the control group even after propensity score matching.
Conclusions: The reinforcement of pancreaticojejunostomy with applying the PGA mesh could prevent pancreatic fistula with lessened overall abdominal complication after pancreatoduodenectomy.
A Case Presentation of Distal Bile Duct Adenocarcinoma: Distinguishing Between Cholangiocarcinoma and Pancreatic Ductal Adenocarcinoma
H. Karasaki, Y. Mizukami, Y. Ono, M. Ogata, D. Yoshikawa, T. Maejima, K. Nagashima, T. Kono. Center for Clinical and Biomedical Research, Sapporo Higashi Tokushukai Hospital, Sapporo, Japan.
Distinguishing between distal cholangiocarcinoma and ductal adenocarcinoma of the pancreatic head is occasionally challenging due to histological similarities and the close anatomical location. Although it may not affect clinical management, it represents an important biological question regarding the entity of the tumor.
Here we describe a case of a 70-year-old man who developed adenocarcinoma primarily located in the distal bile duct. The tumor was a moderately differentiated adenocarcinoma with mutant KRAS, and it completely occluded the distal bile duct and invaded into the pancreatic head with modest superficial spread within bile duct. Multiple pancreatic cysts were evident and the majority were lined with flat/non-papillary epithelium positive for MUC5AC and MUC6, suggesting a neoplastic phenotype rather than retention cysts. Additionally, high-grade PanINs were also demonstrated in intimately associated areas of the resected pancreas. The presence of these multiple lesions with distinct phenotypes supports the presence of a field defect, which is commonly observed in intraductal papillary mucinous neoplasms (IPMN) of the pancreas. Therefore, the lesion in the distal bile duct in the current case may have originated from the "field" generated in the pancreatic tissue. Alternatively, given the similarity of embryological paths during the development of the gastrointestinal system, it is also possible that these tumors may have arisen from a common precursor cell. Genetic and molecular profiling can provide new insights regarding differences and similarities of pancreatobiliary tumors
Candidate Gene Analysis of Russian Pancreatic-Cancer Patients
A.A. Kashintcev,1 E. Imyanitov,2 N. Kokhanenko.1 Departments of 1Surgery; and 2Medical Genetics, Saint-Petersburg State Pediatric Medical University, St. Petersburg, Russia.
Introduction: There are little information about genetic features and target therapy in Russian population with pancreatic cancer.
Methods and results: We had analyzed 253 patients with PC for verification of predisposition genes in two directions. The first was an estimation of KRAS and EGFR status in tissue samples (n-73). In the second part of our study we had analyzed in blood samples (n-178) founder disease-predisposing alleles in BRCA1, BRCA2, PALB2, CHEK2, NBN/NBS1, BLM, genes which play role in DNA repairing and associated with hereditary disease. These genes are characterized by little number of alleles in Eastern Europe population. We hadn’t found positive EGFR mutation, and only 28 carriers (37,3%) positive KRAS in tissue samples. The analysis of BRCA15382insC allele was positive in 2, BRCA2 5197_5198delTC in 1 case and all of them had a familial history. Another candidate gene, associated with BRCA was positive in 2/174 for NBN/NBS1 657del5, in 5/176 for CHEK2. In the last cohort hereditary disease predisposition was absent.
Discussion: Furthermore, this investigation revealed that EGFR and KRAS pathways meet not so frequently in Russian population. Analysis of genes, associated with DNA double-strand repairing may require further consideration.
Risk Factor of Postoperative Pancreatic Fistula After Distal Pancreatectomy Using Triple-Row Stapler
H. Kawaida, M. Watanabe, N. Hosomura, H. Amemiya, H. Kono, M. Matsuda, H. Fujii. First Department of Surgery, University of Yamanashi, Chyuo-shi, Yamanashi, Japan.
Purpose: Postoperative Pancreatic Fistula (POPF) is one of the major complication in patients undergoing distal pancreatectomy (DP). Recently, dividing the pancreas by stapler is common technique but POPF still remains. Therefore, it is important to evaluate risk factors of this complication. The purpose of this study was to determine the risk factors for POPF after DP using triple-row stapler.
Methods: A total of 50 patients underwent DP using triple-row stapler at Yamanashi University between December 2012 and February 2016. The thickness of the pancreatic cutting line was measured by intraoperative ultrasonography and the cartridge height of the stapler was selected according to the thickness. The closure jaw was clamped slowly and carefully and stapler was not released immediately after firing. Risk factors for POPF after DP using triple-row stapler were identified based on univariate and multivariate analyses.
Results: Clinical POPF (ISGPF grade B and C) leaks were seen in 4 of 50 patients (8.0%). Body mass index (BMI) was significantly higher in patients (26.6±0.5kg/m2) with POPF in comparison to patients without POPF (21.2±0.6 kg/m2). A cut-off value for BMI was established as 25.7 kg/m2 based on the receiver operating characteristic(ROC) curve. Additionally, patients that had the POPF was significantly younger than the patients who did not had it (53.0±8.8 vs 67.1±1.9).The Cut-off age was 55 years old. Pancreatic thickness was not a risk factor for POPF in this study.
Conclusion: BMI and younger age was significant risk factors for clinical Postoperative Pancreatic Fistula after DP using triple-row stapler.
Spleen-Preserving Distal Pancreatectomy
T. Kawana, H. Shimamura, K. Takeda. Department of Surgery, Sendai Medical Center, Sendai, Japan.
Introduction: When distal pancreatectomy is performed for benign diseases, preservation of spleen may be favorable in terms of immunity. Spleen-preserving distal pancreatectomy (SpDP) with or without ligation of splenic vessels remains to be discussed. Here we review our SpDP cases as a single institute experience.
Patients and Methods: Between March 2001 and April 2016, patients who underwent SpDP were recruited. Operative outcomes and follow-up results of these patients were retrospectively reviewed.
Results: Fifteen of 111 patients who underwent distal pancreatectomy were enrolled as SpDP cases. Average age was 55.2, and 10 of them were female. Diseases of the patients were pancreatic neuroendocrine neoplasm (pNET, n=5), mucinous cystic neoplasm (MCN, n=2), serous cystic neoplasm (n=2), solid peudopapillary neoplasm (n=2), intraductal papillary-mucinous neoplasm (n=1) and others (n=3). All lesions located in the pancreatic tail. Median operative time and operative blood loss were 260min and 420ml, respectively. No patients required perioperative blood transfusion. Median postoperative hospital stay was 17days. Postoperative pancreatic fistula (Grade B or C, ISGPF) was occurred in 2 cases. Three cases (pNET: 2, MCN: 1) were laparoscopically performed (LAP-SpDP). In all LAP-SpDP, splenic vessels were divided (Warshaw’s procedure). There was no mortality. During follow-up time, we have not experienced spleen-associated disorder. For cases of Warshaw’s procedure, no splenic infarction has been observed.
Conclusions: SpDP is a feasible procedure for benign diseases in the pancreatic tail. Also feasible is Warshaw’s procedure, especially in LAP-SpDP.
Clinical Characteristics of Hospitalized Acute Pancreatitis Patients in Our Hospital
Y. Kawasaki, A. Asakura, M. Mori, T. Tokuda, M. Matsushita, Y. Sato, H. Saiki, Y. Onishi, Y. Tokuda, A. Ishimi, T. Kawai, M. Hamano, M. Chiba, K. Maeda, K. Yamamoto, N. Tatsumi, T. Ito. JCHO Osaka Hospital, Osaka, Japan.
Aim: AP is still high mortal disease in Japan. Japan Society of Pancreas revised a guideline for AP in 2015. Both diagnostic procedures and therapeutic strategies were recommended in this issue. Following treatment of AP base on this guideline, analyses on clinical features should be addressed, but it seemed not enough to be done. In this study, we tried to clarify clinical characteristics of this disease using this guideline.
Method: 110 patients with AP were enrolled. They were hospitalized in our hospital from January 2010 to December 2015. Patients with ERCP related pancreatitis were excluded.
Result: The enrolled patients consisted of 79 males and 31 females. Average age was 57.8 years old (16 to 90 y-o). They consisted of 48 alcoholic, 34 gall stone-related, and 22 idiopathic patients. Etiology of six patients was unknown. Gall stone-related pancreatitis was dominant in patients over 65 years old, while alcoholic pancreatitis was in patients under 65 years old. Using the guideline, 40 patients were diagnosed as severe acute pancreatitis; 7 cases diagnosed with only prognostic factor, 28 cases by only CT grade, and 5 cases with both prognostic factors and CT grade. All of them were alive following the recommended therapy. 8 patients were subjected to hemodialysis and infusion of antibiotics and protease inhibitors into selective pancreatic artery. There was no difference of serum lipase and serum amylase on admission in mild and severe pancreatitis. Maximum CRP and urinary amylase were observed to be higher in the severe pancreatitis. 14 patients had the recurrence of AP consisting of 9 alcoholic, 3 gall-stone related, and 2 idiopathic patients. One patient was diagnosed as severe AP, others as mild. No patient was dead.
Conclusion: Application of this guideline to AP could improve prognosis. In order to prevent the disease, it is important to lead less consumption of alcohol and check gallstones. It is necessary to accumulate more cases for better therapeutic strategies.
Early Surgery is Beneficial for Pain Control and Pancreatic Function Preservation in Chronic Pancreatitis: A Retrospective Study of 297 Consecutive Patients
N. Ke,1 W. Huang,2 Q.M. Nunes,2 X. Liu,1 R. Sutton.2 1West China Hospital, Chengdu, China; 2Royal Liverpool University Hospital, Liverpool, United Kingdom.
Objectives: To determine whether early surgery offers more favourable outcome for pain relief in patients with chronic pancreatitis.
Methods: Symptomatic chronic pancreatitis patients undergoing surgery from 2007 to 2012 were studied. Early surgery was defined as < 3 years of diagnosis, late surgery ≥ 3 years. The primary end point was Izbicki pain score at follow-up for > 3 years. Secondary end points were surgical complications and pancreatic function.
Results: A total of 297 patients with histologically confirmed chronic pancreatitis were analysed, of whom 98 underwent early and 199 late surgery. Patients in the two surgical groups were not significantly different in age, gender, TIGAR-O classification and endocrine function at baseline, except that the early group had less frequent exocrine insufficiency (60.2 vs 72.4%, P = 0.034). The early group had a higher incidence of postoperative pancreatic fistulae (18.4% vs 10%, P = 0.044) and longer hospital stay (14.4 vs 12.2 d, P = 0.009). The early group had significantly more patients with complete (68.7% vs 46.7%, P = 0.01) and overall (91.1% vs 83.8%, P = 0.01) pain relief and lower rates of exocrine (60.2% vs 80.4%, P = 0.005) and endocrine insufficiency (35.7% vs 52.8%, P = 0.033). Subgroup analyses showed Frey and Berne, but not other procedures consistently to be associated with greater pain relief and less compromise of exocrine and endocrine function when performed early (all P < 0.05).
Conclusions: Early surgery resulted in higher rates of pain relief and pancreatic function preservation in chronic pancreatitis although with a cost of a higher incidence of postoperative pancreatic fistulae. Prospective comparisons of early versus late surgery for chronic pancreatitis are warranted.
Pancreatic Mucinous Cystic Neoplasms (MCN) of any Size, Without Worrisome Features or Symptoms Can Be Safely Surveyed in Women but Should Be Resected in Men: A Multinational Cohort Study Including 211 Patients
G. Keane,1 A. Shamili,2 L. Nilsson,3 A. Antila,4 J.B. Millastre,5 M. Marijinissen van Zanten,6 C. Verdejo,7 Y. Vaalavuo,4 T. Hoskins,8 S. Robinson,8 G. Ceyhan,9 M. Abuhilal,10 S. Pereira,11 J. Laukkarinen,4 M. Del Chiaro.12 1Institute for Liver and Digestive Health, University College London, United Kingdom Freeman Hospital, Newcastle, United Kingdom; 2Southampton University Hospital, Southampton, United Kingdom; 3Karolinska Institute, Solna, Sweden; 4Tampere University Hospital, Tampere, Finland; 5Gastroenterology, Miguel Servet University Hospital, Zaragoza, Spain; 6Pathology, Nijmegen University Hospital, Nijmegen, Netherlands; 7Gastroenterology, Hospital General Universitario De Ciudad Real, Ciudad Real, Spain; 8Freeman Hospital, Newcastle, United Kingdom; 9Technische Universität München, Munich, Germany; 10Southampton University Hospital, Southampton, United Kingdom; 11Institute for Liver and Digestive Health, University College London United Kingdom Freeman Hospital, Newcastle, United Kingdom; 12Div. of Surgery, Dept. of Clinical Science, Intervention and Technology (CLINTEC), Karolinska Institute, Stockholm, Sweden.
Introduction: Pancreatic MCNs are rare mucin-producing cystic tumours, defined by the presence of ovarian-type stroma. The aim was to better define the criteria for surgical resection in MCN.
Patients and methods: This multicentre retrospective study included all surgically resected MCNs between 2003–2015. Lesions without ovarian-type stroma were excluded. The diagnosis of MCN in male patients was confirmed by two experienced pancreatic pathologists. Patient characteristics, preoperative findings, histopathology, follow-up data and prognosis were recorded.
Results: 211 patients with a confirmed and surgically resected MCN were included. Median age was 53 (range 18–82) years, and 95.7% (202/211) were in women. Median pre-operative tumour size was 52 (range 12–230) mm. 16.1% 211) were malignant. Rates of malignancy (33.3% (3/9) vs. 15.3% (31/202)) and high-grade dysplasia (33.3% (3/9) vs. 15.8% (32/202) were double in men compared to women. In all cases of malignancy or high-grade dysplasia, at least one of the following characteristics was seen: male patient, symptoms, or a preoperative worrisome feature (solid component, septations, main pancreatic duct dilatation >6mm, elevated serum ca 19–9). A total of five cases of malignant transformation occurred in MCNs less than 4 cm in size. All these cases were associated with features of concern on pre-operative cross-sectional imaging.
Conclusions: In all patients, malignancy or high-grade dysplasia was solely seen in MCNs with symptoms or worrisome features, regardless of the size of the tumour. Unlike in females, in males the rate of malignancy and high grade dysplasia is high, suggesting that operative treatment should be considered in all male patients with a suspected MCN of any size. In female patients conservative management seems to be a safe for suspected MCNs of any size without symptoms or worrisome features.
Risk Factors for Pancreatic Atrophy in Type 1 Autoimmune Pancreatitis: A Nationwide Survey by the Japan Pancreas Society
M. Kitano,1 T. Ito,1 S. Kawa,2 K. Kubota,3 T. Kamisawa,4 K. Okazaki,5 T. Shimosegawa.6 1Gastoenterology, Shinshu University School of Medicine, Nagano, Japan; 2Shinshu University, Nagano, Japan; 3Gastoenterology, Yokohama City University Graduate School of Medicine, Yokohama City, Japan; 4Tokyo Metropolitan Komagome Hospital, Tokyo, Japan; 5Kansai Medical University, Osaka, Japan; 6Tohoku University Graduate School of Medicine, Sendai, Japan.
Background and Aim: As autoimmune pancreatitis (AIP) has the potential to cause pancreatic atrophy, this investigation sought to clarify its risk factors.
Methods: Questionnaires on patients with type 1 AIP were sent to 21 high-volume medical centers across Japan to compare the clinical features of patients with pancreatic atrophy to those without.
Results: Of the completed records of 615 AIP patients (470 male median age [range]: 67 [17–93] years), 139 (22.6%) had experienced pancreatic atrophy (110 male) and 476 had not (360 male). Although there were no remarkable differences in serology results between the groups, BT-PABA findings were significantly lower in patients with pancreatic atrophy (p=0.002). Moreover, imaging results disclosed pancreatic head swelling (p=0.007), pancreatic stone formation (p=0.0004), retroperitoneal fibrosis (p=0.03), and stenosis of the hilar or intrahepatic bile ducts (p=0.008) significantly more frequently in patients with pancreatic atrophy. Prednisolone discontinuation and recurrence were more frequently in this group as well (p=0.0002, p=0.001, respectively). In multivariate analysis, follow-up period (OR: 2.62, p=0.04), prednisolone continuation (OR: 0.24, p=0.002), stenosis of the hilar or intrahepatic bile ducts (OR: 3.53, p=0.01), and pancreatic head swelling (OR: 3.11, p=0.04) were independently associated with pancreatic stone formation.
Conclusion: The increased frequency of pancreatic head swelling in AIP patients experiencing pancreatic atrophy indicated a propensity for pancreatic juice stasis and subsequent atrophy development. Prednisolone continuation and prevention of recurrence suggested the possibility of protection against pancreatic atrophy. The association of hilar or intrahepatic bile duct stenosis with pancreatic atrophy in AIP requires further investigation.
Effect of Intrapancreatic Fat on Diabetes Risk After Total Pancreatectomy With Islet Autotransplantation
M. Kizilgul,1 M. Bellin,1,2 M. Abdulla,1 D. Heller,1 G.J. Beilman,3 S. Chinnakotla,3 T.B. Dunn,3 T.L. Pruett,1 B.J. Hering,1 J.J. Wilhelm.1 1Schulze Diabetes Institute; Departments of 2Pediatrics; and 3Surgery; University of Minneasota, Minneapolis, MN.
Background: While intrapancreatic fat is postulated to impair b-cell mass and function in type 2 diabetes, via local release of non-esterified fatty acids and pro-inflammatory factors, the impact of intrapancreatic fat has never been studied in patients with pancreatitis undergoing total pancreatectomy with islet autotransplantation (TPIAT) for chronic pancreatitis.
Material and Methods: We studied the association between intrapancreatic fat and diabetes outcomes in 87 patients undergoing TPIAT between 2009 and 2016 who met the following criteria: marginal islet mass (2000–5000 islet equivalents/kg), non-diabetic prior to TPIAT, with low (“LPF”, n=58) or high (“HPF”, n=29) intrapancreatic fat content. Intrapancreatic fat was evaluated by using both gross examinations before isolation and banked histology samples. HPF and LPF groups were matched for age, BMI, and etiologic classification.
Results: Insulin independence or low-dose partial insulin supplementation was more frequent at 1 year with LPF (p=0.016); 1 and 2 hour glucose levels during mixed meal tolerance tests were higher in the HPF group (p=0.027, p=0.016) while Beta Score (a composite marker for islet function) was significantly better in the LPF group (6.08 ± 1.73 to 4.58 ± 1.93, p=0.028). Average insulin use, fasting glucose, HbA1c, fasting and stimulated C-peptide levels were similar between groups.
Conclusion: Patients with HPF were more likely to be insulin dependent, with higher post-prandial glucose excursion, suggesting that intrapancreatic fat might lead to b-cell dysfunction with detrimental effects on diabetes outcomes after TPIAT. Alternatively, high intrapancreatic fat may be a marker for patients with greater visceral adiposity and insulin resistance.
Sphincter of Oddi Botulinum Toxin Injection to Prevent Pancreatic Fistula After Distal Pancreatectomy
U. Klaiber,1 P. Sauer,2 T. Kehayova,1 P. Probst,1 P. Knebel,1 M.K.-M. Diener,1 L. Schneider,1 O. Strobel,1 C.W. Michalski,1 A. Ulrich,1 M.W. Büchler,1 T. Hackert.1 1Department of General, Visceral, and Transplantation Surgery; and 2Interdisciplinary Center of Endoscopy; University of Heidelberg, Heidelberg, Germany.
Background: Postoperative pancreatic fistula (POPF) represents the most frequent and potentially life-threatening complication after distal pancreatectomy (DP). The aim of this study was to evaluate the use of a preoperative endoscopic injection of botulinum toxin (BTX) into the sphincter of Oddi to prevent POPF.
Methods: Investigator-initiated, prospective clinical trial with an exploratory study design (German Clinical Trials Register number: DRKS00007885). Included patients underwent preoperative endoscopic sphincter BTX injection (100 units of Botox® reconstituted in 1 mL of 0.9% sodium chloride). Outcomes were the feasibility, safety, and postoperative outcomes including POPF within 30 days following DP. Descriptive statistical analysis was performed.
Results: Between February 2015 and February 2016, 29 patients scheduled for DP for various underlying diseases were included. All patients underwent successful sphincter of Oddi BTX injection within a median of 6 days before surgery. One patient showed asymptomatic, self-limiting (48h) increase in serum amylase and lipase after injection. No other intervention-related side effects or complications occurred. DP was performed in 24 patients. 7 patients (29%) showed elevated amylase levels in drainage fluid on postoperative day 3 (ISGPS definition POPF grade A) without symptoms or need for reintervention. No clinically relevant fistulas (ISGPS grades B/C) were observed.
Conclusion: Preoperative sphincter of Oddi BTX injection is a novel and safe approach to reduce POPF after DP. The results of this pilot trial suggest its efficacy in the prevention of clinically relevant POPF and are currently validated in the Federal Government-sponsored, multicenter randomized controlled PREBOT trial.
Bioglue® Sealed Fish-Mouth Closure of the Pancreatic Remnant as a Feasible Alternative to Stapler Closure During Laparoscopical Distal Pancreatectomy
F. Klein, R. Zorron, J. Pratschke, M. Bahra. Department of General, Visceral and Transplantation Surgery, Charite Universitätsmedizin Berlin, Germany.
Background: Postoperative pancreatic fistula formation (POPF) remains the main problem after open and laparoscopical distal pancreatectomy. Stapler closure of the pancreatic closure is nowadays considered the gold standard technique during laparoscopical distal pancreatectomy but is limited especially in “hard” pancreatic tissue remnants. After promising results during open distal pancreatectomy we have now developed a novel BioGlue® sealed hand sutured fish-mouth closure technique of the pancreatic remnant during laparoscopical distal pancreatectomy. The aim of this study was to analyse the feasibility of our technique and report our first results.
Patients and methods: 12 patients underwent BioGlue® sealed hand sutured fish-mouth closure of the pancreatic remnant during laparoscopical distal pancreatectomy at our institution. Perioperative and postoperative results were analyzed with regard to clinically relevant POPF and overall postoperative morbidity. Essential technical steps will be demonstrated as a video presentation.
Results: Indications for distal pancreatectomy included pancreatic adenocarcinoma (4 patients), IPMN (3 patients), MCN (2 patients), chronic pancreatitis (1 patient), neuroendocrine tumor (1 patient) and SPN (1 patient). 4 out of 12 patients (33%) developed a grade B pancreatic fistula, which could all be treated conservatively. There was no postoperative mortality. The median length of hospital stay was 11 days.
Conclusion: The performance of a BioGlue® sealed hand sutured fish-mouth closure of the pancreatic remnant during laparoscopical distal pancreatectomy was shown to be technically feasible and may thus be considered as a safe alternative to standard stapler closure especially in patients with a “hard” pancreatic tissue remnant.
Comparison of Clinical Course and Outcome of Alcohol Induced and Gallstone Induced Acute Pancreatitis
R. Kochhar,1 J. Samanta,1 N. Dhaka,1 V. Gupta,2 T.D. Yadav,2 S.K. Sinha.1 Departments of 1Gastroenterology; and 2Surgery; Postgraduate Institute of Medical Education and Research, Chandigarh, India.
Introduction: This study was designed to investigate the role of the two main etiological factors, alcohol and gallstones, on the clinical course and outcome of AP.
Methods: Consecutive patients of AP from Jan 2011 to June 2016 of alcohol induced pancreatitis (AIP) and gallstone induced pancreatitis (GSIP) were compared for episodes of recurrent attacks, BISAP score, APACHE II score, CT severity index (CTSI), organ failure (OF), persistent organ failure (POF), need for ICU stay, ventilator and dialysis, hospital stay, need for percutaneous catheter drain (PCD), surgery and mortality.
Results: Of the 614 patientsstudied, 296 (48%) patients had AIP and 188 (31%) had GSIP. 97% AIP patients were males and 67% of GSIP were females. Recurrent attacks of AP were seen in 38 (13%) in AIP and 16 (9%) in GSIP (p=0.18). Comparison of BISAP, APACHE II and CTSI showed no significant difference between the two groups (p-0.70, p-0.84, p-0.14). 158 (53%, out of whom 40% had POF) patients in AIP and 99 (53%, out of whom 42% had POF) patients in GSIP had OF (p=0.92). Similarly, length of hospital stay also was not different (19.70±16.8 vs 21.7±20.2, p=0.23). ICU requirement was seen in 109 (37%) patients of AIP group whereas in GSIP group 74 (39%) required ICU care (p-0.63). 52 (18%) patients of AIP and 42 (22%) in GSIP group needed ventilatory support (p-0.19), while dialysis need was seen in 24 (8%) of AIP patients and 16 (9%) in GSIP group (p-0.86). PCD and surgery requirement were seen in 139 (47%) & 32 (11%) of AIP and 72 (38%) &12 (6%) of GSIP patients respectively (p-0.07& p-0.10). Mortality in AIP and GSIP was 28 (10%) and 26 (14%) respectively (p-0.06).
Conclusion: AIP and GSIP are similar in severity scores, hospital stay, organ support.
Gastrointestinal Fistula in Acute Pancreatitis
R. Kochhar,1 J. Samanta,1 N. Dhaka,1 S. Kochhar,2 R. Prasad,1 S.K. Sinha,1 V. Gupta,3 T.D. Yadav.3 1Gastroenterology, Postgraduate Institute of Medical Education and Research, Chandigarh, India; 2Radiodiagnosis, Govt Medical College and Hospital, Chandigarh, India; 3Surgery, Postgraduate Institute of Medical Education and Research, Chandigarh, India.
Introduction: Gastrointestinal tract (GI) fistulzation occurs rarely in the clinical course of acute pancreatitis (AP).
Methods: Records of all patients of AP from 2006-2015 were analysed retrospectively for the occurrence of fistulzation into GI tract. Details of demographic characteristics, clinical presentation, time interval between pancreatitis and development of fistula, diagnostic studies, treatment (including surgery), and outcome were retrieved and analyzed.
Results: GI fistulization was detected in 62 (5.7%) of the 1079 patiens of AP. Etiology of AP was alcohol in 44 (71%) followed by gall stones in 12 (22.5%). The most common presentation was persistent fever in 35 (56.5%), followed by GI bleed in 21 (33.9%) and pain abdomen in 14 (22.6%) patients. Fistula developed spontaneously in 34 (55%) patients, while 28 (45%) had history of either surgical or percutaneous intervention. Location of fistula was colon in 25, duodenum in 24, and others in rest. CECT abdomen showed air foci within the necrotic collection in 47.5%% and inflammatory involvement of the bowel in 15 % while only necrosis was seen in 27.5% of patients. Out of 27 patients, who underwent endoscopy, 23 (85%) had direct visualization of the fistulous communication. Treatment included surgical intervention in 31 (50%), conservative management in 16 (25.8%), endotherapy in 12 (19.4%) and percutaneous intervention in 3(4.8%). Overall mortality was 15 (24.2%). The need for surgical intervention (p=0.008) and mortality (p=0.003) were significantly higher in colonic than upper gastrointestinal fistulas.
Conclusion: GI fistulas occur in 5.7% of AP patients and colonic location is associated with higher surgical intervention and higher mortality.
Endo180 Regulate Phosphorylation of Myosin Light Chain 2 Activity and Increase the Ability of Extracellular Matrix Remodeling in Leading Pancreatic Stellate Cells
K. Koikawa, K. Ohuchida, S. Kibe, Y. Ando, S. Takesue, H. Nakayama, T. Abe, S. Endo, T. Okumura, T. Moriyama, K. Nakata, Y. Miyasaka, T. Manabe, T. Ohtsuka, E. Nagai, K. Mizumoto, M. Nakamura. Department of Surgery and Oncology, Kyushu University, Fukuoka, Japan.
Background and Aim: Leading cells are the specific cell populations which lead the local invasion in cancer. Regulating the leading cells may control of cancer invasion. We previously reported that pancreatic stellate cells (PSCs) lead the pancreatic cancer cell invasion and that cancer cells frequently invaded following the PSCs. Invading PSCs changed the alignment of collagen fibers in the extracellular matrix (ECM). Our data suggested that PSCs lead the local invasion of pancreatic cancer cells, by physically ECM remodeling. However, the underlying mechanisms of the phenomenon have remained unclear. We elucidated that the mechanisms of physically ECM remodeling and the cancer cell invasion by leading PSCs.
Methods: We established in vitro 3D collagen invasion assay models, in which PSCs and/or pancreatic cancer cells were cultured. We investigated the movement of PSCs and cancer cells in collagen matrix and the factor of ECM remodeling in PSCs.
Results: Endo180 is a collagen binding and internalization receptor. The expression level of Endo180 is higher in PSCs than in pancreatic cancer cells. Endo180 knockdown by RNA interference attenuated the invasive abilities of invading PSCs (P<0.01) and decreased the number of invading cancer cells (P<0.01). Knockdown of Endo180 in PSCs decreased the expression level of phosphorylation of myosin light chain (MLC) 2. We co-transplanted cancer cells and PSCs into nude mice pancreas tail, knockdown of Endo180 in PSCs reduce tumor growth and local invasion compared with control PSCs.
Conclusions: Physically ECM remodeling depends on Endo180 in leading PSCs, possibly via the function of promoting reconstruction of an actin cell skeleton by phosphorylation of MLC 2.
Renalase Protects Against Pancreatitis by Activating a Plasma Membrane Calcium ATPase (PMCA)
T. Kolodecik,1 A. Reed,1 K. Date,1,2 F.S. Gorelick.3 1Internal Medicine, Digestive Diseases, Yale University, New Haven, CT; 2Graduate School of Humanities and Science, Ochanomizu University, Tokyo, Japan; 3VA Connecticut Healthcare, West Haven, CT.
The kidney hormone renalase (RNLS) is secreted into the bloodstream and has been found to be reduced in patients in severe kidney disease, a condition that predisposes to acute pancreatitis. Our lab has shown that renalase has a protective role in the early events of acute pancreatitis: RNLS added to pancreatic acinar cells prevents secretagogue-stimulated zymogen activation and cellular injury. The plasma membrane calcium channel 4b (PMCA-4b) has recently been identified as a RNLS receptor. Here we examine whether RNLS’s protective effects in acinar cells are due to PMCA activation. We first determined that the PMCA isoforms expressed in mouse acini were PMCA 1, 2 and 4B. The PMCA inhibitor, caloxin 1b1, blocks all of the PMCA’s, but has the highest affinity for PMCA-4. Pancreatic lobules were pretreated with caloxoin prior to addition of RNLS and subsequent treatment with cerulein (CER). As expected, RNLS blocked CER stimulated zymogen activation and amylase secretion, but caloxin blocked the protective effect of RNLS. In an assay for cellular injury, caloxin pretreatment prevented the protective effect of RNLS on CER-stimulated cell injury resulting injury similar to that seen with cerulein alone. Since PMCA is a calcium channel we determined the effect of RNLS/caloxin on calcium signaling. Previously, RNLS had caused an increase in the rate of calcium extrusion in cerulein treated acini. Caloxin treatment, however, returned the rate of calcium extrusion back to that seen for CER alone. In summary, the PMCA inhibitor caloxin 1b1 blocked the protective effects of RNLS against cerulein stimulated zymogen activation and calcium extrusion. This suggests that the effects of renalase on acinar cells may be mediated by activation of PMCA-4B.
Inhibition of Jak/STAT Signaling Limits the Activation of Pancreatic Stellate Cells In Vitro and Cerulein-Induced Pancreatitis In Vivo
H. Komar,1 T. Mace,1 G. Serpa,1 O. Elnaggar,1 D.L. Conwell,2 P. Hart,2 C. Schmidt,3 M. Dillhoff,1 J. Ming,1 G. Lesinski.4 1The Ohio State University Comprehensive Cancer Center, Columbus, OH; Departments of 2Gastroenterology, Hepatology, and Nutrition and 3Surgery; The Ohio State University Wexner Medical Center, Columbus OH.
Chronic pancreatitis (CP) is a devastating disease characterized by persistent inflammation and fibrosis of the pancreas. No therapeutic options exist and understanding of the molecular pathology is limited. However, recent findings implicate pancreatic stellate cells (PSC) as prominent mediators of CP pathology. To better understand and target these cells, we cultured a variety of primary and immortalized PSC from backgrounds of both CP and PDAC. In vitro, these cells demonstrated secretion of several immunomodulatory factors including MCP-1, IL-6, and VEGF at levels up to 31,000, 19,000, and 800 pg/mL respectively. All cell lines displayed activation of the pro-survival, pro-inflammatory Jak/STAT and MAPK pathways. Treatment with the Jak1/2 inhibitor Ruxolitinib resulted in reduced pSTAT3 and decreased cell growth (40% growth after 72 hrs of 50 μM). Treated cells remained adherent and did not display PARP cleavage by western blot, suggesting the effects were not pro-apoptotic. Instead, western blot and fluorescent microscopy showed a dose-dependent decrease in α-SMA, a marker of PSC activation. Treatment with a MAPK pathway inhibitor (MEK162) had no effect on growth or activation. These data suggest that the Jak/STAT pathway, and not the MAPK pathway, functions to limit PSC growth and activation. To examine this hypothesis in vivo, we used the caerulein-induced murine model of CP. After 4 weeks mice displayed acinar cell loss, inflammation, and fibrosis of the pancreas as well as decreased serum amylase and lipase. For therapeutic studies, Ruxolitinib was administered at 90mg/kg twice daily by oral gavage during the final week of caerulein injections. After 1 week, Ruxolitinib-treated mice displayed increased serum amylase and lipase compared to control mice. Immunohistochemical analysis of pancreata show reduced fibrosis and inflammation. Consistent with these observations, there was a mild reduction in acinar loss of Ruxolitnib treated mice. Together these data suggest that inhibition of Jak/STAT signaling may limit caerulein-induced pancreatic damage in this model.
An International External Inter-and Intraobserver Variability Study Evaluating Needle Based Confocal Laser Endomicroscopy (nCLE) for Diagnosis of Pancreatic Cystic Lesions (PCLs)
S.G. Krishna,1 W.R. Brugge,2 J.M. Dewitt,3 P. Kongkam,4 B. Napoleon,5 C. Robles-Medranda,6 D. Tan,7 S. El-Dika,1 P. Hart,1 D.L. Conwell.1 1Ohio State University Medical Center, Columbus, OH; 2Department of Gastroenterology, Massachusetts General Hospital, Boston, MA; 3Indiana University, Terre Haute, IN; 4Chulalongkorn University, Bangkok, Thailand; 5Hôpital Privé Jean Mermoz, Lyon, France; 6Ecuadorian Institute of Digestive Disease, Guayaquil, Ecuador; 7Singapore General Hospital, Singapore.
Background: EUS-guided nCLE characteristics of common types of PCLs have been identified; however surgical histopathology was available in a minority of patients.
Aims: To assess the performance characteristics of EUS-nCLE for differentiating mucinous from non-mucinous PCLs in a larger series of patients with a definitive diagnosis.
Methods: Six expert endosonographers (nCLE experience>30 cases) from 4 different continents blinded to all clinical data, reviewed nCLE images (twice with 2 week wash-out; different sequences) from consecutive patients with PCLs. The nCLE images were acquired and edited by a single endoscopist for clarity. A tutorial on published data was provided. The performance characteristics of nCLE and the k statistic for interobserver agreement (IOA) and intraobserver reliability (IOR) were calculated.
Results: A total of 29 (16 mucinous-PCLs, 13 non-mucinous PCLs) nCLE patient videos were reviewed. Surgical histopathology was available in 23 patients. Three patients with serous cystadenomas (SCAs) (characteristic image patterns, CEA < 1 ng/dL) and 3 patients with pseudocysts (history of acute pancreatitis, supportive cytology, >50% reduction in cyst size over 1-year) did not undergo surgical resection.
The overall sensitivity, specificity, and accuracy for the diagnosis of mucinous-PCLs were 95%, 94%, and 95%, respectively. The IOA and mean (±standard deviation [SD]) IOR were k = 0.81, 95% confidence interval [CI] 0.71-0.90 and k =0.86±0.11 respectively.
The overall specificity, sensitivity, and accuracy for the diagnosis of SCAs (n: 4) were 99%, 98%, and 98%, respectively. The IOA and IOR (mean±SD) for recognizing characteristic image pattern of SCA were k =0.83, 95% CI 0.73-0.92 and k=0.85±0.11 respectively.
Conclusions: EUS-guided nCLE can provide virtual histology of PCLs with a high degree of accuracy, and inter-and intraobserver agreement in differentiating mucinous versus non-mucinous PCLs. These results support larger multi-center studies to evaluate EUS-nCLE.
Cholecystectomy (CCY) During Index Admission for Acute Pancreatitis (AP) Decreases the Risk of Recurrences and Readmissions: A National-Level Analysis
S.G. Krishna,1 A. Hinton,1 D. Yadav,2 D. Conwell.1 1Ohio State University Medical Center, Columbus, OH; 2University of Pittsburgh Medical Center, Pittsburgh, PA.
Background: National societies recommend same-admission CCY for mild gallstone-related AP (gallstone-AP). There are no studies evaluating 30-day readmission rates after gallstone-AP at a national level in the United States (US).
Aims: To evaluate the impact of the timing of CCY on recurrences and readmissions in patients with gallstone AP in a national level database in the US.
Methods: We identified all adults (age ≥18 years) with a primary discharge diagnosis of AP from Jan-Nov 2013 in the Nationwide Readmission Database. Patients with concurrent diagnosis of chronic pancreatitis (CP) or pancreatic neoplasms, missing information on length of stay (LOS) or readmission, and those who died during index admission were excluded. Gallstone etiology of AP was identified by associated diagnoses codes using a hierarchical model. Severe AP was defined as presence of either acute renal failure, respiratory failure, or need for intravenous vasopressin. Multivariable Cox regression models assessed independent predictors for 30-day readmission for which unweighted data were utilized.
Results: Among 41,094 patients with gallstone-AP hospitalizations, 21,839 (53.1%) underwent same-admission CCY (11.7% in severe AP, 88.3% in non-severe AP). The risk of readmission for primary diagnosis of AP during a median follow up of 5.5 months was 2.52% and 4.53% for severe and non-severe AP respectively.
On multivariable analyses, after controlling for disease severity and other relevant covariates, the risk of recurrent AP was significantly lower in patients who underwent CCY during index admission when compared to patients who did not undergo CCY during index admission (hazard ratio [HR] 0.30, 95% confidence interval [CI] 0.25-0.35). The risk of readmission for any reason within 30 days (HR 0.47, 95% CI 0.43-0.52) and at anytime during follow up (HR 0.46, 95% CI 0.43-0.49) was also significantly lower in patients who underwent CCY during index hospitalization.
Conclusion: CCY should be considered in all patients with gallstone-AP during index admission or soon afterwards when feasible.
Can Hemostasis Products Prevent Postoperative Pancreatic Fistulas After Distal Pancreatectomy?
C.M. Kühlbrey, S. Kasper, U.T. Hopt, U.A. Wittel. Department of General- and Viszeral Surgery, University Hospital Freiburg, Freiburg, Germany.
Introduction: Postoperative pancreatic fistula (POPF) after distal pancreatectomy remains a pestering challenge for pancreatic surgeons. As many hemostasis products have been applied for sealing the pancreatic stump without showing significant clinical benefit, our aim was to analyze if these existing products are worthwhile for further clinical studies.
Material and methods: In-vitro hemostasis products were exposed to enterokinase activated pancreatic juice for up to 7 days and physical stability was determined at different times. In vivo laparoscopic assisted distal pancreatectomy was conducted in nine pigs. Closure of the stump was either obtained by Sealant A (S_A, based on glutaraldehyd), Sealant B (S_B, based on two polyethylenglycols) or no closure at all (control). The stability of pancreatic stump closure was evaluated by burst pressure experiments. Histology and immunohistochemistry was performed to support the clinical findings.
Results: All hemostasis products based on collagen or fibrin completely lost their integrity in activated pancreatic juice in less than 6 hours. In vitro two sealants showed acceptable stability in activated pancreatic juice on day 7. After laparoscopic distal pancreatectomy in pigs, burst pressure applied to the pancreatic duct showed significant lower burst pressures in pigs treated with S_A than S_B and control animals (S_A 81 ±24 mmHg, S_B 242 ±12 mmHg, control 218 ±7 mmHg p<0.05). Histologically and chemically S_A animals showed inflammation at the closure surface and in serum (control 207%±4 vs. S_A 409%±6; p<0.05). In contrast, animals treated with S_B only showed little inflammation.
Conclusion: Hemostasis products on basis of fibrin or collagen are not suitable to prevent POPF after pancreas resection. Polyethylenglycol based sealants may have the capability to prevent POPF after distal pancreatectomy.
MEK Resistance via Amphiregulin Mediated EGFR-STAT3 Activation in Pancreatic Cancer
P. Lamichhane,1 N.S. Nagathihalli,1 J. Castellanos,2 C. Shi,2 C. Roberts,1 M. Vansaun,1 N. Merchant.1 1Department of Surgery, University of Miami, Miami, FL; 2Department of Pathology, Microbiology, and Immunology, Vanderbilt University Medical Center, Nashville, TN.
Introduction: Targeting KRAS in PDAC has remained an elusive goal. Therefore efforts have focused on targeting downstream effectors of RAS such as MEK. Unfortunately, clinical trials of MAPK-directed therapies have been unsuccessful in PDAC. Here we report a novel mechanism of resistance to MAPK-directed therapies, which is associated with amphiregulin (AREG)-mediated activation of EGFR-STAT3 signaling.
Methods: Effects of MEK inhibition on the phosphorylation of multiple signaling proteins and EGF family ligands was assessed. Activation of MAPK and STAT3 was quantified in human pancreatic tissues. AREG release was measured in the conditioned media of PDAC cells treated with MEK, EGFR, STAT3, and/or TACE inhibitors. Tumorigenicity, cell cycle analysis, and apoptosis assays were performed with human PDAC cells treated with inhibitors. PDAC xenografts and patient derived xenografts (PDXs) were treated with inhibitors. PKT mice (Ptf1acre/+;LSL-KrasG12D;Tgfbr2fl/fl) were treated with inhibitors and assessed for OS and plasma AREG was assessed by ELISA.
