Abstracts of Papers Submitted to the 49th Annual Meeting of the American Pancreatic Association, October 31–November 3, 2018, Miami Beach, Florida

doi: 10.1097/MPA.0000000000001177
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Germline Mutation Status as a Risk Factor for Neoplastic Progression Among High Risk Individuals Undergoing Pancreatic Surveillance

T. Abe,1 K. Tamura,1 M. Ford,1,2 P. McCormick,3 M. Borges,1 J.A. Almario,1,2 A. Blackford,4 E.J. Shin,2 A.M. Lennon,2 R. Hruban,1 M.I. Canto,1,2 M. Goggins.1,2,4 1 Departments of Pathology, 2 Medicine, and The Sol Goldman Pancreatic Cancer Research Center, The Johns Hopkins University School of Medicine, Baltimore, MD; 3 University College Dublin, Leinster, Ireland; 4Department of Oncology, The Sol Goldman Pancreatic Cancer Research Center, The Johns Hopkins University School of Medicine, Baltimore, MD.

Background: Most patients who undergo pancreatic surveillance for their family history of pancreatic cancer have not been tested to determine if they carry mutations in germline susceptibility genes that contribute to pancreatic cancer risk.

Methods: Of 464 individuals undergoing pancreatic surveillance as part of the Cancer of the Pancreas Screening program at Johns Hopkins Hospital, 120 were known to carry germline mutations in pancreatic cancer susceptibility genes when they were enrolled into pancreatic surveillance program; 344 individuals were undergoing pancreatic surveillance because of their family history of pancreatic cancer. To more accurately determine risk factors of neoplastic progression (the development of pancreatic cancer or high-grade dysplasia), we used next-generation sequencing to identify previously unrecognized germline mutations in the 344 individuals undergoing pancreatic surveillance for their familial risk.

Results: Of the 344 HRIs in the familial risk group, next-generation sequencing analysis identified 15 individuals (4.6%) with a new pancreatic cancer susceptibility gene mutation (12 ATM, etc). The cumulative incidence of neoplastic progression was significantly higher in the germline mutation risk (n = 135) group compared to the familial risk group (n = 329) (P = 0.003).

Conclusion: The cumulative incidence of pancreatic cancer/high-grade dysplasia is significantly higher among those with a germline mutation in a pancreatic cancer susceptibility gene than among than those with a familial risk without a known germline mutation. Gene testing individuals who meet criteria for pancreatic surveillance based on their family history may better define those individuals most at risk of neoplastic progression.

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Nutritional Status of Children and Adults Before Total Pancreatectomy With Islet Autotransplantation: Updates From Prospective Observational Study of TPIAT (POST)

M. Abu-El-Haija,1 S.J. Schwarzenberg,2 Y. Yang,2 D. Adams,3 S. Ahmad,4 A.N. Balamurugan,5 G. J. Beilman,2 S. Chinnakotla,2 D.L. Conwell,6 T.B. Dunn,2 M.L. Freeman,2 T.B. Gardner,7 M. Hughes,5 V. Kirchner,2 L.F. Lara,6 L. Long-Simpson,2 K. Morgan,3 J.D. Nathan,1 B. Naziruddin,8 A. Posselt,9 T.L. Pruett,2 V.K. Singh,10 K. Smith,7 M. Wijkstrom,11 P. Witkowski,12 J. Hodges,2 M.D. Bellin.2 1 Cincinnati Children’s Hospital, Cincinnati, OH; 2 University of Minnesota Minneapolis, MN; 3 The Medical University of South Carolina, Charleston, SC; 4 University of Cincinnati Medical Center, Cincinnati, OH; 5 University of Louisville, Louisville, KY; 6 The Ohio State University Medical Center, Columbus, OH; 7 Dartmouth-Hitchcock Medical Center, Lebanon, NH; 8 Baylor Health, Dallas, TX; 9 University of California San Francisco, San Francisco, CA; 10 John Hopkins Medical Institutions, Baltimore, MD; 11 University of Pittsburgh Medical Center, Pittsburgh, PA; 12University of Chicago, Chicago, IL.