Results: MAPK inhibition leads to activation of TACE and EGFR and subsequent activation of STAT3. Combined MEK/STAT3 or MEK/EGFR inhibition blocked MEK, EGFR and STAT3 activation, decreased tumorigenicity in vitro, and decreased growth of PDAC xenografts and human PDX tumors in vivo. OS in combined MEK/STAT3 treated PKT mice was extended to a median of 85 days vs. 52 days for vehicle treatment. AREG release was significantly reduced with combined MEK/STAT3 inhibition. Evaluation of TACE activation and AREG release in response to MEK inhibition demonstrated that resistance to MEK inhibition in PDAC is mediated by reactivation of the STAT3 pathway and is strongly influenced by increased AREG production.
Conclusions: Our study elucidates the molecular mechanism that helps explain the heterogeneous response and therapeutic resistance of PDAC to MAPK pathway inhibition and provide a strong rationale that AREG mediated EGFR-STAT3 pathway activation is a major resistance mechanism that impairs the efficacy of MEK inhibitors.
Diabetes Burden Following Total Pancreatectomy With Islet Autotransplantation (TPIAT)
A. Lane,1 P. Ptacek,1 K.L. Berry,2 T.B. Dunn,2 T.L. Preutt,2 M. Cook,2 S. Chinnakotla,2 M. Freeman,3 S.J. Schwarzenberg,1 G.J. Beilman,2 M. Bellin,1,2,3 Departments of 1Pediatrics; 2Surgery; and 3Medicine; University of Minnesota, Minneapolis, MN.
Background: The physiologic fatigue and stress of diabetes management is assessed using the standardized Diabetes Distress Scale (DDS). Diabetes distress (DD) is common and higher DD scores correlate with poorer glycemic control. DD has never been evaluated in patients undergoing total pancreatectomy with islet autotransplant (TPIAT) for chronic pancreatitis.
Methods: We analyzed annual post-TPIAT DDS results in 259 patients (age 34.2±1.0, 76% F) undergoing TPIAT between 2006 and 2015. Each patient completed one or more DDS questionnaire from 1 to 7 years post-TPIAT (n=523). Using year 1 responses (n=179; 34% insulin independent), we examined variables that predict greater DD after TPIAT. An overall DD score of ≥2 (scale 1–6) signifies at least moderate distress while <2 is considered little to no distress; the inventory contains subscales for Emotional Burden (EmB), Regimen Distress (RegD), Physician Distress (PD), and Interpersonal Distress (ID).
Results: Mean DD scores were lower at year 1 than years 5, 6, and 7 (p ≤ 0.02 for all). At 1 year, 21% of all patients had moderate or high DD based on DDS score ≥2, whereas 79% expressed little to no distress. Insulin users had higher mean DD scores than insulin independent patients (mean±SE 1.74±0.07 vs 1.28±0.07, p<0.0001) and were more likely to have DDS ≥2 (29% vs 7%, p<0.001). DD score had a weak linear correlation with HbA1c (r=0.28 p<0.0001). EmB and RegD subscores were significantly higher than PD or ID subscores (p≤ 0.02). DD scores did not differ by gender or by age category (pediatric/adult).
Conclusion: DD is low at 1 year after TPIAT, but higher DD is observed in insulin users and with poor glycemic control. Distress is characterized by Emotional Burden and Regimen Distress.
Alternative Diagnoses for Mild Elevations in Pancreatic Enzymes: A Case Series Descriptive Study
D. Lew,1 S. Pandol,2 E. Afghani,2 Cedars-Sinai Medical Center, Los Angeles, CA; 1Department of Medicine, 2Division of Gastroenterology, Los Angeles, CA.
Background: Acute pancreatitis (AP) is diagnosed when 2 of the 3 following criteria are met: pancreatic-type abdominal pain, serum lipase and/or amylase ≥3 times upper limit of normal, and characteristic findings on imaging. AP is commonly misdiagnosed based on mild elevations in pancreatic enzymes. In this study, we examined the potential etiologies for serologic elevations in lipase/amylase in patients who were diagnosed with AP at discharge but did not meet the criteria for AP.
Methods: Demographic, clinical, laboratory, and radiographic data of all consecutive adult admissions (age≥18 years) with discharge diagnosis of AP based on ICD 9 code 577 was retrospectively collected from January to December 2014 at Cedars-Sinai Medical Center.
Results: In 2014, a total of 426 patients were diagnosed with AP. 162 (38%) did not meet the criteria for AP. 60 of the 162 were excluded because they had normal enzymes or enzymes ≥3 times upper limit of normal. The remaining 102 of the 162 were those that did not meet the criteria for AP but did have mild elevations in lipase/amylase. 13 of the 102 (12.7%) had no pain but mild elevations in pancreatic enzymes whereas 89 of the 102 (87.3%) had pain and mild elevations in pancreatic enzymes. Upon further investigation, the most common etiologies in the group of patients with no pain but mild elevations in pancreatic enzymes were acute on chronic kidney disease (61.5%) and alcohol intoxication (23.1%). The most common etiologies in patients with pain and mild elevations in enzymes were malignancy (12.3%), chronic pancreatitis (12.3%), alcohol intoxication (12.3%), post-ERCP (10.1%), and acute on chronic kidney disease (9%).
Discussion: Careful consideration should be given to those with mild elevations in lipase and amylase that do not meet criteria for AP to evaluate for other possible clinical conditions.
YAP is Critical Mediator of TGF-β1 Induced EMT and Cell Invasion in Pancreatic Cancer
X. Li,1 Z. Jiang,2 Q. Ma,2 Departments of 1General Surgery; and 2Hepatobiliary Surgery; First Affiliated Hospital of Xi'an Jiaotong University, Xi'an, China.
Background and Aim: Pancreatic cancer (PanCa) is a kind of malignant tumor with extremely high mortality. Our recent studies have found that the YAP (Yes-associated protein) was abnormal high expressed in PanCa tissue and play an important role in PanCa cells proliferation and invasion. TGF-β1 is mainly secreted by PSC and play an important role in pancreatic cancer progression. Thus, we speculate that YAP maybe a critical mediator of TGF-β1-induced epithelial-mesenchymal transition (EMT) and cell invasion in PanCa cells.
Methods: Lentivirus vector YAP-shRNA was used to suppress YAP expression in PanCa cell BxPC3. PSC cells and tumor cells were co-cultured in vitro. The invasion ability was determined by Transwell assay. BxPC3-shYAP and BxPC3-shNC PanCa cells were pretreatment with or without TGF-β1(10ng/ml). The EMT related proteins, SMAD2, phospho-SMAD2 and MMPs were detected by Western blot and Real-time RT-PCR. The invasion ability was determined by Transwell assay. The nuclei SMAD2 level were evaluated with Immunofluorescence staining assay.
Results: Under co-culture condition or with treatment with TGF-β1, N-cadherin, Vimentin, MMP-2, MMP-9 and phospho-SMAD2 level were increased in BxPC3 cells. However, YAP knockdown reversed these pro-invasion effects of PSC co-culture or TGF-β1 treatment. Moreover, YAP knockdown also abolished TGF-β1-induced nuclear accumulation of SMAD2. Additionally, YAP was significantly up-regulated after TGF-β1 treatment and this effect was reversed by TGF-β1 receptor inhibitor SB431542.
Conclusion: YAP was critical mediators of TGF-β1-induced tumorigenic events, including EMT, cell migration, and invasion. YAP also mediates the coupling between stroma and cancer cells in PanCa microenvironment.
Chai-Qin-Cheng-Qi Decoction Improves Intestinal Motility by Regulating Protein Kinase C- and Adenylate Cyclase-Mediated Ca2+ Release in Colonic Smooth Muscle Cells in Rats With Acute Necrotizing Pancreatitis
Z.Q. Lin,1 J. Guo,1 W.W. Chen,1 L.H. Deng,1 X.Y. Zhang,2 W. Huang,2 J.A. Windsor,3 R. Sutton,2 P. Xue,1 Q. Xia.1 1Department of Integrated Traditional Chinese and Western Medicine, Sichuan Provincial Pancreatitis Centre, West China Hospital, Sichuan University, Chengdu, China; 2NIHR Pancreas Biomedical Research Unit, University of Liverpool, Liverpool, United Kingdom; 3Department of Surgery, University of Auckland, Auckland, New Zealand.
Background: Chai-Qin-Cheng-Qi decoction (CQCQD) and its derivatives improve intestinal motility in acute pancreatitis (AP), but the mechanisms require elucidation.
Objective: To investigate the effects of CQCQD on colonic smooth muscle cells (CSMCs) in a rodent model of necrotizing AP.
Methods: The 4 experimental groups were: (1) intraperitoneal normal saline; (2) intraperitoneal L-Arginine (2.5 g/kg, two injections, 1 hour apart); (3) L-Arginine and intragastric CQCQD (20 g/kg, 2 hourly × 3 doses) or (4) L-Arginine and intraperitoneal carbachol (60 μg/kg) after 24 hours. Animals (10 per group) were sacrificed 30 hours after the first injection; CSMCs were collected to measure protein kinase C (PKC) isoforms and adenylate cyclase (AC) (RT-PCR and Western-blot) and intracellular calcium fluorescence intensity ([Ca2+]i) (confocal microscopy with 5 μM Fluo-3AM). Serum motilin and substance P were measured by ELISA.
Results: CQCQD decreased histological severity of AP more than carbachol (mean score 2.5 and 5.0 vs 6.9, P<0.01). Both CQCQD and carbachol significantly up-regulated PKC-α mRNA and PKC protein, and down-regulated AC mRNA (all P<0.05) vs no treatment. Neither CQCQD nor carbachol significantly altered L-Arginine-induced PKC-β1 and PKC-ε mRNA reduction. CQCQD and carbachol significantly increased CSMC [Ca2+]i (3563 and 3046 vs 1087, both P<0.01). CQCQD was equivalent to carbachol in increasing serum motilin (193 and 218 vs 155 ng/l, both P<0.05). CQCQD but not carbachol significantly reduced substance P (1662 vs 3074 pg/l, P<0.01).
Conclusion: These data suggest CQCQD improves intestinal motility in AP by increasing [Ca2+]i in CSMCs by up-regulating PKC and down-regulating AC.
Pancreas Divisum in Pediatric Acute Recurrent and Chronic Pancreatitis
T.K. Lin,1 M. Abu-El-Haija,1 J.J. Palermo,1 J.D. Nathan,1 M.E. Lowe,2 B. Zimmerman,3 I. Insppire,3 A. Uc.4 1Gastroenterology, Cincinnati Children's Hospital Medical Center, Cincinnati, OH; 2Department of Pediatrics, Children's Hosptial of Pittsburgh of University of Pittsburgh Medical Center, Pittsburgh, PA; 3University of Iowa, Iowa City, IA; 4Pediatrics, University of Iowa, Iowa City, IA.
Introduction: The clinical significance of pancreas divisum (PD) is debated, but a higher frequency of PD in adults with idiopathic pancreatitis suggests an association. The significance of PD in children associated with pancreatitis has been understudied. Through sixteen centers of the INSPPIRE (International Study group of PediatricPancreatitis: In search for a cuRE) cohort, we sought to determine the frequency and associations of PD in children.
Methods: Demographic/clinical information of children with acute recurrent pancreatitis (ARP) and chronic pancreatitis (CP) ≤19 years of age were collected.
Results: PD was found in 13.5% (39/288) of subjects. Females were more likely to have PD (72% vs 53% without PD, p=0.025). Children with PD were not different in age at first presentation of acute pancreatitis (AP) or CP, or time from the diagnosis of first AP to CP. PD was no more frequent in those with SPINK1, CFTR or CTRC or with other obstructive, and/or toxic-metabolic risk factors. Subjects with PD were less likely to have a pancreatitis family history (p<0.05) and had a lower rate of PRSS1 (10% vs 36% without PD; p=0.004). Having PD did not impact the frequency/pattern of abdominal pain or school attendance. Children with PD did have a higher number of emergency room visits and hospitalizations (p=0.042 and 0.021, respectively) compared with non-PD. Children with PD were less likely to be exocrine pancreatic insufficient (p=0.032).
Conclusion: The frequency of PD was only slightly higher in our cohort compared with reported rates in the general population. PD did not seem to associate with genetic mutations to influence the risk of disease. PD overall did not appear to impact the risk for pancreatitis or disease progression in childhood.
Knocking Down ZIP4 Inhibits Epithelial-Mesenchymal Transition-Induced Metastasis of Pancreatic Cancer
M. Liu, J. Yang, C. Houchen, R. Postier, M. Li. Department of Medicine, Department of Surgery, The University of Oklahoma Health Sciences Center, Oklahoma City, OK.
Background: A dietary zinc transporter, ZIP4, has been shown to play an important role in tumor progress and metastasis. Our previous studies found ZIP4 is overexpressed in human pancreatic cancer and contributes to cancer cell proliferation, migration and invasion. EMT is considered to be important in initiating the process of tumor metastasis. But how does ZIP4 regulate tumor invasiveness and EMT progress in pancreatic cancer has not been investigated. Our goal is to study the underlying mechanism of ZIP4-regulated EMT in pancreatic cancer.
Methods: Functional analysis of ZIP4 knocking down was investigated in human pancreatic cancer cell lines for invasion and migration. The expression profile of ZIP4 and EMT markers ZO-1, claudin-1, ZEB1, Slug, MMP7, MMP9 in pancreatic cancer cell lines, xenograft tumors and 87 human pancreatic cancer tissue specimens were detected by Western blotting, immunohistochemistry, and the correlation between ZIP4 and the EMT markers was also analyzed.
Results: We found that knocking down ZIP4 inhibited pancreatic cancer migration and invasion both in pancreatic cancer cell lines and orthotopic xenograft tumors. Correlations between ZIP4 and the EMT markers were analyzed in both orthotopic xenografts and human pancreatic cancer tissues, and it’s demonstrated that ZIP4 is positively related with ZEB1, Slug, MMP7, MMP9 but inversely associated with ZO-1 and claudin-1.
Conclusion: Our results indicate that knocking down ZIP4 inhibits pancreatic cancer invasion and migration and metastasis through repressing EMT. This study suggests a new molecular link of ZIP4 and EMT process in pancreatic cancer, and may provide new insight on targeted therapy for this malignant disease.
Pro-Fibrogenic Gremlin is a Novel Marker of Pancreatic Stellate Cells
K. Liu,1 Y. Cao,1 C. Rastellini,2 J. Bailey,1 C. Chao,2 T. Ko.1 1UTHealth, Houston, TX; 2UTMB Health, Galveston, TX.
Introduction: Despite the growing number of markers for pancreatic stellate cells (PSCs), limitations remain regarding specificity. For example, vitamin A droplets, desmin and glial fibrillary acidic protein (GFAP) are detected in quiescent PSCs, and α-smooth muscle actin (SMA) is a marker of activated PSCs. Gremlin (Grem1) is a glycoprotein, important in development and fibrosis. We previously reported that Grem1 is expressed in the pancreas, elevated in chronic pancreatitis (CP), and functions as a pro-fibrogenic mediator. However cellular localization of Grem1 in the pancreas is not clear. This study examines Grem1 expression in the pancreas compared with currently available PSC markers.
Methods: Grem1 cellular localization was detected by immunofluorescence in human and mouse pancreas, cerulein- and pancreatic duct ligation (PDL)-induced mouse CP and isolated human and mouse PSCs. Cellular markers including vimentin (a stromal marker), E-cadherin (E-cad, an epithelial marker), F4/80 (a macrophage marker), and PSC markers including desmin, GFAP and α-SMA were used. Oil Red O (ORO) staining was used for detection of vitamin A droplets.
Results: Increased Grem1 staining is observed in CP compared to normal pancreas. Grem1 staining co-localizes with vimentin (100%), not with E-cad or F4/80, indicating Grem1 positive cells localize to stroma. Grem1 staining co-localizes with desmin (80-90%) in normal and CP, with α-SMA (57-86%) in CP, indicating Grem1 labels both quiescent and activated PSCs. In vitro in PSCs, Grem1 staining is overlapped with ORO (~60%), desmin (~20%), GFAP (~65%) and α-SMA (~90%).
Conclusions: We have identified Grem1 as a novel and unique PSC marker, which has advantage over existing markers as it labels both quiescent and activated PSCs.
Elevated Circulating Histones Associate With Multiple Organ Dysfunction Syndromes in Acute Pancreatitis
W. Huang,2 T. Liu,1 S. Abrams,1 L. Wang,3 P. Szatmary,2 Y. Alhamdi,1 Z.Q. Lin,3 I. Welters,4 G. Wang,1 C.H. Toh,5 R. Sutton.2 1Department of Clinical Infection, Microbiology and Immunology, Institute of Infection and Global Health; and 2NIHR Liverpool Pancreas Biomedical Research Unit, Royal Liverpool University Hospital NHS Trust; University of Liverpool, Liverpool, United Kingdom; 3Department of Integrated Traditional Chinese and Western Medicine, Sichuan Provincial Pancreatitis Centre, West China Hospital, Sichuan University, Chengdu, China; 4Intensive Care Unit, Royal Liverpool University Hospital, Liverpool, United Kingdom; 5Roald Dahl Haemostasis & Thrombosis Centre, Royal Liverpool University Hospital, Liverpool, United Kingdom.
Background & Aims: As circulating histones are elevated in multiple organ dysfunction (MODS) we sought to identify associations between circulating histones and clinical events in patients with acute pancreatitis (AP).
Methods: Consecutive AP patients aged 18–85 y and no advanced co-morbidities with a primary admission at our hospital (n=260, blood sampling <24 h) or referred (n=52) from other hospitals or healthy controls (n=47) were recruited. Referred patients had persistent organ failure (POF, ≥48 h) (blood sampling <24 h of admission to ICU then daily for one week) within 3 weeks of disease onset. Histones, cytokines and routine biochemical markers were measured. Multivariable analyses determined associations between circulating histone levels and age, aetiology, comorbidity (Charlson), cytokines, organ failure, necrosis, infection (P significant <0.05).
Results: There 176 mild (no organ failure), 59 moderate (transient organ failure) and 25 severe (POF) primary admissions; all inter-hospital referrals were severe (POF). Circulating histones were significantly elevated in all severe patients, significantly more so in referral patients; other patients were similar to healthy controls. Circulating histones significantly correlated with SIRS (r=0.383), APACHE II (r=0.434) and SOFA (r=0.408) scores, troponin T, urea and pro-inflammatory cytokines (IL-1β, r=0.376; IL-6, r=0.367; IL-8, r=0.566; all P<0.001). In multivariate analysis only POF (OR=57 primary; OR=121 with referrals) was significantly associated with elevated circulating histones (≥5.4 μg/ml).
Conclusion: Elevated circulating histones occur in POF <24 h and rise to even higher levels during the progression of POF in AP. Histones may have a pathogenic role and might be targeted to treat POF in AP.
Necrotizing Pancreatitis Following Partial Pancreaticoduodenectomy: An Analysis of 1235 Consecutive Cases
M. Loos, M. Dietrich, O. Strobel, U. Hinz, M.W. Büchler, T. Hackert. Department of General, Visceral, and Transplantation Surgery, University of Heidelberg, Heidelberg, Germany.
Background: Postoperative necrotizing pancreatitis (PNPa) is a rare but deleterious complication after partial pancreaticoduodenectomy (PPD). This study aimed to analyze the clinical relevance of PNPa following PPD and to identify laboratory parameters to enable its early detection.
Methods: Patients undergoing PPD between Jan 2010 and Dec 2014 in the Department of Surgery in Heidelberg were identified from a prospective database. Perioperative clinical, surgical, pathological, and laboratory data were analyzed and risk factors were assessed.
Results: Of 1235 consecutive patients (pts), 16 pts developed PNPa (1.29%) and 46 (3.7%) pts had postoperative pancreatic fistula requiring reoperation (POPF C), including all pts with PNPa. In 313 pts, serum lipase activity was at least three times upper limit of normal in the postoperative course (all pts with PNPa (100%); 15 of 30 pts with POPF C without PNPa (POPF C*; 50%), and 106 of 590 pts with normal postoperative courses (NPC; 18%)). Median serum and drain lipase levels on postoperative day 1 (POD 1) were significantly increased in pts with PNPa (513.0 U/l and 14,744 U/l, respectively) compared to pts with POPF C* (155 U/l; 3.541 U/l) and NPC (41 U/l; 203 U/l; p<0.0001). All pts with PNPa had both serum and drain lipase levels increased on POD 1 (POPF C*: 39%; NPC 18%). Despite immediate resection of the pancreatic remnant, 90-day mortality was 38% in pts with PNPa.
Conclusion: Necrotizing pancreatitis of the pancreatic remnant is an underestimated but relevant problem following PPD. The combination of serum and drain lipase levels on postoperative day 1 can be helpful for early detection of PNPa. Early relaparotomy with resection of the necrotic pancreatic remnant is mandatory but still associated with high mortality.
Outcomes of Surgical Management for Pancreatic Neuroendocrine Tumors (PNETs): A Single-Center Experience
X. Lu, B. Hou, Y. Zhou, D. Li. General Surgery, Guangdong General Hospital, Guangzhou, China.
Background: The optimal management for patients with pancreatic neuroendocrine tumors (PNETs) is surgical resection. While the indication for small PNETs (≤2 cm) and the appropriate operation method remains controversial. We summarized our PNETs cases in recent years to assess the outcomes of surgical management for PNETs.
Methods: From March 2003 to December 2015, a total of 71 consecutive patients with PNETs underwent surgery were selected. Data were identified from a prospectively maintained database. Postoperative and survival outcomes were retrospectively analyzed. We also compared different operation method (enucleation and standard resection) to evaluate feasibility and the difference in postoperative complications. 5-year survival rate was investigated between patients with PNETs ≤2 cm and >2 cm.
Results: There were 28 patients (38.4%) who exhibited clinical symptoms. Tumor enucleation was performed in 16 patients (22.5%), pancreatoduodenectomy in 17 (23.9%), distal pancreatectomy in 22 (31%) and partial pancreatectomy in 11 (15.5%). Overall morbidity was 28.2%, while the most common complications were pancreatic fistula (8.5%) and anastomotic bleeding (9.9%). Based on our data, enucleation was regarded as a relatively safe procedure dealing with small tumor located at the surface layer away from the pancreatic duct. The rate of pancreatic fistula between the enucleation group (6.25%) and standard resection group (10.0%) was comparable (P = 0.548). About 40.8% patients had a small PNET (≤2 cm), and these patients had an overall survival advantage over patients with PNETs >2 cm (5-year survival rate: 94.1% vs 81.2%, P = 0.048).
Conclusions: Based on tumor size and location, enucleation may be a safe procedure during resection of small PNET away from the pancreatic duct. Patients with PNETs ≤2 cm have a survival benefit to those with PNETs >2 cm. However, a well-designed randomized controlled trial is necessary to more thoroughly evaluate it.
Human Pancreatitis Organellar Disorders: Ex-Vivo Models
A. Lugea,1 G. Groblewski,2 R.T. Waldron,1 S. Messenger,2 D.D. Thomas,2 E. Jones,2 H.-Y. Su,1 O.A. Mareninova,3 S. Gretler,3 J. Yang,1 I. Gukovsky,2 A.S. Gukovskaya,3 F. Gorelick,4 S.J. Pandol.1 1Cedars-Sinai Medical Center, Los Angeles, CA; 2University of Wisconsin, Madison, WI; 3UCLA/VAGLAHS, Los Angeles, CA; 4Yale University, New Haven, CT.
Introduction: Knowledge of the mechanisms of acute pancreatitis is largely based on studies using rodents. To assess similar mechanisms in humans, we performed ex vivo studies in human pancreatic acini.
Methods: Acini isolated from pancreatic cadaveric tissues from organ donors were incubated for up to 24 hours with carbachol (CCh) to determine functional integrity. Cell and organelle morphology was characterized by electron, light, and fluorescence microscopy. Physiologic and pancreatitis responses to CCh and taurolithocholic acid-3-sulfate (TLCS) were assayed: amylase secretion, trypsin activity, LDH release and propidium iodide uptake (cell viability), mRNA and protein levels of LC3 and p62 (impaired autophagy), sXBP1 andCHOP (ER stress), and cytokine/chemokine release.
Results: Proteomic analysis of acini from donors of diverse ethnicity showed similar profiles of digestive enzymes and proteins involved in translation, protein folding, and endolysosomal function. Muscarinic 3 receptor and bile salt-sensitive transporter/GPCR mRNA were found by qPCR. Human acini maintained physiologic responses to CCh for at least 24 h. As in rodent acini, supraphysiologic concentrations of CCh and TLCS caused trypsinogen activation, decreased cell viability; organelle damage manifest by disordered autophagy (LC3-II accumulation), mitochondrial fragmentation, and pathological ER stress (decreased sXBP1 and increased CHOP). Human acini also secreted inflammatory mediators elevated in acute pancreatitis patients including IL6, TNFα, PAI-1, IL1β, MIF, and chemokines mediating neutrophil and monocyte infiltration.
Conclusions: human cadaveric pancreatic acini maintain physiologic functions ex-vivo and have similar organellar disorders with pancreatitis-causing treatments as observed in rodent acini.
Regulation of Yes-Associated Protein 1 in Activated Pancreatic Stellate Cells
A. Lugea, J. Yang, H.-Y. Su, R.T. Waldron, Q. Chen, Q. Wang, S.J. Pandol. Cedars-Sinai Medical Center, Los Angeles, CA.
Yes-associated protein 1 (YAP), a transcriptional co-activator in the Hippo pathway, regulates cell differentiation and morphology in many cell types, and supports aberrant tumor growth. We reported YAP upregulation in pancreas tissues from PDAC and chronic pancreatitis patients, with YAP largely found in cancer cells and stellate (PaSC) cells in the stroma. We studied here the role of YAP in PaSC activation in PDAC. Pancreas of P48/Cre; KrasG12D (KC) mice displayed YAP upregulation at an early stage (3-weeks-old) in parallel with PaSC activation and proliferation. Consistent with these findings, we found YAP expression in cultured-activated mouse and human PaSC but not in quiescent, freshly isolated cells. PaSC isolated from KC mice or PDAC patient tissues were fully activated and displayed robust nuclear YAP suggesting YAP transcriptional activity. Factors that induce quiescence such as Bromodomain and Extra-Terminal (BET) inhibitors, the vitamin D analogue Calcipotriol and p38 MAPK inhibitor SB203580 reduced YAP levels in cultured PaSC. Moreover, selective mTOR inhibition also decreased YAP expression and reduced cell growth in PaSC. Experimental evidence indicates that YAP phosphorylation at Ser127 confines it to the cytoplasm and reduces its nuclear activity. In PDAC-associated PaSC, TGFβ1 promoted fibrosing responses but had little effect on cell proliferation, levels of Ser127-phosphorylated YAP or YAP nuclear location. In contrast, IGF1 and PDGF that exert potent mitogenic effects in PaSC, induced marked YAP Ser127 phosphorylation, an effect likely related with actin reorganization.
In conclusion, our data suggest complex crosstalk between distinct signaling systems including YAP, TGFβ1 and mitogenic signals regulating the activation state of PaSC in PDAC.
Xbp1 Genetic Deletion Accelerates Tumor Progression in Kras-Induced Pancreatic Tumorigenesis
A. Lugea, H.-Y. Su, R.T. Waldron, J. Yang, C. Hu, H. Hurley, S.J. Pandol. Cedars-Sinai Medical Center, Los Angeles, CA.
Pancreatic adenocarcinoma (PDAC) originates from neoplastic transformation of pancreatic acinar cells driven by Kras activating mutations. We reported that XBP1s, a transcription factor that regulates ER function and protein trafficking, is essential for the secretory phenotype of the acinar cell. XBP1s also exhibits pro- or anti-tumor effects depending on the cancer type. We studied whether XBP1s acts as tumor suppressor delaying Kras-induced PanIN formation and tumor progression.
Materials & Methods: We used acinar cell specific (Ela1-cre/ERT2; XBP1[INCREMENT]ac) Xbp1 KO, pancreas specific (Ptf1a-Cre) Xbp1 partial genetic deletion (XBP1[INCREMENT]pn), and Ptf1a-Cre; KrasG12D (KC) mice, and mouse and human acinar cells treated with a specific inhibitor of XBP1s (IRE1-I) or infected with Ad-XBP1s.
Results: XBP1[INCREMENT]ac mice exhibited extensive acinar cell loss, acinar-ductal-metaplasia (ADM) and inflammation. XBP1s levels declined in long-term cultured acinar cells in parallel with loss of secretory capacity and upregulation of ADM markers. Similarly, XBP1 deficient acinar cells had less CCK-induced amylase secretion; this effect was reversed by Ad-XBP1s infection. These data demonstrate that XBP1 maintains acinar cell secretory function and differentiation. XBP1s protein levels were higher than wild-type in pancreas of young KC mice (<8 week-old), but significantly lower in KC mice exhibiting PDAC (6–9 months). XBP1 genetic deletion in KC mice markedly hastened desmoplasia, inflammation and PanIN progression. Compared to age-matched KC mice, 4-month-old KC;XBP1[INCREMENT]pn mice had almost complete loss of exocrine parenchyma, widespread fibrosis, pancreatic adipose tissue infiltration, and PDAC.
Conclusion: Our data suggest that XBP1 acts as tumor suppressor by preventing Kras-induced acinar cell neoplastic transformation.
IL-6 and PD-L1 Antibody Blockade Combination Therapy Limits Tumor Progression in Murine Models of Pancreatic Cancer
T. Mace,1 R. Shakya,1 J.R. Pitarresi,1 B. Swanson,1 C. McQuinn,1 S. Loftus,1 L. Yu,1 G. Young,1 X. Zhong,2 T. Zimmers,2 M. Ostrowski,1 T. Ludwig,1 M. Dillhoff,1 C. Schmidt,1 D.L. Conwell,1 T. Bekaii-Saab,3 G. Lesinski.1 1The Ohio State University, Columbus, OH; 2Indiana University, Indianapolis, IN; 3Mayo Clinic, Phoenix, AZ.
Objective: Pancreatic ductal adenocarcinoma (PDAC) is the fourth leading cause of cancer in America with few efficacious therapies and is accompanied by profound systemic immunosuppression that renders this disease non-responsive to immunotherapy. Limited efficacy of immune checkpoint inhibitors in PDAC has prompted investigation into combination therapy. We hypothesized that IL-6 blockade would modulate immunologic features of PDAC and enhance the efficacy of anti-PD-L1 checkpoint inhibitors therapy in PDAC.
Design: In vivo efficacy and mechanistic studies were conducted with antibodies (Ab) targeting IL-6, PD-L1, CD4, or CD8 in the subcutaneous Panc02 and MT-5 (KPC derived) models; in an orthotopic luciferace KPC model; and the aggressive, genetically engineered PDAC model [KrasLSL-G12D, Trp53LSL-R270H, Pdx1-cre, Brca2F/F (KPC-Brca2 mice)]. Systemic and local changes in immunophenotype were measured by flow cytometry or IHC as appropriate.
Results: In vivo studies demonstrated that combined IL-6 and PD-L1 antibody blockade limited tumor progression in KPC-Brca2 mice (p<0.001). Histological analysis following a 2 week treatment demonstrated a marked shift in the proportion of pancreata with PanIN1/2 lesions versus PanIN3 or adenocarcinoma, and significantly increased intratumoral CD3+ T cells (p=0.003). This treatment combination also increased splenic Th1 cells. IL-6 and PD-L1 blockade elicited CD8+ T cell-dependent efficacy in mice bearing subcutaneous MT5 (p<0.02) and Panc02 tumors (p=0.046) and was accompanied by increased intratumoral effector T lymphocytes (CD62L−CD44+). Additionally, combination IL-6 and PD-L1 antibody blockade limited tumor growth of orthotopic KPC-luciferase expressing tumor cells compared to isotype controls (p=0.05). Further, administration of CD8-depleting, but not CD4-depleting Ab abrogated the efficacy of combined IL-6 and PD-L1 blockade in mice bearing Panc02 tumors (p=0.002).
Conclusions: These pre-clinical results indicate that targeted inhibition of IL-6 may enhance the efficacy of anti-PD-L1 in PDAC.
Tissue Immunohistochemistry Differentiates Diabetic Exocrine Pancreatopathy From Chronic Pancreatitis
S. Majumder,1 N.A. Philip,1 Y. Zen,2 L. Zhang,3 R.P. Sah,1 W.S. Harmsen,1 F.T. Enders,1 T.C. Smyrk,3 S.T. Chari.1 1Mayo Clinic, Rochester, MN; 2Kobe University, Kobe, Japan; 3Laboratory Medicine & Pathology, Rochester, Mayo Clinic.
Background: Diabetic exocrine pancreatopathy (DEP) is a term used to describe fibro-atrophic changes in the exocrine pancreas in asymptomatic patients with diabetes mellitus (DM). To determine if DEP could be distinguished from CP we performed tissue immunohistochemistry (IHC) for markers of inflammation and fibrosis.
Methods: From the Mayo Clinic autopsy database we identified subjects with i) DEP (n=9) defined as DM patients without clinical pancreatic disease with moderate-to-severe fibrosis in the exocrine pancreas and ii) CP (n=7), based on clinical history and histopathology. Pancreatic tissue sections were stained with antibodies against CD3 (T cell), CD20 (B cell), CD68 (monocyte/macrophage), CD138 (plasma cell) and alpha smooth-muscle actin (αSMA) (myofibroblast/activated stellate cell). Unaware of clinical history or case distribution, an expert pathologist (YZ) reviewed this IHC panel on a digital pathology platform using a semi-quantitative score (0–12; product of distribution [0–4] and quantitative scores (0–3)).
Results: Mean (SD) CD3 and CD20 scores were lower in DEP compared to CP (CD3: 8.9 (3.3) vs 3.8 (3.2); CD20: 7.4 (3.0) vs 1.1 (1.9)). A one point increase in total score was associated with significantly decreased odds of DEP for 3 of the 5 IHC stains; CD3 (OR =0.64, 95% CI 0.43-0.96; p=0.03), CD20 (OR=0.54, 95% CI 0.33-0.88; p=0.01 and aSMA (OR=0.5, 95% CI 0.26-0.97; p=0.04). The aSMA immunostaining pattern also differed; thin peri-acinar and intralobular network in DEP (“chicken-wire”), compared to broad, predominantly interlobular distribution in CP.
Conclusion: Tissue IHC highlights key differences between DEP and CP. DEP is characterized by relative paucity of lymphoplasmacytic cells and a pattern of fibrosis that is distinct from that of CP.
Risk of Subsequent Pancreatic Cancer After Resection of Main-Duct Intraductal Papillary Neoplasms (MD-IPMN)
S. Majumder, N.A. Philip, N. Takahashi, R.P. Sah, K.C. Mara, S.T. Chari. Mayo Clinic, Rochester, MN.
Background: In subjects with surgically resected main duct IPMN (MD-IPMN), there is a significant risk of subsequent cancer in the remnant pancreas. In this study we aimed to identify predictors of subsequent pancreatic cancer (PC) in MD-IPMN.
Methods: The Mayo Clinic surgical pathology database was searched for all cases of MD-IPMN between 1997 and 2014. Cases with histologically confirmed main pancreatic duct (MPD) involvement either isolated or in a mixed pattern with branch-duct involvement were included. Outcomes of subsequent PC, and death related to MD-IPMN were assessed with survivorship analyses (Kaplan-Meier and Cox regression).
Results: In the182 patients with MD-IPMN included in this study the prevalence of concomitant PC was 22%. The mean (SD) duration of follow-up was 4.05 (3.5) years. Subjects who underwent total pancreatectomy or partial pancreatectomy with surgical margins positive for cancer were excluded from the analysis of long-term outcomes. For the remaining 142 cases, overall 5-year incidence of PC in the remnant pancreas was 14%. The risk of subsequent PC was higher in those with concomitant PC than those without (HR=12.87, 95% CI: 4.57-36.2, p < 0.001). In patients without concomitant PC, risk of subsequent PC was higher for those with intermediate or high-grade dysplasia (IGD/HGD) in the resected pancreas compared with low-grade dysplasia (LGD) (IGD vs LGD: HR = 21.8, 95% CI: 1.89-250.1, p=0.01; HGD vs LGD: HR= 17.01, 95% CI: 1.97-146.8, p=0.01). Age, maximal diameter of MPD and the extent of MPD dilation (diffuse vs segmental) were not risk factors of subsequent PC. The overall 5-year cumulative incidence of death due to MD-IPMN was 7.2%.
Conclusion: For patients with MD-IPMN undergoing segmental pancreatectomy, the presence of concomitant PC or IGD/HGD in the resected pancreas is associated with significantly increased risk of subsequent PC in the remnant pancreas. These patients would benefit from careful post-operative surveillance.
Does Unavailability of Biliary Intervention at Rural and Small Hospitals Impact Immediate Patient Outcomes in Biliary Acute Pancreatitis?: A National Analysis
A. Malli,1 S. El-Dika,2 S. McCarthy,1 J.R. Groce,2 A. Hinton,2 D.L. Conwell,2 S.G. Krishna.2 1Ohio State University-Wexner Medical Center, Columbus, OH; 2Ohio State University-Wexner Medical Center, Columbus, OH.
Background and Aims: The in-hospital availability of biliary intervention (BI) may favorably impact the outcomes of patients with biliary-acute pancreatitis (biliary-AP). We aimed to evaluate the impact of the unavailability of (BI) in US hospitals on the immediate outcomes of biliary-AP.
Methods: Ten years (2004–2013) of the Nationwide Inpatient Sample was reviewed to identify all adult inpatients with a principal diagnosis of biliary-AP. Hospitals were categorized into centers performing BI vs. not (non-BI). The main outcomes were in-hospital mortality, length of stay, and cost. Univariate and propensity score-matched cohort analyses were performed to compare the outcomes of both groups.
Results: A total of 339,472 (BI 331,518; non-BI 7,954) admissions for biliary-AP were identified. In the BI group, the most frequent intervention was ERCP (32.5%) while a minority (2.2%) had non-endoscopic BI.
The majority of hospitals in the non-BI group were rural (73.3% vs. 12%, p<0.01) and small volume hospitals (55% vs. 12%, p<0.01) compared to the BI group. Non-BI hospitals transferred out significantly more biliary-AP patients (18% vs. 2.8%, p<0.01). There was no difference in mortality between BI vs. non-BI hospitals [1% (n: 3,218) vs. 0.75% (n: 59); p 0.39].
A propensity-score matched cohort analysis did not reveal an association between non-BI hospitals and mortality (OR 1.29, p: 0.32). However, biliary-AP patients incurred shorter length of stay (by 0.6 days; p<0.01) and lower hospital costs (by $1,400; p<0.01) at non-BI hospitals.
Conclusion: A majority of rural and smaller hospitals lack availability of in-hospital biliary intervention without adversely impacting immediate biliary-AP outcomes. This is likely due to patient transfers to larger hospitals.
Targeted Polyplex Nanoparticle to Gastrin Inhibits Growth and Decreases Metastases of Pancreatic Cancer
C. Mankongpaisarnrung,1 J. Burks,2 S. Nadella,1 J. Wang,1 J.-I. Hahm,3 R. Tucker,4 A. Mahmaud,5 S. Stern,5 J. Smith.1 1Dept. of Medicine, Gastroenterology, Georgetown University, Washington, DC; 2Department of Medicine, Div. of Oncology, Georgetown University, Washington, DC; Department of 3Chemistry; 4Comparative Medicine; Georgetown University, Washington, DC; 5NIH, Nanotechnology Characterization Lab, Frederick, MD.
Introduction: Gastrin stimulates growth of pancreatic ductal adenocarcinoma (PDAC) in an autocrine fashion through the CCK-B receptor. RNAi techniques that downregulate gastrin expression inhibit PDAC growth in vitro; however, delivery of siRNA to tumors in vivo is difficult due to degradation by RNAses.
Aim: The aim of this study is to develop a novel 2nd generation nanoparticle (NP) that selectively targets PDAC and inhibits growth by downregulating gastrin in vivo.
Methods: We designed a thiol functionalized polyethylene glycol-block-poly(L-lysine) (SH-PEG-PLL) polymer that targets the CCK-B receptor. NPs were reacted with gastrin siRNA (GSi) to form a polyplex micelle. Three human PDAC cell lines that express gastrin (AsPc1, BxPC-3, and PANC-1) were cultured in vitro and treated with NP-GSi (120, 240, or 480nM), PBS, or NP-scrambled RNA control (Csc). Viable cell counts were done and gastrin expression was measured by qRT-PCR. Athymic nude mice bearing orthotopic luciferase-tagged BxPC-3 tumors were treated three times weekly with 0.1ml ip of targeted or untargeted NP-Gsi, NP-Csc, or PBS. Tumor volumes were measured by IVIS imaging weekly and after 4 weeks of therapy mice were euthanized, tumors and metastases dissected.
Results: Growth on PDAC in vitro was inhibited by NP-Gsi but not by NP-Csc or PBS controls (p=0.005). Gastrin expression was significantly downregulated by qRT-PCR in cells treated with NP-Gsi but not in control treated cells. Mice treated with CCK-receptor targeted NP-GSi (240nM) had no metastases whereas PBS, untargeted NP-GSi, and targeted NP-Csc all had metastases to liver and mesenteric nodes.
Conclusion: A novel NP that delivers gastrin siRNA decreases growth and metastases of PDAC.
Changes in the Expression of RRM Subunits Induce Gemcitabine Resistance in a Growth Dependent Manner
K.S. Mann, S. Brumskill, P. Ghaneh, W. Greenhalf. Department of Molecular and Clinical Cancer Medicine, University of Liverpool, Liverpool, United Kingdom.