Background: Chronic pancreatitis (CP) and recurrent acute pancreatitis (RAP) cause significant morbidity including nutritional deficiency. Nutritional status and associated risks in patients undergoing TPIAT are poorly understood.

Methods: Consenting patients undergoing TPIAT at 9 centers in the U.S. were prospectively enrolled in POST (A Prospective Observational Study of TPIAT). Pre-surgery, we evaluated body mass index, pancreatic enzyme replacement therapy (PERT), vitamin deficiency, and exocrine pancreatic insufficiency (EPI), comparing children with adults and effects of CP and genetic risk factors. Overweight was defined as >85th percentile or >25 kg/m2 and underweight as <10th percentile or <18.5 kg/m2 for children or adults respectively.

Results: Currently 112 participants are enrolled (25% children; 66% female). Known genetic risk factors for pancreatitis included PRSS1 (n = 24), SPINK1 (n = 17), and CFTR (n = 32). A CP diagnosis was more frequent in adults (85%) than children (68%, P = 0.054), while ≥1 AP episode was more common in children (27, 96% vs 65, 77% of adults, P = 0.02). Similar proportions of children and adults used PERT (75% and 80% respectively, P = 0.5), with a similar dose in units/kg/meal (P = 0.9). Prevalence of overweight (46% vs 52%, P = 0.6), underweight (0% and 6%, P = 0.33), and EPI by fecal elastase (40% and 46%, P = 0.7) were similar in children vs adults.

Vitamin D deficiency (<20 ng/mL) was present in 18% of adults and 4% of children (P = 0.1). PRSS1 mutation was associated with underweight (29% vs 8% of those without it or not tested, P = 0.01), and with fecal elastase <100 (80% vs. 29%, P < 0.01), despite a similar duration of disease to non-PRSS1 (9.01 ±11.55 vs 6.49 ±5.85, P = 0.31). There were no differences in nutritional status by CFTR or SPINK1 mutation status.

Conclusion: When considering TPIAT in CP patients, special consideration should be given to nutritional risks. Those with PRSS1 mutations may be at higher risk for underweight status and exocrine pancreatic insufficiency.

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Lowest Costs and Mortality Rates Among Pancreatic Resections in High-Volume Centers: A Nationwide Study in Finland 2012-2014

R. Ahola,1 J. Sand,1 J. Laukkarinen.1,2 1 Department of Gastroenterology and Alimentary Tract Surgery, Tampere University Hospital, Tampere, Finland; 2University of Tampere, Tampere, Finland.

Introduction: Centralization of pancreatic surgery has proceeded gradually in Finland. The aim of this study was to analyze the effect of operation volumes on short-term prognosis and costs of the operative treatment during the first 90 days after the procedure.

Methods: Patients who had undergone pancreatoduodenectomy or total pancreatectomy in Finland between 2012 and 2014 were selected from the national register. Demographic, operative and complication data was recorded up to ninety days postoperatively. Readmissions as well as possible treatment in other hospitals than the operative hospital were included. Complications were classified according to Clavien-Dindo. The operation volume was defined according to the yearly rate of PDs (pancreatoduodenectomy) as high (≥20, HVC), intermediate (6-19, MVC) and low (≤5, LVC) volume centers. Costs based on the 2012 billing list of Tampere University Hospital and prices were adjusted with the cost of one post-operative day in surgical ward.

Results: 501 patients were included into the study. Demographics did not differ between the centers. 30-day and 90-day mortality were significantly the lowest in HVCs (0.7% vs 8.8- 12.1% and 1.8% vs 10.4-15.2%; P < 0.01). Operation volume and age were significant factors in multivariate analysis. Median costs were at lowest in the HVC group among all patients (P = 0.014), among cancer patients with Clavien-Dindo classes 0-II (P = 0.002), among patients over 75 years (P = 0.002) and among patients who survived over 5 days (P = 0.010).

Conclusion: This nationwide study showed the safest and the most cost-effective results in pancreatic surgery in high-volume centers. Mortality rates were at lowest in HVCs both in 30- and 90-days postoperatively as well as overall costs were lowest in the HVC-group. Better short-term prognosis and lower overall costs favor centralization of pancreatic surgery to high-volume centers.

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Risk Stratification Score for Post-Endoscopic Retrograde Cholangiopancreatography (ERCP) Pancreatitis

V.S. Akshintala,1 A. Kamal,1 J. Elmunzer,2 A. Andriulli,3 R. Talukdar,4 M.K. Goenka,5 R. Kochhar,6 M.A. Khashab,1 A.N. Kalloo,1 D.N. Reddy,4 V.K. Singh.1 1 Johns Hopkins Hospital, Baltimore, MD; 2 Medical University of South Carolina, Charleston, SC; 3 Casa Sollievo della Sofferenza, San Giovanni Rotondo FG, Italy; 4 Asian Institute of Gastroenterology, Hyderabad, India; 5 Apollo Gleneagles Hospitals, Kolkata, India; 6Postgraduate Institute of Medical Education and Research, Chandigarh, India.

Background: Pancreatitis is the most common serious complication of endoscopic retrograde cholangiopancreatography (ERCP). Patient and procedure related risk factors for the development of post-ERCP pancreatitis (PEP) were well described but a risk assessment score is not available.

Methods: Model derivation and validation was based on data from participants of prior randomized controlled trials (RCT). The predictors were identified by univariable analysis followed by ridge regression analysis to incorporate shrinkage as a model fitting procedure. The PEP risk score was developed using regression coefficients obtained from the model. Model performance was evaluated using 10 fold cross validation. Classification and regression tree (CART) modeling was used to determine cutoff values for high, intermediate, and low risk based on the total PEP score.

Results: A total of 2994 patients (mean age, 54.38 years; 62% females) were included in the model among whom 10.05% developed PEP. Thirteen variables were identified as important predictors of PEP which were included in the risk score. Prophylactic strategies such as rectal indomethacin and pancreatic duct (PD) stent were included in the regression model to adjust for confounding but not in the risk score. The discriminatory power of the model based on the cross validation was 0.90. The CART modelling identified three strata of risk categories as low (0–12), intermediate (13–38) and high risk (39–100). The PEP risk estimate of each participant undergoing ERCP can be assessed using a log transformation calculator based on the total score derived from regression coefficients of the model.

Conclusion: This simple risk stratification tool can predict the risk of development of PEP. Data from future RCTs and prospective studies can be used to further train the model, validate externally and evaluate the risk reduction from prophylaxis strategies. This will assist the provider to suitably choose among intravenous hydration, rectal indomethacin and PD stent.

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Does Choice of Approach to Pancreatectomy Affect Postoperative Outcomes in Patients Undergoing Resection for Chronic Pancreatitis: A NSQIP Study

E.M. Aleassa,1,2 G. Morris-Stiff.1 1 Section of Hepato-Pancreato-Biliary Surgery, Digestive Diseases and Surgery Institute, Cleveland Clinic Foundation, Cleveland, OH; 2Department of Surgery, College of Medicine and Health Sciences, United Arab Emirates University, Al-Ain, UAE.

Background: Pancreatic resection is the optimal treatment for a subset of patients with chronic pancreatitis (CP). Whilst open and minimally invasive techniques (MIS) are applicable, the optimal approach has not been defined. The aim of the current study was to evaluate the influence of surgical approach (open versus MIS) on outcome in patients undergoing resection for chronic pancreatitis.

Methods: The American College of Surgeons - National Surgical Quality Improvement Program (ACS-NSQIP) Pancreatectomy Targeted Participant Use Data File was queried to identify patients undergoing pancreatoduodenectomy (PD) or distal pancreatectomy (DP) for CP. Patients converted from an MIS procedure were excluded. Surgical site infection (SSI), postoperative pancreatic fistula (POPF), and delayed gastric emptying (DGE) rates were evaluated as were length of stay (LOS), 30-day readmission (30-R), and 30-day mortality (30-M).