Background: Inhibition of Ribonucleotide Reductase (RRM) pauses the cell cycle in G1, while incorporation of fluorinated nucleotides in DNA causes damage and cell death. The literature suggests high RRM1 levels are associated with gemcitabine resistance. The p53R2 subunit is a p53-regulated homologue of RRM2. We've selected resistant Suit-2 cell lines that all have RRM1/RRM2 levels that vary from parent lines, some of which indicate reduced RRM activity. Hypothesis: perturbation of the RRM complex will increase resistance to gemcitabine.
Methods: Suit-2, Suit-2R & Suit-2R2 cell lines were transfected with RRM1 siRNA and cDNA. After 24hrs, varying doses of gemcitabine were added for 48hrs. An MTS assay was performed and IC50 calculated. Western analyses of RRM1, RRM2 and p53R2 were performed on cell lysates. Cell cycle analysis was carried out by FACS.
Results: The initial Suit-2, Suit-2R2 and Suit-2R assays demonstrated a gemcitabine IC50 of 25nm, 257nm and 160nm, but the knockdown assay demonstrated a decrease in IC50 of all cell lines; 13nm, 132nm and 95nm respectively. In resistant lines cells gemcitabine caused accumulated in G1 while in the parent cells accumulated in S-phase. Overexpression gave IC50s of 23nm, 250nm and 190nm respectively: non-significant changes. Western analysis of the parent SUIT-2 showed the expected reduction in RRM1 but no significant change to RRM2/p53R2. Both Suit-2R and Suit-2R2 exhibited increases in RRM2 and maintained high levels of p53R2 compared to the parent line.
Discussion: Changes in expression of RRM subunits are variable in distinct resistant clones of the Suit-2 cell line. This has an impact on cell cycle progression causing an arrest in G1. These clones seem to have developed a different regulatory response to changes in RRM1 level: the increase in p53R2 suggest a p53-mediated process instigating resistance and affecting DNA transcription and translation.
Conclusion: alterations in RRM are a consequence of gemcitabine resistance but not a mechanism for it.
Downregulation of Atg4B Stimulates Autophagy and Ameliorates Alcohol-Induced Pancreatic Injury
J.M. Elperin,1 S. Suriany,1 G.E. Lee,1 S.W. French,2 A.S. Gukovskaya,1 I. Gukovsky,1 O.A. Mareninova.1 1Veterans Affairs Greater Los Angeles Healthcare System and University of California at Los Angeles, Los Angeles, CA; 2Southern California Research Center for ALPD and Cirrhosis, Los Angeles, CA.
Introduction: Atg4B is a cysteine protease that regulates autophagy by delipidating LC3-II, a key mediator of autophagosome formation. Autophagy is increasingly implicated in the pathogenesis of non-alcoholic pancreatitis, but how it is affected by alcohol, a major risk factor for pancreatitis, is largely unknown. Here, we investigate ethanol’s effects on Atg4B and its role in alcoholic pancreatitis.
Methods: Mice were pair fed for 6 weeks ethanol or control Lieber-DeCarli diet followed by low-dose cerulein. In ex-vivo model, mouse and human acinar cells were incubated with 50–100 mM ethanol followed by 100 pM CCK-8. We measured autophagy and pancreatitis parameters, Atg4B expression and its activity with immunoblotting, light and electron microscopy, and enzymatic assays. Effects of Atg4B overexpression or knockdown were assessed in the ex-vivo model using adenoviral vectors expressing mCherry-Atg4B or Atg4B shRNA.
Results: Ethanol inhibited autophagy in pancreatitis models, evidenced by decreased LC3-II level, accumulation of p62/SQSTM1, and reduced autophagic flux. We found that ethanol markedly increased Atg4B level and activity, both in tissue and cells, by inhibiting caspases that degrade Atg4B. Atg4B overexpression inhibited autophagy in acinar cells and increased trypsin activity and necrosis, thus mimicking the effects of alcoholic pancreatitis. Silencing Atg4B increased LC3-II level and p62 degradation, and prevented ethanol-induced trypsinogen activation and necrosis in acinar cells.
Conclusions: Alcohol exposure inhibits pancreatic autophagy by stimulating Atg4B-mediated LC3-II delipidation. Downregulation of Atg4B alleviates pathologic responses of alcoholic pancreatitis. The results reveal a novel mechanism by which ethanol predisposes to pancreatitis.
Clinical Evaluation and Management of Exocrine Pancreatic Insufficiency (EPI) After Pancreatic Resection: A Retrospective Analysis
S. Masood, V.T. Kommineni, A.K. Mathur, N.N. Katariya, A.A. Moss, C.C. Nguyen, L.J. Miller, D.O. Faigel, R. Pannala. Mayo Clinic, Scottsdale, AZ.
Background/Aims: We evaluated the prevalence of clinically recognized EPI and its management in patients who underwent resection for pancreatic neoplasms.
Methods: We included all patients who underwent pancreatic resection for neoplasms at our institution from January 2010 -December 2013 (n=141). Patients with a pathologic diagnosis of chronic pancreatitis without neoplasm were not included. Demographic and clinical data including steatorrhea, EPI testing (72 hour stool fat testing), and pancreatic enzyme replacement therapy (PERT) prescriptions were abstracted. Fecal elastase test was not available at our institution in that time period. Data are presented as means±SD or median (interquartile range, IQR). Chi Square test was performed to assess association between EPI and other categorical clinical variables.
Results: Mean age and BMI were 65±13 years and 28±6 kg/m2; 54% were male. 68 (48%) patients underwent a pancreaticoduodenectomy (PD), 59 (41%) had a distal pancreatectomy, and 14 (10%) had enucleation/other surgeries. Pathological diagnosis was ductal adenocarcinoma (56, 40%), neuroendocrine tumor (32, 23%), and cystic/other neoplasms (53, 38%). Mean tumor size was 3±2 cm; 48% of tumors were located in the pancreatic head. Diabetes (DM) was noted preoperatively in 12 (9%) and postoperatively in 38 (27%) patients. Clinical steatorrhea was noted in 34 (24%) patients at a median duration of 31 (IQR 13–123) days; only 2 (1%) patients had 72 hr stool test of which one test was abnormal. PERT was prescribed in 39 (28%) patients; 32 of these patients (82%) had a documented clinical response. Per meal median PERT dosage was 24,000 (IQR 12,000-42,000) lipase units. EPI prevalence was significantly higher in PD patients (24/68, 35%), compared to distal pancreatectomy (9/59, 15%, p<.01).
Conclusions: EPI is common after surgery for pancreatic neoplasms but objective testing is rarely performed and PERT dosing appears lower than recommended. Pre- and postoperative evaluation of EPI and adequate PERT dosing based on objective testing in this population is warranted.
High-Grade PanIN/Carcinoma In Situ of the Pancreas Associated With Cystic Changes and Fibrosis
Y. Matsuda,1 T. Furukawa,2 S. Yachida,3 M. Nishimura,4 A. Seki,1 K. Nonaka,1 J. Aida,5 K. Takubo,5 T. Ishiwata,5 W. Kimura,6 T. Arai,1 M. Mino-Kenudson.7 1Department of Pathology, Tokyo Metropolitan Geriatric Hospital, Tokyo, Japan; 2Institute for Integrated Medical Sciences, Tokyo Women's Medical University, Tokyo, Japan; 3Division of Cancer Genomics, National Cancer Center Research Institute, Tokyo, Japan; 4Department of Endoscopy, Tokyo Metropolitan Geriatric Hospital, Tokyo, Japan; 5Department of Aging and Carcinogenesis, Research Team for Geriatric Pathology, Tokyo Metropolitan Institute of Gerontology, Tokyo, Japan; 6Department of Gastroenterological, General, Breast & Thyroid Surgery, Yamagata University, Yamagata, Japan; 7Department of Pathology, Massachusetts General Hospital, Boston, MA.
Introduction: Recently we have reported that most primary pancreatic tumors found in 8,399 autopsy cases were invasive ductal adenocarcinomas, and approximately 8% of pancreatic invasive ductal adenocarcinomas had progressed asymptomatically and were discovered incidentally at autopsy; however, distant metastasis present in 27% of these occult cancers. Thus, it is important to detect pancreatic cancers at an early stage, preferably at a preinvasive stage. This study aimed to investigate clinicopathological features associated with high-grade PanIN/carcinoma in situ of the pancreas (HD-PanIN).
Methods: Tissues obtained from 173 consecutive autopsies without invasive cancer or IPMN performed at a single tertiary hospital between 2012 and 2015 were used in this study. The entire pancreas was sliced with a 5 mm interval and all the sections were processed for microscopic examination. Ductal lesions as well as the extent and distribution of fibrosis and fatty changes in both intra- and extralobular regions were evaluated in each case.
Results: HD-PanIN was found in 4% of the 173 cases, and was more frequently identified in patients with diabetes mellitus and/or older age. HD-PanIN lesions showed the following characteristics: multifocality, cystic changes, the pancreatic body and tail location, and small interlobular/intralobular ducts involvement. HD-PanIN was also accompanied by higher-grade extralobular fibrosis, but was not associated with pancreatic fat infiltration.
Conclusions: HD-PanIN lesions were identified in 4% of our study cohort, and were associated with several unique clinicopathological features. The cystic changes along with fibrotic pancreatic parenchyma that could be depicted by imaging studies such as endoscopic ultrasound may facilitate the detection of HD-PanIN.
Intracellular Trypsinogen Activation in Phagocyting Macrophages Acts as DAMP Fueling Severe Acute Pancreatitis
J. Mayerle,1 M. Sendler,1 F.-U. Weiss,1 T. Wartmann,2 W. Halangk,3 M.M. Lerch.1 1Department of Medicine A, University Medicine, Ernst-Moritz-Arndt-University Greifswald, Greifswald, Germany; 2Division of Experimental Surgery, Otto-von-Guericke University Magdeburg, Magdeburg, Germany; 3Division of Experimental Surgery, Otto-von-Guericke University Magdeburg, Magdeburg, Germany.
Introduction: Premature, intra-acinar cell trypsinogen activation is cathepsin B(CTSB)-dependent and an initial event in acute pancreatitis. A second peak of trypsinogen activation is thought to be mediated by infiltrating leukocytes. We have studied the role of protease mediated macrophage (MΦ) activation in experimental pancreatitis.
Methods: Bone marrow derived macrophages (BMDΦ) were isolated from CTSB−/− and C57Bl6-WT mice and cultured under MCSF stimulation. After 7d cells were incubated with trypsinogen, isolated acinar cells or were used for adoptive transfer to mice. Trypsin activity was measured by R110-Ile-Pro-Arg, cytokine release was measured by cytometric beads. Severe acute pancreatitis was induced by partial duct ligation.
Results: In pancreatitis MΦs but not neutrophils were found to contain intracellular trypsinogen and trypsin. BMDΦ exposed to either trypsinogen or co-incubated with CCK-stimulated acinar cells developed intracellular trypsin activity. Trypsinogen activation could be blocked with bafilomycin A1 and cytochalasin B and was absent in MΦs from CTSB−/− animals. On confocal imaging intracellular trypsin activity was confined to a vesicular compartment and co-localised with CTSB activity. In response to intracellular trypsin activation MΦs secreted pro-inflammatory cytokines acting as M1-Φ, and this process was blocked by nafamostat. In vivo adoptive transfer of WT MΦ to CTSB−/− mice restored trypsinogen activation in the pancreas during pancreatitis.
Conclusion: Intracellular protease activation is not restricted to pancreatic acinar cells. In phagocytosing macrophages trypsinogen is activated in a CTSB-dependent manner. MΦ activating trypsinogen polarise to M1Φ, release pro-inflammatory cytokines and contribute to disease severity. Intracellular active trypsin in MΦ acts as a danger-associated molecular pattern molecule (DAMP).
EGFR1 Targeted Delivery of 5-Fluorouracil Using Tumor Specific Theranostic Aptamers in Pancreatic Ductal Adenocarcinoma
U.M. Mahajan,1 J.P. Kühn,2 T. Marschall,1 B. Appel,3 F. Lämmerhirt,1 M. Sendler,1 P.R. Wagh,1 S. Müller,3 F.-U. Weiss,1 M.M. Lerch,1 J. Mayerle.1 Departments of 1Medicine A; and 2Diagnostic Radiology and Neuroradiology; University Medicine, Ernst-Moritz-Arndt-University Greifswald, Greifswald, Germany; 3Institute of Biochemistry, Ernst-Moritz-Arndt University, Greifswald, Germany.
Introduction: Pancreatic ductal adenocarcinoma (PDAC) is projected to be the second leading cause of cancer related death by 2030. The fluoropyrimidine 5-fluorouracil (5FU) has been used to treat PDAC in the context of neoadjuvant, adjuvant and palliative chemotherapeutic regimens. In spite of combinations with other agents 5FU efficiency remains temporary and limited. One explanation for the inadequate response is an insufficient and nonspecific delivery of 5FU to the tumor.
Aim: We constructed and evaluated theranostic aptamers targeting pancreatic cancer cells via the EGFR1 combined with tumor specific delivery of 5-Fluorouracil as well as a [18F]-FDG residue for Positron-Emission-Tomography (PET). This theranostic approach allows for a companion diagnostic imaging.
Materials and methods: 5FU-incorporated SELEX-selected EGFR1 specific aptamers using in-vitro transcription were synthesized followed by 5’ labeling of [18F]-FDG to the 5' end of the aptamers for PET to monitor tumor size and to explore the possibility of aptamers to be selectively taken up by tumors and metastases.
Results and discussion: 5FU-aptamers reduced proliferation in a concentration-dependent manner in various mouse and human pancreatic cancer cell-lines. Time-lapsed live cell imaging revealed specific uptake of 5FU-aptamers as early as 5 minutes. Pretreatment with cetuximab (an anti-EGF antibody) lead to significantly reduced uptake of 5FU-aptamers confirming the specificity towards the EGFR1. 5FU-aptamer treatment was equally effective in 5FU-sensitive and 5FU-refractory PDAC cell-lines. 5FU-aptamer treatment in krasG12D,p53R172H,pdx.creTg+/− mice (KPC) lead to uptake quantified by diffusion weighted MRI as well as significant reduction in tumor volume.
Conclusion: EGFR1 targeted delivery of 5 Fluorouracil using tumor specific theranostic aptamers shows high tumor specificity, overcomes the resistance mechanism of conventional 5FU chemotherapy and represents a successful treatment for pancreatic cancer in KPC mice.
Statin Use Is Not Associated With SIRS or Outcomes in Acute Pancreatitis
J. McNabb-Baltar,1 V. Antoine-Gustave,2 V. Kadiyala,3 D.X. Jin,3 S.L. Suleiman,3 D.L. Conwell,4 P.A. Banks.3 1Center for Pancreatic Disease, Division of Gastroenterology, Hepatology, and Endosocopy, Brigham and Women's Hospital, Boston, MA; 2Gotham Medical Associates, New York, NY; 3Center for Pancreatic Disease, Division of Gastroenterology, Hepatology, and Endoscopy, Brigham and Women's Hospital, Boston, MA; 4Ohio State University-Wexner Medical Center, Columbus, OH.
Introduction: The association between statin use and outcomes of acute pancreatitis is controversial, with some reports suggesting a protective effect and others the opposite. On the basis of these considerations, we assessed the impact of statin use on outcomes of acute pancreatitis.
Methods: Retrospective cohort study of patients, 18 years or older, admitted to our institution with AP (01/01/99 - 12/31/09). Smoking status was classified as never or ever smoker. Outcomes included systemic inflammatory response syndrome (SIRS) on admission, presence of pancreatic necrosis, intensive care unit (ICU) stay, length of stay greater than 7 days (LOS) and death. We performed multivariable analysis to assess the impact of statins on outcomes controlling for covariates including age, gender, race, smoking status, etiology of pancreatitis.
Results: Overall, 2066 patients presented with AP, including 53.1% men. 63.6% were white. 7.1 % of patients were on a statin; 70 on simvastatin, 55 on atorvastatin and 22 on another statin. 7.3% of patients had SIRS on admission, 2.7% had pancreatic necrosis, 3% were admitted to the ICU, 20.7% had LOS > 7 days, and 1.2 % died. More patients on statins were ever smokers (53.4 vs. 12.5%, p < 0.001) and men (56.5 vs. 46.2%, p=0.01). Patients on statins were older (Mean age 72 vs. 61, p < 0.001). In multivariable analysis, statin use was not a predictor of SIRS on admission or poor outcomes. After controlling for confounders, smoking status remained a predictor of poor outcomes (SIRS: OR 3.6, p < 0.001, necrosis: OR 2.2, p=0.034, ICU: OR 2.5, p=0.006, LOS: 1.5, p=0.021, Died: OR 2.9, p=0.049).
Conclusions: In this large cohort study, statin use is not a predictor of SIRS on admission or poor outcomes in AP after controlling for confounders. However, smoking status is.
The Impact of Diabetes on Outcomes in Acute Pancreatitis: A Report From the Nationwide Inpatient Sample
J. McNabb-Baltar,1 A. Hinton,2 D.L. Conwell.3 1Center for Pancreatic Disease, Division of Gastroenterology, Hepatology, and Endosocopy, Brigham and Women's Hospital, Boston, MA; 2Ohio State University-Wexner Medical Center, Columbus, OH; 3Ohio State University Medical Center, Columbus, OH.
Introduction: Diabetes may affect the course of acute pancreatitis (AP). We sought to evaluate the impact of diabetes on AP-related clinical outcomes and health care utilization in the US.
Methods: The Nationwide Inpatient Sample (2008–2013) was reviewed to identify all adult inpatients (≥18 years) with a principal diagnosis of AP (ICD-9-CM 577.0). Uni- and multivariate analyses were used to examine primary clinical outcomes (mortality, renal failure, respiratory failure and hospital-acquired complications (HACs)) and secondary resource outcomes (length of stay and hospital charges). Propensity score-matched analysis was also performed to compare the outcomes in patients with and without diabetes.
Results: Diabetes was associated with 36.1% (423,469/1,172,752) of all AP admissions. The mean age of presentation was higher in patients with diabetes (Mean(SE) 55,8(0.07) vs. 50.8(0.06), P < 0.001). Etiology of pancreatitis also differed. Biliary (18.5 vs. 22.5%, P < 0.001) and alcohol (17.9 vs. 32.5%, P <0.001) pancreatitis were less common and chronic pancreatitis (16.8 vs. 14.4%, P < 0.001) was more common in the diabetes group. Diabetes was associated with increased HACs on both multivariate (OR 11.2, 95% CI 10.4, 12.2, P < 0.001) and propensity score-matched cohort analysis (OR 9.4, 95% CI 8.0, 11.1, P < 0.001). Acute kidney failure was more common in diabetes (12.8 vs.10.8%; P < 0.001). Diabetes was associated with a decreased inpatient mortality on both multivariate (OR 0.7, 95% CI 0.6, 0.8, P < 0.001) and propensity score matched analysis (OR 0.8, 95% CI 0.6, 0.9, P < 0.001). Hospital charges and length of stay were not increased in multivariate and propensity score-matched cohort analysis.
Conclusion: Although, diabetes is associated with adverse clinical outcomes of hospital acquired complications and renal failure, it is associated with decreased inpatient mortality. Costs and length of stay are not increased in patients with diabetes in acute pancreatitis. This suggests that diabetes may have a limited role in severity in acute pancreatitis.
Kras Mutation Imparts Neoplastic Potential on Duct Cells but Not Acinar Cells in a Mouse Model of Obstructive Chronic Pancreatitis
A.L. Means,1, 2 F.C. Pan,2 J. Kim,1 C. Shi,3 M.K. Washington,3 J. Kopp,4 M. Sander,4 M. Gannon,6 R.D. Beauchamp,1, 2 C.V. Wright.2 Deparments of 1Surgical Sciences; 2Cell and Developmental Biology; 3Pathology, Microbiology, and Immunology; Vanderbilt University Medical Center, Nashville, TN; 4Pediatrics and Cellular and Molecular Medicine, University of California, San Diego, La Jolla, CA; 6Medicine, Vanderbilt University Medical Center, Nashville, TN.
Chronic pancreatitis is the highest non-familial risk factor for pancreatic cancer. In order to understand the role of chronic pancreatitis in development of pancreatic cancer, we developed a mouse model that combines Kras mutation, an established initiator of pancreatic cancer, with obstructive chronic pancreatitis. The cell of origin for pancreatic ductal adenocarcinoma (PDAC) has been controversial but recent data indicate that both acinar cells and duct cells can give rise to PDAC when both copies of Trp53 gene are deleted in the background of Kras mutation. To address the issue of the cell of origin, we initiated Kras mutation in adult mice in either acinar cells or duct cells. To model obstructive chronic pancreatitis, we performed pancreatic duct ligation one month later, and followed the mice over time. We found that KrasG12D expression in duct cells but not in acinar cells led to progression from metaplastic ducts to eventual dysplasia and cancer. While Kras mutation in acinar cells increased acinar-to-ductal metaplasia (ADM), the cells upregulated p53 and underwent apoptosis, resulting in loss of most acinar-derived cells. Kras mutation in duct cells did not trigger increased p53 expression and had a lower rate of apoptosis. By 6–10 months’ post-ligation, all 6 duct-derived Kras mutant mice exhibited atypia and local invasion characteristic of PDAC. The microenvironment around these tumors displayed features characteristic of human PanIN3 and PDAC. Additionally, one mouse had lymph node involvement and one had metastasis to the liver. In summary, in the setting of obstructive chronic pancreatitis, Kras mutation in acinar cells leads to ADM with an increased rate of apoptosis, while Kras mutated duct cells progress from ADM to pancreatic ductal adenocarcinoma.
AdipoRon Suppresses Cytokine Mediated STAT3 Activation Through SOCS3 to Inhibit Pancreatic Cancer Growth
F. Messaggio, N. Nagathihalli, N. Merchant, M.N. VanSaun. Department of Surgery, University of Miami Miller School of Medicine, Sylvester Comprehensive Cancer Center, Miami, FL.
Introduction: The rising incidence of pancreatic cancer is associated with an increased prevalence of obesity, a documented risk factor for the disease. Obesity harbors a systemic chronic inflammatory disorder characterized by increased production and secretion of pro-inflammatory adipokines leptin and IL-6; while exhibiting a decrease in the anti-inflammatory adipokine; adiponectin. Dysregulation of these factors is thought to be a key mechanism of obesity-associated cancers, contributing to increased activation of mitogenic pathways, such as STAT3.
Hypothesis: We have demonstrated that adiponectin inhibits pancreatic cancer proliferation and tumor growth, however, the molecular mechanisms by which adiponectin regulates these processes are unknown. We hypothesize that Adiponectin Receptor (AdipoR) agonist, AdipoRon, suppresses STAT3 through modulation of SOCS3.
Results: In vitro assessment showed that AdipoRon was highly effective at inhibiting cell proliferation and increasing apoptosis. Immunoblot analysis of treated PDAC cell lines revealed that AdipoRon effectively suppresses pSTAT3 while increasing pAMPK in a dose dependent manner. Additionally, we demonstrated that AdipoRon can suppress STAT3 activation in response to cytokine-mediated stimulation such as Leptin or IL-6. Treatment with AdipoRon or with 15-PGJ2 (PPARγ agonist) results in an increased expression of SOCS3, which binds receptor tyrosine kinases and Jak2 to block the downstream phosphorylation of STAT3.
Conclusions: Our results demonstrate that adiponectin receptor activation increases SOCS3 expression, which correlates with suppression of pSTAT3 and decreased proliferation.
Malnutrition and Pancreatic Enzyme Supplementation in Chronic Pancreatitis Patients
M. Min,1 B. Patel,2 S. Han,3 J. Kheder,4 L. Bocelli,4 W. Wassef.4 1Department of Internal Medicine, University of Massachusetts Medical Center, Worcester, MA; 2Internal Medicine Residency, UMass Medical School, Worcester, MA; 3Gastroenterology, University of Colorado School of Medicine, Aurora, CO; 4Gastroenterology, University of Massachusetts Medical Center, Worcester, MA.
Background: Patients with chronic pancreatitis often develop pancreatic exocrine dysfunction, resulting in steatorrhea, malnutrition and maldigestion symptoms. Reducing fat intake remains the 1st line of therapy, but pancreatic digestive enzyme supplementation is often required for symptom relief and nutritional support. This study evaluates whether nutritional status and the amount of enzyme supplementation needed are related.
Methods: 36 patients with chronic pancreatitis on digestive enzyme supplementation were given the Malnutrition Universal Screening Test (MUST). The MUST scores were compared to the amount of enzyme therapy they were taking along with their body mass index (BMI).
Results: 22.2% of our patients were malnourished based on MUST scoring. The average BMI was 28.9. There were no differences between the malnourished group and the normal nourished group based on BMI (23.8 vs. 30.5, p<0.11) or pancreatic enzyme doses (57500 vs. 51666.7 units of lipase, p<0.27; 184500 vs. 168851.9 units of protease, p<0.31; 293000 vs. 267222.2 units of amylase, p<0.30). There was no significant correlation between MUST scores and pancreatic enzyme levels (p<0.83 for lipase, p<0.77 for protease, p<0.76 for amylase) or MUST scores with BMI (p<0.11).
Conclusion: Pancreatic insufficiency and fat malabsorption remains a challenging aspect of managing chronic pancreatitis. This study evaluated whether there was a correlation of nutritional status with pancreatic enzyme supplementation and found that there was none, suggesting that nutritional status alone cannot be used as a guide for pancreatic enzyme supplementation dosing. Further studies will be needed to identify the relationship of exocrine function with disease progression to better treat this disease.
Invasive Size Predicts Recurrence and Survival of Small Invasive Carcinoma Arising in Intraductal Papillary Mucinous Neoplasm of the Pancreas
M. Mino-Kenudson,1 V. Morales-Oyarvide,2 K. Date,3 T. Ohtsuka,3 Y. Omori,4 M. Tanino,5 Y. Mizukami,6 S.-M. Hong,7 D.W. Hwang,8 S.C. Kim,8 G. Zamboni,9 P. Castelli,10 R. Higuchi,11 M. Yamamoto,11 K. Shimizu,12 M. Nakamura,3 H. Maguchi,13 C. Fernandez-Del Castillo,14 T. Furukawa.15 1Department of Pathology, Massachusetts General Hospital, Boston, MA; 2Dana Faber Cancer Center, Boston, MA; 3Department of Surgery and Oncology, Kyushu University, Fukuoka, Japan; 4Department of Pathology, Teine Keijinkai Hospital, Sapporo, Japan; 5Department of Cancer Pathology, Hokkaido University Graduate School of Medicine, Sapporo, Japan; 6Center for Clinical and Biomedical Research, Sapporo Higashi Tokushukai Hospital, Sapporo, Japan; 7Pathology, Asan Medical Center, University of Ulsan College of Medicine, Seoul, Korea; 8Division of Hepatobiliary and Pancreas Surgery, Department of Surgery, Asan Medical Center, University of Ulsan College of Medicine, Seoul, Korea; 9Pathology, University of Verona and Don Calabria Hospital, Verona, Italy; 10Pathology, Don Calabria Hospital, Negrar, Italy; 11Surgery, Institute of Gastroenterology, Tokyo Women’s Medical University, Tokyo, Japan; 12Gastroenterology, Institute of Gastroenterology, Tokyo Women’s Medical University, Tokyo, Japan; 13Center for Gastroenterology, Teine Keijinkai Hospital, Sapporo, Japan; 14Department of Surgery, Massachusetts General Hospital, Boston, MA; 15Institute for Integrated Medical Sciences, Tokyo Women's Medical University, Tokyo, Japan.
Aim: To evaluate clinical outcomes of small invasive carcinomas arising in intraductal papillary mucinous neoplasms of the pancreas (small invasive IPMNs) stratified by the proposed size cut-offs (Ta: ≤0.5cm, Tb: >0.5cm - ≤1.0cm, Tc: >1.0cm - ≤2.0cm).
Methods: Pancreatic surgical databases from 6 hospitals were queried for IPMNs, with invasive components measuring ≤2.0cm (the largest diameter of invasive foci), resected since 2000. Clinicopathologic features were analyzed to identify predictors of recurrence and survival.
Results: A total of 92 small invasive IPMNs were found (34 [37%] Ta, 26 [28%] Tb and 32 [35%] Tc). Of those, 39% were multifocal and the most common histologic subtype was tubular (60%). Lymph node metastases were present in 13% and adjacent organ involvement in 9.8%. The invasive recurrence was seen in 20%, and the median time to recurrence was 18 months (2–69 months). The 5-year disease specific survival (DSS) rate was 90% and median DSS was not reached. In univariate analysis, Tc was associated with shorter recurrence-free survival (RFS) (5-year RFS rate: Ta 93%, Tb 86% and Tc 63%, p=0.004). The other predictors of shorter RFS included female gender (p<0.001), tubular invasive histology (p=0.006), gastric or pancreatobiliary IPMN epithelial type (p=0.023), nodal involvement, lymphatic, vascular and perineural invasion (p<0.001) and positive resection margin (p=0.041), but not adjacent organ involvement (p=0.097). Similarly, invasive size reversely correlated with RFS in the T1 subset (no adjacent organ involvement, p=0.016), the tubular carcinoma subset (p=0.017), node negative entire cohort (p=0.029), node negative T1 (p=0.049) and node negative tubular carcinoma (p=0.034) subsets, and also with DSS in the entire cohort (p=0.008), the T1 (p=0.035) and tubular carcinoma (p=0.040) subsets.
Conclusions: In this multi-institutional cohort of resected small invasive IPMNs, invasive recurrence was seen in 1/5. Along with other well-known predictors of survival, invasive size was predictive of invasive recurrence and survival.
Liquid Biopsy for Early Detection of Pancreatic Cancer
Y. Mizukami,1 Y. Ono,1 H. Karasaki,1 M. Ogata,1 A. Sugitani,1 K. Koizumi,2 S. Asahara,3 K. Kawakubo,4 K. Takahashi,5 H. Maguchi,5 K. Nagashima.1 1Center for Clinical and Biomedical Research, Sapporo Higashi Tokushukai Hospital, Sapporo, Japan; 2Center for Gastroenterology, Shonan Kamakura General Hospital, Kamakura, Japan; 3Department of Gastroenterology, Chiba Tokushukai Hospital, Funabashi, Japan; 4Department of Gastroenterology and Hepatology, Hokkaido University Graduate School of Medicine, Sapporo, Japan; 5Center for Gastroenterology, Teine Keijinkai Hospital, Sapporo, Japan.
The genetic alterations that are responsible for the initiation and progression of pancreatic ductal adenocarcinoma (PDA) could serve as immediate biomarkers for diagnosis of the disease. Circulating cell-free DNA (cfDNA) shed from tumors into the general circulation has been studied for monitoring tumor genetics and it offers the promise of a sensitive and non-invasive method to trace genomic evolution of cancer systematically. There are two main types of pathologically and genetically distinct precursors for PDA, pancreatic intraepithelial neoplasias (PanINs) and intraductal papillary mucinous cystic neoplasias (IPMNs), and the key driver mutations in KRAS and GNAS regulate disease initiation and progression. We established protocols for super-sensitive and absolute quantification of very low concentrations of the target mutant alleles using a droplet digital PCR platform (Bio-Rad; QX200) with a detection limit of 0.05%. The role of cfDNA genotyping to identify mutations in KRAS and GNAS were investigated in Japanese patients who have pancreatic tumors (UMIN000012810). Thus far, we recruited 60 PDA and 140 IPMN patients with either benign or malignant disease. In this meeting, data from surgically resected PDA/IPMN cases and concordance of genotyping between tumor tissue and plasma cfDNA will be presented. The major technical challenge of widespread implementation of this clinical test is the coverage of uncommon forms of KRAS mutation, as well as sensitivity for early stage cancer where the amount of cfDNA may be limited. Another endpoint of this multi-center prospective analysis is to evaluate whether such an approach can accurately monitor the risk of IPMN progression and detect localized early-stage PDA non-invasively. The early results of these studies will be discussed.
Main Pancreatic Duct Size Independently Predicts Histological Main Duct Involvement, Intestinal Phenotype, and Malignancy in Intraductal Papillary Mucinous Neoplasm
V. Morales-Oyarvide,1 I. Pergolini,1 C.R. Ferrone,1 M. Mino-Kenudson,2 A.L. Warshaw,1 K.D. Lillemoe,1 C. Fernandez-Del Castillo.1 Departments of 1Surgery; and 2Pathology; Massachusetts General Hospital, Boston, MA.
Background: Main pancreatic duct (MPD) dilation is a high-risk feature in IPMN, but its association with other aspects of IPMN disease biology remains unclear.
Methods: We evaluated 318 cases of resected IPMN. Patients were classified based on the radiological MPD size as: no dilation (<5mm), mild dilation (5-9mm), and severe dilation (≥10mm). MPD size was assessed to identify pathological IPMN type, epithelial subtype, and malignancy.
Results: There were 183 (58%) patients with no MPD dilation, 81 (25%) with mild dilation, and 54 (17%) with severe dilation. Mild dilation was associated with a 10.1-fold higher risk of mixed/MD-IPMN (P<0.001), 4.8-fold higher risk of intestinal subtype (P<0.001), and 3.7-fold higher risk of malignancy (P<0.001) compared with no dilation. Severe dilation was associated with a 5.4-fold higher risk of mixed/MD-IPMN (P=0.003), 2.4-fold higher risk of intestinal subtype (P=0.022), and 3.6-fold higher risk of malignancy (P=0.001) compared with mild dilation. Multivariate analysis adjusting for high-risk stigmata and worrisome features revealed that mild dilation predicts mixed/MD-IPMN (OR 9.3, 95% CI 4.17-20.60), intestinal subtype (OR 4.1, 95% CI 2.02-8.30) and malignancy (OR 3.5, 95% CI 1.82-6.68) compared with no dilation, while severe dilation predicts mixed/MD-IPMN (OR 14.1, 95% CI 2.16-92.75) and malignancy (OR 3.4, 95% CI 1.36-8.54) compared with mild dilation. Among invasive IPMN, a MPD size ≥5mm was associated with higher risk of colloid carcinoma (OR 6.1, 95% CI 1.04-35.81).
Conclusion: MPD dilation is an independent predictor of major histological MPD involvement, intestinal subtype and malignancy in IPMN, suggesting that MPD size does not only aid in clinical decision-making but also provides insight into the biology of IPMN.
Intraductal Papillary Mucinous Neoplasms in Young Patients Exhibit Distinct Biology, Clinicopathological Characteristics, and Favorable Prognosis
V. Morales-Oyarvide,1 M. Mino-Kenudson,2 C.R. Ferrone,1 A.L. Warshaw,1 K.D. Lillemoe,1 I. Pergolini,1 M. Attiyeh,3 N. Rezaee,4 P.J. Allen,3 C.L. Wolfgang,4 C. Fernandez-Del Castillo.1 Departments of 1Surgery; and 2Pathology; Massachusetts General Hospital, Boston, MA; 3Department of Surgery, Memorial Sloan Kettering Cancer Center, New York City, NY; 4Department of Surgery, Johns Hopkins University School of Medicine, Baltimore, MD.
Background: Intraductal papillary mucinous neoplasms (IPMN) are most commonly diagnosed in the seventh decade of life. We sought to determine the clinicopathological profile of young patients with resected IPMN.
Methods: We evaluated 1,564 resected IPMN from the database of the Pancreatic Surgery Consortium. Patients were classified as “young” (<50 years of age) or “older” (≥50 years). Clinicopathological characteristics and survival were compared between groups.
Results: There were 78 (5%) young patients and 1,486 (95%) older patients. Median age among young patients was 44 yrs (range 19–49) and 70 yrs (range 50–93) in older patients. There was no difference in sex between age groups. In univariate analysis, young patients were less likely to present with jaundice (0% vs. 11%, P<0.001), and had higher risk of intestinal (52% vs. 31%; OR 2.4, 95% CI 1.14-4.90, P=0.017) and oncocytic (16% vs. 3%, OR 5.3, 95% CI 1.85-15.33, P=0.006) subtypes. There was no significant difference in recurrence/progression rates (13% in both groups), but young patients were more likely to have a reoperation (67% vs. 13%, OR 13.4, 95% CI 2.86-62.54, P<0.001) than older patients. Young patients had 64% lower risk of invasive cancer after adjusting for sex and IPMN type (95% CI 0.15-0.87, P=0.023). Among invasive IPMN, young patients had a non-significant higher frequency of colloid phenotype (55% vs. 32%, P=0.189) and a favorable prognosis (5-year overall survival 71% vs. 38%, log-rank P=0.036).
Conclusion: IPMN is uncommonly resected in patients under age 50, but when this occurs, it has distinct clinicopathological features. These include a higher frequency of intestinal and oncocytic phenotypes, a lower risk of invasive cancer, and a more favorable prognosis among invasive cases, suggesting there are underlying biological differences between young and older patients.
Pathophysiological Modulation of Pancreatic Acinar Cell Bioenergetics by Cholecystokinin
J. Morton,1,2 J. Armstrong,2 N. Cash,1 Y. Ouyang,1 A. Tepikin,1 R. Sutton,2 D. Criddle.1,2 1Dept. of Celullar and Molecular Physiology, Institute of Translational Medicine, University of Liverpool, Liverpool, United Kingdom; 2NIHR Liverpool Pancreas Biomedical Research Unit, Royal Liverpool University Hospital University of Liverpool, Liverpool, United Kingdom.
Background & Aims: Cholecystokinin (CCK) modifies pancreatic acinar cell (PAC) Ca2+ signalling and mitochondrial function dependent on concentrations, although bioenergetic changes underlying pathophysiology are unclear. The aim was to elucidate effects of CCK on PAC bioenergetics.
Methods: The actions of CCK on murine PACs O2 consumption and extracellular acidification rates (OCR, ECAR) were evaluated with a Seahorse XF24 Flux Analyzer, NADH/FAD+ autofluorescence and Ca2+-activated Cl− currents (IClCa) using confocal microscopy with whole-cell patch clamp, and cell death with plate-reader.
Results: 100ρM-10nM CCK increased basal OCR within 5mins (n=4; p<0.01), while 10ρM had no effect. At 100ρM CCK OCR remained elevated at 30mins, whereas at 1-10nM it returned to control levels. CCK (10-100ρM) elevated spare respiratory capacity, with an increase of ATP turnover capacity at 100ρM (p<0.01). In contrast, high CCK (1-10nM) significantly reduced ATP turnover (p<0.01). CCK (10pM-10nM) had no effect on proton leak. 10pM CCK reduced ECAR, whereas 100ρM-10nM CCK raised ECAR within 5mins, remaining significantly elevated at 30mins at 1-10nM (p<0.01). In single PACs, 5pM CCK induced oscillatory IClCa with concomitant ↑NADH/↓FAD+, whereas 5nM caused sustained IClCa with transient ↑NADH then a prolonged decrease. 10nM CCK induced time-dependent PAC death; apoptosis was evident within 2hrs, whereas both apoptosis and necrosis were elevated at 7 and 13hrs (p<0.01).
Conclusion: Low CCK (pM) caused oscillatory Ca2+ signals and ↑NADH, spare respiratory and ATP turnover capacities, consistent with demand for secretion. However, pathophysiological levels inducing Ca2+ overload and ↓NADH, reduced spare respiratory capacity and ATP turnover, effects accompanied by a shift toward glycolysis.
Prevalence and Outcomes of Acute Pancreatitis (AP) in Older Adults: Results From a Propensity Matched Analysis of Nationwide Inpatient Sample (2008–2012)
S. Munigala,1 A. Duvvuri,2 K.C. Kottapalli,3 D. Subramaniam,4 D. Subramaniam,5 G. Trikudanathan,6 D.L. Conwell.7 1Saint Louis University Center for Outcomes Research (SLUCOR), St. Louis, MO; 2Internal Medicine, Kansas City Veterans Affairs Medical Center, Kansas City, MO; 3Internal Medicine, Wheaton Franciscan Healthcare St. Franscis Hospital, Milwaukee, WI; 4Internal Medicine, Saint Louis University Center for Outcomes Research, St. Louis, MO; 5Health Service Research, Internal Medicine, University of Kansas Medical Center, Kansas City, KS; 6Medicine, GI, University of Minnesota, Minneapolis, MN; 7Ohio State University-Wexner Medical Center, Columbus, OH.
Background: Improvements in healthcare have led to increasing age of the general population. However, there is limited data on AP in older adults. The aim of this investigation was to evaluate the prevalence, etiology and clinical outcomes of AP in older adults > 65 years of age.
Methods: The Nationwide Inpatient Sample (2008–2012) was used to identify all adult inpatients (≥18 years) with primary diagnosis of AP (Cases: AP patients ≥ 65 years; Controls: AP patients 18–65 years). Primary clinical outcomes [inpatient mortality, acute kidney injury (AKI), acute respiratory failure (ARF), severe AP] & secondary outcomes [hospital length of stay (LOS), total charges ($)] were analyzed using univariate & multivariate comparisons. Propensity matched analysis was performed to compare outcomes between cases & controls.
Results: 23.5% of AP admissions were associated with older adults (307,743/1,310,176). Multivariate analysis showed older adults have increased inpatient mortality [odds ratio (OR): 3.65, confidence interval (CI) 3.39, 3.94; p<0.001], AKI (OR: 2.07, 95%CI 2.02, 2.12, p<0.001), ARF (OR: 1.48, 95%CI 1.43, 1.54, p<0.001), severe AP (OR:1.40, 95%CI 1.37, 1.43, p<0.001), increased LOS (0.19 days, p<0.001) & total charge ($1735, p<0.001). Propensity matched analysis confirmed the increased risk of inpatient mortality (2.2% vs 0.6%), AKI (13.6% vs. 7.6%), ARF (7.0% vs. 4.4%), severe AP (18.6% vs. 12.8%), LOS (mean difference: 0.20 days; p<0.001) and total charge (mean difference $2244; p<0.001).
Conclusions: Approximately 25% of all AP admissions in older adults, and are associated with more severe course and high risk of mortality irrespective of comorbidity status.
Risk of Diabetes Mellitus in Pancreatic Cancer: Results From a Propensity Matched Study (2008–2012)
S. Munigala,1 D.L. Conwell.2 1Saint Louis University Center for Outcomes Research (SLUCOR), St. Louis, MO; 2Ohio State University-Wexner Medical Center, Columbus, OH.
Objectives: Population-based data on association between diabetes mellitus (DM) & Pancreatic Cancer (PaCa) are limited. We evaluated the prevalence & clinical outcomes of DM in PaCa patients in a propensity matched analysis.
Methods: The Nationwide Inpatient Sample (2008–2012) was used to identify all adult inpatients (≥18 years) with primary diagnosis of PaCa & associated secondary diagnosis codes for DM for identifying cases & controls. Primary clinical outcomes [inpatient mortality, acute kidney injury (AKI), acute respiratory failure (ARF)] & secondary outcomes [hospital length of stay (LOS), total charges ($)] were analyzed using univariate & multivariate comparisons. Finally propensity matched analysis was performed to compare outcomes between cases & controls.
Results: DM is associated with 34.5% of all PaCa admissions. Univariate analysis showed that DM patients were more likely to be male (58.2% vs. 47.3%, p<0.001), black (15.4% vs. 11.6%, p<0.001), increased co-morbid conditions (AHRQ-Elixhauser index 3 3, 53.3% vs. 45.5%, p<0.001), associated with higher percentage of AKI and total charge. In addition, DM was associated with a lower percentage of mortality (7.4% vs 9.4%, p<0.001), ARF (6.5% vs. 7.4%, p=0.002). Multivariate analysis showed DM is associated with decreased mortality [odds ratio (OR): 0.77, confidence interval (CI) 0.74, 0.81; p<0.001], ARF (OR: 0.81, 95%CI 0.77, 0.85, p<0.001), LOS (−0.41 days, p<0.001) & total $ (p=0.006) but no change in AKI (p=0.16) or surgery for pancreatectomy (p=0.77). Propensity matched analysis confirmed the decreased risk of ARF (OR: 0.87, 95%CI 0.77, 0.98; p=0.023) & mortality (OR: 0.88, 95% CI 0.79, 0.99; p=0.03).
Conclusions: Pancreatic cancer patients with associated DM have less respiratory failure and lower mortality when compared to PaCa without DM. Further studies need to validate these findings, evaluate the duration and timing of DM in relation to PaCa development. Furthermore, the role of anti-diabetic medications on the natural history of PaCa needs clarification.
Risk of Diabetes Mellitus in Chronic Pancreatitis: Results from a Propensity Matched Study (2008–2012)
S. Munigala,1 D.L. Conwell.2 1Saint Louis University Center for Outcomes Research (SLUCOR), St. Louis, MO; 2Ohio State University-Wexner Medical Center, Columbus, OH.
Background: Population-based data on association between diabetes mellitus (DM) & chronic pancreatitis (CP) are limited. We evaluated the prevalence & clinical outcomes of DM in CP patients in a propensity matched analysis.
Methods: The Nationwide Inpatient Sample (2008–2012) was used to identify all adult inpatients (≥18 years) with primary diagnosis of CP (ICD-9 codes 577.1). Secondary diagnosis codes for DM were used to identify cases (CP with DM) & Controls (CP without DM). Primary clinical outcomes [inpatient mortality, kidney injury (KI), respiratory failure (RF)] & secondary outcomes [hospital length of stay (LOS), total charges ($) were analyzed using univariate & multivariate comparisons. Finally propensity matched analysis was performed to compare outcomes between cases & controls.
Results: DM is associated with 31.7% of all CP admissions. Univariate analysis showed that DM patients were older (50 vs 47 years; p<0.001), had more males (58.2% vs. 47.3%, p<0.001), associated with higher percentage of KI, total $ & ≥ 1 centers for Medicare & Medicaid Services (CMS) hospital acquired conditions (p<0.001). Multivariate analysis showed DM is associated with increased RF [odds ratio (OR): 1.46, 95% confidence interval (CI) 1.36, 1.58; p<0.001] & decreased mortality (OR: 0.52, 95% CI 0.39, 0.69; p<0.001) but no change in LOS (−0.18 days, p=0.083) & total $ (p=0.55). Propensity matched analysis confirmed the increased risk of RF [odds ratio (OR): 1.55, 95% confidence interval (CI) 1.25, 1.92; p<0.001] & showed a trend for decreased in-hospital deaths (OR: 0.50, 95% CI 0.24, 1.03; p=0.059)
Conclusions: DM is associated with adverse clinical outcomes and hospital acquired conditions. Further studies need to evaluate the duration of DM on the disease burden of CP patients.
Acute Pancreatitis (AP) Early Readmission Rates in United States: Results From a Nationwide Hospital Readmissions Data
S. Munigala. Saint Louis University Center for Outcomes Research (SLUCOR), St. Louis, MO.
Background: Hospital readmissions can have negative consequences. Reducing readmissions would reduce costs and improve quality of life. Data on recurrent AP and late readmissions is well established; however, data on acute pancreatitis (AP) hospital early readmissions (<30 days) is limited.
Methods: The Healthcare Cost and Utilization Project (HCUP) 2013 Nationwide Readmissions Database (NRD) was used. We identified all index admissions with a primary diagnosis of AP. Early readmission was defined as admission to the hospital or emergency department within 30 days of index event. Demographic, etiological factors and clinical outcomes associated with early readmissions were assessed using univariate & multivariate comparisons. Following patients were excluded: patients died in the hospital, age < 18 years, missing LOS, pregnant cases, discharged in January, December and against medical advice.
Results: There were 211,223 hospital discharges with primary AP diagnosis in 2013 of which 33,398 (15.8%) were readmitted to hospital within 30 days (all cause readmissions). The median LOS for readmissions was 4 days (interquartile range (IQR) 2–6) and median days to readmit were 11 days (IQR 5–19). A multivariate logistic regression analysis showed that AP readmissions were more common in patients aged 18–44 years, in males [odds ratio (OR) 1.06, 95% confidence interval (CI) 1.03, 1.09, p<.001], in large hospitals (OR 1.09, p<.001), those with pseudocyst complications (OR 1.63, 95%CI 1.56, 1.70, p<.001), severe AP (OR 1.34, 95%CI 1.29, 1.40, p<.001), discharged to home health care (OR 1.56, p<.0001) and those transferred to short-term hospital (OR 1.53, p<0.001). AP patients 65+ years (OR 0.58, p<.001), with private insurance (OR 0.65, p<.001) and those who had cholecystectomy during index event reported significantly less readmissions (OR 0.54, 95%CI 0.50, 0.57, p<.001).
Conclusions: Early readmission (<30 days) rates for AP in our study is less than what was previously reported. Cholecystectomy during index event is associated with significantly less readmissions.
CCK Receptor Knock-Out Prevents Pancreatic Cancer Growth From Dietary Fat
S. Nadella,1 J. Burks,2 G. Inyang,1 J. Wang,1 R. Tucker,1 J. Smith.2 1Department of Medicine, Gastroenterology, Georgetown University, Washington, DC; 2Medicine/Gastroenterology, Georgetown University, Washington, DC.
Introduction: Epidemiologic studies have found that the incidence of pancreatic ductal adenocarcinoma (PDAC) is greatest in countries that consume diets high in fat. The gastrointestinal peptide cholecystokinin (CCK) that is released in the blood response to dietary fat, has been shown to stimulate growth of PDAC through the CCK receptor (CCKR).
Aim: The aim of this investigation was to determine if dietary fat promotes growth of PDAC by activation of the CCKR.
Methods: Murine Panc02 PDAC cells underwent genetic knockout (KO) of the CCKR by CRISPR/Cas9 technology. Clonal CCKR KO was confirmed by RT-PCR. Panc02 wild type (WT) cells with CCKR and KO cells were (25,000 per well) were plated in vitro and treated with CCK (0.1, 1.0, 10nM) for five days and viable cell counts were performed. C57BL/6 mice (N=40) were injected sc with 2x106 Panc02 WT or KO cells. After 1 wk, half the mice in each group were placed on a high fat or isocaloric regular fat diet. Tumor growth was monitored and animals were euthanized after 42 days before any became obese.
Results: RT-PCR and gel electrophoresis for the CCKR revealed the appropriate PCR product for WT cells but not from the KO cells, with positive internal control (HPRT gene). KO cells treated with CCK in vitro showed no evidence of stimulated growth compared to diluent treated control cells. In contrast, Panc02-WT cells significantly increased in cell number (p= 0.032) at a concentration of CCK (1.0nM). In vivo, a high fat diet significantly stimulated growth of WT tumors compared to mice on an isocaloric diet. In contrast, mice with Panc02 KO tumors failed to response to dietary fat (p=0.002).
Conclusion: Dietary fat mediates growth of PDAC via the CCK receptor and this effect is independent of obesity.
L-carnitine Supplementation Improved Hepatic Steatosis After Pancreatectomy
M. Nakamura,1 K. Nakata,1 K. Yoshida,2 K. Hino.2 1Department of Surgery and Oncology, Kyushu University, Fukuoka, Japan; 2Department of Hepatology and Pancreatology, Kawasaki Medical School, Kurashiki, Japan.
Objectives: Hepatic steatosis is one of the most frequent long-term complications after pancreatectomy influencing even survival rate. However, its treatment has not been established except pancreatic enzyme. The purpose of this study was to clarify the effect of L-carnitine on hepatic steatosis after pancreatectomy. The influence of Acyl/free carnitine ratio on hepatic steatosis after pancreatectomy was also analyzed.
Methods: In this retrospective study of 21 patients who had undergone pancreatectomy, serum carnitine concentrations, fractions of carnitine, and hepatic attenuation on computed tomography images were analyzed with the aim of identifying risk factors for hepatic steatosis. Eight of 31 patients were given L-carnitine after pancreatectomy. The effect of L-carnitine on hepatic steatosis was analyzed using attenuation of the liver on CT images.
Results: Thirteen (61.9%) of the 21 patients were diagnosed as having hypocarnitinemia after pancreatectomy. Average hepatic attenuation was as low as 48.7 (±20.4 SD). A high ratio of acyl/free carnitine was significantly correlated with less pronounced hepatic attenuation according to both univariate and multivariate analysis. Administration of L-carnitine significantly improved hepatic attenuation.
Conclusion: A high ratio of acyl/free carnitine is a significant risk factor for hepatic steatosis after pancreatectomy, and L-carnitine supplementation may be a new treatment option for it.
Radical Carbon-Ion Radiotherapy for Locally Advanced Unresectable Pancreatic Cancer
K. Nakata,1 T. Ohtsuka,1 Y. Mori,1 Y. Miyasaka,1 S. Makoto,2 E. Nagai,1 M. Nakamura.1 1Department of Surgery and Oncology, Kyushu University, Fukuoka, Japan; 2Ion Beam Therapy Center, SAGA HIMAT Foundation, Tosu, Japan.
Most of the pancreatic cancer (PC) patients are diagnosed as having locally advanced PC. In an effort to improve outcomes, various multidisciplinary approaches have been employed. Carbon-ion beam has been recently reported to demonstrate increased relative biological effect and carbon-ion radiotherapy (CIRT) followed by salvage surgery is expected to improve the prognosis in patients with locally advanced PC. We have recently experienced 6 patients who underwent radical CIRT for unresectable locally advanced PC in the pancreas head involving the superior mesenteric artery. Among them, two patients were converted to undergo salvage operation, while in the remaining 4, two patients had liver metastasis despite the sufficient local control of PC and the other two had limited effect of CIRT. In the 2 operated patients, decreased uptake value of PET-CT after CIRT was noted in the first patient and the normalization of serum CA19-9 level was observed in the second patient. Both patients received R0 pancreatoduodenectomy. Operation time, intraoperative blood loss, and postoperative hospital stay in the first and second patient were 538 min, 1220 g, 17 days and 468 min, 950 g, 30 days, respectively. The first patient showed pathological complete response (Evans grade IV) and the second had pathological minimal residual tumor (Evans grade III). The first patient has been alive for 30 months after the initial diagnosis without any findings of recurrence, while the second patient had liver metastasis at 9 months after the operation (19 months after the initial diagnosis). In conclusion, CIRT may bring beneficial effect of local control of PC in selected patients, although control of distant metastasis remains unresolved problem.
A Hungarian Family With Hereditary Pancreatitis and the p.L104P Mutation in the Human Cationic Trypsinogen
B.C. Nemeth,1 A.V. Patai,2 M. Sahin-Tóth,3 P. Hegyi.4 1First Department of Medicine, University of Szeged, Szeged, Hungary; 22nd Department of Internal Medicine, Semmelweis University, Budapest, Hungary; 3Department of Molecular and Cell Biology, Boston University Medical Campus, Boston, MA; 4Department of Translational Medicine, University of Pecs, Pecs, Hungary.
Introduction & Aims: The most frequent mutations in the human cationic trypsinogen (PRSS1) cause early activation of trypsinogen inside the pancreas. Carriers develop chronic pancreatitis usually in childhood. A subset of rare mutations such as c.311T>C (p.L104P) seem to exert their effect by causing trypsinogen misfolding and endoplasmatic reticulum stress. However, whether misfolding-causing mutations casue hereditray pancreatitis or sporadic disease only has remained unclear. Our aim was to establish the association between the PRSS1 p.L104P mutation and the development of hereditary chronic pancreatitis in a Hungarian family.
Methods: Genetic testing by Sanger sequencing was performed in all family members who signed informed consent. Diagnosis of chronic pancreatitis was based on abdominal CT scan, abdominal ultrasound, clinical signs and functional tests.
Results: Three members of a Hungarian family with clinically documented pancreatolithiasis, idiopathic recurrent acute and chronic pancreatitis carried the rare p.L104P mutation in the PRSS1 gene in the heterozygous state. Disease onset, as defined by the first episode of acute pancreatitis, was over 18 years of age in all cases. Five members of the family also carried the same mutation without acute or chronic pancreatitis, however, three of them are under 18 years of age and one is a young adult. Family members without the investigated mutation did not develop pancreatitis. Four members of the family including one affected patient carried the heterozygous p.G60G mutation in the chymotrypsinogen C (CTRC) gene.
Conclusion: This is the first study showing clear association between the PRSS1 p.L104P mutation and clinically documented hereditary chronic pancreatitis with unusual features of later age of onset and pancreatolithiasis.
ESPAC-4: A Multicenter, International, Randomized Controlled Phase III Trial of Adjuvant Combination Chemotherapy of Gemcitabine (GEM) and Capecitabine (CAP), Versus Monotherapy Gemcitabine in Patients With Resected Pancreatic Ductal Adenocarcinoma
J.P. Neoptolemos,1 D. Palmer,1 P. Ghaneh,1 J. Valle,2 D. Cunningham,3 J. Wadsley,4 T. Meyer,5 A. Anthoney,6 B. Glimelius,7 S. Falk,8 P. Lind,9 J. Izbicki,10 G. Middleton,11 P. Ross,12 H. Wasan,13 A. McDonald,14 T. Crosby,15 E. Psarelli,1 P. Hammel,16 M.W. Büchler.17 1University of Liverpool, Liverpool, United Kingdom; 2University of Manchester & The Christie, Manchester, United Kingdom; 3Royal Marsden Hospital, London, United Kingdom; 4Weston Park Hospital, Sheffield, United Kingdom; 5Royal Free Hospital, London, United Kingdom; 6St James's University Hospital, Leeds, United Kingdom; 7University of Uppsala, Uppsala, Sweden; 8Bristol Haematology and Oncology Centre, Bristol, United Kingdom; 9Karolinska University Hospital, Stockholm, Sweden; 10University of Hamburg Medical Institutions UKE, Hamburg, Germany; 11Royal Surrey County Hospital, Guildford, United Kingdom; 12Guy's Hospital, London, United Kingdom; 13Hammersmith Hospital, London, United Kingdom; 14The Beatson West of Scotland Cancer Centre, Glasgow, United Kingdom; 15Velindre Hospital, Cardiff, United Kingdom; 16Hopital Beaujon, Clichy, France; 17University of Heidelberg, Heidelberg, Germany.
Aim: To determine whether combination chemotherapy with GEM/CAP improved survival compared to GEM alone for resected pancreatic cancer.
Background: The ESPAC-3 trial compared adjuvant GEM with 5-fluorouracil/folinic acid. GEM is the standard of care based on similar survival and less toxicity.
Methods: Patients with pancreatic cancer were randomized within 12wk of to have either six 4wk cycles of IV GEM alone or GEM with oral CAP. The primary endpoint was overall survival; secondary endpoints included toxicity and 5 year survival. 722 patients (480 expected events), 361 in each arm, were needed for a 10% difference in 2y survival rate with 90% power (log-rank test with 5% 2-sided alpha).
Results: Between Nov 2008 and Sep 2014, 732 patients were randomized with 730 included in the full analysis set (366 GEM, 364 GEM/CAP). Median age was 65 years, 57% were men. WHO performance status was 0, 1 or 2 in 42% 55% and 3%respectively. Postoperative median CA19-9 was 19 kU/L. Median maximum tumor size was 30 mm, 60% were R1 resections, 80% were node positive and 40% were poorly differentiated. On Dec 11 2015 the Independent Trial Monitoring Committee requested that the trial proceed to full analysis. Stratified log-rank for overall survival produced an HR=0.82 [95% CI, 0.68 – 0.98]; χ 2 (1) = 4.61, P=0.032. Median survival (months) for patients treated with GEM/CAP was 28.0 (95% CI, 23.5 – 31.5) and 25.5 (22.7 – 27.9) for GEM. The 5y survival rate for the GEM/CAP was 28.8 (22.9 – 35.2)% and 16.3 (10.2 – 23.7)% for GEM. 196 out of 366 GEM patients in the safety set reported 481 grade 3/4 adverse events vs 226 out of 359 GEM/CAP patients reporting 608 grade 3/4 adverse events (P=0.242).
Conclusion: Adjuvant GEM/CAP is the standard of care for resected pancreatic cancer.
Should Patients With Established Chronic Pancreatitis Undergo Testing for Celiac Disease?
A.S. Nett, E.-J. Wamsteker, M.J. Dimagno. Department of Medicine, University of Michigan, Ann Arbor, MI.
Objectives: The strength of the known association of celiac disease (CD) with chronic relapsing pancreatitis (CRP) or established chronic pancreatitis (ECP) is uncertain. To investigate this association, we determined the frequency of new diagnoses of CD in patients who had a duodenal biopsy during EUS evaluation for pancreatitis. We hypothesized that this frequency would be greater than in the general population (0.75%).
Methods: We retrospectively reviewed 583 EUS exams (2005–2015) performed by EJW after referral for evaluation of suspected or established pancreatitis; 160 had duodenal biopsies performed and 126 of 160 (79%) fulfilled criteria for diagnoses of pancreatitis and were the subjects of this investigation. Criteria for diagnoses of pancreatitis were the Atlanta Criteria for acute pancreatitis (AP) and Mayo Score >/=4 for ECP, allowing EUS to fulfill imaging criteria. CD was defined as abnormal histology (villous atrophy, crypt hyperplasia, intraepithelial lymphocytic [IEL] infiltration of lamina propria) plus positive TTG antibody.
Results: Of the 126 patients with pancreatitis and duodenal biopsies, 68 [54%] had ECP, 26 [20.6%] had AP, 18 [14.3%] had CRP, and 14 [11.1%] had recurrent AP due to a specific cause. Of the 126 with pancreatitis, 5 (4%) met criteria for CD. All 5 patients with CD had ECP, representing 7.4% (5/68) of patients with ECP.
Conclusions: In patients with pancreatitis undergoing duodenal biopsy at EUS, CD was present in at least 4% overall and 7.4% with ECP, much higher than compared to the general population. Testing for CD should be considered in all patients with ECP.
The Impact of Integrated Molecular Pathology Analysis on EUS Gastroenterologist Management Decisions for Pancreatic Cystic Lesions
J. Nieto,1 S. Jackson,2 N. Toney,2 A. Lankarani.1 1Borland-Groover Clinic, Jacksonville, FL; 2Clinical Development, Interpace Diagnostics Corporation, Pittsburgh, PA.
Background and Aim: To determine if DNA-based Integrated molecular pathology (IMP) results change endoscopic ultrasound (EUS) Gastroenterologist management recommendations.
Methods: Two EUS Gastroenterologists reviewed 220 de-identified patient cases. Cases were randomly selected from a database of 12,331 to include a combination of profiles with and without clinically worrisome features (WF). Physicians reviewed all clinical result reports without and then with comprehensive IMP result reports (PancraGEN, Interpace Diagnostics), including tumor suppressor LOH mutations, KRAS mutations, and DNA quantity analysis. They recorded their management recommendations for surgery or surveillance and length of surveillance in each instance.
Results: 52% (114/220) of recommendations changed after viewing IMP test results. Recommendations changed to more relaxed surveillance in 34% (75/220) of cases (p <0.0002), with 97% (73/75) having low risk IMP results. Recommendations changed to closer surveillance or surgery in 18% (39/220) of cases (p=0.006), with 69% (27/39) of these cases having high risk IMP results. In cases with no WF, close surveillance or surgery of patients was recommended in 37%; after viewing IMP results, this was reduced to only 14%. In cases with one WF, close surveillance or surgery of patients was recommended in 60%; after viewing IMP results, this was reduced to 45%. In cases with two WF, close surveillance or surgery of patients was recommended in 69%; after viewing IMP results, this was reduced to 61%.
Discussion: IMP results significantly change EUS physician management recommendations. IMP low risk diagnoses often result in longer surveillance intervals; IMP high risk diagnoses result in closer surveillance or surgery recommendations.
Activation of IL-1 Signaling in CD133+ Pancreatic Cancer Cells
A. Nomura,1 V.K. Gupta,1 P. Dauer,1 V. Dudeja,1 A.K. Saluja,2 S. Banerjee.1 1University of Miami, Miami, FL; 2Dept. of Surgery, University of Miami, Leonard M. Miller School of Medicine, Miami, FL.
Introduction: Cancer stem cells (CSCs) have been implicated in pancreatic cancer in tumor initiation, progression, and metastasis. The CD133 positive population has reliably been described in these processes and its expression has recently been described to functionally promote tumorigenicity and metastasis through NF-kB activation. However, the process by which CD133 increases NF-kB signaling is unclear.
Aim: The aim of the study is to determine the mechanism of NF-kB activation in pancreatic cancer, specifically in CD133+ cancer stem cells.
Methods: Various established pancreatic ductal adenocarcinoma cancer cell lines of differing CD133 expression were utilized for this study. Silencing and overexpression of CD133, NF-kB, and IL-1 signaling were used in cell lines with high and minimal populations of CD133, respectively.
Results: The expression levels of IL-1β significantly correlated with CD133 expression in several pancreatic cancer cell lines. Upon overexpression of CD133, expression and secretion of IL-1β are increased, leading to increased NF-kB signaling, induction of epithelial-mesenchymal transition (EMT), and cellular invasion. These effects were found to be mediated by IL-1 signaling, as the blocking or silencing of IL-1R1, via IL-1Ra or siRNA, respectively, significantly inhibited NF-kB activity, EMT transcription factor gene expression, mesenchymal marker expression, and cellular invasiveness. However, this decrease was significantly more pronounced in cells overexpressing CD133 or cell lines with higher populations of CD133.
Conclusion: CD133 expression in pancreatic cancer stimulates IL-1 signaling to promote NF-kB activation and cellular invasiveness.
The Heparin-Binding Proteome in Murine Experimental Acute Pancreatitis
Q.M. Nunes,1 D. Su,1 P.J. Brownridge,2 C. Sun,2 Y. Li,2 W. Huang,1 D.J. Rigden,2 R.J. Beynon,2 R. Sutton,1 D.G. Fernig.2 1NIHR Liverpool Pancreas Biomedical Research Unit, Liverpool, United Kingdom; 2Department of Biochemistry, University of Liverpool, Liverpool, United Kingdom.
Background: Heparin-binding proteins (HBPs) play a central role in cell communication in the extracellular space (ECS). We used tissue heparin affinity chromatography (HAC) and mass spectrometry (MS) to identify extracellular HBPs in acute pancreatitis (AP). Furthermore, we developed a method to isolate HBPs from plasma.
Methods: Pancreases were obtained from 6–8 week old male adult CD1 mice with caerulein-induced AP. Tissue was processed using differential centrifugation, density gradient separation, HAC, TCA precipitation and subjected to MS. Label free quantification was performed using the ‘Top3’ method. HBP networks were analysed with Cytoscape 2.8.1. Functional analyses were undertaken using QIAGEN’s Ingenuity Pathways Analysis. Plasma was obtained from CD1 mice and stored in 3.8% trisodium citrate anticoagulant. After albumin depletion with HAC and 2M NaCl elution, the HBP fraction was adsorbed onto StrataClean resin for MS.
Results: Many new HBPs (460) were discovered more than doubling their total number to 883, and forming highly interconnected protein interaction networks in AP. Some such as NDUFS4 may have ‘moonlighting’ roles in the ECS. Label-free quantification identified 102 HBPs, including known biomarkers of AP, e.g. carboxypeptidases (CPB1 and CPB2) and pancreatic amylase (AMY2A), which were overexpressed. Reduced expression of klotho beta (KLB) and NDUFA9 in AP point to a possible disturbance in glucose homeostasis and underlying mitochondrial dysfunction. HBPs were successfully isolated from plasma.
Conclusions: HBPs, by virtue of their extracellular location and heparin binding property, are easily accessible and are potential drug targets in AP. They can be easily isolated from plasma for potential biomarker development in AP patients.
Accurate Admission Transcriptomic Signature of the Severity of Acute Pancreatitis
Q.M. Nunes,1 B. Lane,1 W. Huang,1 K. Altaf,1 L. Rainbow,2 J. Armstrong,1 W. Greenhalf,1 D. Fernig,3 C. Hertz-Fowler,2 A. Cossins,2 F. Falciani,2 S. Maskell,4 A. Morris,5 R. Sutton.1 1NIHR Liverpool Pancreas Biomedical Research Unit, Royal Liverpool and Broadgreen University Hospitals NHS Trust, Liverpool, United Kingdom; 2Functional and Comparative Genomics; and 3Department of Biochemistry; Institute of Integrative Biology, University of Liverpool, Liverpool, United Kingdom; 4Department of Electrical Engineering and Electronics, Faculty of Science and Engineering, University of Liverpool, Liverpool, United Kingdom; 5Department of Biostatistics, Institute of Translational Medicine, University of Liverpool, Liverpool, United Kingdom.
Background: Accurate identification of severe acute pancreatitis (AP) on admission enables timely and effective targeting of resources to improve outcome, but current predictors are inaccurate. We have used transcriptional change in blood to define a molecular fingerprint to predict AP severity.
Methods: Patients (>500) with AP were recruited from July 2010 at our institution with blood taken on admission (PAXgene tubes). Whole transcriptome gene expression was measured in blood from 92 patients to compare disease severities using Affymetrix HGU133plus2 arrays. Revised Atlanta and Determinant-Based Classifications (RAC, DBC) were used to classify patients as mild vs non-mild, pooling other categories (RAC, 32 vs 60; DBC, 43 vs 49). A set of probes and machine learning algorithm that best distinguished severity were selected to define a fingerprint. Independent training and test sets were generated by data split resampling. Feature finding was by probe importance in random forest models with training set data. The test set was used to calculate accuracy, sensitivity and specificity of models built with the training set. Average model performance was estimated over multiple resamples.
Results: Random forest models had the lowest misclassification error of all algorithms. This identified a fingerprint of three transcripts that predicted RAC moderately severe or severe AP with 87% accuracy, 91% sensitivity and 78% specificity, and of six transcripts that predicted DBC moderate, severe or critical AP with 78% accuracy, 81% sensitivity and 74% specificity. A NanoString panel incorporating these signatures was validated successfully.
Conclusions: The defined molecular fingerprint predicts severity of AP accurately, more so for RAC; independent cohort validation is underway.
Low Serum Pancreatic Amylase and Lipase Level Values are Simple and Useful Predictors to Diagnosis of Chronic Pancreatitis
H.-C. Oh,1 C.-I. Kwon,1 J. Easler,1 I. El Hajj,1 J. Watkins,1 E. Fogel,1 L. McHenry,1 S. Sherman,1 M. Zimmerman,2 G. Lehman.1 1Division of Gastroenterology and Hepatology; and 2Department of Pathology and Laboratory Medicine; Indiana University School of Medicine, Indianapolis, IN.
Background: Early diagnosis with a simple method is essential to prevent the progression of disease to an irreversible state, especially in chronic pancreatitis. This study aimed to evaluate the diagnostic role of low serum pancreatic amylase and lipase values in the detection of chronic pancreatitis.
Methods: From Dec 2012 to Nov 2015, patients diagnosed as non-calcific (NCCP, n=113) and calcific (CCP, n=112) chronic pancreatitis with serum amylase and lipase values were compared with healthy controls (H, n=170).
Results: Mean age of H, NCCP, and CCP groups were 48.1, 50.7, and 53.7, respectively. Mean serum amylase (normal range, 19–86 U/L) and lipase values (7–59 U/L) (±SD) were 48.1 (±13.2) and 26.4 (±11.3) for H, 41.4 (±22.3) and 28.3 (±22.9) for NCCP, and 31.5 (±16.0) and 12.8 (±11.5) for CCP, respectively. The optimal cut-off values for discriminating NCCP from H were 40 U/L for amylase and 20 U/L for lipase, respectively, and for CCP from H were 38 U/L for amylase and 15 U/L for lipase, respectively. For diagnosis of NCCP, with a criterion of serum amylase < 40 and lipase < 20 U/L, sensitivity, specificity, positive predictive value, negative predictive value, and accuracy were 32.7%, 88.8%, 66.1%, 66.5%, and 66.5%, respectively. For diagnosis of CCP, with a criterion of serum lipase < 15, sensitivity, specificity, positive predictive value, negative predictive value, and accuracy were 67.0%, 86.5%, 76.5%, 79.9%, and 78.7%, respectively.
Conclusions: Low serum pancreatic amylase (30 percentile of normal range) and lipase (15 percentile) values were simple and useful diagnostic predictors for the discrimination of chronic pancreatitis.
Circumportal Pancreas, a Rare Congenital Anomaly, Increases a Risk of Pancreatic Fistula After Pancreatectomy
T. Ohtsuka,1 Y. Mori,1 K. Ishigami,2 T. Fujimoto,1 Y. Miyasaka,1 K. Nakata,1 K. Ohuchida,1 T. Manabe,1 E. Nagai,1 Y. Oda,3 M. Nakamura.1 Departments of 1Surgery and Oncology; 2Clinical Radiology; and 3Anatomical Pathology; Kyushu University, Fukuoka, Japan.
Background and Aim: Circumportal pancreas is a rare congenital pancreatic anomaly, which may develop via the aberrant embryogenesis where the ventral and dorsal pancreatic primordium fuse over the portal vein. Most patients with circumportal pancreas are asymptomatic, while during pancreatectomy at the level of portal vein, there will be two transection plate in both ventral and dorsal side of portal vein, which may increase the risk of pancreatic fistula. The aim of this study was to clarify the clinical characteristics of surgical cases with circumportal pancreas.
Methods: Medical records of 508 patients including 362 with pancreatoduodenectomy and 146 with distal pancreatectomy were retrospectively reviewed. The prevalence of cirmuportal pancreas and related anatomical variation, surgical procedure, and postoperative outcome were compared between the patients with and without circumportal pancreas.
Results: Circumportal pancreas was observed in 9 patients (1.7%), all of whom had the portal vein completely encircled by the pancreatic parenchyma above the level of the spleno-portal junction, and main pancreatic duct ran ventral to portal vein. Seven patients with circumportal pancreas underwent pancreatoduodenectomy and the remaining 2 did distal pancreatectomy. The rate of the presence of variant hepatic artery in patients with circumportal pancreas was not significantly different from that with standard pancreas (44% vs. 23%, p=0.23). Pancreatic fistula developed more frequently in patients with circumportal pancreas than those with standard pancreas (44% vs. 14%, p=0.03). The other clinical parameters were not different between the patients with and without circumportal pancreas.
Conclusions: Clinician should pay careful attention to the presence of circumportal pancreas during pancreatectomy despite its rarity because this anomaly increases the risk of postoperative pancreatic fistula.
Participation of Crk-Associated Substrate (CAS) in Human Pancreatic Cancer Cell Migration, Invasion and Metastatic Processes
H. Okamoto,1 T. Kusama,1 J. Itakura,1 H. Fujii.2 1Gastrointestinal, Breast & Endocrine Surgery; and 2First Department of Surgery; Uiniversity of Yamanashi, Chuo, Japan.
Introduction: Crk-associated substrate (CAS, p130Cas) is a major tyrosine phosphorylated protein in cells transformed by v-crk and v-src oncogenes. In non-transformed cells, CAS localizes to focal adhesions and undergoes tyrosine phosphorylation in response to integrin-mediated signaling. In the current study, we evaluate the role of CAS in cell migration, invasion and metastatic processes in human pancreatic cancer cells.
Materials and Methods: We investigated that CAS tyrosine kinase activity and tumor cell migration, invasion to endothelial cell monolayer in human pancreatic cancer cell lines, in vitro. CAS tyrosine phosphorylation were measured by immunoblotting and immunoprecipitation using anti-CAS monoclonal antibody and anti-phosphotyrosine antibody. Tumor cell migration was measured with Matrigel-coated modified-Boyden’s chamber assay. Tumor cell invasion to endothelial cell monolayer was assayed with a co-culture invasion bioassay.
Results: Tyrosine phosphorylation of CAS in human pancreatic cancer cells were induced during tumor cell migration and invasion. Phosphorylated tyrosine level of CAS was coincident with invasion capability in human pancreatic cancer cell lines, PSN-1, MiaPacaII, Panc1, despite of similar protein expression level. Protein tyrosine kinase inhibitor, Herbimycin A, had an inhibitory effect on migration and invasion as well as CAS tyrosine phosphorylation in tumor cells.
Conclusion: These results indicated that CAS tyrosine phosphorylation in pancreatic tumor cells was correlated with invasive capability, suggesting a pivotal role of CAS in migration, invasion and metastatic processes.
Fatty Acid Uptake via CD36 Enhances Invasiveness of Pancreatic Cancer Cells
T. Okumura, K. Ohuchida, T. Moriyama, K. Nakata, Y. Miyasaka, T. Manabe, T. Ohtsuka, E. Nagai, K. Mizumoto, M. Nakamura 1Department of Surgery and Oncology, Kyushu University, Fukuoka, Japan.
Background: Pancreas is the retroperitoneal organ surrounded by adipose tissue all around. But little is known about synergy between extra-pancreatic tumor invasion and the adipose microenvironment.
Aim: To investigate the expression of CD36/fatty acid translocase in pancreatic cancer cells and examine the relationship between fatty acid uptake and invasiveness of cancer cells.
Methods: Immunohistochemistry, Real time RT-PCR, Western blotting, Transwell migration assay
Results: Immunohistochemistry revealed that expression of CD36 was heterogeneous in pancreatic cancer cells. Human pancreatic cancer cells including Panc1 and SW1990 cells highly expressed CD36 protein. Oleic acid, palmitoleic acid, and linoleic acid enhanced invasiveness of cancer cells. Sulfo-N-succinimidyloleate (SSO), known as CD36 inhibitor, attenuated both uptake of fatty acids and invasiveness of cancer cells (p<0.001).
Conclusion: Uptake of fatty acids via CD36 enhances invasiveness of pancreatic cancer cells. Investigation about adipocyte-cancer cell interaction might lead a novel therapeutic strategy.
Exploring the Protein Profile of Pancreatic Cancer-Associated Diabetes
L. Oldfield,1 C. Jenkinson,1 T. Purewal,2 R. Sutton,1 J. P. Neoptolemos,1 W. Greenhalf,1 E. Costello.1 1National Institute for Health Research, Liverpool Pancreas Biomedical Research Unit, Royal Liverpool University Hospital, Liverpool, UK; 2Department of Diabetes and Endocrinology, Royal Liverpool University Hospital, Liverpool, UK.
Introduction: At the time of PDAC diagnosis over 40% of patients have diabetes mellitus (DM). PDAC-associated DM (PDAC-DM) can precede cancer diagnosis by up to 4 years. Only 1% of patients with new-onset DM have PDAC, it is therefore necessary to differentiate PDAC-DM (type 3c) from the more common type 2, for screening to be feasible in this high-risk group.
Aim: To investigate whether differences in aetiology between new-onset DM and PDAC-DM will allow a means of earlier detection of PDAC amongst the high-risk group of new-onset DM.
Methods: Candidate biomarkers were selected from a comprehensive mass spectrometry based discovery program. Promising markers were assessed using immunoassays on 140 serum and/or plasma samples categorised into various disease groups; PDAC (with/without diabetes), chronic pancreatitis (with/without diabetes), long-term type 2 DM, new-onset type 2 DM and healthy controls.
Results: A discovery program identified 45 PDAC markers with links to metabolic disease pathways. Twenty-five of these were significantly enriched for an association with diabetes. 17 candidate markers have been evaluated to-date revealing differences in the protein profile of PDAC and PDAC-DM patients to that of type 2 DM patents.
Conclusions: Proteins associated with metabolic disease appear to behave differently in type 2 and type 3c diabetes, further study among well-defined disease groups will be necessary to identify a type 3c specific protein panel suitable for development into a diagnostic tool.
Protective Effects of Necrostatin-1 in Experimental Acute Pancreatitis
Y. Ouyang,1,2 L. Wen,1,2 D. Latawiec,1 J. Armstrong,1 M. Awais,1 P.J. Gough,3 J. Bertin,3 R. Mukherjee,1 R. Sutton,1 D. Criddle.2 1NIHR Pancreas Biomedical Research Unit; and 2Cellular and Molecular Physiology, University of Liverpool, Liverpool, United Kingdom; 3GlaxoSmithKline, Pattern Recognition Receptor Discovery Performance Unit, Immuno-Inflammation Therapeutic Area, Collegeville, PA.
Background & Aim: Receptor-interacting protein kinase 1 (RIPK1) contributes to programmed necrosis, an effect inhibited by necrostatin-1 (Nec-1). We sought to determine involvement of RIPK1 in experimental acute pancreatitis (EAP) and its potential as a therapeutic target.
Methods: Pancreatic acinar cells (PAC) from wild type (WT, C57BL/6J) with/without Nec-1 or transgenic kinase dead Ripk1K45A mice were exposed to taurolithocholic acid 3-sulphate (TLCS, 500 μM) or palmitoleic acid ethyl ester (POAEE, 100 μM) and examined by confocal microscopy (propidium iodide readout). Nec-1 was given by minipump in WT vs without Nec-1 or Ripk1K45A mice after induction of FAEE-AP (1.35g/kg ethanol and 150 mg/kg POA), TLCS-AP (ductal infusion of 3 mM TLCS) or CER-AP (50 μg/kg caerulein, 7 ip injections).
Results: In PAC assays Nec-1 (10 μM) fully prevented both TLCS- and POAEE-induced necrosis. In Ripk1K45A mice POAEE-induced necrosis was fully inhibited, whereas TLCS-induced cell death was partially inhibited (<50%). In all EAP models Nec-1 treatment greatly inhibited AP development, with biochemical parameters and histopathological scoring significantly reduced compared to controls (≥6 mice/group, p<0.05). EAP parameters in Ripk1K45A mice were similar to WT controls.
Conclusion: Nec-1 treatment afforded pronounced protection against PAC necrosis and markedly ameliorated EAP in three models, despite more modest effects of Ripk1K45A on PAC cells and minimal effects in EAP. These data indicate RIPK1 is not an effective target in EAP, but Nec-1 acts on another yet to be defined.
Anacardic Acid Inhibits Cell Growth and Synergizes With Chemotherapeutics by the Activation of Chmp1A
M. Park,1 D. Upton,2 V. Eversole,2 M. Blackmon,1 S. Craver,1 D. Perkins.2 1KYCOM; and 2Department of Biology and Chemistry; University of Pikeville, Pikeville, KY.
Background: Pancreatic cancer is the 4th leading cause of death from cancer and its incidence has risen steadily. The survival rate of pancreatic cancer patients remains the lowest among all cancer types, signifying the great need for the development of new therapeutic interventions. We have shown that Chromatin Modifying Protein 1A (Chmp1A) functions as a pancreatic tumor suppressor by the activation of the tumor suppressors, Ataxia Telangiectasia Mutated (ATM) and p53, which we define as Chmp1A – ATM – p53 signaling pathway in this study.
Specific aim: Specific aim of this study is to investigate the anticancer effect of Anacardic Acid (AA) alone or in combination with chemotherapeutics, 5-Fluorouracil (5-FU) or Gemcitabine (GEM), and to explore the underlying mechanisms in pancreatic cancer cells.
Methods: For functional studies, we used cell viability assays using MTT, and three dimensional spheroid formation assays. To study mechanisms, we used Western blot and immunocytochemical analyses and examined expression and/or activation of Chmp1A, ATM and p53 proteins. We also silenced Chmp1A and tested its need for the anticancer action of AA.
Results: Our data shows that AA induces growth inhibition of pancreatic cancer cells, and potentiates the anti-cancer activity of 5-FU and GEM. Mechanistically, AA exerts its anticancer activity via the activation of Chmp1A – ATM – p53 signaling pathway. Interestingly, cells treated with 5-FU and GEM shows an increase in Chmp1A protein and activation of ATM and p53, demonstrating the significance of Chmp1A for the anticancer activity of the chemotherapeutics. In addition, knockdown assays show that Chmp1A is required for the anticancer function of AA.
Conclusion: Collectively, our data suggests that cashew nuts and mangos, which contain a number of Anacardic Acids, may be promising complementary supplements to slow the progression and possibly the initiation of pancreatic cancer.