Results: From 2014–2016, 1258 patients (DP = 396 [31.5%]; PD = 862 [68.5%]) underwent resection. The rates of superficial (1.7% vs. 5.4%; P = 0.17), deep (0.9% vs. 1.8%; P = 0.68), and organ space SSI (4.3% vs.13.6%; P = 0.007) were less in the MIS-DP cohort as was DGE (1.7% vs 6.9%; P = 0.038). The rate of POPF was higher after MIS-DP (21.7% vs. 18.6%; P = 0.48).

For PD, the superficial (4.3% vs. 8.7%; P = 0.42), deep (2.2% vs. 1.8%; P = 0.59), and organ space SSI (10.9% vs. 9.1%; P = 0.6) rates were not different by approach as were, DGE (13% vs. 14%; P = 0.86) and POPF (11.1% vs. 12.5%; P = 0.78). LOS was less for MIS-DP (5 [interquartile range {IQR}, 4- 6] vs. 7 [IQR, 7-10] days; P < 0.001) and MIS-PD (6 [IQR, 5-10] vs. 8 [IQR, 7-13] days; P = 0.002).

30-R rates were clinically but not statistically significant in favor of MIS for DP (19.4% vs. 11.3%; P = 0.053) and PD (23.9% vs. 13.7%; P = 0.055). The mortality rates for DP (0.9% vs 1.1%; P > 0.999) and PD (2.2% vs 2%; P = 0.61) patients were comparable.

Conclusion: The choice of surgical approach influences the early postoperative outcomes in patients undergoing DP for chronic pancreatitis, favoring an MIS technique. However, the benefits of an MIS approach were not seen for PD except for a shorter length of hospital stay.

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Necroptosis at Invasion Front of Pancreatic Cancer Can Promote Cancer Cell Migration and Invasion

Y. Ando,1 K. Ohuchida,1 A. Yonenaga,1 A. Sagara,1 S. Kibe,1 S. Takesue,1 M. Nakayama,1 K. Shindo,2 T. Moriyama,1 K. Nakata,1 Y. Miyasaka,1 T. Ohtsuka,1 K. Mizumoto,1 M. Nakamura.1 1 Department of Surgery and Oncology, Graduate School of Medical Sciences Kyushu University, Fukuoka, Japan; 2Department of Endoscopic Diagnostics and Therapeutics, Kyushu University Hospital, Fukuoka, Japan.

Background: Necroptosis is regulated necrosis, which depends on the formation of the necrosome, consisting of receptor-interacting serine/threonine protein kinase-1/3 (RIPK1/3), and mixed lineage kinase domain-like (MLKL). It leads to rapid plasma membrane permeabilization and to the release of cellular contents. Pancreatic cancer has potential inducer of necroptosis, such as tumor necrosis factor receptor-1 (TNFR1), TNF- related apoptosis-inducing ligand (TRAIL) receptors and reactive oxygen species. Recent studies suggested that necroptosis plays both positive and negative roles in the development of cancer, but its role in pancreatic cancer is unclear. Here we aimed to elucidate the siginificance of necroptosis in pancreatic cancer.

Methods: We performed immunohistochemical analysis and evaluated the expression and localization of RIPK3 and MLKL in pancreatic cancer. In the in vitro experiment, we induced necroptosis in pancreatic cancer cells (AsPC-1, BxPC-3, PANC-1), then investigated the changes in the ability of migration and invasion of cancer cells by transmembrane migration and matrigel invasion assays.

Results: RIPK3 and MLKL were highly expressed in human pancreatic cancer than in the surrounding normal tissue. Interestingly, RIPK3 intensity was significantly higher at the invasion front of the tumor than in the core. We induced necroptosis in AsPC-1 and BxPC- 3 cells after the treatment of TNF-α and Second Mitochondrial-Derived Activator of Caspase mimetic (TS treatment) with pan-caspase inhibitor zVAD-fmk, but not in PANC-1 cells. On the other hand, TS treatment induced apoptosis in PANC-1 cells. The conditioned medium (CM) of necroptosis-induced BxPC3 and AsPC1 cells promoted migratory and invasive behavior in cancer cells, but CM of apoptosis-induced PANC-1 did not.

Conclusions: The principal components of necrosome were expressed in human pancreatic cancer. Particularly, RIPK3 was highly expressed at invasion front of tumor and the present findings suggest that necroptosis in pancreatic cancer promotes cancer cell migration and invasion.

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Management of Postoperative Complications May Favor the Centralization of Also Distal Pancreatectomies

A. Antila,1 R. Ahola,1 J. Sand,2 J. Laukkarinen.1,3 1 Department of Gastroenterology and Alimentary Tract Surgery, Tampere University Hospital, Tampere, Finland; 2 Päijät-Häme Central Hospital, Lahti, Finland; 3Faculty of Medicine and Life Sciences, University of Tampere, Tampere, Finland.

Background and Aim: Centralization of pancreatic surgery has proceeded in many countries including Finland during the past few years. Larger hospital volume has been shown to improve post-operative morbidity and mortality, as well as long-term survival after pancreatoduodenectomy (PD). However, information about the effect of hospital volume specifically on distal pancreatic resections (DP) is lacking.

The aim of this study was to investigate the effect of hospital volume on the postoperative complications in DP patients in Finland.

Methods: All DP operations performed in Finland during 2012-2014 were analyzed from national registers. Hospital volumes and all post-operative complications were graded. Postoperative pancreatic fistulae (POPF) were graded according to the new ISGPF classification and overall complications according to Clavien-Dindo classification. Reoperations and 90-day mortality were recorded. Hospital volume was defined to reflect the number of pancreatic resections (PR) in general and DP in particular as HVC (>40 PR/yr or >20 DP/3 yr, MCV (16–40 PR/yr or 10–19 DP/3 yrs) and LVC (<8 PR/yr or <5 DP/3 yrs).

Results: 199 DP were performed nationwide in Finland during 2012–2014, overall in 18 different hospitals. 41% (82) were performed in HVC (2 hospitals), 44 % (87) in MVC (6 hospitals) and the rest 15% (30) in LVC (10 hospitals). Patient demographics did not differ between the hospital volume groups. The rate of clinically relevant POPF, Clavien-Dindo grade 3-5 complications and 90-day mortality were without significant difference between the different hospital volumes. Significantly more reoperations were performed in LVC (10%) than in HVC (1.2%) or MCV (1.1%); P = 0.036.

Conclusion: Even though the rate of postoperative complications after DP is not affected by the hospital volume, reoperations are performed ten times more often in the low-volume centres. Optimal management of postoperative complications may favor centralization of not only PD, but also DP.

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Plasma suPAR May Help to Distinguish Malign and Benign Pancreatic Diseases

A. Aronen,1 J. Aittoniemi,2 R. Huttunen,3 A. Nikkola,1 I. Rinta-Kiikka,4,5 J. Nikkola,1 O. Limnell,4 I. Nordback,1 J. Sand,1 J. Laukkarinen.1,4 1 Department of Gastroenterology and Alimentary Tract Surgery, Tampere University Hospital, Finland; 2 Fimlab Laboratories, Tampere, Finland; 3 Department of Internal Medicine, Tampere University Hospital, Finland; 4 University of Tampere, Faculty of Medicine and Life Sciences, Tampere, Finland; 3 Department of 5Radiology, Tampere University Hospital, Finland.