Validation Study of the IAP/APA Management Guideline in Acute Pancreatitis on Prospectively Collected Hungarian Data
A. Parniczky,1 B. Kui,2 A. Szentesi,2 A. Balazs,3 A. Szucs,4 D. Mosztbacher,5 J. Czimmer,6 P. Sarlos,6 J. Bajor,6 S. Godi,6 A. Vincze,6 A. Illes,6 I. Szabo,6 G. Par,6 T. Takacs,2 L. Czako,2 Z. Szepes,2 Z. Rakonczay,2 F. Izbeki,7 J. Gervain,7 A. Halasz,7 S. Crai,8 J. Novak,8 I. Hritz,9 C. Gog,10 J. Sumegi,11 M. Varga,12 B. Bod,13 J. Hamvas,14 M. Varga-Muller,15 Z. Papp,15 M. Sahin-Tóth,16 P. Hegyi.17. 1Heim Pal Children's Hospital, Budapest, Hungary; 2First Department of Medicine, University of Szeged, Szeged, Hungary; 3First Department of Medicine, University of Szeged, Szeged, Hungary; 4First Department of Surgery, Semmelweis University, Budapest, Hungary; 5Department of Pediatrics, Balassa János Hospital of County Tolna, Szekszard, Hungary; 6First Department of Medicine, University of Pécs, Pécs, Hungary; 7Szent György University Teaching Hospital of County Fejér, Székesfehérvár, Hungary; 8Pándy Kálmán Hospital of County Békés, Gyula, Hungary; 9Bács-Kiskun County University Teaching Hospital, Kecskemét, Hungary; 10Healthcare Center of County Csongrád, Makó, Hungary; 11Borsod-Abaúj-Zemplén County Hospital and University Teaching Hospital, Miskolc, Hungary; 12Dr. Réthy Pál Hospital, Békéscsaba, Hungary; 13Dr. Bugyi István Hospital, Szentes, Hungary; 14Bajcsy-Zsilinszky Hospital, Budapest, Hungary; 15Institute for Translational Medicine, University of Pécs, Pécs, Hungary; 16Department of Molecular and Cell Biology, Boston University Medical Campus, Boston, MA; 17Institute for Translational Medicine, University of Pecs, Pecs, Hungary.
Background: The Hungarian Pancreatic Study Group has established a national registry for prospective data collection of patients suffering from acute pancreatitis (AP). Validation of the IAP/APA guideline’s recommendations in cohorts has not been performed yet.
Aims: The main goals of our study were to 1) analyse the course of AP in a multicentre large prospectively collected cohort and 2) to validate the major recommendations of the IAP/APA evidence-based guidelines for the management of AP.
Methods: 600 patients were prospectively enrolled from 14 Hungarian center between 1 January, 2013 until 1 January, 2015 organized by HPSG. Diagnosis of the patients were based on the’A1’recommendation of the IAP/APA guideline.
Results: The cohort was summarized in 50 statements of 6 main questions (etiology, diagnosis and sympthoms, physical examination, imaging, laboratory parameters, complications).Severe AP occured in 8.8% of the 600 patients. The mortality rate was 28.3% in severe AP. We identified WBC number above 23G/l, level of CRP above 200U/l, PCT level above 10U/l, calcium level below 2mmol/l and triglyceride level above 40mmol/l as associated factors for severe AP. Fluid replacement in the first 24 hours out of the range 1500-3000mL elevates the risk of severe AP with 6.27% and the mortality rate with 2.85%. Antibiotic therapy was administered in 77.1% of our cohort. There were no significant differences neither in mortality nor in severity between the groups of prevention and infection-indicated therapy suggesting that preventive antibiotic therapy is not beneficial.
Conclusion: The IAP/APA guidelines should be translated to all languages and distributed to all centers where patients suffering from AP are treated. Supported by NKFI and HAS.
A Quality of Life Comparison in Chronic Pancreatitis Patients Between Smokers and Non-Smokers
B. Patel,1 M. Min,1 S. Han,2 J. Kheder,3 L. Bocelli,3 A. Wachholtz,4 W. Wassef.3 1Department of Internal Medicine, University of Massachusetts Medical School, Worcester, MA; 2Division of Gastroenterology and Hepatology, University of Colorado, Aurora, CO; 3Department of Gastroenterology, University of Massachusetts Medical Center, Worcester, MA; 4Psychiatry, University of Massachusetts, Worcester, MA.
Introduction: Chronic pancreatitis is an inflammatory condition within the pancreas that remains difficult to treat and has been found to be associated with a poor quality of life. Recent data suggests that smoking may accelerate disease progression in chronic pancreatitis. This study aims to evaluate how smoking may affect quality of life in patients with this disease.
Methods: Several previously validated quality of life instruments including the PANQOLI (Pancreatitis Quality of Life Instrument), the Hospital Anxiety and Depression Scale (HADS), and the Brief Cope 24 were given to patients with chronic pancreatitis (previously diagnosed by endoscopic ultrasound and pancreatic function testing). Quality of life scores were then compared between smokers and non-smokers with chronic pancreatitis.
Results: There were 30 smokers and 33 non-smokers with chronic pancreatitis in this study (n=63). Compared to non-smokers, smokers had a significantly worse quality of life in terms of the PANQOLI (48.6 vs. 61.9, p<0.0001) and higher rates of anxiety and depression based on the HADS. Smokers also had worse coping skills based on the Brief Cope 24 (87.0 vs. 103.6, p<0.02).
Discussion: While it is known that smoking is an independent risk factor for the development of chronic pancreatitis, this is one of the first studies to demonstrate the difference in quality of life that smoking may bring in this patient population. Given the difficulty in managing this disease, smoking cessation should be heavily emphasized in this patient group, and future studies should elucidate the impact of smoking cessation on quality of life.
Bile Acids (BA) in Human Pancreatic Necrosis (PN) Worsen Acute Pancreatitis (AP) via a Non-Micellar Interaction with Fatty Acids (FA)
K. Patel,1 B. Khatua,1 J.R. Yaron,1 C. De Oliveira,1 R.J. Singh,2 G. Papachristou,3 D. Yadav,3 K. Lee,4 F. Murad,5 V.P. Singh.1 1Department of Medicine, Mayo Clinic, Scottsdale, AZ; 2Lab Medicine and Pathology, Mayo Clinic, Rochester, MN; 3Department of Medicine, University of Pittsburgh, Pittsburgh, PA; 4Department of Surgery, University of Pittsburgh, Pittsburgh, PA; 5NorthShore University Health System, Evanston, IL.
Background: Being amphiphilic, BAs may regulate biliary AP severity by stabilizing hydrophobic FAs released from fat necrosis in our aqueous in vivo environment. We tested this hypothesis by studying interactions between BAs and FAs in the progression of peripancreatic fat necrosis (PPFN), which in isolation is known to be milder in humans than when it is associated with pancreatic necrosis (PN).
Methods: BA in human PN collections (n=22), pancreatic ducts of rats with biliary AP were studied using LCMS-MS. In vitro, interactions of the main BA(taurocholate) and FA(oleate; OA) in human biliary AP were studied at relevant concentrations(100-300μM) by ultracentrifugation, electron microscopy, and high voltage flow cytometry. Acinar end points included injury (LDH leakage), intracellular calcium (Cai) and mitochondrial membrane potential (ψm). In vivo, PPFN progression in AP was studied using the triglyceride of OA[glyceryl trioleate;GTO, given IP, as in PMID 25500204] during biliary(biliopancreatic duct ligation;DL) and non-biliary (cerulein;CER) AP. A p<0.05 was considered significant.
Results: Cholic acid conjugates(taurocholate) were higher in rat(89.5±2.0%) and human biliary AP(n=14; 37.7±5.8%) vs. other BAs, with concentrations up to 250μM. In vitro, sodium taurocholate between 100-200μM, dose dependently retained OA in the micelle free 105000g infranatent, and enhanced OA but not caerulein induced Cai, ψm and LDH leakage. In vivo, DL alone increased BAs in ascites, serum, and caused mild AP. AP with DL+ GTO was worse with higher PN(55.2±6.4 vs. 0.6±0.1 %) extending inwards from the PPFN in the DL+GTO but not CER+GTO groups(1289±143 vs. 116±28 microns). DL+GTO had higher serum FA(3.3±0.5 vs 1.3±0.2 mM), earlier mortality(100% within 20 hours vs 72 hours), earlier elevations in serum IL-6, IL-1β, MCP-1, TNF-α, BUN, along with kidney and lung injury vs. the CER+GTO group.
Conclusions: BAs relevant to human pancreatic necrosis enhance acute lipotoxicity of FAs by stabilizing them in the aqueous environment via a non-micellar interaction, and thus worsen AP outcomes.
Histone Demethylase KDM3A Regulates Cancer Stem Cells for Pancreatic Cancer Progression
S. Paul,1 C. Ghosh,1 D. Subramaniam,2 K. Palaniyandi,1 T. Iwakuma,1 S. Anant,2 A. Dhar.1 Departments of 1Cancer Biology; and 2Surgery, The University of Kansas Medical Center, Kansas City, KS.
Introduction: Pancreatic ductal adenocarcinoma (PDAC) is the major leading cause of cancer related human death in USA. There is no possible treatment or target available in PDAC. It was proposed that the cancer stem cells (CSCs) can regulates malignancy in PDAC. DCLK1 is one of the quiescent cancer stem cell marker in PDAC that can regulate tumor progression. Identification of the key factors that influence stemness will help to target PDAC. Histone lysine demethylase KDM3A is an enzyme/protein which can regulate stem cell renewability and tumor progression, thereby KDM3A can interact with DCLK1 for tumor progression.
Aim: KDM3A influences tumor growth and regulates stemness. Moreover, we will determine the regulation of tumor progression in PDAC interacting with DCLK1.
Methods: We observed expression of KDM3A in both PDAC patients’ samples and cells. Knockdown and overexpression of KDM3A were executed by using lentiviral vector. Tumor progression were also observed in orthotopic mice model. ChIP and RNA seq were performed to validate the data.
Results: KDM3A was overexpressed in human PDAC patient tissues and human pancreatic cancer cells with concomitant increase of CSC marker, DCLK1. Moreover, DCLK1 and KDM3A was found to be co-localized in patient tissue samples and identified binding sites of KDM3A with DCLK1 using ChIP-seq. Knockdown of KDM3A abrogates oncogenic potential whereas, overexpressed KDM3A in transformed HPNE cells showed malignant properties with enhanced invasive property, pancosphere formation, foci formation and tumor formation in mouse. Moreover, ChIP-seq and RNA seq suggested that KDM3A regulated DCLK1 expression in tumor progression.
Conclusion: KDM3A regulated DCLK1 for stemness and enhance tumor progression in PDAC.
Risk of Malignant Transformation in Suspected Branch Duct Intraductal Papillary Mucinous Neoplasms Extends Beyond 5 Years
I. Pergolini,1 K. Sahora,1 C.R. Ferrone,1 W.R. Brugge,2 M. Patino,3 K.D. Lillemoe,1 A.L. Warshaw,1 C. Fernandez-Del Castillo,1 Departments of 1Surgery; 2Gastroenterology; and 3Radiology; Massachusetts General Hospital, Boston, MA.
The natural history and risk of malignancy of branch duct intraductal papillary mucinous neoplasms (BD-IPMNs) are incompletely understood, and therefore management remains controversial. We sought to evaluate long-term outcomes in a large cohort of patients followed for BD-IPMN in a single institution.
588 patients (median age 66, 59% female) with suspected BD-IPMN were followed for a minimum of 6 months and a median of 84. 60% of patients had at least one EUS/FNA. 319 patients (54%) underwent surveillance for more than 5 years (median of 103 months, range 60–329), and 95 (16%) for > 10 years. By 5 years, 82 patients (14%) underwent resection, and 24 (29%) had malignancy; 2 other patients had unresectable tumor; overall progression to invasive cancer within 5 years was 2.6% (15/588). Of the 319 patients followed for 5 years or more, 31 (10%) underwent resection a median of 89 months after initial diagnosis, and in 11 (35%) either invasive cancer (7) or high-grade dysplasia (4) was found. In addition, 5 patients developed inoperable pancreatic cancer; total progression to invasive cancer after 5 years was 3.8%. None of cysts with stable size ≤ 1.5 cm after 5 years of follow-up (n=102) developed malignancy within the cyst and only one (1%) a distinct ductal adenocarcinoma 65 months after initial diagnosis of BD-IPMN, whereas in patients whose cysts were > 1.5 cm after 5 years (n=217), 15 (7%) developed malignancy a median of 105 months after initial diagnosis (p=0.024). 5 of these developed malignancy after > 10 years of follow-up.
Conclusion: Risk of progression to malignancy in presumed BD-IPMNs under surveillance is at least 7%, and persists after 5 and even 10 years. Stability of small cyst (≤1.5 cm) may be a valuable parameter to safely discriminate low-risk BD-IPMN.
Zinc Mediates Pancreatitis Responses in In Vitro and In Vivo Mouse Models of Acute Pancreatitis
M.Y. Phadke,1 F. Gorelick.2,3 1Pediatric Gastroenterology, Yale University School of Medicine, New Haven, CT; 2Digestive Diseases, Yale University, New Haven, CT; 3Veterans Administration CT Healthcare, West Havenn, CT.
Background: Zinc is an essential trace mineral that plays important roles in immunity and other biologic processes. Zinc deficiency in rodents is associated with increased inflammation and zinc supplementation may reduce injury in acute inflammatory processes. We hypothesized that zinc levels would affect acute pancreatitis (AP) responses, and that zinc deficiency would be associated with worse disease.
Methods: In vitro, isolated mouse pancreatic acini were pre-treated with zinc chloride (ZnCl2) for 1 h followed by cerulein (100nM) to induce AP. Zymogen activation, amylase secretion and cell injury were then measured. In vivo, mice were fed a zinc-deficient (<1 ppm) or zinc-adequate (50 ppm) diet for 3 weeks. Pancreatitis was then induced with 6 hourly IP injections of ceruelin (40 μg/kg). Zymogen activation, serum amylase and other markers of pancreatitis were measured.
Results: We found that zymogen activation was significantly decreased in acini pretreated with 10 and 50 μM of ZnCl2. Amylase secretion was significantly increased in zinc-treated acini. Zinc treatment in vitro may also reduce cell injury. In vivo, zinc-deficient mice had significantly increased ceruelin-stimulated zymogen activation and serum amylase compared to zinc-adequate mice. Zinc had no significant effect on pancreatitis-related edema or histology.
Conclusion: Zinc supplementation in pancreatic acinar cells reduced ceruelin-induced zymogen activation. In vivo, mice with zinc deficiency exhibited some features of more severe pancreatitis. These findings may have therapeutic implications in humans with acute or chronic pancreatitis – conditions for which current treatment in both adult and pediatric populations remains largely supportive.
MutY-Homolog Modulates Pancreatic Cancer Cell Survival and Chemoresistance
P. Phillips,1 G. Sharbeen,1 J. Youkhana,1 A. Mawson,1 J. McCarroll,2 A. Akerman,1 D. Goldstein.1 1Pancreatic Cancer Translational Research Group; and 2Children's Cancer Institute; Lowy Cancer Research Centre, University of New South Wales, Sydney, Australia.
Background: Pancreatic cancer (PC) is the 4th leading cause of cancer-related deaths. It is characterised by extensive fibrosis, which creates a hypoxic and nutrient-deprived microenvironment. The PC microenvironment and chemotherapeutics increase intracellular oxidative stress, causing DNA damage. DNA repair proteins help PC cells survive under these conditions and therefore we postulate may be therapeutic targets for PC. MutY-Homolog (MYH) is a key DNA repair protein that repairs oxidative DNA damage and its function is unknown in PC cells. Our hypothesis is that MYH is an essential pro-survival protein for PC cells.
Aims: Our aims were to determine the effect of silencing MYH on PC cell survival, chemosensitivity and metastatic potential in vitro.
Methods: PC cells (MiaPaCa-2, AsPC-1) were treated with control siRNA (ns-siRNA) or MYH-siRNA. siRNA effects on PC cell colony forming ability, chemosensitivity (gemcitabine, paclitaxel, vincristine), apoptosis (AnnexinV staining + flow cytometry) and anchorage-independent survival, were then measured.
Results: Knockdown of MYH (relative to ns-siRNA): 1) reduced PC cell proliferation (MiaPaCa-2 = 54.8±6.5% reduction, n=3, p<0.05; AsPC-1 = 39.2±1.3% reduction, n=3, p<0.05) and clonogenic capacity (MiaPaCa-2 = 54.5±4.9% reduction, n=5, p<0.0001; AsPC-1 = 55.3±3.7% reduction, n=6, p<0.0001); 2) significantly chemosensitised MiaPaCa-2 to all tested drugs and AsPC-1 to paclitaxel and vincristine; 3) decreased MiaPaCa-2 anchorage-independent growth (34.3±8% decrease relative to ns-siRNA, n=7, p<0.01) and increased anoikis (67.9±5.8% increase relative to ns-siRNA, n=3, p<0.01).
Conclusions: We have demonstrated that knockdown of MYH reduces PC cell survival, metastatic potential, and chemoresistance in vitro, and warrants further investigation in an in vivo setting.
Translation: Therapeutic targeting of MYH in PC patients could potentially reduce PC growth and increase chemotherapy drug efficacy, thus improving patient outcome.
Exploring Machine Learning Methods to Determine Predictors of Pancreas Function
V. Pidlaoan,1 D.L. Conwell.2 1Gastroenterology, Hepatology and Nutrition, Ohio State University, OH; 2Ohio State University-Wexner Medical Center, Columbus, OH.
Background: Pancreatic function testing (PFT) is considered the non-histologic gold standard for the diagnosis of pancreatic dysfunction often seen in chronic pancreatitis (CP). The aim of this study is to determine if machine learning (ML) methods can be used on patient symptoms, risk factors, comorbidities and imaging to predict a normal or abnormal PFT.
Methods: Demographics, smoking history, alcohol use, symptoms, PFT and imaging data were collected from patients referred for evaluation of CP at a tertiary referral center. All patients had undergone an MRI, endoscopic ultrasound (EUS) and PFT evaluation. Abnormal PFT thresholds were tailored to the length of the test (<80 meq/L for 1 hour,<75 meq/L for a 45 minute test). MRI findings were coded as normal, equivocal, mild, moderate or severe. EUS grade was coded as normal, mild/equivocal or moderate/marked. Data analysis was performed using the Weka software package (University of Waikato, New Zealand) which implements machine learning methods and visualization aids. We evaluated two ML methods- Naïve Bayes (NB) and Decision Trees (DT).
Results: There were 126 patients in the cohort with an average age of 47 years. The majority of were female (62%) and 90% had abdominal pain. Sixty patients (48%) had an abnormal PFT. Using NB, a combination of abdominal pain, diabetes, a previous cholecystectomy, honeycombing on EUS, mild to marked changes of CP on MRI were the features predictive of an abnormal PFT. The model correctly classified 64.3% of instances and had a higher specificity (83%) than sensitivity (43%). Using DT, mild to marked changes on EUS and MRI were predictive of an abnormal PFT. The model correctly classified 57% of instances and had almost equal sensitivity (56.7%) and specificity (57.6%).
Conclusion: The use of machine learning methods can help us understand which combinations of clinical features predict a normal versus an abnormal PFT. NB appeared to be more robust when predicting patients with a normal PFT. The absence of these significant features may help triage patients who may not benefit from further invasive testing.
MicroRNA-345 in Pancreatic Cancer Pathogenesis
S. Rachagani,1 M. Kalaga,1 R. Pothuraju,1 S.K. Batra.1,2,3 1Department of Biochemistry and Molecular Biology, University of Nebraska Medical Center, Omaha, NE; 2Fred and Pamela Buffett Cancer Center, Omaha, NE; 3Eppley Institute for Research in Cancer and Allied Diseases, Omaha, NE.
Background: Pancreatic cancer (PC) is characterized by an extensive desmoplasia, which limits the delivery and efficacy of chemotherapy, and is primarily driven by over expressed sonic hedgehog (SHH). SHH also plays an important role in the epithelial to mesenchymal transition and cancer stem cells resulting in increased tumor growth, recurrence and resistant to therapy. Hence, there is a need to discover molecular targets to abrogate SHH signaling to enhance efficacy of therapy in PC patients. Herein, we proposed to explore the effect of miR-345 on SHH signaling and its impact on PC pathogenesis.
Methods: Expression of miR-345 in PC cell lines and tissues was assessed using TaqMan assay. The miR-345-GFP construct was overexpressed in PC cell lines using lentivirus and its overexpression was validated using TaqMan assay. Microarray and immunoblotting analyses were carried out in the vector and miR-345 transfected PC cell lines to decipher the miR-345 mediated signaling mechanism(s).
Results: We observed that miR-345 expression is significantly decreased in PC tissues and cell lines. The microarray analysis on miR-345 overexpressed PC cells revealed down regulation of SHH and its downstream targets of SHH: MYC, CCND1, CTNNB1, AXIN1, HES1, Notch1/2/3and other target genes such MUC4, Kras, EGFR, HIF1A, AIF1, ALDH1A1, HMGA1, ADAM9, BCL2, and FGFR1. Ingenuity pathway analysis of microarray data revealed down regulation of signaling pathways related to cell cycle regulation/DNA damage, mTOR, PI3K/AKT, ERK/MAPK1, RAC, VEGF, SHH and oxidative stress response. Functionally, overexpression of miR-345 in PC cell lines resulted in diminished growth and motility. Immunoblot analysis confirmed down regulation of SHH, cMYC, XIAP, c-FLIP, pAKT, pSTAT3, C1GALT1 and up regulation of cleaved caspase 7/3, PTCH2, and PARP in miR-345 overexpressed PC cells.
Conclusions: Restoration of miR-345 resulted in down regulation of SHH signaling and other important genes like MUC4, KRAS, which are known to play important roles in PC pathogenesis.
Targeting Cancer Stem Cells With Combined Inhibition of MEK and STAT3 in Pancreatic Cancer
C. Roberts, M. Vansaun, P. Lamichhane, F. Messaggio, K. Kovacs, N. Nagathihalli, N. Merchant. Department of Surgery, University of Miami, Miami, FL.
Background: Pancreatic ductal adenocarcinoma (PDAC) tumors are very heterogeneous, composed of a variety of cells including tumor cells and CD133+ cancer stem cells (CSCs). Many anti-cancer therapies are able to kill tumor cells, but are unable to eradicate CSCs due to their inherent chemoresistance resulting in tumor recurrence. Their self-renewal process is regulated by multiple signaling pathways including: Oct4, Sox2, c-MYC, and Nanog. These genes are regulated by STAT3 and MAPK signaling pathways.
Methods: To understand the effect of MEKi (AZD6244) and STAT3i (AZD1480) of PDAC cells, MiaPaCa-2, Panc-1, BxPC3 were used. To determine in vivo effects, Ptf1acre/+;LSL-KrasG12D;Tgfbr2fl/fl (PKT) mice were treated with either vehicle, MEK inhibition, STAT3 inhibition, or the combination for 2 weeks. Post-treated pancreatic tissue was extracted and examined for pancreatic integrity using immuno-histological. The pancreas from the mice was also examined with flow cytometry for EpCAM, CD24, CD44, and CD133.
Results: Treatment with combined MEKi and STAT3i treatment led to a significant decrease in spheroid size and metabolic activity. Combination treatment also reduced proliferation and colony formation in 2-D cultured cells. In PKT mice, treatment with combined MEKi and STAT3i resulted in the enhanced suppression of tumor formation. Histological analysis of combined MEK/STAT3 maintained a higher percentage of pancreatic integrity, displaying increased percentage of normal acini, reduced CK-19 staining and collagen deposition. We have found that in the mice, MEKi and STAT3i treatment decreased CD133+ and CD24+CD44+ cell populations after two weeks of treatment.
Conclusions: Combined MEKi and STAT3i dramatically reduced tumor burden and cancer stem cell populations in a PDAC mouse model.
Quality of Life Predictors in Chronic Pancreatitis: A European Cohort Study
S.M. Robinson,1,2 S. Rasch,3 S. Beer,4 A. Mickevicius,5 I. Valantiene,6 R. Charnley,1 J. Rosendahl.7 1Department of HPB Surgery, Newcastle upon Tyne Hospitals, Newcastle Upon Tyne, United Kingdom; 2Institute of Cellular Medicine Fibrosis Research Group, Newcastle University, Newcastle Upon Tyne, United Kingdom; 3II. Medizinische Klinik und Poliklinik, Klinikum rechts der Isar, Technische Universität München, Munich, Germany; 4Department für Innere Medizin, Neurologie und Dermatologie, Universitätsklinikum Leipzig, Leipzig, Germany; 5Center of Hepatology, Gastroenterology and Dietetics, Vilnius University Hospital Santariskiu Klinikos, Vilnius, Lithuania; 6Lithuanian University of Health Sciences, Kaunas, Lithuania; 7Universitatsklinik und Poliklinik fur Innere Medizin I, Universitatsklinikum Halle (Saale), Halle (Saale), Germany.
Background: Chronic pancreatitis is characterised by inflammation and fibrosis of the pancreas that lead to the clinical sequelae of chronic pain, pancreatic exocrine insufficiency and diabetes mellitus all of which might have a negative impact on quality of life. The aim of this study was to identify those factors that predict quality of life in these patients.
Methods: Quality of life was assessed in patients (n=197) with chronic pancreatitis presenting to 5 European centres using the EORTC QLQ-C30 and PAN-28 questionnaires. Clinical details were recorded and baseline blood tests taken. Statistical analysis was performed in SPSS with a p<0.01 considered significant.
Results: Gender and age had no impact on quality of life. Those with pancreatitis of alcohol etiology had a similar quality of life to those of other etiologies other than for increased alcohol related guilt (p=0.003). Inadequate glycaemic control (HbA1C≥53mmol/mol) was associated with poorer sexual function (p=0.005); increased night pain (p=0.004) and weight loss (p=0.01).
Current smokers (n=97) had a poorer quality of life scores across 18 assessed domains in areas as diverse as pain (p=0.001); physical (p=0.001) and social role functioning (p<0.001); body image (p<0.001); weight loss (p=0.002) and fear for their future health (p=0.001).
A CRP>5mg/ml was linked with diminished quality of life in 10 of the assessed domains including pain (p<0.001); body image (p<0.001) and weight loss (p=0.004). Smokers had a higher CRP (7vs.5mg/ml; p=0.004).
Conclusion: Smoking is extremely common amongst patients with chronic pancreatitis and is associated with increased inflammation and a poorer quality of life across multiple domains. Targeted smoking cessation services should be developed for this patient group.
Risk Factors for Asparaginase Associated Pancreatitis: A Systematic Review
F.T. Rose,1 J.-A. Oparaji,2 A. Orabi,1 A.S. Howard,3 D.C. Okafor,3 R. Turner,4 M.E. Lowe,1 K.A. Ritchey,1 S.Z. Husain.1 1Department of Pediatrics, Children's Hosptial of Pittsburgh of University of Pittsburgh Medical Center, Pittsburgh, PA; 2Pediatrics, Walter Reed National Military Medical Center, Bethesda, MD; 3School of Medicine, University of Pittsburgh, Pittsburgh, PA; 4University of Pittsburgh, Pittsburgh, PA.
Background: Drug-induced pancreatitis is a major problem. Asparaginase, which is a primary treatment for the most common childhood cancer, acute lymphoblastic leukemia (ALL), is a well-described cause of pancreatitis. Asparaginase associated pancreatitis (AAP) has an incidence between 2-18% and is one of the most common reasons for early termination of asparaginase treatment. The risk factors for AAP are unclear making predictions of which patients will develop pancreatitis impossible.
Methods: To evaluate possible risk factors for the development of AAP, we performed a systematic review of the current literature through May 2015.
Results: In an expansive screen, 1,842 citations were funneled into a review of 59 full articles, of which only 10 were deemed eligible based on the predetermined inclusion criteria including greater than 9 patients with AAP, standard Atlanta criteria definition for the diagnosis of pancreatitis, and English language. Two studies showed that children older than 10 years of age had a greater than 2-fold risk of AAP compared to younger children. Patients placed in high risk ALL categories had a greater incidence of pancreatitis in two studies. Additionally, use of PEG asparaginase, compared with the other formulations of asparaginase, resulted in a higher incidence of AAP in one study.
Conclusion: In this extensive systematic review, age, ALL risk stratification, and asparaginase formulation appear to play a role in the development of AAP. However, the risk of AAP cannot be determined confidently based on the identified studies. The work does suggest though the potential for yet to be determined underlying factors that may contribute to the development of pancreatitis in patients receiving asparaginase.
AC3/AC-Associated Protein 1 Complex Regulates Actin Filament Dynamics in Pancreatic Cancer Cells
M.E. Sabbatini, H. Hassan, A. Newsom, S. Mehratra. Biological Sciences, Augusta University, Augusta, GA.
Background: An activation of adenylyl cyclase (AC) by forskolin (FSK) inhibits migration of pancreatic cancer cells. However, the mechanism responsible for the inhibitory effect is not fully understood. Our previous studies have shown that both AC1 and AC3 are highly expressed in human pancreatic cancer tissues. Hence, we conducted studies to identify proteins that interact with AC1 and AC3, and define the pathophysiological significance of such interaction. AC associated protein-1 (CAP1) is a scaffolding protein involved in the regulation of filopodium formation and, in consequence, cell migration.
Aim: To study the extent to which CAP1, by linking AC1 and AC3 to G-actin, inhibits filopodium formation.
Methods: PANC-1 and HPAC cells were stimulated with FSK. Studies of protein interactions were carried out using co-immunoprecipitation (IP). Cell lysates were rotated with anti-AC1 or anti-AC3 antibody followed by complex collection. In another experiment, AC1 or AC3 was knocked down using small interference RNA. Cell lysates were rotated with anti-actin antibody followed by complex collection. The immunocomplexes were probed with anti-CAP1 antibody. Studies of filopodium formation were carried out using immunocytochemistry.
Results: Both AC1 and AC3 interacted with CAP1. In scrambled siRNA cells stimulated with FSK, the association between CAP1 and actin was not affected. The CAP1-actin complex formation was impaired in AC3-deficient cells, but was not modified in AC1-deficient cells. The lack of AC3, but not AC1, impaired the inhibitory effect of FSK on filopodium formation.
Conclusion: CAP1 acts as a scaffolding protein by holding the complex between G-actin and AC3. In the presence of AC3, CAP-1, by sequestering G-actin, inhibits filopodium formation.
Paraneoplastic Weight Loss in Pancreatic Cancer (PC) With Selective Reduction in Subcutaneous Relative to Visceral Fat Mediated by PC Exosomes
R.P. Sah,1 S. Nagpal,1 A. Sharma,1 N. Ahmed,1 S. Mohapatra,1 N. Takahashi,1 D. Mukhopadhyay,2 S.T. Chari.1 1Mayo Clinic, Rochester, MN; 2Mayo Clinic, Jacksonville, FL.
Background: Our previous studies suggest that weight loss (WL) accompanies development of diabetes in pancreatic cancer (PC) and lipolysis is induced in Subcutaneous Adipose Tissue (SAT) by PC through exosomes (Exo). We aimed to characterize WL occurring prior to PC diagnosis (DX) and explore its mechanisms by analyzing changes in Visceral Adipose Tissue (VAT) and SAT using CT scans and studying effect of PC Exo on VAT and SAT in-vitro.
Methods: Timelines of pre-diagnostic WL were compared in PC and colon cancer (CC) patients. In 83 PC and 39 CC patients with modest WL, we compared VAT and SAT area (measured at L3-L4 in abdominal CTs) at DX and ≥ 1 year prior. Precursor adipocytes (Ad) isolated from VAT and SAT of renal transplant donors (N=4) were differentiated in presence of Exo from PC (N=4) and CC (N=2) cell lines.
Results: Mean WL (Kg) for PC vs CC was 3.3 vs 1.3 (P<.001; N=635 PC, 1220 CC) between 6 months and DX, 1 vs 0.3 (P<.001; N=391 PC, 741 CC) between 6 mth and 1yr, and no WL seen ≥18 mth. There was 6% greater loss of SAT than VAT in PC Vs 4% greater loss of VAT than SAT in CC (P=0.06). VAT/SAT ratio increased by.08 in PC Vs decreased by.05 in CC (P=0.01). PC (but not CC) Exo increased lipid content of VAT Ad to 220% of that in control without Exo (P=0.01). In contrast, PC and CC Exo both reduced lipid content in SAT Ad (P<0.01 each). Transcriptome of PC Exo-treated VAT Ad revealed induction of adipogenesis pathways.
Conclusion: Significant WL beginning 1 yr prior to cancer diagnosis occurs in PC with relative preservation of VAT and preferential shedding of SAT which is distinct from Non-PC cachexia. This seems to be mediated by PC exosomes. Preservation of VAT with loss of SAT might explain insulin resistance and worsening diabetes despite WL in PC.
Interleukin (IL) 10 and Pyridone 6 (P6) Modify the Migration of Pancreatic Cancer Cells in Co-Cultures With Macrophages
A. Salmiheimo,1,2 H. Mustonen,1,2 S. Vainionpää,1,2 E. Kemppainen,1,2 P. Puolakkainen,1,2 H. Seppänen.1,2 1Helsinki University Hospital, Helsinki, Finland; 2University of Helsinki, Helsinki, Finland.
Introduction: The prognosis of pancreatic ductal adenocarcinoma (PDAC) is very grim and there are few tools to restrict an inoperable disease. Macrophages participate to the migration of PDAC cells in co-cultures. The aim of this study was to examine the possibilities of modifying the migration of PDAC through macrophages.
Methods: We studied the migration rate of fluorescein stained pancreatic cancer (MiaPaCa-2) cells in Matrigel cultured alone or with M-CSF (50ng/ml) differentiated macrophages isolated from healthy human subjects. We supplemented the cell cultures with either anti-inflammatory IL10 (25ng/ml) or JAK/STAT inhibitor P6 (500nM). We used flow cytometry to measure the activation of STAT 3 and NFkB in macrophages.
Results: Macrophages increased the migration rate of MiaPaCa cells from 10.1μm/h to 13.3μm/h (p<0.001). JAK/STAT-inhibitor P6 was able to completely inhibit the macrophage induced increase in the MiaPaCa migration rate. Oppositely, IL10 increased the migration of MiaPaCa in macrophage co-cultures from 10.4μm/h to 17.8μm/h (p<0.001). In single MiaPaCa cell cultures the migration rate was unaffected by P6 and IL10. Co-culture increased STAT3 and NFkB activation in macrophages. In co-cultures NFkB activation decreased with both IL10 and P6. STAT3 activation was inhibited with P6 and remained unchanged with IL10.
Conclusion: In PDAC cells IL10 activates and P6 inhibits the migration rate through macrophages.
Comparison of Clinical Course and Outcome of Acute Pancreatitis, Recurrent Acute Pancreatitis and Acute on Chronic Pancreatitis
J. Samanta,1 D.J. Sharma,1 N. Dhaka,1 V. Gupta,2 A. Gulati,3 S.K. Sinha,1 R. Kochhar.1 Departments of 1Gastroenterology; 2Surgery; and 3Radiodiagnosis; Postgraduate Institute of Medical Education and Research, Chandigarh, India.
Introduction: There is lack of published data regarding comparative studies between the natural course of disease and outcome of patients of acute pancreatitis (AP) vis-à-vis that of recurrent acute pancreatitis(RAP) and acute on chronic pancreatitis(ACP).
Methods: Patients admitted between July 2014 and June 2016 with AP (first attack only), RAP (more than 2 attacks) or ACP (acute episode with radiological evidence of chronic pancreatitis) were compared for severity, organ failure (OF), persistent organ failure (POF), need for ICU stay, ventilator and dialysis, hospital stay, need for percutaneous catheter drain (PCD), surgery and mortality.
Results: Of the 248 patients evaluated,158 (64%) patients had AP, 45(18%) had RAP and 45 (18%) patients had ACP. 86 (54%) of AP, 4 (9%) of ACP and none of the patients in RAP group had severe AP (p<0.001). 101 (63%;of whom 54% had POF) of AP, 6 (13%; of whom 9% had POF) of ACP and none of RAP patients developed OF (p<0.001). PCD and surgery requirement were seen in 89 (56%) & 9 (6%) of AP, 5(11%) & 4 (9%) of ACP and none of RAP patients respectively (p<0.001 & p=0.44). Length of hospital stay was significantly higher in AP and ACP as compared to RAP (18.80±14.5, 8.82±11.2,3.36±4.4 days,p<0.001) whereas ICU requirement was seen in significantly more patients in AP group (37%) than in ACP (11%) and RAP group (0%)(p<0.001). 41 (26%) of AP, 2 (4.4%) of ACP, but none in RAP group needed ventilatory support (p<0.001), while13 (8%) of AP patients, 1 (2.2%) of ACP and none of RAP required dialysis (p<0.05). Mortality in AP, ACP and RAP was 29 (18%), 2 (4.4%), 0 (0%) respectively (p<0.05).
Conclusion: First attack of AP has more severe course as compared to that of RAP and ACP.
Surgical Resection Following Neoadjuvant Chemotherapy for Borderline Resectable Pancreatic Cancer: Redefining the Standards
M. Sandini,1 G. Marchegiani,1 L. Maggino,1 E. Viviani,1 A. Montresor,1 A. Binco,1 G. Malleo,2 R. Salvia,1 C. Bassi.1 1Department of Surgery, Pancreas Institute, University of Verona Hospital, Verona, Italy; 2Surgery B, AOUI Verona Hospital Trust, Verona, Italy.
Background: The optimal management of borderline resectable (BR) pancreatic cancer (PC) is controversial. As the development of new neo-adjuvant therapies is emerging, there is scarcity of data regarding the outcome of surgical resection following chemo-radiotherapy.
Methods: From a prospective database, we retrospectively analyzed all BR cancer patients who underwent surgical exploration from January 2013 to December 2015 after neoadjuvant chemo (CT) or chemo/radiation (CRT).
This cohort (group 1) was compared with a group of BR pancreatic patients who underwent upfront surgery plus venous vascular resection (group 2, n=98) and with a third group of resectable patients (group 3, n=98). Among-group comparisons were made with propensity score. BR PC was defined according to ISGPS classification.
Results: 111 subjects underwent surgical exploration after neoadjuvant CT/CRT. Eighty of them (72.1%) were resected. Patients in group 2 experienced higher rates of overall complication (50.5%, 64.3% and 48.0% respectively, p=.05), reoperation (5.4%, 13.3.% and 3.1% respectively, p=.014) and postoperative death (0.0%, 3.1% and 0.0% respectively, p=.04).
At pathology, group 1 had significantly lower rates of nodal involvement (median node ratio of.04,.11 and.11 respectively, p < 0.001) and lymphatic/vascular and perineural involvement (p < 0.001 and p=.001).
R0 was 57.5%, 43.9%, and 64.3% respectively in the groups (p=.008) and median overall survival 66.0, 28.8 and 32.8 months (p=.002).
Conclusion: Surgical resection following neoadjuvant CT/CRT for BR PC is associated with similar postoperative outcomes of upfront surgery for resectable tumors. Neoadjuvant CT/CRT for BR PC improves the pathological outcome and seems to provide encouraging results on long-term survival.
NF-kB Activation With Radiocontrast Exposure During Post-ERCP Pancreatitis is Dependent on Bcl10 in the Scaffolding CBM Complex
S. Sanker, A.I. Orabi, D. Hu, L.M. McAllister-Lucas, P.C. Lucas, S.Z. Husain. Department of Pediatrics, Children’s Hospital of Pittsburgh of UPMC, University of Pittsburgh, Pittsburgh, PA.
Pancreatitis is the most common iatrogenic complication of ERCPs, but the mechanisms that transduce inflammation in post-ERCP pancreatitis are unclear. We recently showed that exposure of the pancreas, both in vivo and ex vivo, to the radiocontrast commonly used during ERCPs induced marked nuclear translocation of the central inflammatory transcription factor NF-kB. This activation was dependent on the calcium phosphatase calcineurin. In this preliminary study, we hypothesized that the point of intersection between calcineurin and NF-kB is through calcineurin-mediated dephosphorylation of Bcl10, a component of the trimeric complex CBM (Carma1/3, Bcl10, Malt1). CBM serves as a scaffold to permit the activation of the upstream kinase IKK in the NF-kB canonical pathway. Firstly, we found that the mouse pancreatic acinar cell expresses Carma3, but not Carma1, as well as the other two proteins in the complex. Secondly, among nine disparate tissues cell lines, radiocontrast caused NF-kB translocation only in the pancreatic AR42J cell and its close cousin the liver hepG2 cell. The latter is faster growing and thus easier to manipulate. From this cell line, we generated a CRISPR/cas9-mediated stable Bcl10 knockout and found that it was largely protected against radiocontrast-induced NF-kB, compared to over a twenty-fold increase with the control Bcl10-sufficient cell. These data suggest a novel mechanism, through Bcl10 in the CBM complex, to trigger inflammatory NF-kB signals during post-ERCP pancreatitis.
Autoimmune Pancreatitis in Children: Working Guidelines for Diagnosis and Management
I. Scheers,1 J.J. Palermo,2 S. Freedman,3 M. Wilschanski,4 U. Shah,5 M. Abu-El-Haija,2 B. Barth,6 D. Fishman,7 C. Gariepy,8 M. Giefer,9 M. Heyman,10 R. Himes,7 S.Z. Husain,11 T.K. Lin,2 Q. Liu,12 M.E. Lowe,11 M. Mascarenhas,13 V. Morinville,14 C.Y. Ooi,15 E. Perito,10 D.A. Piccoli,13 J. Pohl,16 S.J. Schwarzenberg,17 D. Troendle,6 S. Werlin,18 B. Zimmerman,19 A. Uc,19 T. Gonska.1 1Hospital for Sick Children, Toronto, Canada; 2Cincinnati Children's Hospital Medical Center, Cincinnati, OH; 3Harvard Medical School, Beth Israel Deaconess Medical Center, Boston, MA; 4Hadassah Hebrew University Hospital, Jerusalem, Israel; 5Harvard Medical School, Massachusetts General Hospital for Children, Boston, MA; 6University of Texas Southwestern Medical School, Dallas, TX; 7Baylor College of Medicine, Houston, TX; 8Nationwide Children’s Hospital, Columbus, OH; 9Seattle Children's Hospital, Seattle, WA; 10University of California at San Francisco, San Francisco, CA; 11Children's Hospital of Pittsburgh of UPMC, Pittsburgh, PA; 12Keck School of Medicine, University of Southern California, Children's Hospital Los Angeles, Los Angeles, CA; 13The Children's Hospital of Philadelphia, Philadelphia, PA; 14Montreal Children’s Hospital, McGill University, Montreal, Canada; 15Discipline of Paediatrics, School of Women’s and Children’s Health, Medicine, University of New South Wales and Sydney Children’s Hospital Randwick, Sydney, Australia; 16University of Utah, Salt Lake City, UT; 17University of Minnesota Masonic Children’s Hospital, Minneapolis, MN; 18Medical College of Wisconsin, Milwaukee, Wi; 19University of Iowa Carver College of Medicine, Iowa City, IA. *T. Gonska and A. Uc are equal senior authors.