Background: SuPAR (soluble urokinase plasminogen activator receptor) is a marker associated with inflammation and certain malignancies. Previously we have shown that it is possible to predict severity of acute alcohol-induced pancreatitis (AAP) and long-term survival after recovery from first AAP using P-suPAR levels. Urinary suPAR/creatinine ratio is elevated both in chronic pancreatitis (CP) and in pancreatic cancer (PC). We studied levels of P-suPAR in CP during long-term prospective follow-up and in patients operated because of malignant-suspicious pancreatic mass to investigate whether P-suPAR could be used to distinguish malign and benign diseases.

Methods: Two groups were studied. Firstly, 83 patients (median age, 47.5; range, 25–71 years, 90% male) diagnosed with first AAP were prospectively followed for median 7.0 (range, 0.3-9.8) years with repeated P-suPAR measurements. Development of CP and other covariates during the follow-up were registered. Secondly, P-suPAR levels were preoperatively measured from 23 patients (median age, 69.0; range, 34–84 years; 52% male) who were operated because of malign-suspicious pancreatic mass.

Results: Twelve patients from AAP group developed CP during follow-up. P-suPAR levels were low after recovery from AAP and stayed low during follow-up, despite of development of CP. P-suPAR of patients with CP was 2.6 (1.8–3.6) ng/mL (median [IQR]). 18 patients operated for a pancreatic mass were diagnosed with pancreatic cancer, and the final histology was benign in five patients. P- suPAR levels of PC patients (3.5 [2.7–4.6] ng/mL, n = 18) were significantly higher than in patients with benign histology (2.0 [1.8–2.8]; ng/mL, n = 5), P = 0.024.

Conclusion: P-suPAR levels remain low after AAP during long-term follow-up despite of development of CP. P-suPAR levels are significantly higher in PC compared to patients with benign histology. Thus P-suPAR may possibly be used to distinguish CP from PC.

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The Role of ATF3 for PDAC Initiation and Progression

N. Azizi,1,2 C. Pin.1,2,3,4 1 Children’s Health Research Institute; Departments of 2 Physiology and Pharmacology, 3 Pediatrics, and 4Oncology, Schulich School of Medicine & Dentistry, Western University, London, Canada.

Background: Pancreatic Ductal Adenocarcinoma (PDAC) is the 3rd leading cause of cancer- related death in North America with a 5-year survival rate <8%. Despite identifying key events in PDAC progression, including activating mutations in KRAS (KRASG12D) and acinar-to-duct cell metaplasia (ADM), survival rates has remained low. Further characterization of specific, early events during PDAC are needed to identify diagnostic or therapeutic targets. We recently showed loss of Activating Transcription Factor 3 (ATF3) reduced ADM during acute pancreatic injury. The goal of this project was to determine if ATF3 is required for KRASG12D-mediated ADM and PDAC. We hypothesize that the absence of ATF3 reduces KRASG12D-mediated ADM and progression to PDAC.

Methods: Atf3-/- mice were mated to Ptf1acreERT/+ loxP-stop-loxP KRASG12D (Ptf1acreERT/+ LSL-KRASG12D) mice which harbor inducible KRASG12D and lineage-tracing (ROSA26-LSL-YFP) alleles, and creERT targeted to the Ptf1a gene. The resulting mice (referred to as APK), Ptf1acreERT/+ LSL-KRASG12D mice, and appropriate control genotypes, were treated with tamoxifen (Tx) at 2–4 months of age, followed one-week later by cerulein injections to induce pancreatic injury. Tissue morphology and gene expression were assessed 14 and 35 days post-cerulein treatment. Acinar cell cultures were also established and characterized following Tx treatment.

Results: APK and Ptf1acreERT/+ LSL-KRASG12D mice developed fibrotic pancreatic tissue with significant ADM and PanIN lesions post Tx treatment. However, APK pancreata were significantly smaller, with fewer SOX9+ (ADM marker) and Ki67+ (proliferation marker) cells relative to Ptf1acreERT/+ LSL-KRASG12D mice. Preliminary results in cultures suggest the absence of ATF3 reduced ADM in vitro as well.