Background & Aims: Autoimmune pancreatitis (AIP) is an increasingly recognized disease entity, but data in children are limited. Pediatric gastroenterologists relied on adult AIP guidelines but disease presentation and outcome of AIP in children might differ from the adult experience. We aim to develop a working definition and diagnostic approach for AIP in children.
Methods: Clinical data, imaging, histology, and treatment modalities were collected using 2 different approaches: (1) a systematic literature search and (2) children with an AIP diagnosis from the largest multicenter study of chronic pancreatitis in children (INSPPIRE) and from Cliniques St-Luc (CUSL). We then sought expert opinion from pediatric pancreatologists.
Results: We identified 44 AIP cases, 26 from literature review, 14 from the INSPPIRE and 4 from CUSL cohort. The median age at diagnosis was 13.2 years. Abdominal pain (39/44, 87%) and/or obstructive jaundice (20/44, 45%) were the most reported symptoms at diagnosis. Elevated IgG4 levels was seen in only 8/38 (21%). Cross-sectional imaging was abnormal in all children mainly showing hypointense global or focal gland enlargement (35/43, 81%), irregularity of the main pancreatic duct (29/43, 67%) and common bile duct stricture (25/43, 58%). Lymphoplasmacytic inflammation, pancreas fibrosis and ductal granulocyte infiltration were the main histologic findings (18/25, 72%). Children with AIP had a prompt clinical response to steroids. Complications of AIP included impaired exocrine (4/25,16%) and/or endocrine (3/27,11%) function.
Conclusion: AIP in children is a distinct subtype of pancreatitis. Based on these observations, we established working guidelines to help identification and management of children with AIP and pave the way for future studies.
Zoledronic Acid as a Novel Radiosensitizer for Pancreatic Cancer
P. Seshacharyulu,1 R.K. Nimmakayala,1 S. Rachagani,1 S. Kaur,1 M.P. Ponnusamy,1,2 M. Jain,1,2 C. Lin,2,3 S.K. Batra.1,2. 1Department of Biochemistry & Molecular Biology, University of Nebraska Medical Center, Omaha, NE; 2Fred & Pamela Buffet Cancer Center, Eppley Institute for Research in Cancer and Allied Diseases, University of Nebraska Medical Center, Omaha, NE; 3Department of Radiation Oncology, University of Nebraska Medical Center, Omaha, NE.
Aim: To characterize and target novel pathways involved in radioresistance of pancreatic cancer.
Background: Due to the existence of intrinsic and acquired resistance to radiotherapy (RT), >80% of pancreatic cancer (PC) patients do not achieve objective response. Recently, we observed that differential expression of multiple members of cholesterol biosynthesis (CBS) pathway plays a central role in imparting radioresistance (RR) to PC cells. Herein, we characterized radioselected (RS) PC cells and evaluated the impact of Zoledronic acid (ZOL) on RT of PC.
Methods: Radioresistant cell lines were derived from CD18/HPAF and Capan-1 PC cell lines using fractionated irradiation (a total of 20 Gray). Clonogenic survival, cellular morphological, cell cycle and side population (SP) analyses were performed to evaluate response of RS and parental PC cells to RT. RT-PCR and immunoblotting studies were carried out to identify the molecular mechanism(s) of radiosensitization by ZOL.
Results: Chronic radio selection (RS) of PC cells resulted in altered cellular morphology [spindle like elongated (Capan-1) or cobble stone like phenotype (CD18/HPAF)], increased survival accompanied by G2/M arrest, and 1.5 fold increase in side population in comparison to unselected control cells. Immunoblotting studies illustrated increased expression of FDPS and SC4MOL along with Cyclin D1, Cyclin E, ALDH1, CD133 and ESA in RS PC cells compared to controls. Interestingly, 24 h pre-treatment of PC cells with 5 μM ZOL prior to irradiation (5 Gy) effectively sensitized radioselected PC cells (p<0.05). Further, molecular signaling studies illustrated that ZOL exerted its radiosensitization effect via modulation of FDPS.
Conclusions: PC radioresistance is accompanied by upregulation of FDPS and can be targeted using ZOL.
Laser Capture Microdissection and Proteomic Characterization of Acinar Cells From Cerulein Treated Mice Reveals Significant Up-Regulation of Cytoskeletal Proteins
J.P. Shapiro,1 H. Komar,2 B. Hancioglu,3 L. Yu,4 P. Hart,1 Z. Cruz-Monserrate,1 D.L. Conwell,1 G. Lesinski.1,2 Divisions of 1Gastroenterology, Hepatology and Nutrition; and 2Medical Oncology; The Ohio State University Wexner Medical Center, Columbus, OH; 3Biomedical Informatics, The Ohio State University, Columbus, OH; 4Center for Biostatistics, Department of Biomedical Informatics, The Ohio State University, Columbus, OH.
Introduction: The underlying mechanisms for the development of chronic pancreatitis (CP) are not well understood. We hypothesized that acinar cells from pancreata at difference stages of CP express unique proteome signatures that would provide a mechanistic understanding of the molecular pathophysiology of early stage CP.
Methods: C57BL/6 mice were injected ip with cerulein or PBS (6x/day, 3x/week) for 1, 3 or 6 weeks. At each time point, 10,000 acinar cells were collected using laser capture microdissection (LCM). LCM retrieved proteins were trypsin digested and peptides analyzed with liquid chromatography combined with tandem mass spectrometry (LC-MS/MS). LC-MS/MS identified peptide counts were used to quantify protein expression in cerulein vs. control mice.
Results: We detected expression of proteins involved in hallmarks of fibrosis, exocrine insufficiency, and protease mediators of CP. Among these, fibronectin, collagen α2, trypsin-1 and chymotrypsinogen were overexpressed at 1, 3 and 6 weeks (p<0.05). Amylase and lipase levels were down at 6 weeks (p<0.05). Interestingly, we found nineteen cytoskeletal-associated proteins (CKPs) to be significantly over-expressed at 3 and 6 weeks of cerulein treatment (p<0.05). These included: keratins [7,8,18,19,20]; microtubule associated proteins: [stathmin,CKAP4, MAP4]; actin associated proteins: [capping protein, vinculin, talin1, and α-actinin1]; and cytoskeletal proteins; [α-filamin, vimentin, moesin, tropomyosin-α4, desmin, cortactin and plectin].
Conclusions: CKPs are known to facilitate secretion of proteins such as zymogen granules. The identified changes in CKP expression may help explain the aberrant protein secretion of acinar cells and provide a novel understanding CKPs contribution to the progression of CP.
Risk for Pancreatic Cancer in Patients With Pancreatic Cysts and Family History of Pancreatic Cancer
A. Sharma, S. Mukewar, N. Philip, S.S. Vege, S.T. Chari. Gastroenterology and Hepatology, Mayo Clinic, Rochester, MN.
Background and Aims: Whether family history of pancreas cancer (PC) in first-degree relative (FDR) of subjects with pancreatic cystic lesion (PCL) confers higher risk of PC is unknown.
Methods: From Mayo Clinic database, we selected all the patients from 2000 to 2013 with family history of PC in FDR of PC and reviewed their cross sectional imaging for pancreas cyst. Using data from medical records we compared the risk of undergoing pancreatic resection and risk of PC among subjects 3 groups: Group I (PCL+ FH+, n=304), Group II (PCL+ FH-, n=1195) and Group III (PCL- FH+, n=744).
Results: The 5-year risk of developing PC was significantly higher for group I compared to group III (5.9% vs. 1.7%; p=0.0064) but not group II (5.9% vs. 2.8%; p=0.65). Among patients with PCLs and FDR of PC, the 5-year risk for PC was higher in those with single FDR than multiple FDRs (6.6% vs. 0.0%; p=0.01). When stratified based on Fukuoka criteria, having FDR with PC did not increase the 5-year risk of PC compared to cysts without FH of PC (p=0.61). There was no difference in 5-year risk of undergoing pancreas cyst surgery in group I vs. II (14.7% vs. 11.8%; p=0.43). For Fukuoka negative cysts, the 5-year risk of undergoing surgery was higher in group I than group II (11.3% vs. 5.9%; p=0.01) but not for Fukuoka positive cysts (p=0.67).
Conclusion: Family history of PC does not alter the risk of PC in subjects with PCLs, suggesting that more intense surveillance is not required for such patients.
Oral Administration is as Effective as Intraperitoneal Administration of Minnelide Against Pancreatic Cancer
N.S. Sharma, S. Modi, B. Giri, J. George, B. Garg, V. Sethi, S. Banerjee, V. Dudeja, A.K. Saluja. Department of Surgery, Sylvester Comprehensive Cancer Center, University of Miami, Miami, FL.
Introduction: Pancreatic cancer is an aggressive disease. The current chemotherapy only marginally increases the survival of patients with PDAC and therefore requires novel therapy.
We have shown that Minnelide administered parenterally (currently in phase I trials) is very effective against pancreatic cancer. We aimed to evaluate the anti-cancer effects of low doses of Minnelide administered orally alone or in combination with standard therapy in PDAC.
Methods: Xenograft, subcutaneous and orthotopic models were established using S2-VP10 and MIA PaCa-2 cell lines. Immunocompetent subcutaneous model was established using KPC derived cells. Minnelide was used in doses ranging from 0.15-0.6 mg/kg/day either oral or i.p. and standard chemotherapy was Gemcitabine 250 mg/kg/week + nab-paclitaxel 25 mg/kg/week. Due to the development of anaphylactic reaction to human derived albumin in nab-paclitaxel, equivalent doses of paclitaxel were used in immunocompetent mice. The tumor burden was compared between various treatment groups.
Results: Minnelide i.p significantly decreased tumor mass in KPC and MIA PaCa-2 derived tumors and oral administration had equipotent effects. In the S2-VP10 model, Combination of low doses of orally administered Minnelide with Gemcitabine + nab-paclitaxel significantly inhibited tumor progression and was effective in decreasing the tumor burden leading to an increased survival of tumor bearing mice (p<0.05). Combination therapy significantly reduced cancer related morbidity by decreasing ascites and metastasis.
Conclusion: Oral administration of Minnelide is as effective as parenteral administration and can potentially lead to better patient compliance and easy administration in future.
Misdiagnosis of Chronic Pancreatitis (CP) in a UK Regional Pancreas Center
A.R.G. Sheel,1,2 C. Halloran,1,2 P. Ghaneh,1,2 R.D. Baron,1 M. Raraty,1 J. Kleeff,1,2 V. Yip,1 J. Evans,3 F. Campbell,4 R. Sutton,1,2 J. Ramesh,5 J.P. Neoptolemos.1,2. 1Pancreatobiliary Surgery, Royal Liverpool and Broadgreen University Hospitals Trust, Liverpool, United Kingdom; 2NIHR Pancreas Biomedical Research Unit, University of Liverpool, Liverpool, United Kingdom; Departments of 3Radiology; 4Histopathology; and 5Gastroenterology and Endoscopy; Royal Liverpool and Broadgreen University Hospitals Trust, Liverpool, United Kingdom.
Introduction: The 2014 APA Practice Guidelines for CP (defined as chronic, progressive, irreversible parenchymal damage) highlighted the limits of previously accepted diagnostic criteria for early CP.
Aims: An audit of evidence for CP in 805 patients referred and reviewed in our HPB outpatient clinic between 2003–2016.
Patients and Methods: Clinical, genetic, radiological and histological details of 526 men and 276 women, median age 57 (IQR 48–66) years were reviewed by a consultant led team during 2015–2016.
Results: The diagnosis of CP was rejected in 118 (14.7%) patients: chronic abdominal pain syndrome (CAPS) = 36; minimal change CP (MCCP) on EUS, no disease progression = 29; post-AP sequelae = 22; RAP = 12; IPMN only = 5, other = 14. In the MCCP group, 22 (71%) reported pain as their main symptom.
The 65 (37M: 28F) patients reclassified as CAPS or MCCP underwent a total of 246 investigations including: 166 CT’s, 61 EUS’s, 18 MRI’s and 1 ERCP. Twenty two (61%) CAPS underwent a minimum of one EUS. A median of 4 (IQR 2–5) radiological scans were performed per patient over a median of 4 (IQR 2–6.5) years, all without evidence of radiological progression. The median age at first symptoms in this subgroup was 40.5 (IQR 14–46) years. Previous hyperamylasaemia was confirmed in 23 patients. Median pain duration was 10 (IQR 5.25-14.75) years. Twelve consumed ≥62 units/week of ETOH ≥1 year; 36 were “ever” smokers; diabetes = 9 (IDDM=2); unemployed = 53 (82%). Four required repeated ketamine infusions and 30 (46%) were dependent on daily morphine.
Conclusion: The diagnosis of CP requires careful consideration; certain patients may warrant a period of observation prior to making a formal diagnosis. CAPS can be misdiagnosed as CP. Newer classifications of CP could incorporate a time limit for progression of MCCP EUS findings in the diagnostic algorithm.
Induction of the Artery First Approach in Pancreaticoduodenectomy for Pancreatic Head Cancer
H. Shimamura, H. Kodama, A. Endo, K. Takeda. Department of Surgery, Sendai Medical Center, Sendai, Japan.
Purpose: Recently focused technic in pancreaticoduodenectomy (PD) for pancreatic head cancer is the artery first approach (AFA). There have been many reports that this procedure avoids congestion of uncinate process resulting in decrease of blood loss. Here we review our cases and discuss about this procedure.
Patients and Methods: Between January 2005 and December 2015, patients with pancreatic head cancer who underwent PD in our hospital were retrospectively reviewed. We induced AFA since December 2012. Patients underwent PD by AFA were recruited as AFA group (n=18), whereas patients underwent PD by former procedure were recruited as historical control (Control group, n=44). Operative outcomes between these two groups were compared, using Student’s t-test or Chi-squared test.
Results: Between two groups, there was no significant difference in terms of backgrounds. There was no significant difference on operative time between the two groups. But, operative blood loss of AFA group (average 882ml) was significantly fewer than that of Control group (average 1188ml). Perioperative blood transfusion units also seemed fewer in AFA group, though not significant. There was no difference in achieving rate of R0 resection. Occurrence rate of postoperative pancreatic fistula (Grade B and C, ISGPF) in AFA group was smaller than that in Control group, though not significant. On the other hand, postoperative hospital stay in AFA group was significantly shorter than that of Control group.
Conclusion: There are a couple of merits in PD using AFA for pancreatic head cancer in terms of fewer blood loss and shorter postoperative hospital stay.
Genetic Deletion of AMPK Results in Greater Baseline and Secretagogue-Stimulated Enzyme Activity and Cellular Injury
C.A. Shugrue,1 A.J. Ceplenski,1 E.J. Foglio,2 V. Patel,1 M. Foretz,3 B. Viollet,3 F.S. Gorelick.1,4 Departments of 1Internal Medicine, Section of Digestive Diseases; and 2Pediatrics; Yale University School of Medicine, New Haven, CT; 3Institut Cochin, INSERM, Paris, France; 4VA Connecticut Healthcare, West Haven, CT.
We have shown that AMP-activated protein kinase (AMPK) has a protective role in acute pancreatitis; pretreatment of pancreatic acini with AMPK activators decreases zymogen activation. We hypothesized that genetic deletion of AMPK would worsen pancreatitis responses. In AMPK-γ1−/− (knockout, KO) mice, α and β subunit levels are also reduced. KO mice given cerulein (CER; 6x i.p.) had 3–4 fold greater pancreatic trypsin/chymotrypsin activity than C57BL/6 (Wild-type, WT).
Basal trypsin activity in KO acini tended to be higher, but chymotrypsin activity was significantly increased (4.2 fold) greater than WT. Pancreatic lobules treated with CER or carbachol were probed for trypsinogen activation peptide (TAP) immunoreactivity by IF. KO lobules exhibited greater TAP labeling at baseline and with stimulation than WT; TAP labeling was found in the basolateral cytoplasm as well as the expected punctate pattern.
Basal amylase secretion (as percent total) in KO acini was similar to WT but stimulated secretion in KO acini was reduced. KO acini had higher total amylase levels, but total trypsin (enterokinase activated) was only slightly higher in KO acini, suggesting that KO acini may not have greater enzyme content.
MTT assay showed that KO acini had more basal and stimulated injury than WT. Basal KO pancreas showed more acinar cell content-containing vacuoles and neutrophil infiltration than WT and had discrete parenchymal perilipin-positive fat deposits.
In summary, KO acini have greater basal and stimulated enzyme activity and cellular injury than WT. KO acini also have higher total amylase levels and secrete less upon stimulation. Taken together, this suggests that genetic deletion of AMPK results in basal injury and a more severe pancreatitis.
The Rate of Post-ERCP Pancreatitis Associated With Single Operator Peroral SpyGlass Cholangioscopy (SOC) is Well Tolerated With an Established Practice
A. Siiki,1 M. Ukkonen,1 J. Laukkarinen.1,2 1Dept. of Gastroenterology and Alimentary Tract Surgery, Tampere University Hospital, Tampere, Finland; 2School of Medicine, University of Tampere, Tampere, Finland.
Background: Single operator SpyGlass cholangioscopy (SOC) with visual intra-ductal image is an adjunct to standard endoscopic retrograde cholangio-pancreatography (ERCP) and may improve the diagnostics of indeterminate biliary strictures.
Aim: To study the rate of adverse events after SOC ERCP during the learning curve and after establishment of a routine in SOC practice in a tertiary referral center.
Methods: The study included all SOC ERCPs performed from the beginning or SOC practice in our hospital, during 2012–2015. The data was collected prospectively for indications, procedure details and adverse events, and supplemented from patient records for eventual clinical outcome.
Results: Out of the total of 1920 ERCPs performed during the study period, 113 patients (5.9%; mean age 58 y, 57% male) underwent SOC ERCP. Median follow-up was 19 (range 1–48) months. The indication for SOC was primary sclerosing cholangitis in 43%, indeterminate stricture in 30% and stone disease in 24%. 92% of SOCs were performed in outpatient setting. In the very first 25 SOCs, the rate of adverse events was 20%: all 5 patients had a severe post-ERCP pancreatitis (PEP), including one mortality. In the next 88 SOCs rate of adverse events was 8% (PEP 3%, cholangitis 3%, bleeding 1%, mortality 0%, none severe). In indeterminate strictures, the sensitivity and specificity of SOC biopsies were 75% and 87% for detecting cancer, respectively.
Conclusion: The risk of PEP after SOC is high in the beginning of practice. When experience accumulates and procedure becomes routine, the risk decreases to the level seen in standard ERCP. SOC may be considered as a safe adjunct to ERCP even in outpatient setting. It may bring additional value to the diagnostics of biliary strictures.
Alternative Lengthening of Telomeres and Loss of DAXX/ATRX Expression Predicts Metastatic Disease and Poor Survival in Patients With Pancreatic Neuroendocrine Tumors
A.D. Singhi,1 T.-C. Liu,2 J.L. Roncaioli,3 H.J. Zeh,4 A.H. Zureikat,4 A. Tsung,4 J.W. Marsh,4 K.K. Lee,4 M.E. Hogg,4 N. Bahary,5 R.E. Brand,5 K. McGrath,5 A. Slivka,5 K.L. Cressman,1 K. Fuhrer,1 R.J. O'Sullivan.3 1Department of Pathology, University of Pittsburgh Medical Center, Pittsburgh, PA; 2Department of Pathology, Washington University, St. Louis, MO; 3Department of Pharmacology and Chemical Biology, University of Pittsburgh, Pittsburgh, PA; Departments of 4Surgery; and 5Medicine; University of Pittsburgh Medical Center, Pittsburgh, PA.
Background: Pancreatic neuroendocrine tuamors (PanNETs) are a heterogeneous group of neoplasms with increasing incidence and unpredictable behavior. Whole exome sequencing has identified recurrent mutations in the genes DAXX and ATRX, which correlate with loss of protein expression and alternative lengthening of telomeres (ALT). Both ALT and loss of DAXX/ATRX were initially reported to be associated with a favorable prognosis; however, recent studies suggest the contrary. Our aims were to assess the prevalence and prognostic significance of ALT and DAXX/ATRX in both primary and metastatic PanNETs.
Experimental Design: Telomere-specific fluorescence in situ hybridization and DAXX/ATRX immunohistochemistry were performed on a multi-institutional cohort of 321 patients with resected PanNET and 191 distant metastases from 52 patients. These results were correlated with clinicopathologic features including disease-free survival (DFS) and disease-specific survival (DSS).
Results: The prevalence of ALT and loss of DAXX/ATRX in resected PanNETs was 31% and 26%, respectively, and associated with larger tumor size, higher WHO grade, lymph node metastasis and distant metastasis (p < 0.001). The 5-year DFS and 10-year DSS of patients with ALT-positive and DAXX/ATRX-negative PanNETs were 40% and 50%, respectively, as compared to 96% and 89%, respectively, for wild-type PanNETs (p < 0.001). Among distant metastases, the prevalence of ALT and loss of DAXX/ATRX was 67% and 52%, respectively, and only occurred in the setting of an ALT-positive and DAXX/ATRX-negative primary PanNET. By multivariate analysis, ALT and DAXX/ATRX loss were negative, independent prognostic factors for DFS (p < 0.001).
Conclusions: ALT and loss of DAXX/ATRX in PanNETs were associated with shorter DFS and DSS. Based on an analysis of paired primary and metastatic PanNETs, ALT and loss of DAXX/ATRX are late events in the pathogenesis of PanNETs, occurring prior to the development of metastatic disease and likely play a significant role in driving distant metastases in patients with PanNETs.
Blocking DNA Damage Repair Improves Efficacy of Therapeutics in Pancreatic Cancer
S. Srinivasan,1 C. Shi,2 C. Roberts,1 M. Vansaun,1 N. Merchant,1 N.S. Nagathihalli.1 1Department of Surgery, University of Miami, Miami, FL; 2Vanderbilt University Medical Center, Nashville, TN.
Aim: To investigate whether blocking of DNA damage repair proteins (RAD51 and BRCA2) improves efficacy of therapeutics in pancreatic cancer (PDAC).
Background: The repair process involves DNA homologous recombination system that requires recombinase RAD51 and BRCA2, which co-localize to replication centers within the damaged cell nucleus. The defective DNA repair mechanisms in cancer cells can be exploited for therapy, when abrogated DNA double-strand breaks (DSB) repair. Targeted therapies are challenged by emergence of tumor cell resistance associated with downstream components of KRAS signaling pathways in PDAC. It is, therefore, essential to develop novel therapeutic strategies to overcome chemoresistance.
Methods: RAD51 expression was determined in human pancreatic tissues from a tissue microarray (TMA) and analyzed for overall survival (OS). Cell lysates from mouse cell lines derived from the LSL-KrasG12D/+;Pdx1Cre/+ (PanIN) and LSL-KrasG12D/+; Trp53R172H/+;Pdx1Cre/+ (PDA and LMP) genetic mouse models of PDAC were immunoblotted for RAD51 expression. In vitro cellular proliferation and tumorigencity were analyzed in PDAC cells treated with inhibitors for RAD51 (B02) and/or AKT (MK2206). In vivo (xenograft and mouse models) response to RAD51 and AKT inhibition were determined. In vitro sensitivity of BRCA2 and RAD51 deficient cells to PI3K/AKT therapy was determined.
Results: RAD51 expression confirmed a stepwise increase from normal pancreas to chronic pancreatitis through advancing grade and stage of PDAC. OS in patients with high RAD51 expression was significantly lower than those with low expression (15 vs 37 months respectively). RAD51 inhibition decreased cellular proliferation, tumorigenicity in vitro and tumor growth in vivo. BRCA2 and RAD51 deficiency increased PDAC cells sensitivity to AKT inhibition.
Conclusions: DNA damage repair deficiency increases PDAC cell sensitivity to AKT inhibition. RAD51 and BRCA2 expression levels in PDAC tumors may be useful in identification of PDAC patients who will benefit from this therapy.
Pancreatic Intraepithelial Neoplasia Alter Macrophage Populations to Mediate Pancreatic Tumorigenesis
P. Storz,1 G.-Y. Liou,1 B. Edenfield,1 L. Zhang,2 D. Dawson,3 N. Bardeesy.4 1Cancer Biology, Mayo Clinic, Jacksonville, FL; 2Laboratory Medicine & Pathology, Mayo Clinic, Rochester, MN; 3Pathology & Laboratory Medicine, UCLA, Los Angeles, CA; 4Department of Medicine, Harvard Medical School, Boston, MA.
The contributions of the innate immune system to the development of pancreatic cancer are still ill-defined. Inflammatory macrophages can initiate metaplasia of pancreatic acinar cells to a duct-like phenotype (ADM), which then give rise to pancreatic intraepithelial neoplesia (PanIN) when oncogenic KRas is present. However, it remains unclear when and how this inflammatory macrophage population is replaced by tumor-promoting macrophages. We here show that a stem cell-like cell type present in PanIN lesions named Tuft cells initiates such as switch in macrophage populations. We show that Tuft cells express interleukin-13 (IL-13), which can convert inflammatory into alternatively-activated macrophages. We further show that macrophages altered in their polarization by IL-13 release factors such as IL-1ra, CCL2 and TIMP1 to drive pancreatic fibrogenesis and tumorigenesis. Treatment of mice expressing oncoenic KRAs under an acinar cell-specific promoter with a neutralizing antibody for IL-13 significantly-decreased the accumulation of alternatively-activated macrophages at these lesions, resulting in decreased fibrosis and lesion growth.
Notch4 Acts as an Oncogenic Signal in Pancreatic Tumorigenesis
G.H. Su,5 W. Qiu,1 S. Chadi,1 N. Tsay,1 A.R. Chambers,1 D.D. Suh,1 P.A. Sims,2 C.J. Shawber,3 J. Kitajewski,4 H.E. Remotti.5 1Herbert Irving Comprehensive Cancer Center; Departments of 2Systems Biology; and 3Obstetrics and Gynecology; Columbia University, New York, NY; 4Department of Physiology & Biophysics, University of Illinois at Chicago, Chicago, IL; 5Pathology, Columbia University, New York, NY.
The Notch signaling network is an evolutionarily conserved intercellular signaling pathway which regulates interactions between adjacent cells. The Notch family receptor and its signaling pathways are involved in the development of numerous tissues in multicellular organisms. Recent studies from our Acvr1b flox/flox;LSL-Kras G12D ;Pdx1-Cre mice suggest that Notch4 plays an oncogenic role in dictating cell fate and contributes to pancreatic tumorigenesis, particularly in the development of pancreatic intraductal papillary mucious neoplasms (IPMNs). The perinuclear localization of the activated Notch4 (but not other Notch members) intracellular domain (ICD) in the apical cytoplasm of neoplastic cells appeared to be associated with the expansion of the IPMN lesions. The unique role of Notch4 signaling pathway in IPMN development was also confirmed by unbiased RNA-Seq analyses. The deletion of Notch4 in the p16 flox/flox;LSL-Kras G12D ;Pdx1-Cre mice prolonged survival, further supporting its oncogenic role in pancreatic tumorigenesis.
Impact of Postoperative Short-Term Outcomes on the Survival of Pancreatic Head Cancer
T. Sugiura, Y. Okamura, T. Ito, Y. Ito, R. Ashida, K. Uesaka. Shizuoka Cancer Center, Nagaizumi, Japan.
Objective: The aim of this study was to evaluate the influence of postoperative short-term outcomes on the survival in patients with pancreatic head cancer.
Patients and methods: A total of 232 patients undergoing surgical resection for pancreatic head cancer between 2007 and 2014 were enrolled in this study. Perioperative variables including postoperative complication and length of postoperative hospital stay were collected. Multivariate analysis was performed to identify the prognostic factors and evaluate the impact of postoperative short-term outcomes on long–term survival.
Results: Postoperative complications were occurred as follows; Clavien-Dindo grade I in 10 patients, grade II in 49, grade IIIa in 48, and grade IIIb/IV in 13, respectively. Median postoperative hospital stay was 20 days (9–189 days). Median survival time according to Clavien-Dindo grading was 24 months in grade 0, 21 months in grade I, 26 months in grade II, 23 months in grade IIIa, and 48 months in grade IIIb/IV, respectively. There were no significant differences between each group. As for the postoperative hospital stay, median survival time were 24 months in patients with hospital stay <6 weeks and 16 months in patients with hospital stay ≥6 weeks (p=0.023). Multivariate analysis revealed lymph node metastases (odds ratio (OR) =2.74, p<0.001), absence of adjuvant chemotherapy (OR=1.77, p=0.008), CA19-9 ≥300 U/ml (OR=1.63, p=0.005), age ≥70 years old (OR=1.52, p=0.022), and postoperative hospital stay ≥6 weeks (OR=1.75, p=0.022) as significant prognostic factors.
Conclusion: Clavien-Dindo grading did not influence the postoperative survival in patients with pancreatic head cancer. However, longer postoperative hospital stay adversely affected the patients’ survival.
TFF1 Might Inhibit Invasion but Accelerate Lymph Node Metastasis of Pancreatic Ductal Adenocarcinoma
M. Sunagawa, J. Yamaguchi, Y. Yokoyama, T. Kokuryo, M. Nagino. Surgical Oncology, Nagoya University Graduate School of Medicine, Nagoya, Japan.
Background: Trefoil Factor Family proteins (TFFs) are secreted from mucus-secreting cells of the gastrointestinal tract and seems to be the tumor suppressor gene in gastric carcinogenesis. The aim of this study is to investigate the expression status of TFF1 and their role in pancreatic ductal adenocarcinoma (PDAC).
Methods: We assessed the expression of TFF1 by immunohistochemistry in 79 surgically resected specimens of PDAC obtained at our institution between 2006 and 2013.
Results: TFF1 expression in the primary tumor can be detected in 74.3% (n=58/79) of PDAC specimens; however, TFF1 expression are lost or decreased at the invasive front of the tumor in 50% (n=29/58), suggesting that loss of TFF1 might be associated with invasive transformation of PDAC. While the expression status of TFF1 in primary tumor does not correlate with clinicopathological features including survival rate, the loss of TFF1 in invasive front has some impact: the cases with TFF1-positive invasion show significantly lower survival rate than the cases with TFF1-negative invasion (3-year DFS are 20.1% vs 38.5% respectively, p < 0.01; 3-year OS are 25.7% vs 49.1% respectively, p < 0.05), suggesting that TFF1-positive invasion is the risk factor for patient survival. In addition, the presence of lymph node metastasis are significantly higher in the cases of TFF1-positive invasion than TFF1-negative invasion (66.7% vs 33.3% respectively, p < 0.01), indicating that TFF1-positive invasion might result in the lymph node metastases.
Conclusion: While TFF1 is frequently lost or decreased in invasive front of PDAC, TFF1-positive invasion are associated with poor survival and high lymph node metastasis. These data suggest that TFF1 might inhibit invasion but accelerate lymph node metastasis of PDAC.
Carboxyl Ester Lipase Hybrid Gene and the Unfolded Protein Response: A Novel Trypsin Independent Model of Injury in Pancreatic Acinar Cells
W.M. Sunseri, G. Jones, X. Xiao, M.E. Lowe. Department of Pediatric Gastroenterology, Children's Hospital of Pittsburgh of the University of Pittsburgh Medical Center, Pittsburgh, PA.
Chronic pancreatitis (CP) is an inflammatory process causing irreversible damage to the pancreas. Gene mutations make up 67% of pediatric cases. Most known mutations are in the gene encoding trypsinogen or linked to the activation or degradation of trypsin causing CP by premature or unabated trypsin activation and auto-digestion of the gland. Recent association of alterations in the carboxyl ester lipase (CEL) gene with CP suggests a novel, trypsin-independent mechanisms. One CEL variant associated with CP in European case–control studies is a product of genomic recombination between CEL and its pseudogene (CEL-HYB). Little is known about the mechanism in which CEL-HYB increases risk for CP. We hypothesized that CEL-HYB protein is misfolded and accumulates intracellularly as aggregates triggering a maladaptive up-regulation of the ER stress and unfolded protein response causing inflammation and eventual CP. To investigate this theory, we studied the biochemical characterization of purified recombinant CEL-HYB and CEL WT. Then we studied the behavior of CEL-HYB protein compared to CEL WT by adenovirus transduction in AR42J cells, a rat pancreatic acinar cell line. AR42J cells were pretreated with dexamethasone to induce an acinar phenotype. First, the biochemical characterization of CEL-HYB and CEL WT showed comparable enzymatic properties. Second, in AR42J cells, less CEL-HYB was secreted in the medium and more CEL-HYB was retained intracellularly compared to CEL WT. A greater fraction of intracellular CEL-HYB was present as detergent-insoluble aggregates compared to CEL WT. There was no significant difference in BiP protein levels, BiP mRNA levels or XBP-1 mRNA splicing between CEL WT and CEL-HYB. In conclusion, CEL-HYB causes a reduction in CEL secretion and an increase in accumulation of insoluble protein aggregates compared to CEL WT in AR42J cells. There is no increase in the investigated cell signaling pathways in response to the accumulation of CEL-HYB. We plan to further investigate the cellular response under various forms of metabolic stress.
Progression of Acute Pancreatitis to Acute Recurrent Pancreatitis in the Pediatric Population: A Single Center Prospective Database Report
K.F. Sweeny, T.K. Lin, J.D. Nathan, J.J. Palermo, L. Hornung, T. Thompson, M. Abu-El-Haija. Cincinnati Children's Hospital Medical Center, Cincinnati, OH.
Introduction: Acute Pancreatitis (AP) in the pediatric population remains poorly understood with a rising incidence to a rate of 1/10,000. Research is lacking on AP progression to Acute Recurrent Pancreatitis (ARP). We aimed to study AP progression from the first episode to ARP using our comprehensive prospective database.
Methods: Eighty-five AP patients were enrolled in the registry since 2013, a total of 83 patients had a minimum of 3 months follow up. Physician surveys were entered in REDCap (Research Electronic Data Capture).
Results: 17 of the 85 (20%) AP patients have ARP as of April 2016. Risk factors for the first AP attack were: toxic/drug (19%), viral/systemic (18%), gallstone/biliary (18%), and trauma (9%). Eleven of the 17 (65%) patients with ARP had their second attack within 3 months of the first attack. To identify factors associated with rapid recurrence, patients who progressed to ARP within 3 months (n=11) were compared to patients who had only a single attack with at least 3 months of follow-up (n=72). A univariate analysis of clinical and laboratory characteristics between the two groups showed that the weight percentile for age was significantly higher in the rapid progression group (p=0.03). The presence of pancreatic necrosis during the first AP attack significantly differed between the two groups, 2/11 cases (18%) in the rapid progression versus no cases in the no progression group (p=0.02).
Conclusions: Through a prospective database we can further study outcomes of pediatric AP and progression to ARP. A higher weight percentile for age and pancreatic necrosis during the first AP attack were associated with a rapid progression to ARP within 3 months. Future studies are needed to better understand factors that lead to pediatric ARP progression.
Novel Methods for the Measurement of Blood Flow in Experimental Acute Pancreatitis
P. Szatmary,1 A. Taylor,1 T. Leather,1 H. Poptani,1 D. Criddle,1 A. Tepikin,1 R. Sutton.2 1Cellular and Molecular Physiology, University of Liverpool, Liverpool, United Kingdom; 2NIHR Liverpool Pancreas Biomedical Research Unit, Royal Liverpool University Hospital, University of Liverpool, Liverpool, United Kingdom.
Background: An aim of drug development for acute pancreatitis (AP) is maintenance of pancreatic blood flow, loss of which contributes to necrosis, but blood flow assessment is not routine. We have developed a novel method to assess murine pancreatic blood flow in experimental (E)AP.
Methods: Assessments of blood flow were undertaken in naïve and pancreatitis CD1 mice (caerulein, 50 mg/kg ip hourly x12) 1 hour after induction by: 1) aortic arch injection of 15 mm dia fluorescent microspheres then pancreatectomy, tissue lysis and spectro-photometry; 2) post-mortem arterial pancreas perfusion with 1 mm dia fluorescent microspheres, pancreatectomy and epifluorescent analysis; 3) post-mortem arterial pancreas perfusion with Gd-DTPA and iron oxide nanoparticles, pancreatectomy and magnetic resonance imaging. Tissues were then fixed, embedded and sectioned for histological comparison.
Results: Slow injection of large fluorescent microspheres was effective in delivery to the abdominal organs. Concurrent measurement of femoral arterial blood allowed estimation of total blood flow to the pancreas at 1.2 ml/minute. Following induction of AP, pancreatic blood flow dropped to 43% of normal (p=0.0002). Perfusion with small microspheres similarly revealed less fluorescence in the inflamed pancreas, and patchy organ perfusion. Using MRI, good contrast could be achieved with both Gd-DTPA (T1 weighting) and iron oxide nanoparticles (T2 weighting), characterizing vasculature to 0.2 mm dia.
Conclusions: Disruption of microvascular blood flow is critical in the pathogenesis of EAP. We present novel methods for its targeted assessment in EAP that could be adapted for clinical trial use.
Effect of Heparins on Histone Catabolism and Cellular Injury in Acute Pancreatitis
P. Szatmary,1 T. Liu,1 D. Criddle,1 A. Tepikin,1 R. Sutton.2 1Cellular and Molecular Physiology, University of Liverpool, Liverpool, United Kingdom; 2NIHR Liverpool Pancreas Biomedical Research Unit, Royal Liverpool University Hospital, University of Liverpool, Liverpool, United Kingdom.
Background & Aim: Heparins have negative surface charges that may ameliorate acute pancreatitis (AP) by neutralisation of the positive charge-dependent effects of histones on pancreatic necrosis and distant organ failure. We have tested the impact of a range of heparins on experimental (E)AP.
Methods: Standard, clinical grade unfractionated (UFH), low molecular weight (tinzaparin, LMWH) and O-desulfated, non-anti-coagulant (ODSH) heparins were tested in isolated murine pancreatic acinar cells (PAC, propidium iodide readout) exposed to histones and in experimental AP induced by either 4 or 12 hourly cerulein (50 mg/kg) intraperitoneal injections in CD1 mice. EAP was assessed by biochemistry, immunology and histopathology.
Results: All three heparins rescued PAC from histone-induced cell death. 25mg/kg ODSH reduced amylase levels to near control in mild EAP, indicating the effect is independent of anticoagulant properties of heparin. The same dose in severe EAP, however, dramatically increased plasma amylase, IL-6 and histone H3 beyond cerulein alone, but had no effect on the saline control. Pancreatic trypsin and pancreatic and lung myeloperoxidase were similarly elevated. This detrimental effect was not seen with 10mg/kg ODSH, UFH or LMWH. Serial quantification of recombinant histone H3 spiked into murine plasma revealed histone H3 is degraded at room temperature in <10 min. Addition of UFH delays degradation by >30 min, and ODSH by >60 min.
Conclusions: ODSH paradoxically increases the concentration of extracellular histones at high concentrations and contributes to severity of AP in mice, likely through inhibition of histone degradation. These data suggest that careful dosing would be required were heparin to be used in clinical AP.
Haploinsufficiency of Beclin1 Inhibits PanIN Development in a KrasG12D Mouse Model of Pancreatic Tumorigenesis
K. Takakura,1,2 E. Mascarinas,3 B. Decant,3 D. Dawson,4 G. Eibl,5 A. Gukovskaya,1,2 P. Grippo.3 1Department of Medicine, David Geffen School of Medicine at UCLA, Los Angeles, CA; 2VA Greater Los Angeles Healthcare System, Los Angeles, CA; 3Department of Medicine,University of Illinois-Chicago, Chicago, IL; Departments of 4Pathology and Laboratory Medicine; and 5Surgery, David Geffen School of Medicine at UCLA, Los Angeles, CA.
Background & Aims: The function of autophagy in pancreatic neoplasia is controversial, with reports supporting both tumor promoting and suppressing roles. Here, we evaluate the role of Beclin1, a key autophagy mediator, in pancreatic intraepithelial neoplasia (PanIN) development in an oncogenic KrasG12D (KC) mouse model.
Methods: We generated KC transgenic mice heterozygous for Beclin1, termed KCB, by crossing Pdx1-Cre/LSL-Kras with Beclin+/− mice (Beclin1 null mice die in utero). We examined the effects of Beclin1 haploinsufficiency on PanIN progression and the underlying pathways. Using tissue microarray, we evaluated the levels of Beclin1 and LC3-II in 150 pancreatic cancer patients.
Results: Compared with wild type, the expression of oncogenic Kras increased the number of autophagic vacuoles in PanINs by 3.5±0.1 fold (n=6). Beclin1 haploinsufficiency inhibited autophagy in KC mice, manifest by markedly decreased LC3-II. We found a clear distinction between KC and KCB mice, revealing a pronounced inhibitory effect of Beclin1 haploinsufficiency on PanIN development. By 5–6 months of age, more than half of the pancreatic parenchyma of KC mice was disturbed with 30-60+ PanIN lesions at all grades and neighboring acinar-to-ductal metaplasia (ADM). In contrast, KCB mice displayed mostly normal parenchyma, with 10–15 small predominantly grade1 PanIN lesions and a few areas of ADM. Beclin1 haploinsufficiency significantly decreased cell proliferation, 2.5 fold increased active caspase-3, decreased levels of both phosphorylated Akt and phosphorylated ERK. We found that the expression for LC3 and Beclin1 increased in human pancreatic tumor compared to normal ducts. However, both markers showed no statistically significant effect in predicting survival.