Conclusion: APK mice still show significant deficits in acinar cells and increased tissue fibrosis suggesting ATF3 enhances cell survival in response to injury. However, ADM is reduced in the absence of ATF3. Future work will use cell-specific deletions of Atf3 in macrophages or stellate cells to delineate the cell types that contribute to the APK phenotype.

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Impaired Bacterial Clearance Underlies the Progression of Sterile to Infected Necrosis and Sepsis

A. Bag, B. Khatua, C. de Oliveira, V.P. Singh. Department of Medicine, Mayo Clinic, Scottsdale, AZ.

Background: Necrotizing acute pancreatitis (NAP) is commonly complicated by infection and sepsis. In contrast, severe inflammatory bowel disease (IBD) despite extensive mucosal ulceration, and fistulae causing abdominal abscesses is rarely associated with sepsis. These observations, and the frequent but transient bacteremia after dental procedures suggests that impaired pathogen clearance perhaps due to the high fatty acids (FA) in NAP may cause infections and sepsis in NAP.

Methods: Peritoneal macrophages and the J774A.1 (macrophage) cell line were exposed to the most prevalent FAs in NAP, and endpoints described under results were measured. In vivo the most prevalent FA (oleate) was administered to mice at concentrations relevant to NAP (0.3% body weight) and clearance of intraperitoneal GFP E.coli, circulating CD68 positive injury (annexin V) and bacteremia were measured.

Results: The unsaturated FAs oleate and linoleate (<200 nM unbound concentrations) unlike the saturated FA palmitate rapidly integrated into cell membranes, increased cytosolic calcium, caused mitochondrial depolarization, reduced oxygen consumption, reduced ATP levels, globally reduced protein tyrosine phosphorylation, impaired uptake of E. coli, lysosomal acidification, phagocytosis, and increased LDH leakage significantly (in call cases by > 3 folds) vs. controls. In vivo oleate increased unbound FAs from 6 ± 3 nM to 40 ± 10 nM (P < 0.01), increased circulating annexin V positive (8.5 ± 5.2% vs. 1.5 ± 0.7%, P < 0.02) and % GFP E.coli positive WBCs (15.6 ± 7.8% vs. 0.03 ± 0.04%, P < 0.01) and peritoneal E. coli by >105 fold, along with causing renal failure and shock, thus fulfilling criteria of sepsis.

Conclusion: The enriched unsaturated FA environment of NAP results in impaired bacterial clearance due to lipotoxic impairment of innate immune cell function. This results in infected NAP and sepsis. Therefore impaired pathogen clearance is more important in the progression of sterile NAP to septic NAP than bacterial translocation which commonly occurs in IBD and dental procedures.

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Fentanyl Exacerbates the Severity of L-ornithine-induced Experimental Acute Pancreatitis

E.R. Bálint,1 Z. Balla,1 B. Kui,1 L. Kiss,1 G. Für,1 V. Venglovecz,2 P. Pallagi,3 J. Maléth,2 P. Hegyi,4,5 Z. Rakonczay, Jr.1 Departments of 1 Pathophysiology, 2 Pharmacology and Pharmacotherapy, 3 First Department of Medicine, University of Szeged, Szeged, Hungary; 4 Institute for Translational Medicine, University of Pécs, Pécs, Hungary; 5MTA-SZTE Momentum Translational Gastroenterology Research Group, University of Szeged, Szeged, Hungary.

Background: Opioids are widely used for the management of pain associated with acute pancreatitis (AP). However, Barlass et al have demonstarted that morphine treatment worsened the severity of AP in mice. Due to its good safety profile, the application of fentanyl is increasing. In our study, we aimed to assess the effect of fentanyl on the severity of AP in rats.

Methods: Wistar rats (200-250 g) were administered 3x0.1 or 3x0.2 mg/kg fentanyl intraperitoneally (i.p.) at 10 hourly intervals. AP was induced by i.p. injection of 3 g/kg L- ornithine 1 hour before (pre-treatment) or after the first fentanyl injection (post-treatment). Rats treated with L-ornithine and/or fentanyl were compared to their respective saline-treated controls. To determine AP severity, pancreatic water content, serum amylase and pancreatic myeloperoxidase activities were measured, and histological parameters were evaluated.