Conclusion: Beclin1-mediated autophagy plays a pro-tumorigenic role, facilitating PanIN progression in the KC model; pharmacologic/molecular approaches to inactivate Beclin1 could be beneficial in pancreatic cancer treatment.
Pancreas Divisum is Associated With a Higher Risk of Recurrent Acute Pancreatitis Only in the Presence of PRSS1 and Cathepsin B Polymorphisms
R. Talukdar,1,2 M. Aslam,1 S. Avanthi,2 V.V. Ravikanth,2 B. Govardhan,2 N. Zaheer,1 D.N. Reddy.1 1Medical Gastroenterology, Asian Institute of Gastroenterology, Hyderabad, India; 2Basic Sciences Division, Asian Healthcare Foundation, Hyderabad, India.
Aim: Relationship between pancreas divisum (PD) and pancreatitis is not clear. We earlier reported SPINK1, CTRC, PRSS1(5’UTR), CTSB and Claudin2 polymorphisms in idiopathic recurrent acute(IRAP) and chronic(ICP) pancreatitis. Now we evaluate association of PD with IRAP/ICP in presence of these polymorphisms.
Methods: We enrolled 116 IRAP, 228 ICP, 244 matched controls from May-December 2015. IRAP & ICP patients were divided into those with/without PD (confirmed with MRCP/ERCP). We recorded demographic & disease characteristics; & genotyped SPINK1(N34S), PRSS1(5’UTR), CTRC(180C>T), CTSB(L26V) & CLDN2 genes. We compared clinical data between patients with/without PD using Mann–Whitney U and Chi Square/Fischer’s Exact test. We expressed association of gene polymorphisms with IRAP/ICP as odd’s ratio (OR) with 95%CI. Bonferroni corrected two-tailed ‘p’ value of ≤0.05 was considered statistically significant.
Results: 25 & 70 patients with IRAP & ICP respectively had PD. While mean age (yrs) of symptom onset was similar in both IRAP & ICP patients with & without PD, patients without PD in the ICP category presented earlier [age(yrs) 18.4(10.3)v/s21.8(12.6);p=0.03]. Other demographic/disease characteristics were similar, except for family history, which was higher among patients without PD [IRAP:0(0%) v/s 18(15.5%); p=0.02 & ICP 5(2.2) v/s 42(18.4); p=0.003].
Genotyping revealed significant association of PRSS1(5’UTR) and CTSB(L26V) with IRAP [OR(95%CI) 3.2(1.1-9.3); p=0.03 & 3.9(1.3-11.3); p=0.01 respectively] among patients with PD. No association was observed in ICP. Presence of combination of PRSS1 and CTSB polymorphism increased the OR (95%CI) of IRAP in the presence of PD to 4.01(1.6-10.3)(p=0.004).
Conclusion: PD is associated with increased risk of IRAP in presence of PRSS1(5’UTR) and CTSB(L26V) polymorphisms.
Altered Gut Microbiota in Patients With Chronic Pancreatitis is Associated With Endocrine Dysfunction
R. Talukdar, S.M. Jandhyala, M. Arutla, D. Govardhan, D.N. Reddy. Medical Gastroenterology and Basic Sciences, Asian Institute of Gastroenterology, Hyderabad, India.
Aim: We aimed to evaluate the intestinal microbiota & functional alterations in CP.
Methods: 8 controls, 16 CP without & 14 with diabetes were enrolled. Disease morphology, glycemic & nutritional (clinical, biochemical) parameters were recorded. PEI (fecal elastase) & plasma endotoxin (LAL) was quantified.
V3-V4 sequencing of 16SrDNA amplicon was performed on Illumina MiSeq. Diversity indices & functional abundances (KEGG) were evaluated on MG RAST platform. Principal component analyses (PCA) with ANOSIM (10,000 Monte Carlo simulation) & hierarchical clustering (Euclidean similarity matrix) was performed in the PAST package. Differences in relative abundances of taxa & functional pathways were evaluated with Kruskall-Wallis test. Pearson & Spearman coefficients were used for correlation between disease characteristics & bacterial/functional abundances. Bonferroni corrected ‘p’value of ≤0.05 indicated statistically significant.
Results: PCA revealed distinct clustering of the groups at species level. There was reduced richness (Chao1) & alpha-diversity (Shannon index) in CP patients with diabetes. There was significant progressive reduction in abundance of Faecalibacterium prausnitzii (p=0.03) in CP without & with diabetes. There was significant progressive increase in the LPS synthetic pathways (p=0.03) from controls to CP without & with diabetes, with a corresponding increase in plasma endotoxin level (EU/ml)[0.30(±0.05), 0.51(±0.11), 1.11(±0.21) respectively (p<0.001). Plasma endotoxin levels correlated positively with blood glucose (r2=0.7;p<0.001) & negatively with Faecalibacterium prausnitzii abundance (r2=0.2;p=0.01)
Conclusion: Reduced Fecalibacterium prausnitzii results in mucosal barrier dysfunction and endotoxemia that could contribute to diabetes in patients with CP.
Contribution of Activating Transcription Factor 3 to Development of Acinar-To-Ductal Cell Metaplasia
J. Toma,1 C. Young,1 K. Berger,1 C. Pin.1,2 1Physiology and Pharmacology, University of Western Ontario, Children's Health Research Institute, University of Western Ontario, London, Canada; 2Paediatrics, University of Western Ontario, London, Canada.
Pancreatic ductal adenocarcinoma (PDAC) is the 4th leading cause of cancer death in Canada with 5-year survival rates <5%. The highest risk factor for PDAC is recurrent pancreatitis, a debilitating disease defined by inflammation and fibrosis of the exocrine pancreas. While the link between PDAC and pancreatitis are unknown, de-differentiation of acinar cells is common to both diseases. Our lab has shown that Activating Transcription Factor 3 (ATF3), a factor upregulated during pancreatic injury, contributes to the development of acinar-to-ductal cell metaplasia (ADM), a precursor phenotype of PDAC. The goal of this study was to identify how ATF3 contributes to ADM. We hypothesized that ATF3 transcriptionally targets genes and pathways that affect acinar cell differentiation. To address this hypothesis, acinar cells were isolated from Atf3 −/− and wild type (WT) mice, and treated pharmacologically with agents that promote (e.g. TGFα) or inhibit (e.g. rottlerin) ADM. In addition, the expression of Atf3 and its putative targets were assessed in genetic mouse and acinar culture models exhibiting various levels of ADM. Consistent with the requirement of ATF3 for ADM, we observed decreased ADM in Atf3 −/− acinar cultures along with changes in gene expression suggesting ATF3 targets genes involved in the differentiation process. These results suggest that ATF3 is required for ADM. Further studies are aimed at elucidating the whether ATF3 affects oncogenic KRAS’ ability to promote PDAC. Identifying these mechanisms are important in defining early markers for PDAC in a susceptible population.
Prevalence of Deep Vein Thrombosis (DVT) and Pulmonary Embolism (PE) in Hospitalized Acute Pancreatitis (AP) Patients: A Population Based Cohort Study
G. Trikudanathan,1 C. Umapathy,2 S. Munigala,3 D.L. Conwell,4 S.G. Krishna.4,5. 1Medicine, GI, University of Minnesota, Minneapolis, MN; 2Internal Medicine, University of Pittsburgh, PA. 3Saint Louis University Center for Outcomes Research (SLUCOR), St. Louis MO; 4Ohio State University Medical Center, Columbus, OH; 5Ohio State University-Wexner Medical Center, Columbus, OH.
Background and aims: In AP, prolonged hospitalization and the pro-inflammatory cascade serve as a milieu for the development of venous thromboembolism (VTE). Our aim was to estimate the prevalence of DVT/PE (VTE) in hospitalized patients with AP and to ascertain its impact on morbidity and mortality.
Methods: The Nationwide Inpatient Sample (2002–2011) was reviewed to identify all adult (≥18 years) inpatient discharges with a primary diagnosis of AP and a concomitant diagnosis of VTE. The primary clinical outcomes [mortality, acute kidney injury (AKI) and respiratory failure] and secondary resources outcomes (length of stay and total hospital charges) were analyzed using univariate and multivariate comparisons.
Results: Among 2,453,997 discharges with AP, 23,614 (1%) were associated with VTE. Multivariate analysis showed that VTE in AP was associated with whites (OR 1.12), Medicare insurance (OR 1.07), urban hospitals (non-teaching OR 1.61, teaching OR 2.01), medium and large hospitals (OR 1.21 and 1.44), higher comorbid conditions [AHRQ-Elixhauser index > 3] (OR 1.63), AKI (OR 1.74), acute respiratory failure (OR 4.45), and pseudocyst (OR 2.88). Multivariate analysis confirmed that VTE was independently associated with higher mortality (OR 1.39), prolonged hospitalization (9.1 days, p<0.001) and higher hospital cost ($60,585, p<0.001).
Conclusion: Presence of VTE portends poor clinical outcome in AP patients resulting in more resource utilization. VTE prophylaxis and ambulation should be strongly encouraged in AP inpatients.
Portal Vein Thrombosis (PVT) in Acute Pancreatitis (AP) is Associated With Poor Clinical Outcomes and Increased Resource Utilization: A Population Based Cohort Study
G. Trikudanathan,1 S. Munigala,2 M. Arain,1 K.C. Kottapalli,3 R. Attam,1 S. Amateau,1 S. Mallery,1 M.L. Freeman.1 1Medicine, GI, University of Minnesota, Minneapolis, MN; 2Saint Louis University Center for Outcomes Research (SLUCOR), St. Louis, MO; 3Internal Medicine, Wheaton Hospital, Milwaukee, WI.
Aim: Portal vein thrombosis (PVT) may adversely influence the clinical course of AP; however there are no population-based studies assessing its impact in AP. We evaluated the prevalence and clinical outcomes of PVT in AP by a propensity matched analysis.
Methods: The national inpatient sample (1998–2012) was reviewed to identify all pts hospitalized with AP with a concomitant diagnosis of PVT. The primary clinical outcome [mortality, acute kidney injury (AKI), respiratory failure and pseudocyst] and secondary resources outcomes [length of stay (LOS) and total hospital cost] were analyzed using univariate and multivariate comparisons. Propensity score analysis (matched for patient demographics, hospital characteristics, etiology, and AHRQ-Elixhauser comorbidities) was performed to compare the outcomes in pts with and without PVT.
Results: Among 3454400 pts with AP, 5681 (0.2%) were associated with PVT with a trend towards increased frequency over the years. Multivariate analysis showed PVT is associated with increased mortality [odds ratio (OR): 1.3, p=0.023], AKI (OR 1.5, p<0.001), respiratory failure (OR: 3.1, p<0.001), prolonged LOS (median 5.4 days, p<0.001) and higher hospital cost (median: $36,509, p<0.001). Propensity matched analysis confirmed that PVT is associated with AKI (OR 1.8; p=0.002), respiratory failure (OR: 2.9; p<0.001), pseudocyst formation (OR: 4.5; p<0.001), increased LOS (median: 5.5 days, p<0.001) and higher hospital cost (median: $38,123, p<0.001).
Conclusions: PVT is associated with adverse clinical outcomes like organ failure, prolonged LOS and increased hospital cost which negatively impacts inpatient healthcare utilization.
Magnetic Resonance Cholangiopancreatography in Assessing Pancreatic Function Testing in Pediatrics
A.T. Trout,1 D.B. Wallihan,2 M. Abu-El-Haija.1 1Cincinnati Children's Hospital Medical Center, Cincinnati, OH; 2Charlotte Radiology, Charlotte, OH.
Introduction: Secretin-enhanced magnetic resonance cholangiopancreatography (S-MRCP) may provide a non-invasive way to assess pancreatic exocrine function. The purpose of this study was to assess the accuracy and interrater reproducibility of measurements of enteric fluid volume on S-MRCP.
Methods: Accuracy: Phantoms with varying, known fluid volume (47–206 ml) were imaged using the clinical quantification sequence (see below). Fluid volume was measured by segmenting images to select only regions of fluid signal (ImageJ), measuring the area occupied by fluid and multiplying by slice thickness. Reproducibility: Pediatric patients with suspected pancreatic disease underwent S-MRCP with coronal fat-saturated T2-weighted single shot fast spin echo sequences acquired pre- and post-secretin injection (0.2 mcg/kg, max 16 mcg). Secreted fluid volume (post minus pre-secretin) was independently measured by two blinded reviewers using the same technique as the phantom measurements. Measured volumes were compared using paired t-tests and Spearman’s correlation with bias calculated by Bland-Altman analysis.
Results: Phantom fluid volumes were accurately measured by MRCP with absolute percent errors ranging from 0.03% to 9.0%. Nineteen patients (mean age 13.2 y, range: 0.6-25.3y) were included in this study. Measured secreted fluid volumes were highly correlated between reviewers (r=0.93). Mean secreted volumes measured by reviewers 1 and 2 were 99.6 and 93.9 mL (p=0.23) with a bias between measurements of 5.7 mL (95%CI:-40-51.5 mL).
Conclusions: Measurement of fluid volume by MRI is highly accurate with less than 10% absolute error in measured volume. Measurements of secreted fluid volume on S-MRCP are highly reproducible with a bias of less than 10mL between reviewers.
Obesity as a Risk Factor in Pediatric Acute Recurrent and Chronic Pancreatitis
A. Uc,1 M. Abu-El-Haija,2 B. Barth,3 M. Bellin,4 D. Fishman,5 S. Freedman,6 C. Gariepy,7 M. Giefer,8 T. Gonska,9 M. Heyman,10 R. Himes,5 S. Husain,11 T.K. Lin,2 Q. Liu,12 V. Morinville,13 J.D. Nathan,2 C.Y. Ooi,14 J.J. Palermo,2 E. Perito,10 J. Pohl,15 S. Rhee,10 S.J. Schwarzenberg,4 D. Troendle,3 S. Werlin,16 M. Wilschanski,17 B. Zimmerman,1 M.E. Lowe.11. 1University of Iowa, Iowa City, IA; 2Cincinnati Children's Hospital Medical Center, Cincinnati, OH; 3UTSW, Dallas, TX; 4Pediatrics, University of Minnesota, Minneapolis, MN; 5Baylor College of Medicine, Houston, TX; 6Harvard Medical School, Boston, MA; 7Nationwide Children’s Hospital, Columbus, OH; 8Seattle Children's Hospital, Seattle, WA; 9Hospital for Sick Children, Toronto, Canada; 10UCSF, San Francisco, CA; 11Children's Hospital of Pittsburgh of University of Pittsburgh Medical Center, Pittsburgh, PA; 12Children's Hospital Los Angeles, Los Angeles, CA; 13Montreal Children’s Hospital, Montreal, Canada; 14UNSW, Kensington, Australia; 15University of Utah, Salt Lake City, UT; 16MCW, Milwaukee, WI; 17Hadassah Hebrew University Hospital, Jerusalem, Israel.
Introduction: Obesity is associated with a heightened inflammatory response in acute pancreatitis (AP). It is not known whether obesity is a risk factor in acute recurrent pancreatitis (ARP) and chronic pancreatitis (CP).
Objective: To study the impact of obesity on pediatric ARP and CP in the well-phenotyped INSPPIRE (INternational Study group of Pediatric Pancreatitis: In search for a cuRE) cohort.
Methods: 373 children with ARP (n=186) or CP (n=187) ≤19 y/o were enrolled at 16 pediatric centers. CDC Growth Charts and pediatric age- and gender-specific BMI were used. Seventeen children (4.5%) were underweight (<5th percentile), 221 (66%) of normal weight (5th-<85th), 57 (15%) overweight (85th-<95th) and 78 (23%) obese (≥95th). Categorical variables and associations were analyzed using Cochran-Armitage trend test and Jonckheere-Terpstra test.
Results: The groups were not different in gender, race, age at presentation of AP or CP, time from diagnosis of AP to CP, frequency or pattern of abdominal pain, school attendance, emergency room visits or hospitalizations. Obese children were more likely to be of Hispanic ethnicity (p=0.004) and less likely to have CP (p=0.028), with imaging studies showing acute inflammatory changes (p<0.0001) rather than chronic duct damage (p=0.038). Genetic, obstructive and toxic metabolic risk factors were distributed equally among the groups, except SPINK1 mutations were less common and hypertriglyceridemia was found exclusively in overweight and obese (p=0.013 and p=0.021). Obese children were less likely to undergo medical or endoscopic interventions and total pancreatectomy and islet autotransplantation compared to others (p=0.012, 0.022 and 0.012). Finally, overweight and obese children were less likely to be exocrine pancreatic insufficient (p=0.004).
Conclusion: Obese children with recurrent pancreatitis have a proinflammatory rather than a profibrotic phenotype. The impact of obesity as a risk factor on pancreatic disease progression and severity needs to be further investigated.
Differentiating Branch Duct IPMN From Mixed Duct IPMN: Test Characteristics of Preoperative Imaging Modalities
E.E. Ugbarugba, C. Grieco, B. Swanson, P. Hart, S. El-Dika, J. Walker, S. McCarthy, A. Manilchuk, M. Dillhoff, C. Schmidt, D.L. Conwell, S.G. Krishna. Ohio State University-Wexner Medical Center, Columbus, OH.
Background: Follow up guidelines for mixed-duct IPMN (IPMN-mixed) is different from branch duct IPMN (IPMN-BD) due to a higher risk of progression to pancreatic cancer. There is limited literature on test characteristic of imaging modalities in differentiating IPMN-BD from IPMN–mixed.
Objective: To determine pre-operative imaging and EUS characteristics in differentiating IPMN-BD from IPMN-Mixed.
Methods: A retrospective review of the pathology database of pancreatic resections at a single tertiary academic center was performed from 2000–2013. The accuracy of preoperative imaging for predicting main duct involvement in IPMN-BD was compared to histology (the “gold standard”). A standard 2 x 2 contingency table was constructed for each imaging modality and used to calculate their test characteristics.
Results: 83 subjects with IPMN-BD (n: 60, 72%) or IPMN-mixed (n: 23, 28%) were identified.
IPMN-mixed were larger than IPMN-BD (mean±SD 4.14±2.9 mm vs. 2.74±1.9 mm; p=0.03) with more frequent high-grade dysplasia/adenocarcinoma (50% vs. 20%, p=0.003).
As a single modality for the detection of IPMN-mixed, EUS (sensitivity 80%; specificity 78%; accuracy 79%) had the best diagnostic performance followed by MRI (sensitivity 83%, specificity 63%, accuracy 68%). Combined EUS and MRI had a sensitivity of 100%, but specificity and accuracy were 64% and 67%, respectively.
The optimal cyst size to predict IPMN-mixed based on ROC curve analysis was 3 cm. Utilizing cyst size as a predictor of IPMN-mixed, diameter > 3 cm reached a sensitivity, specificity, and accuracy of 75%, 72%, and 73%. Combining EUS, MRI, and cyst size (>3 cm), the sensitivity, specificity, and accuracy reached 77.8%, 70.6% and 73.7% respectively.
Conclusion: The combination of MRI and EUS, achieves a high sensitivity for detection of main duct involvement in IPMNs that otherwise would be assumed to have pure branch duct involvement. This combination should be considered prior to enrolling patients in a surveillance only strategy.
Potential Targets and Role of Ezh2 in Pancreatic Cancer
S. Urayama, A. Habib, W. Pan, N. Alzofon, S. Wang. Internal Medicine, University of California, Davis, Davis, CA.
Background: Pancreatic ductal adenocarcinoma (PDAC) is known for its early development of metastasis and poor patient prognosis. Prior results show that PDACs demonstrate increased expression of Ezh2, and potentially involved in invasiveness and chemoresistance characteristics. In order to delineate the potential targets of Ezh2, we have investigated the effect of Ezh2 gene expression on several of the previously reported genes regulated by Ezh2 (ADRB2, BMPR1B, Dkk1, RAD51, INK/ARF, VASH1 and DAB2IP). In this study, we have investigated the possible connection between Ezh2 expression and the effect on these gene and protein expressions in PDAC.
Method: Based on previous assays of Ezh2 protein expression in PDAC cells, we have chosen two cell lines: BxPC-3, with low constitutive Ezh2 expression, and MiaPaCa-2, with high constitutive Ezh2 expression. We have constructed Ezh2 knockdown (shRNA) and overexpression lentiviral vectors. Each vector construct as well as control was transduced into target cells using a lentiviral kit. After selection processes, total RNAs were harvested from each cells and the gene expression levels were assayed by RT-qPCR. The protein expressions were assayed with Western blot. Global effects of Ezh2 expression on epithelial-to-mesenchymal transition (EMT) was assayed with protein expression levels of E-cadherin and N-cadherin in respective cells.
Results: Ectopic expression of Ezh2 induced N-cadherin expression and decreased E-cadherin expression. Knock-down of Ezh2 decreased N-cadherin expression. Of the 7 different genes analyzed, 4 genes (ADRB2, Dkk1, INK4A/ARF, and RAD51) demonstrated inverse mRNA expression levels with the reversed Ezh2 expression levels. For the protein expression, RAD51 and Dkk1 showed corresponding changes with the varying Ezh2 expression.
Conclusion: Our data indicates that Ezh2 affects EMT. Ezh2 plays more direct role in regulating RAD51 and Dkk1 expression, while other factors may be at play in Ezh2’s mediation of INK/ARF and DAB2IP. Further studies are in progress.
Characteristics and Long-Term Survival of Resected Pancreatic Cystic Neoplasms (PCN) in Finland 2000–2008: The First Nationwide Study
Y. Vaalavuo, A. Antila, R. Ahola, A. Siiki, M. Vornanen, J. Sand, J. Laukkarinen. Department of Gastroenterology and Alimentary Tract Surgery, Tampere University Hospital, Tampere, Finland.
Introduction: In the 2000s, most PCNs were still diagnosed and operated on due to symptoms. The aim of this study was to determine the nationwide characteristics of these resected PCNs and long-term survival in Finland.
Patients and methods: All PCNs operated [ZERO WIDTH SPACE][ZERO WIDTH SPACE]in Finland 2000–2008 were identified by combining data from national operation register and patient archives. Survival was registered in 5/2013. Median follow-up time was
Results: Out of the 2042 pancreatic resections performed, 225 (11.0%) involved a PCN, and all these patients (age 55 (14–87) years, 31% men) were included in the study. 68% were symptomatic. The median PCN size in imaging was 51 (8–220) mm. A distal pancreatic resection was performed in 59%. In final histopathology, there were 22.2% MD-IPMN (main-duct), 13.3% Mx-IPMN (mixed-type), 6.2% BD-IPMN (branch-duct), 17.8% MCN (mucinous cystic neoplasm), 3.6% SPN (solid pseudopapillary neoplasm), and 18.2% SCN (serous cystic neoplasm). Overall, 24.8% were malignant (MD-IPMN 60%, Mx-IPMN 33%, MCN 33%) and 6.2% with high-grade dysplasia (HGD; MD-IPMN 12%, Mx-IPMN 10% and SPN 25%) All SCNs were benign. Out of the symptomatic patients, 28% had malignant and 6% HGD tumor, compared to 9.4% and 7.8% in the asymptomatic patients. In patients with operated malignant PCN, 5-year survival was 34% and median survival 2.79 (0–10.25) years. In patients with operated benign PCN, mortality to pancreatic cancer was 3/173 during the follow-up time.
Conclusions: In this first nationwide study from Finland, 31% of the operated PCNs were malignant or HGD. The median survival after operated malignant PCN was 2.79 years. The proportion of asymptomatic and premalignant operated PCN should increase in the future due to the current follow-up and treatment recommendations.
Predicting Pancreatitis Phenotype in Three Siblings Based on a Shared Genotype
D. Vitale, M. Abu-El-Haija, T.K. Lin. Gastroenterology, Cincinnati Children's Hospital Medical Center, Cincinnati, OH.
Background: A primary risk factor for the development of acute recurrent pancreatitis (ARP) and chronic pancreatitis (CP) in children is genetic mutations. SPINK1 mutations have been identified in up to 2% of the general population with a minority of those that will develop ARP/CP.
We describe three siblings with nearly identical manifestations of early onset ARP, all with the same SPINK1 mutation.
Case presentation: Pancreatitis began at age 9 in the 12-year-old male sibling. A subsequent episode was complicated by a pseudocyst within the head of the pancreas (HOP) that spontaneously resolved. His 9-year-old identical twin sisters both presented with a chronic history of mild, intermittent abdominal pain. Pain severity peaked at age 7 and 8 years, respectively, corresponding to their first documented occurrence of pancreatitis. Both developed an HOP pseudocyst. One twin underwent endoscopic ultrasound with cystduodenostomy drainage and stent placement. Exocrine pancreatic insufficiency was diagnosed by direct pancreatic function testing. The second twin has had ongoing abdominal pain with a stable yet persistent 4 cm pancreatic pseudocyst. Hereditary pancreatitis testing in each sibling identified the same heterozygous SPINK1 mutations (c.101A>G, c.55+1G>A).
Discussion: We report three pediatric-aged siblings presenting with ARP, all possessing the same SPINK1 mutation with all developing the complication of a pancreatic pseudocyst in the HOP. The mirrored phenotypic disease manifestation among the siblings including their age of onset and complication of pseudocyst formation is remarkably notable. More research is needed related to inherited genetic mutations and the factors that influence individual phenotypic manifestations in children with pancreatic disorders.
Human Primary Acinoductal Cell Culture as a Model of ADM and Epithelial-Mesenchymal Interactions
R.T. Waldron, O. Shauly, H.-Y. Su, H. Hurley, A. Lugea, S.J. Pandol. Cedars-Sinai Medical Center, Los Angeles, CA.
In pancreatic ductal adenocarcinoma, wound healing responses such as acinar-ductal metaplasia (ADM), epithelial-mesenchymal transition (EMT), and myofibroblast activation get hijacked and serve cells with abnormally high survival and proliferation. Differentiated/low grade tumors have discrete ductal and stromal areas, whereas high grade and metastatic capacity are associated with mesenchymal cell type. Pancreatic acini predominate upon removal of islets from human pancreas. We found that culturing human acini at low density in 10% FBS DMEM/F12 media initiated dramatic and highly reproducible phenotypic plasticity. Acinar cell type specific differentiation was unstable, and was rapidly (in 2–4 days) replaced by highly proliferative ductal type cells. In turn, ductal cells on tissue culture dishes were unstable to passage, and underwent EMT accelerated by harvesting/trypsinization and replating. These cultures also had mesenchymal type cells that expanded in parallel to ADM/EMT. We exploited this system as a model to correlate distinct phases (acini-ADM-EMT-myofibroblast expansion), identified via marker gene expression, with the dynamic emergence of transcription factors, ligands and receptors that mediate distinct transitions and intercellular communication by qPCR. For example, EMT corresponded closely with abrupt rises in TGFβ, CTGF, and the Notch target gene Hes-1, and with loss of TGFα that had escalated during ADM, while Mbnl1, MrtfA and Yap escalated and sustained. We also detected gene expression of the fusogenic protein, syncytin-1 and its fusion receptor, the amino acid transporter SLC1A4/ASCT1, suggesting cell-cell fusion. In sum, we have derived a novel system for genetic analysis of dynamic phenotypic transitions to provide insight into pancreatic ADM/EMT.
Statin Use Shows Increased Overall Survival in Patients Diagnosed With Pancreatic Cancer: A Meta-Analysis
D. Wang,1 E. Rodriguez,1 E. Donath,1 J. Barkin,2 A. Pakravan.1 1Department of Medicine, University of Miami Miller School of Medicine, Atlantis, FL; 2Department of Gastroentrology, University of Miami Miller School of Medicine, University of Miami Pancreas Center, Miami, FL.
Introduction: Pancreatic ductal adenocarcinoma is currently the fourth leading cause of all cancer related death in the United States. The prognosis for patients diagnosed with pancreatic cancer remain poor with estimated 5 year survival rate of 7.2%. It has been proposed that statins may inhibit tumor growth by stimulating apoptosis, inhibiting angiogenesis, and suppressing metastatic invasion. The aim of this meta-analysis was to examine if the use of statin therapy leads to increase overall survival in patients diagnosed with pancreatic cancer.
Methods: A comprehensive evaluation of all eligible studies was identified by systematically searching the electronic databases PubMed, EMBASE and Cochrane. A total of 230 studies were initially identified, of which five were ultimately included in the meta-analysis. The primary endpoint of interest was overall survival assessed by hazard ratio. The pooled hazard ratio (HR) and 95% confidence intervals (CI) were calculated by using the Mantel-Haenszel method (fixed effects model). Statistical heterogeneity was assessed using the forest plot and inconsistency statistic. If any heterogeneity existed (I-squared > 50%), it was to be explored by subgroup analyses, meta-regression and sensitivity analyses.
Results: A total of five retrospective cohort studies, involving 10,095 patients were included. Administration of statins to patient’s prior and during pancreatic cancer diagnosis reduced the risk of overall mortality by 23% (HR 0.77, 95% Cl 0.73, 0.81). Significant heterogeneity was detected among these studies (I-squared 64%). Therefore, sensitivity analysis was conducted, which indicated the study by Jeon et al contributed most to the variability among all studies, while other studies demonstrated a statistical homogeneity (I-squared 30%, P-value 0.232).
Conclusions: Statin use is associated with increased overall survival in patients diagnosed with pancreatic cancer. This is the first meta-analysis looking at statin’s role in patient’s diagnosed with pancreatic adenocarcinoma.
HSF1-AMPK Negative Feedback Loop Promotes Invasion and Metastasis of Pancreatic Cancer
Z. Wang,1 M Lei,1 K. Chen,1 Q. Xu,2 Z. Wu,1 Q. Ma.1 Departments of 1Hepatobiliary Surgery; and 2Geriatric Surgery, First Affiliated Hospital of Xi'an Jiaotong University, Xi'an, China.
Aim: PDAC is a malignant tumor with high incidence of mortality. The study is to explore the interaction between HSF1 and AMPK and possible mechanism.
Methods: Immunohistochemical staining were applied to detect expression of HSF1 and P-AMPK in KPC mice. HSF1siRNA pool and KRIBB11 were used to inhibit expression of HSF1. The heat shock model was applied to improve HSF1 expression. AICAR and metformin were applied to increase P-AMPK and AMPK expression. Real-time PCR and western blot were used to detect expression of HSF1, AMPK, N-cadherin, E-cadherin, Vimentin. Immunofluorescence was performed to detect localization of HSF1. Transwell assay was applied to detect invasion of pancreatic cancer cells.
Results: In PDAC tissue of KPC mice, negative correlation between high level of HSF1 and low level of P-AMPK was described. HSF1 siRNA or KRIBB11 inhibited N-cadherin, vimentin, snail expression and increased the level of E-cadherin, which decreased invasion ability of pancreatic cancer cells. Heat shock increased the expression of HSF1, HSP70, N-cadherin, and vimentin, in accordance with increased invasion ability. P-AMPK was increased by KRIBB11 but decreased by heat shock model. AICAR or metformin increased the P-AMPK level whereas decreased the level of P-HSF1 and HSP70. Through immunofluorescence assay, it showed impaired translocation of HSF1 from cytoplasm to nucleus. When pretreated with AMPK siRNA, the effects of AICAR on HSF1 expression was reversed. Combined treatment decreased tumor invasion ability.
Conclusion: In pancreatic cancer, HSF1 is abnormally activated but AMPK is inactivated. The activation of HSF1 could inhibit the function of AMPK. Moreover, inactivated AMPK further promotes the activation of HSF1. HSF1-AMPK forms a vicious feedback loop and promotes the invasion and metastasis of pancreatic cancer.
Pancreatic Ductal Adenocarcinoma Can Be Generated From Human Acinar Cells
P. Wang,1 N. Akanuma,1 J. Liu,1 F.E. Sharkey,2 A.D. Singhi,3 H. Crawford.4 Departments of 1Cellular and Structural Biology; and 2Pathology; UT Health Science Center at San Antonio, San Antonio, TX; 3Department of Pathology, University of Pittsburgh Medical Center, Pittsburgh, PA; 4Department of Molecular and Integrative Physiology & Internal Medicine, University of Michigan, Ann Arbor, MI.
Pancreatic ductal adenocarcinoma (PDAC) is one of the most deadly human malignancies characterized by the accumulation of a series of genetic mutations during disease progression. Few models are available to study the molecular mechanism of the tumorigenesis process of human PDAC. To model human PDAC development, we developed a genetic manipulation system to transduce normal human primary pancreatic exocrine cells (hPECs) with lentiviral vector expressing KRASG12D-mCherry and lentiviral CRISPR targeting CDKN2A/p16, p53 and SMAD4. We have confirmed the efficiency of the p16 CRISPR, p53 CRISPR and SMAD4 CRISPR constructs in editing the genome to delete the respective gene loci. To define the cellular origin of PDAC initiation, we developed a flow cytometry-based method to sort viable ductal cells and acinar cells from primary normal hPECs. We identified the signaling pathway that promotes acinar to ductal metaplasia (ADM) from hPECs. To generate PDAC from hPECs, we sorted acinar cells from hPECs and induced ADM. Two million of the transduced acinar derived ductal like cells were transplanted subcutaneously or orthotopically into NOD-SCID mice. The xenografts were harvested one month after transplantation. Pathological analysis found that invasive and metastatic PDAC were generated. We confirmed that the xenografts are derived from human cells by anti-human nuclear antigen antibody (anti-HuNu). Anti-Pan cytokeratin staining proved that the lesions were made up of epithelial cells. Thus, for the first time, we have generated PDAC from human acinar cells, suggesting acinar cells are one of the origins for PDAC. Further comparison with ductal cell derived tumor is ongoing. Our study will reveal the cellular origin of PDAC and provide insight to human PDAC development.
Clinicopathological Analysis and Probability Prediction of Invasive Carcinoma in Patients With Intraductal Papillary Mucinous Neoplasm
A. Wei,1 D. He,2 W. Hu,1 Departments of 1Pancreatic; and 2Pathology; West China Hospital, Chengdu, China.
Aim: To investigate the clinicopathological factors and predictors of malignancy of main pancreatic duct (MPD)-intraductal papillary mucinous neoplasm (IPMN) and branch pancreatic duct (BPD)-IPMN in West China Hospital according to the new guidelines in 2012.
Methods: Data of 124 patients with pathologically confirmed IPMN after surgery from West China Hospital were analyzed. MPD diameters of MPD-IPMN and cyst sizes of BPD-IPMN for malignancy were calculated with receiver operating characteristic curve.
Results: A total of 124 IPMN patients received pancreatic resection at West China Hospital from January 2009 to December 2014. Among these patients, 29 (23%), 84 (68%), and 11 (9%) had MPD-, BPD-, and mix-type IPMNs, respectively. The 5-year overall survival rates were 88.7% for all patients, 78.9% for malignant patients, and 93% for benign patients. Thirty-eight (31%) patients had a pathologically confirmed invasive carcinoma and the incidence of malignancy was higher in MPD-IPMN (p = 0.015). The maximal AUC was at an MPD size of 6.5 cm. The cut-off value of cyst size in BPD-IPMN patients was 4.95 cm. The logistic regression analysis showed that cyst size >5 cm (OR = 2.592, 95% CI: 1.656–10.244, p = 0.028), MPD diameter >5 mm (OR = 3.676, 95% CI: 1.450–17.882, p = 0.043), the presence of mural nodule (OR = 3.318, 95% CI: 1.641–7.176, p = 0.037), and enhanced thick wall (OR = 3.749, 95% CI: 2.402–31.987, p = 0.046) were the predictors of malignancy in BPD-IPMN patients.
Conclusion: The current morphologic classification of MPD-IPMN could be MPD dilation ≥6.5 mm and MPD <6.5 mm. For BPD-IPMN patients, cyst size >5 cm with MPD dilation >5 mm, mural nodules, and enhanced thicken wall could play an important role to predict malignant changes. Patients with risk factors are recommended for surgery.
Key Word: Intraductal Papillary Mucinous Neoplasm, Main pancreatic duct-IPMN; Branch pancreatic duct-IPMN; Mix-type IPMNs; invasive carcinoma.
Epithelial Cell-Specific Calcineurin Signaling Mediates Inflammation in the Context of Pancreatitis
L. Wen, A. Orabi, T.A. Javed, S. Sanker, K. Boggs, J.F. Eisses, S.Z. Husain Children's Hospital of Pittsburgh of UPMC, Pittsburgh, PA.
Cn is a central Ca2+ responsive signaling molecule in many inflammatory disorders. However, Cn is ubiquitously expressed, and it is unclear whether epithelial sources of Cn that are intrinsic to an inflamed organ contribute to the disease. Here we show that Cn within the epithelial cells of the exocrine pancreas, the acinar and ducts cells, mediates inflammation in pancreatitis. In this study, we mostly focused on a mouse model of post-ERCP pancreatitis (PEP). We used a novel intra-pancreatic ductal adeno-associated virus (AAV) gene transfer of promoter-driven Cre recombinase into a Cn floxed mouse line to delete pancreatic-wide Cn or specifically acinar or duct cell Cn. Pancreatic-wide deletion of Cn in the pancreas (using AAV6-CMV-iCre) resulted in a 73% reduction in PEP outcomes. Acinar-specific deletion of Cn (using AAV6-Ela-iCre) led to an 85% reduction in PEP. Ductal-specific Cn deletion (using AAV6-Sox9-iCre) partially reduced PEP by 55%. As a complement, we generated an inducible acinar-specific Cn deleted mouse line (Ela-CreERT2/CnB1f/f), and these mice manifested a 73% reduction in PEP. The finding that pancreatic Cn mediates PEP prompted us to deliver the Cn inhibitor FK506 (1 uM) along with the ERCP contrast dye. This novel formulation reduced PEP by 61% down to sham levels and 100% down to levels of duct manipulation only, without contrast infusion. The findings were corroborated by reduced serum amylase as well as pancreatic granulocyte infiltration and necrosis. Acinar cell Cn deleted mice were also protected against bile infusion pancreatitis. This study highlights a pivotal role for epithelial cell Cn signaling in inflammation and provides the impetus to devise translationally relevant therapies for pancreatitis that target pancreatic Cn.
Radiocontrast Induces Mitochondrial Dysfunction and Impaired Mitophagy in Post-ERCP Pancreatitis Through the Dephosphorylation of Drp1 by Calcineurin
L. Wen,1 N. Shalbueva,2 O.A. Mareninova,2 A.I. Orabi,1 T.A. Javed,1 A.S. Gukovkaya,2 S.Z. Husain.1 1Department of Pediatric, University of Pittsburgh and the Children's Hospital of Pittsburgh of UPMC, Pittsburgh, PA; 2Veterans Affair Greater Los Angeles Healthcare System, University of California Los Angeles and Southern California Research Center for Alcoholic Liver and Pancreatic Diseases and Cirrhosis, Los Angeles, CA.
Radiocontrast (RC) is used during endoscopic retrograde cholangiopancreatography (ERCP), and RC exposure to the pancreas is a risk factor for the burdensome, iatrogenic complication of post-ERCP pancreatitis (PEP). How RC induces pancreatic injury and inflammation is not clear. Using a novel mouse model of PEP, we recently showed that PEP is dependent on activation of the calcium phosphatase calcineurin (Cn). In the current study, we examined the downstream effectors of calcineurin on pancreatic injury through mitochondrial dysfunction. We observed that RC—at a final dilution of 25-33%, which resembles in vivo exposure to the pancreas—induced a rapid and marked loss of mitochondrial membrane potential in mouse pancreatic acinar cells. Within five minutes of exposure, RC caused dephosphorylation of dynamin-related protein 1 (Drp1), a key mediator of mitochondrial fragmentation and a calcineurin phosphatase substrate. In both mouse and human pancreatic acinar cells, inhibition of Cn with FK506 prevented this early Drp1 dephosphorylation. In vivo, intra-pancreatic ductal infusion of RC to induce PEP in mice led to mitochondrial fragmentation followed by mitochondrial autophagy (mitophagy) within the acinar cells. However, acinar cell conditional knockouts of the critical calcineurin subunit CnB1 had reduced mitophagy following PEP, compared with matched wild type controls. These acinar Cn-deficient mice were also protected against PEP. In summary, the findings suggest that RC induces mitochondrial dysfunction and impaired mitophagy through Cn-mediated Drp1 dephosphorylation and subsequent mitochondrial fragmentation. Overall, the work opens up new strategies to prevent PEP by targeting critical acinar cell pathways that intersect calcium, mitochondria, and autophagy.
Adjuvant Chemotherapy After Resection of Pancreatic Ductal Adenocarcinoma: A Retrospective Single Center Analysis of 251 Consecutive Patients in a Non-Selected Cohort
U.A. Wittel, M. Reinmuth, F. Makowiec, O. Sick, R.M. Fritsch, U.T. Hopt. University of Freiburg Medical Center, Freiburg, Germany.
Introduction: The recommendation for postoperative chemotherapy following resection of pancreatic ductal adenocarcinoma (PDAC) is based on prospective randomized trials. However, patients included in clinical trials often do not reflect the overall patient population treated in clinical practice.
Methods: We retrospectively analyzed the clinical course of patients after resection of PDAC in the context of adjuvant treatment in an unselected cohort of 251 consecutive patients. 251 patients after pancreas resection for PDAC between 2001 and 2013 were included in our analysis. Follow-up data from oncologists, general practitioners or hospital patient files were available for 92% of patients.
Results: The median age was 67 years. Only 55.4% of patients received postoperative chemotherapy mostly with gemcitabine, despite treatment had been recommended in 89%. The reasons for not receiving adjuvant treatment were early recurrence of PDAC (33%) and reduced general health condition (20.8%). Patients undergoing adjuvant chemotherapy showed increased disease free survival (4.1 vs. 10.9 months; p<0.01) and overall survival (14.3 vs. 25.6 vs. months; p<0.001). Dose reduction (29% of patients) did not affect survival. Only 60.4 % of treated patients received all 6 cycles of chemotherapy. Reasons for discontinuation were side effects (40.7%) reduced general condition (24.1%) and disease recurrence (33.3%). Discontinuation of chemotherapy before the 4th cycle was associated with decreased survival (14.0 vs. 26.3 months; p<0.05).
Conclusion: Only a small fraction of patients currently completes the recommended 6 months of adjuvant chemotherapy. Future strategies should incorporate more efficacious chemotherapy regimens but also preserve regimens with low side effects for patients with low performance status.
Effect of the Medicare Severity-Diagnosis Related Group Classification System on Predicting Hospital Readmission After Distal Pancreatectomy
D. Xourafas, C. Fernandez-Del Castillo, A.L. Warshaw, K.D. Lillemoe, C.R. Ferrone. Department of Surgery, Massachusetts General Hospital, Harvard Medical School, Boston, MA.
Background: Accurate prediction of readmission is essential to minimize hospitalization costs. Our aim was to determine if the Medicare Severity-Diagnosis Related Group coding system (MS-DRG), which accounts for patient’s principal diagnosis, procedure, comorbidities and complications can accurately predict readmission after distal pancreatectomy (DP).
Methods: Clinicopathologic features and MS-DRG codes pertinent to DP [407: without complication/co-morbidity (CC), 406: with CC, and 405: with major CC] were collected and compared between readmitted and nonreadmitted patients who underwent a DP. Univariate analysis and logistic regression models were used to evaluate outcomes.
Results: Among 393 patients who underwent a DP between 2008 and 2015, 77 (19.5%) were readmitted within 30- or 90-days of discharge. Of all patients for whom an MS-DRG 407 (without CC) was assigned, only 3% were subsequently readmitted. This percentage of readmission increased significantly to 25% for code MS-DRG 406 (with CC) and to 37% for MS-DRG 405 (with major CC, P<0.01). On multivariable analysis, MS-DRG 407 combined with any clinicopathologic factors was not predictive of readmission. In contrast, MS-DRG 406 linked to an estimated blood loss (EBL) of more than 400 ml [odds ratio (OR)=3.2, 95% Confidence Interval (CI)=1.3-7.8, P=0.009] and a length of hospital stay (LOS) of more than 5 days (OR= 5.4, 95%CI=2.1-13.7, P=0.0003) predicted readmission. MS-DRG 405 combined with discharge to a facility was also predictive of readmission (OR=5.3, 95%CI=1.3-21.6, P=0.01). The association of increased EBL, LOS and MS-DRG 406 was specifically predictive of 30-day readmission [Likelihood ratio (LR)=0.01], whereas the combination of MS-DRG 405 with disposition to facility was more predictive of 90-day readmission (LR=0.02).
Conclusions: Individual MS-DRG codes can be combined with specific clinicopathologic factors to create strong predictive models for 30 or 90-day readmission after DP. Hospitals can use this clinical information to accurately identify patients at a higher risk of readmission.
Metformin Inhibits Hypoxia Induced PSC Activation and Pancreatic Cancer Cell Viability and Invasion
Q. Xu. Department of Hepatobiliary Surgery, Xi’an Jiaotong University, Xi'an, China.
Background: Metformin, a widely used anti-diabetic drug, is emerging as a potential anticancer agent but the mechanisms involved remain incompletely understood. Pancreatic stellate cells (PSCs) as a novel and important member of tumor microenvironment, has gained much more focus lately. Tumor hypoxia is associated with enhanced tumor invasion, angiogenesis, and distant metastasis. In this study, we investigated the role of metformin in hypoxia-driven PCa progression.
Methods: Human PSCs were isolated from normal pancreas tissues. The purity of the PSCs was assessed by morphology, oil red O staining, and immunofluorescence of a-smooth muscle actin. qRT-PCR and ELISA were performed to quantify the expression of IL-6, VEGF-A and SDF-1.
Results: We observed that hypoxia increased PSC activation; PSCs cultured under hypoxic conditions exhibit higher levels of IL-6, VEGF-A and SDF-1 transcription and secretion; these factors are known to be involved in modulating the response of tumor cells to activated PSCs. Hypoxia was mildly active in promoting the invasiveness of PCa cells. In addition, hypoxia Conditional media (CM) from PSCs significantly decreased the E-cadherin level and increased the vimentin level of PCa cells, which indicates that hypoxia can drive PCa cells to undergo EMT. Exposure of PSCs to hypoxia during their activation enhanced their ability to affect PCa motility and invasiveness. Conditional media (CM) from PSCs significantly decreased the E-cadherin and increased the vimentin of PCa cells under hypoxic conditions, which indicates that CM from PSCs can drive PCa cells to undergo EMT. Moreover, treatment with the metformin or down-regulated expression of HIF1 with siRNA, abrogated the hypoxia-induced effect on PSCs and PCa cells.
Conclusion: These findings suggest that PSCs are sensitive to hypoxia, which enhances their promotion of PCa invasiveness. Metformin has a protective effect against hypoxia in the pancreatic tumor-stromal interaction. Metformin might be a potential anticancer agent for the treatment of PCa.
Increased Semaphorin 3c Promotes Tumor Growth and Metastasis in Pancreatic Ductal Adenocarcinoma by Inactivating PI3K/AKT Signaling Pathway
X. Xu, H. Wang. Institute of Hepatopancreatobiliary Surgery, Southwest Hospital, Third Military Medical University, Chongqing, China.
Purpose: Semaphorins have been recognized as a key contributor such as axon guidance, immune responses and tumor progression. We investigated the clinical significance of semaphorin 3c and its role in the growth and metastasisi in pancreatic ductal adenocarcinoma (PDAC).
Methods: Expression of semaphorin 3c in 118 PDAC, 67 paracancerous and 26 normal pancreatic tissues were measured. Impact of semaphorin on invasion and metastasis was evaluated in PDAC cells by expressed-plasmid transfection or shRNA lentivirus.
Results: Aberrant Expression of semaphoring 3c is positively associated with tumor stage and correlated with poor overall survival in PDAC patients. Knockdown of sema 3c attenuates the migration and invasion in pancreatic cancer cell line and reduces PDAC cell metastasis by xenotransplantation into nude mice. Semaphorin 3c exerts an promotion effect on the phosphatidylinositol 3-kinase (PI3K)/AKT signaling pathway.
Conclusion: Aberrant expression of semaphorin 3c correlates with PDAC patient’s poor prognosis and promotes tumor growth and metastasis.
Comparison of the International Consensus Guidelines for Predicting Malignancy in Intraductal Papillary Mucinous Neoplasms
S. Yamada, T. Fujii, H. Takami, M. Hayashi, H. Sugimoto, Y. Kodera. Gastroenterological Surgery, Nagoya University Graduate School of Medicine, Nagoya, Japan.
Background: To evaluate the predictors of malignancy in the 2012 international consensus guidelines for intraductal papillary mucinous neoplasms (IPMNs) and validate their diagnostic value relative to the 2006 guidelines.
Methods: Between 2002 and 2014, 177 consecutive patients who underwent curative resection of IPMN were enrolled. Based on the 2012 guidelines, high-risk stigmata (mural nodule with enhancement, main pancreatic duct [MPD] ≥ 10 mm and obstructive jaundice) and worrisome features (cyst size ≥30 mm, thickened cyst wall, mural nodule without enhancement, MPD 5–9 mm, an abrupt change in MPD diameter and lymphadenopathy) were assessed, and predictive and diagnostic values were statistically analyzed.
Results: Multivariate analysis identified obstructive jaundice (OR, 23.9; P < 0.0001), abrupt change in MPD diameter (OR, 3.01; P = 0.017) and lymphadenopathy (OR, 5.84; P = 0.027) as independent predictive factors, and accuracy was 69.8, 67.4 and 66.3%, respectively. Surgery was indicated in 156 (94.0%) patients using the 2006 guidelines, and in 130 (78.3%) using the 2012 guidelines. The accuracy of the 2006 guidelines was 35.5% compared with 44.8% for the 2012 guidelines. The area under the curve (AUC) for the 2006 and 2012 guidelines was 0.65 and 0.67, respectively; ΔAUC was 0.02, which was not significant. When the worrisome features were combined with high-risk stigmata, the AUC increased to 0.79.
Conclusions: Obstructive jaundice, abrupt change in MPD diameter and lymphadenopathy were independent predictive factors in the 2012 guidelines, with high accuracy. Using the new guidelines, the number of observational patients with IPMN and predictive accuracy increased.
TFF1 (Trefoil Factor Family 1) act as Tumor Suppressor to Inhibit Invasive Transformation of PanIN Into PDAC In Vivo
J. Yamaguchi, Y. Yokoyama, T. Kokuryo, M. Nagino. Surgical Oncology, Nagoya University Graduate School of Medicine, Nagoya, Japan.
Background: Trefoil Factor Family (TFF) is secretory proteins abundantly expressed in gastrointestinal mucosa. While TFF contribute to the restitution of damaged mucosa, recent reports suggest the tumor-suppressive role of TFF in gastric carcinogenesis. It has been shown that loss of TFF2 results in the development of IPMN; however, TFF1 is expressed more extensively in premalignant lesions and its function is still to be clarified.
Methods: The expression of TFF1 was assessed by IHC in surgically resected specimen of human pancreas. Human pancreatic cancer cells (panc1 and PK9) were treated by siRNA against TFF1 to investigate the role of TFF1 in vitro. TFF1 knockout mice were bred with KC (Pdx1-Cre; LSL-KRASG12D) mice to clarify the function of TFF1 in vivo.
Results: While TFF1 is expressed abundantly in premalignant lesions such as PanIN and non-invasive IPMN, it is frequently lost in invasive adenocarcinoma. The suppression of TFF1 in human pancreatic cancer cell lines does not have any impact on their proliferation, but results in the development of invasive ability of cancer cells. In addition, the loss of TFF1 upregulated the expression of SNAIL, suggesting that TFF1 can inhibit EMT (epithelial-mesenchymal transition) of cancer cells. TFF1KO mice show no neoplastic lesions in their pancreas, and KC mice show scattered PanIN lesions which express abundant TFF1. While TFF1KO and KC mice show favorable survival, TFF1-KO; KC mice were found to have pancreatic adenocarcinoma and to have a poor survival rate, indicating that TFF1 can inhibit invasive transformation of PanIN into PDAC in vivo.
Conclusion: TFF1 found in pancreatic premalignant lesions might function as tumor suppressor to inhibit invasive transformation of tumor cells.
PR55α Subunit of PP2A Supports the Tumorigenic and Metastatic Potential of Pancreatic Cancer Cells
Y. Yan, A.L. Hein, P. Seshacharyulu, S. Rachagani, M. Ouellette, Y.M. Sheinin, M.P. Ponnusamy, S. Batra. University of Nebraska Medical Center, Omaha, NE.
PP2A holoenzyme consists of a catalytic subunit, a scaffold subunit, and a regulatory subunit. Loss of function analysis using PP2A catalytic inhibitors or inhibition via tumor viral proteins suggest PP2A as a putative tumor suppressor. However, PP2A has also been shown to facilitate the activation of oncogenic signaling pathways when associated with specific regulatory subunits. In this study, we investigated the possible oncogenic role of PP2A in pancreatic cancer. We found a striking increase in the expression of PR55α, a PP2A regulatory subunit, in pancreatic cancer cells compared to normal pancreatic epithelial cells. Consistently, PR55α expression was markedly elevated in pancreatic ductal adenocarcinoma tissues compared to adjacent normal pancreatic tissues (P<0.0001) and correlated with poor survival of pancreatic cancer patients (P<0.0003). RNAi-mediated depletion of PR55α in pancreatic cancer cells resulted in diminished phosphorylation of both AKT and ERK1/2 and decreased protein levels of β-catenin. Accordingly, pancreatic cancer cells with reduced PR55α expression exhibited significantly impaired properties of transformation, including attenuated cell growth, clonogenicity, mobility, and anchorage-independent growth. Moreover, orthotopic implantation of PR55α-depleted pancreatic cancer cells into nude mice showed a marked reduction in tumorigenicity (P<0.001) and distant metastases. These results suggest that PR55α promotes pancreatic cancer development by sustaining hyperactivity of multiple oncogenic signaling pathways, including AKT, ERK and Wnt.
Functional and Non-Functional Pancreatic Neuroendocrine Tumors
M. Yang. Department of Pancreatic Surgery, West China Hospital, Sichuan University, Chengdu, China.
Background and Aim: Currently, there are 2 main TNM staging systems proposed by ENETS and AJCC for the survival analysis of p-NETs, which have not been well assessed, especially for those with different functional status. We investigated the prognostic value of the 2 systems in p-NETs, as a whole, and more interestingly in functional and non-functional sub-groups separately, with a view to ascertaining any potential clinical benefits of using one system over the other.
Methods: Data from patients with surgically resected p-NETs were retrospectively reviewed. The overall survival (OS) and prognostic predictors were respectively analyzed.
Results: In the whole group of 165 patients, both TNM systems successfully discriminated OS differences, when comparing stages I and II with stages III and IV (P<0.05); ENETS stage III patients had a significantly better OS than those in stage IV (P=0.003). Patients with functional p-NETs in ENETS stage II showed a statistically better OS than those in stages III and IV (P<0.05). For non-functional tumors, the AJCC staging system could effectively discriminate between the OS differences of patients in stage I with stages III and IV, or stage II with III and IV (P<0.05). Multivariate analyses by Cox Regression proportional hazards model revealed both systems were effective predictors of death for p-NETs, the AJCC staging manual indicated a relatively imprecise predictive ability for the whole group of p-NETs and for functional tumors but more accurate one for non-functional p-NETs.
Conclusions: The ENETS system might have potential advantages when applied to all p-NETs and to the functional sub-group, while the AJCC system might be clinically more useful in non-functional p-NETs, which supported their more proper use in clinical practice.
Clinical Allograft Islets Post-Transplant 3 Year for Type 1 Diabetes Mellitus: 10 Cases Reported
Y. Yao, L. Wei, M. Yang, L. Luo, H. Xue, L. Luo, G. Xiang, H. Zou, G. Wang, C. Lai, S. Deng, X. Huang. Center of Cell Transplantation & Center of Hepatobiliary and Pancreatic Surgery, Affiliated Hospital of University of Electronic Science and Technology, Sichuan Provincial People's Hospital, Chengdu, China.
Objective: To evaluate the effect of 3-years followed-up of allograft islet transplantation for patients with type 1 diabetes mellitus (T1DM).
Method: 10 cases of islet transplantations were performed on 10 patients with type 1 DM. The pancreases were digested by Liberase collagenase enzyme and high activity of islets were purified using continuous gradients of Ficoll-diatrizoic acid on a refrigerated COBE 2991 centrifuge. Cultured islets were infused by minimally invasive surgical approach to the liver via portal vasculature. After islet cell transplantation, a modified Edmonton immunosuppresion protocol containing antithymocyte globulin (ATG) and Etanercept tacrolimus and mycophenolate mofetil was used,and the changes in blood glucose, C peptide and glycated hemoglobin were monitored regularly during a follow-up period of 3 years.
Result: The glucocorticoid-free immunosuppressive regimen was used. During the follow-up period (36 months), 6 recipients remained insulin-independent. The dosage of insulin decreased by 60% in 4 patients. The levels of blood glucose and HbA1c were all within normal range and liver and renal functions were normal. C-peptide level was normal. No complications related to islet infusion were observed.
Conclusion: Medium term clinical effect of islet transplantation is effective and safe for treating T1DM. Long-term effect needs further observation.
Extracellular Ca2+ Contributes to the Beneficial Effects of Lactated Ringer's During Acute Pancreatitis
J.R. Yaron, K. Patel, B. Khatua, C. De Oliveira, V.P. Singh. Department of Medicine, Mayo Clinic, Scottsdale, AZ.
Background: Recent studies suggest that lactated Ringer’s (LR) use is associated with a milder acute pancreatitis (AP) course than with normal saline. The mechanisms underlying this benefit are unclear. Hypocalcemia is a part of the Ranson’s and Glasgow criteria for severe AP (SAP) and LR contains 3 mEq Ca2+. Here, we tested the effects of increasing doses of extracellular Ca2+ on acinar cell injury in two in vitro models of pancreatic injury.
Methods: 100 nM caerulein (CER) or 100 μM linoleic acid (LA) induced cell injury (LDH leakage), intracellular Ca2+ (Cai; Fura-2AM) and mitochondrial depolarization ratio (Ψm; JC-1) were measured in mouse pancreatic acini. The impact on these of increasing doses of Ca2+ (0, 1, 2 mM), or inhibiting ryanodine receptors (dantrolene; 50 μM), IP3 signalling (2-APB; 50 μM, IP3) and BAPTA-AM (50 μM, intracellular Ca2+ chelator) were studied.
Results: Extracellular Ca2+ (2 mM vs. 1 mM) reduced LA-induced Cai elevation by 34.2% (p < 0.05) and Ψm by 0.74 from 4.1 to 3.3 (p < 0.05), BAPTA-AM reduced Cai elevation by 87% (p < 0.0001) and Ψm by 0.62 (p < 0.05). While dantrolene reduced LA induced Cai elevation (42%; p < 0.0001), it did not affect the Ψm increase. CER-induced Cai elevation was unaffected by extracellular Ca2+. Extracellular Ca2+ dose-dependently protected against LA-induced cell death at 2 hours (0 mM: 30.8% ± 6.9%; 1 mM: 13.4% ± 5.1%; 2 mM: 11.7% ± 4.7%) and 4 hours (0 mM: 60.7% ± 15.1%; 1 mM 49.6% ± 14.2%; 2 mM: 42.7% ± 17.0%) [p < 0.05 to 0.01] but provided no protection against CER-induced cell death at 2 (0 mM: 4.5% ± 1.9%; 1 mM 3.9% ± 4.1%; 2 mM: 5.7% ± 3.2%) or 4 hours (0 mM: 8.1% ± 5.6%; 1 mM: 12.8% ± 10.7%; 2 mM: 11.1% ± 8.6%) [N=9]. BAPTA (40.4% ± 11.8%) reduced LA-induced cell death at 4 hours while dantrolene (50.7% ± 9.4%) and 2-APB (65.1% ± 32.3%) conferred no protection versus 1 mM Ca2+.
Conclusions: Extracellular Ca2+ reduces Ψm, Cai increase and protects against lipotoxic injury in pancreatic acini. This provides a possible mechanism for how the Ca2+ containing LR may have a protective effect in SAP. The relation between lipotoxic injury and Cai needs to be explored further.
C-Src is Involved in Physiologic Zymogen Synthesis and Packaging Through the Golgi in Pancreatic Acinar Cells
J.R. Yaron, G. Singh, K. Patel, R.N. Trivedi, C. De Oliveira, V.P. Singh. Department of Medicine, Mayo Clinic, Scottsdale, AZ.
Background: We have previously shown that Src family of protein tyrosine kinases is involved in trypsinogen activation. However, the role of specific members is poorly understood. In this study we aimed at identifying the role of c-Src in zymogen synthesis and trafficking using a physiologic glucocorticoid stimulus of zymogen granule production, and adenoviral overexpression of c-Src to study its role in zymogen production and packaging.
Methods: The AR42J rat pancreatic acinar cell line was stimulated with 1 μM dexamethasone or transduced with c-Src adenovirus for 24 hours alone of with the clinically used Src family inhibitor Dasatinib at a concentration of 10 μM. Golgi phenotype was assessed by structured illumination fluorescence microscopy for GM130. Amylase and chymotrypsin protein levels were determined by western blot. Activation of Src was assessed by immunoprecipitation and western blot (PY416). Amylase gene expression was performed by RT-qPCR using SYBR chemistries and normalized to 18S rRNA. Total amylase activity was assessed by liquid kinetic assay and normalized to total cell mass.
Results: Dexamethasone activated Src (PY416) between 1–12 hours, induced a > 2 fold increase in amylase and chymotrypsin protein levels, and total amylase activity, all of which were inhibited by Dasatinib. In contrast, adenoviral expression of c-Src which activated Src in a Dasatinib-sensitive manner did not increase amylase or chymotrypsin levels and only caused fragmentation of the Golgi. Dexamethasone also caused Golgi expansion from 8.9 ± 3.5 μm2 to 13.9 ± 5.9 μm2 (p < 0.0001; N=41-43 single cells) which was reduced to 10.21 ± 3.6 μm2 (p < 0.001; N=41-43 single cells) by Dasatinib.
Conclusions: Src family kinases are involved in regulating physiologic zymogen synthesis and trafficking at the mRNA and Golgi levels. c-Src is specifically involved in these phenomena at the Golgi level.
DCLK1 Regulates BMI-1 and is Associated With Clinical Outcome of Pancreatic Cancer
O. Yongsheng, H. Wang. Institute of Hepatopancreatobiliary Surgery, Southwest Hospital, Third Military Medical University, Chongqing, China.
Background: Recent evidence suggests that DCLK1 is a novel candidate marker for cancer stem cells and regulates epithelial to mesenchymal transition (EMT). We have previously demonstrated that BMI-1 is overexpressed and associated with the clinical stages and poor prognosis of pancreatic cancer (PC) and up-regulation of BMI-1 resulted in the inhibition of EMT related transcription factor E-cadherin. Here we aim to investigate whether DCLK1 is an upstream regulator of BMI-1 and its correlation with prognosis.
Methods: We used qRT- PCR and IHC to determine levels of DCLK1 in PC cell lines and patient samples. To assess the function of DCLK1, DCLK1 overexpression or knockdown pancreatic cancer cell lines were established and it was conducted significant changes in malignant properties including migration, invasion, EMT in vitro and metastasis in mouse xenograft molds. These oncogenic effects of DCLK1 are obviously on account of BMI-1 expressed in PC, verified through reporter assay.
Results: DCLK1 in PC cell lines was elevated at both mRNA and protein levels. IHC showed that 124 of 210 (59.0%) paraffin-embedded archival PC specimens exhibited high expression of DCLK1, Up-regulation of DCLK1 was significantly correlated with the clinical stages, distant metastasis, perineural invasion and vascular invasion and that patients with high DCLK1 level exhibited shorter survival time. Knockdown of DCLK1 reduced the invasion and migration, and resulted in the inhibition of BMI-1 and EMT related transcription factors in PC cell lines. Overexpression of BMI-1 enhanced the DCLK1 induced effects on cell migration. Reporter assay revealed DCLK1 increased BMI-1expression. Furthermore, Levels of DCLK1 correlated with activation of BMI-1 and EMT related transcription factors in PC samples.
Conclusions: DCLK1 is up-regulated in PC cell lines and samples from patients. These data also support a potential master regulatory role for DCLK1 in BMI-1 expression, EMT and cancer metastasis.
A Novel Strategy of Targeting Interferon Gamma-Induced Protein 10 to Inhibit Instant Blood Mediated Inflammatory Reaction in Islet Transplantation
G. Yoshimatsu,1 M. Takita,1 C. Darden,1 C. Chang,1 P.S. Saravanan,1 M.C. Lawrence,1 B. Naziruddin.2 1Islet Cell Laboratory, Baylor Research Institute, Dallas, TX; 2Baylor University Medical Center, Dallas, TX.
Introduction: Islet transplantation is a promising treatment to achieve normoglycemia in type 1 diabetes. However instant blood mediated inflammatory reaction (IBMIR) causes early loss of islet graft after islet transplantation. Interferon gamma-induced protein (IP-10 or CXCL10) is a chemokine triggered by cytokine/chemokine component of IBMIR. Here, we demonstrate the effect of CXCL10 blocking to improve islet graft survival.
Method: Streptozotocin-induced diabetic CXCL10 knockout (KO) and wild type (WT) mice underwent islet transplantation into portal vein. Graft function was monitored by daily blood glucose level and intraperitoneal glucose tolerance test (IPGTT) after one month. Cytokine reaction in blood mixture with islet was investigated in vitro. Further, transplanted islets were treated with CXCL10 neutralizing antibody, and blood glucose, IPGTT, and histology were assessed.
Result: IL-1β stimulation induced up-regulation of CXCL-10 mRNA gene and secretion of CXCL10 chemokine in MIN6 cells. Proinflammatory cytokine cocktail induced CXCL10 production in human islets. The transplantation of CXCL10-KO islets resulted in better glucose control through one month. AUC of IPGTT was significantly lower in KO islet transplants when compared to WT (p = 0.002). CXCL10 secreted from islets was blocked by CXCL10 neutralizing antibody and its treatment inhibited IL-6 secretion in islet and blood mixture assay. Anti-CXCL10 antibody treatment group showed significantly higher normoglycemia rate than no CXCL-10 treatment group (75%, 43%, p = 0.04).
Conclusion: Targeting CXCL10 is a promising treatment to protect islets from damage inflicted by IBMIR. CXCL10 inhibition improved islet engraftment resulting in superior glycemic control.
The Loss of ATRX Promotes Susceptibility to KRAS-Mediated Pancreatic Disease
C. Young,1 C. Howlett,2 C. Pin.1 1Department of Physiology & Pharmacology, Schulich School of Medicine & Dentistry, University of Western Ontario, Children's Health Research Institute, London, Canada; 2Department of Pathology and Laboratory Medicine, Schulich School of Medicine & Dentistry, University of Western Ontario, London, Canada.
Pancreatic ductal adenocarcinoma (PDAC) accounts for more than 30,000 deaths in North America each year. More than 90% of PDAC cases demonstrate a constitutively active mutation in the KRAS oncogene. However, additional factors are required to promote progression of PDAC. Loss of epigenetic mediators that affect genomic stability, including DNA repair mechanisms, may increase susceptibility to KRAS-mediated PDAC. ATRX is a Swi/Snf chromatin remodeling protein with known roles in DNA replication, telomere maintenance and DNA repair. In this study, we propose a novel role for ATRX in the exocrine pancreas. It is hypothesized that ATRX loss in pancreatic acinar cells will increase susceptibility to activated KRAS, promoting the initiation of pancreatic disease.
We generated a genetically-altered mouse model expressing an inducible cre-recombinase in pancreatic acinar cells, along with a floxed Atrx gene (Mist1 creERT Atrx flx). Tamoxifen was administered to 2–4 month old Mist1 creERT Atrx flx mice, in the presence/absence of inducible constitutive KRAS activation (KRAS G12D). Mice were monitored for 60 days, and pancreatic tissue was collected for histological, biochemical and molecular analysis at this time. It is demonstrated that loss of Atrx, in the presence of KRAS activation, produced increased pancreatic damage and precancerous lesions within two months. Furthermore, increased levels of DNA damage and proliferation were observed due to Atrx loss. Taken together, these findings indicate that the loss of ATRX enhances pancreatic damage in response to activated KRAS, effectively promoting progression into PDAC.
RNA Sequencing Reveals Therapeutic Effects of Multiple Statins on Pancreatic Cancer Cells
J. Yu,1 S.-H. Liu,1 R. Sanchez,1 W. Fisher,2 F.C. Brunicardi.1 1Department of Surgery, UCLA, Los Angeles, CA; 2Baylor College of Medicine, Houston, TX.
Introduction: Pancreatic cancer (PC) is one of the most deadly forms of cancer. Our recent high-throughput drug screening identified statins that significantly inhibit PC cell growth in vitro. Statins, primarily prescribed for treatment of hypercholesterolemia and to prevent cardiovascular diseases, have been documented to exhibit anti-cancer properties. However, the mechanism remains to be elucidated.
Methods: The aim of this study was to determine the anti-cancer effects and to evaluate their therapeutic potentials of the three statins (fluvastatin, lovastatin, and simvastatin) on human PC cell lines MiaPaCa2 and PANC1. The in vitro effects of statins on the viability of PC were evaluated using CelltiterGlo and IC50 values were calculated. To determine whether statins inhibit PC via shared or via independent molecular targets, RNA-Seq was performed on PC cells treated with statins at either high (50uM) or low (100nM) doses for 72 hours.
Results: The dose–response curves of statins proved that statins inhibited PC cell proliferation in a dose-dependent manner. Global gene expression alterations upon treatment of the statins were determined using RNA-Seq. The transcriptome data indicated that the statins at 100nM had little effects on PANC1 cells, but were able to decrease “cell cycle” gene expressions and stimulate genes involved in “glycolysis” in MiaPaca2 cells, suggesting that different genetic background may influence the outcome of statin treatment. The statins at 50uM showed a significant inhibition of cell proliferation in both MiaPaca2 and PANC1 cells, as well as expression of genes involved in “cell cycle”. 134 genes including BIRC5, TPX2 and CCNB1 in common were significantly inhibited by treatment of statins in both cell lines. RT-PCR proved the significant inhibition of gene expressions for BIRC5, TPX2 and CCNB1.
Conclusion: Taken together, these results demonstrated the anti-cancer effects of statins at high doses on the human PC cells was via the inhibition of cell proliferation gene expression. Statin treatment may provide a novel therapeutic choice for PC.
Opposing Effects of TGF-β and BMP2 on MicroRNA-200b in the Pancreas
P. Yu, K. Liu, Y. Cao, T. Ko. UTHealth, Houston, TX.
Introduction: MicroRNA (miR)-200b is an anti-fibrogenic regulator in the lungs and liver, but its effects in the pancreas are not clear. We reported that bone morphogenetic protein (BMP) 2 opposes the pro-fibrogenic effects of transforming growth factor (TGF)-β in the pancreas. We hypothesize that during chronic pancreatitis (CP) progression, TGF-β suppresses BMP2’s action by downregulating miR-200b, resulting in pancreatic fibrosis. This study examines miR-200b level in mouse CP and miR-200b regulation in pancreatic cells.
Methods: CP was induced in C57BL/6 mice by cerulein (50μg/kg, 5 IP injections/day, 3 days/wk for 4wks). Control mice received normal saline (n=4/group). Pancreatic fibrosis was assessed by Sirius red staining and qPCR for collagen and fibronectin expression. In vitro, primary human pancreatic stellate cells (hPSCs) were treated with vehicle, TGF-β (1ng/ml), BMP2 (50ng/ml) or TGF-β & BMP2 for 24 hrs. The same except the combined treatment was done on pancreatic epithelial AR42J cells. miR-200b level was measured by qPCR.
Results: In CP, miR-200b level decreased by 70% compared to control and inversely correlated with fibrosis (p<0.05). In hPSCs, miR-200b level was not altered by TGF-β alone, but increased by BMP2 alone by 150% compared to vehicle, and the BMP2’s effect was abolished with the combined treatment (p<0.05). In AR42J cells, TGF-β decreased miR-200b level by 60%, while BMP2 increased miR-200b by 110% compared to vehicle.
Conclusions: TGF-β and BMP2 have opposing effects on miR-200b in the pancreas in a cell-specific context. The suppressed miR-200b level in CP indicates a predominantly fibrogenic milieu, which may result from TGF-β overwriting BMP2’s effects on miR-200b.
Genetic Ablation of Mitochondrial Deacetylase Sirtuin 3 Exacerbates Cerulein Pancreatitis
J. Yuan,1 Y. Qin,1,2 S.R. Malla,1 M. Geng,1,3 R.T. Waldron,1,4 O.A. Mareninova,1 A. Lugea,1,4 S.J. Pandol,1,4 A.S. Gukovskaya.1 1VA Greater Los Angeles Healthcare System, University of California Los Angeles, and Southern California Research Center for ALPD and Cirrhosis, Los Angeles, CA; 2The Division of Gastroenterology and Hepatology, Youjiang Medical University for Nationalities, Baise, China; 3Frank Netter H. MD School of Medicine at Quinnipiac University, North Haven, CT; 4Cedars-Sinai Medical Center, Los Angeles, CA.
Background & Aims: Mitochondrial depolarization caused by the opening of the mitochondrial permeability transition pore (mPTP) is a key pathologic event leading to pancreatitis. Mitochondrial resident protein Sirtuin 3 (SIRT3) is known to inactivate mPTP by deacetylating its key component cyclophilin D. Here, we investigated the effect of SIRT3 genetic ablation on experimental pancreatitis induced by cerulein injections in mice.
Methods: We measured changes in protein acetylation, mitochondrial complex activities, mitochondrial membrane potential and oxygen consumption, parameters of autophagy, ER stress response and pathologic responses of cerulein pancreatitis in wild type and SIRT3 knockout mice. Protein acetylation was measured by immunoblot using antibody against acetylated lysine as well as with mass spectrometry.
Results: SIRT3 genetic ablation markedly increased the levels of acetylated proteins in pancreatic mitochondria. This resulted in a decreased level of transcription factor PGC-1alpha mediating mitochondrial biogenesis, decrease in mitochondrial ATP production, and inhibition of mitochondrial dynamics. The latter was manifested by downregulation of mediators of both mitochondrial fusion (MFN1) and fission (FIS1). SIRT3 genetic ablation aggravated cerulein pancreatitis, evidenced by marked increases in serum amylase and lipase levels, increased necrosis, and neutrophil infiltration. At the same time, SIRT3 ablation had little effect on acinar cell vacuolization and trypsinogen activation, indicating SIRT3-independent pathways.
Conclusions: The data indicate critical roles of mitochondrial dynamics and acetylation in regulating pathologic responses of acute pancreatitis.
Sodium Sulfate Suppresses Multiple Damage-Associated Molecular Patterns In Vitro
Z. Yuan, X. Wang. Shanghai Key Laboratory of Pancreatic Diseases & Department of Gastroenterology, Shanghai General Hospital, Shanghai, China.
Background & Aims: Damage-associated molecular patterns (DAMPs) play a central role in the pathogenesis of acute pancreatitis. However, no clinically proven DAMPs inhibitor has been discovered yet. Dachengqi Decoction (DCQD) has been used in treating acute abdomen related disorders including acute pancreatitis in China for thousands of years. The aim of the current study is to determine which active compound(s) of DCQD can attenuate DAMPs of acute pancreatitis.
Methods: Nuclear DNA (nDNA) and mitochondrial DNA (mitDNA) were incubated with active compounds of DCQD-Na2SO4, Emodin, Magnolol-and other related compounds such as Ca2SO4, CaCl2 for 30min at 37°C, and then used in real time PCR to detect presence of nDNA and mitDNA. Acinar cell was used to test extracelluar adenosine triphosphate (ATP) release.
Results: 10mM Na2SO4 significantly degraded nDNA and mitDNA, which showed a similar results to DNase. Emodin and magnolol had no effects on degradation of nDNA and mitDNA. 10mM Na2SO4, 10mM CaCl2, 100μM emodin and 100μM magnolol, all showed significant suppression of extracellular ATP release of acini induced by supermaximal caerulein. Animal studies are ongoing.
Conclusion: Na2SO4 is a promising DAMPs inhibitor. Active ingredients of DCQD may synergistically ameliorate severity of acute pancreatitis via inhibiting DAMPs.
Inverted U Dose–response of Nonspecific Cyclophilin Inhibitor Cyclosporin A on Murine Pancreatic Acinar Cell Injury and Experimental Acute Pancreatitis
X.Y. Zhang,1 M. Chvanov,2 D. Latawiec,1 L. Wen,1 Y. Ouyang,2 R. Mukherjee,1 W. Huang,1,3 A. Tepikin,2 D. Criddle,2 R. Sutton.1 1NIHR Liverpool Pancreas Biomedical Research Unit, Royal Liverpool University Hospital, University of Liverpool, Liverpool, United Kingdom; 2Department of Cellular and Molecular Physiology, University of Liverpool, Liverpool, United Kingdom; 3Sichuan Provincial Pancreatitis Centre, Department of Integrated Traditional Chinese and Western Medicine, West China Hospital, Sichuan University, Chengdu, China.
Background & Aim: Cyclophilin D inhibition protects against acute pancreatitis (AP) but all drugs with this action (Cyclosporin A (CsA), DEB025, NIM811) inhibit other cyclophilins non-specifically, which may be deleterious at higher doses. We determined the concentration- and dose-dependence of the protective effects of CsA in pancreatic acinar cells (PAC) and AP.
Methods: Fresh isolated murine PAC were exposed to taurolithocholic acid 3-sulfate (TLCS, 500 μM) or palmitoleic acid (POA, 100 μM) or cerulein (CER, 10 nM) with or without CsA (100 nM to 50 μM) and cell fate determined with propidium iodide or caspase 3/7 substrate fluorescence over 8 h. Mice received retrograde pancreatic ductal infusion of TLCS (3 mM) or two hourly intraperitoneal injections (ipi) of POA (150 mg/kg) and ethanol (1.35 g/kg) or seven hourly ipi of CER (50 μg/kg) to induce AP, with or without CsA (0.5 to 75 mg/kg) after AP induction. AP was assessed at 24 h (12 h with CER) by biochemistry, immunology and histopathology.
Results: CsA reduced toxin-induced necrotic cell death pathway activation in PAC more effectively at lower concentrations with little effect on toxin-induced apoptotic cell death pathway activation. CsA at 2 or 5 mg/kg was most protective in all AP models, consistently reducing pancreatic myeloperoxidase (all P<0.01) and overall histopathology score (all P<0.05). High doses of CsA were less effective, increasing serum interleukin-6 and lung myeloperoxidase in POA- and CER-AP.
Conclusion: We found the non-specific cyclophilin inhibitor CsA to show an inverted U concentration- and dose–response protective effect in PAC and AP respectively. This work identifies a potential challenge in use of this class of drug in patients and points to the need to develop specific cyclophilin D inhibitors.
Sennoside A Affect Intestinal Motility by Reducing IL-1β and TNF-α to Increase CPI-17 in Small Intestinal Smooth Muscle Cells in Rats With Acute Necrotising Pancreatitis
C. Zhang,1 Z.Q. Lin,1 W. Zhang,2 X. Zhang,1 C. Du,1 P. Xue,1 Q. Xia.1 1Department of Integrated Traditional Chinese and Western Medicine, Sichuan Provincial Pancreatitis Centre, West China Hospital, Sichuan University, Chengdu, China; 2Department of Critical Care Medicine, West China Hospital, Sichuan University, Chengdu, China.
Background: Sennoside A is main active ingredient of Rheum officinale Baill.used in ChaiQinChengQi decoction to treat severe acute pancreatitis (SAP) for many years in West China Hospital, and it improves intestinal motility in SAP, but the mechanism is not very clear.
Aim: To investigate how sennoside A affect intestinal motility on small intestinal smooth muscle cells (SISMCs) in rodent model of acute necrosis pancreatitis(ANP).
Methods: Seventy five male Sprague–Dawley rats were randomly and equably divided into five groups:control, model, model+6h, carbachol(Cch), and sennoside A(SA); The model was constructed by injecting intraperitoneally with 30% L-ornithine(3g/kg) and was sacrificed at the 24th hour; Carbacholine(60ug/kg) and SA(6mg/kg) were used respectively by intraperitoneal injection and tail intravenous injection after successfully making model which was sacrificed at the 30th hour. The pancreas was inspected with hematoxylin and eosin staining. SISMCs were seperated by enzyme digestion and mechanical isolation, and detected by anti-α-SMA staining. The protein and the mRNA expression of proteinkinase C-potentiated phosphatase inhibitor of l7 ku (CPI-17), interlukin-1β (IL-1β) and tumor necrosis factor alpha(TNF-α) in SISMCs of rats were examined by RT-PCR and Western-blot.
Results: Histopathologicaly, there were very significant differences among control versus model groups, Cch, and SA (p<0.01).The expression of CPI-17 mRNA in SA was remarkable higher than it in model groups and Cch (p<0.05) and as well as CPI-17 protein expression; the mRNA expression of IL-1β and TNF-α in SA were obviously lower than it in model groups (p<0.05), it was the same as IL-1β and TNF-α protein expression.
Conclusion: These data suggest Sennoside A might affect intestinal motility in SISMCs in ANP by reducing IL-1β and TNF-α to up-regulate CPI-17.
Chemotherapy-Induced Senescence Phenotype Converse Drug Resistance in Pancreatic Cancer
Y. Zhang,1 Y. Wang,2 B. Ji.1 1Deparment of Cancer Biology, Mayo Clinic, Jacksonville, FL; 2Biochemistry and Molecular Biology, Mayo Clinic, Rochester, MN.
Background: Despite of current advances of diagnosis and treatment, pancreatic cancer patients still suffer from the worst survival rate of all cancers and have limited benefits from most chemo-therapies. Recent studies suggest that cellular senescence is a mechanism of chemotherapy-induced tumor inhibition/regression. However, senescent cells are still dormant living cells and these cells may contribute cancer remission that kills cancer patients. Here we test our hypothesis that chemotherapy incudes senescence through upregulation of cell cycle inhibitors and targeting these pathways overcomes chemo-resistance.
Methods and Results: Pancreatic cancer cell lines (AsPC-1, BxPC-3, MIA PaCa-2 and PANC-1) were treated with gemcitabine and MLN. These drugs inhibited cell growth as measured by MTT and BrDU assays. Within the clinically relevant concentrations, they induced cell apoptosis but failed to kill all the cells. The residual surviving cells exhibited senescent phenotype. This phenotype was associated with increased expression of cell cycle inhibitor genes including Rb, p57 and Myt1. Lentiviruses expressing shRNAs was introduced to knockdown these genes in pancreatic cancer cell lines. Our results showed that shRNA group attenuated both gemcitabine and MLN-induced senescence and increased apoptotic cell death. In orthotopic models, pancreatic cancer cells with stable expression of the shRNAs exhibited increased sensitivity to both gemcitabine and MLN. The mice bearing cancer cells expressing the shRNAs had an improved survival rate and decreased occurrence of ascites.
Conclusion: Our results showed that a combination of shRNAs targeting Rb, p57 and Myt 1 decreased chemotherapy drug-induced cellular senescence and sensitized pancreatic cancer cells to chemotherapy drugs. These data suggested targeting senescence mechanisms may potentiate the effects of chemotherapy in pancreatic patients. This novel concept provides potential insights for developing new combinational cancer therapeutics for pancreatic cancer to overcome chemotherapy resistance.