Results: In the pre-treatment group, 3x0.2 mg/kg fentanyl significantly increased serum amylase and pancreatic myeloperoxidase activities, as well as pancreatic necrosis in L- ornithine-induced AP. However, in case of pancreatic leukocyte infiltration 3x0.1 mg/kg fentanyl administration induced significant elevation compared to the AP group without analgesia, while the higher dose did not cause significant difference. Fentanyl injected after AP induction did not alter any of the histological or laboratory parameters. Fentanyl in itself did not significantly influence any of the measured parameters compared to the respective control groups.

Conclusion: Fentanyl treatment exacerbated the severity of experimental AP in rats when administered before AP induction, whereas it did not change the outcome of the disease when injected after AP induction. Timing and dosage of analgesic administration may be crucial in determining disease severity.

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World-Wide Variance in Reporting of Elective Surgery for Chronic Pancreatitis

M. Baltatzis, S. Jegatheeswaran, A.K Siriwardena. Regional Hepato-Pancreato-Biliary Unit, Manchester Royal Infirmary, Manchester, UK.

Background: One of the difficulties facing clinicians seeking to treat patients with chronic pancreatitis (CP) is the selection of an appropriate procedure from the range of reported operations. Local or national practices may influence choice of procedure. This study explores geographical variation in reporting of elective surgery for CP.

Methods: A systematic search of the literature was performed for reports of five selected procedures for chronic pancreatitis: Duodenum-preserving pancreatic head resection (DPPHR), Total pancreatectomy with islet auto-transplantation (TPIAT), Frey pancreaticojejunostomy, thoracoscopic splanchnotomy and the Izbicki V-shaped resection. The keyword and MESH heading “chronic pancreatitis” was used. 144 papers met inclusion criteria and are the study population. Data were extracted independently by two authors. For each procedure, the first reported citation was identified and reported. All subsequent reports are then identified. The principal focus is on the country of origin of these reports Risk of bias in reporting was assessed using the Cochrane collaboration tool and the study was passed by institutional review board.

Results: There were 33 reports of DPPHR. 21 (64%) were from Germany. There were 60 reports of TPIAT, 53 (88%) from the United States. Over a 20-year period only two reports of TPIAT are from outwith the United States and UK. The 34 reports of the Frey pancreaticojejunostomy originate from 12 countries. There were 20 reports of thoracoscopic splanchnotomy originating from 9 countries. All 3 reports of the Izbicki “V” procedure are from Germany. These latter two procedures are less frequently reported in the last 10 years.

Conclusion: This report highlights the existence of dramatic world-wide variations in reporting of elective surgery for chronic pancreatitis. These variations do not necessarily correlate with disease phenotype. There is a need for greater standardization in the selection and reporting of surgery for patients with painful chronic pancreatitis.

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Presence of Pancreatic Specialty Centers is Associated With Increased Treatment of Exocrine Pancreatic Insufficiency in Chronic Pancreatitis

J.A. Barkin,1 J.C. Tsai,2 M. Alsante,3 D.A. Sussman,1 J.S. Barkin.1 1 Division of Gastroenterology, Department of Medicine, University of Miami Miller School of Medicine, Miami, FL; 2 AbbVie, North Chicago, IL; 3National Pancreas Foundation, Bethesda, MD.

Background: Exocrine pancreatic insufficiency (EPI) in chronic pancreatitis (CP) results in multiple nutritional deficiencies & clinical symptoms. Overall prevalence of EPI in CP is 35-50%. Pancreatic enzyme replacement therapy (PERT) is the mainstay of EPI treatment. The National Pancreas Foundation (NPF) has accredited 39 specialty pancreas centers (NPF-C) nationwide since 2015.

Aim: To assess the effect of NPF-C on PERT prescriptions (PERT-Rx) for EPI in CP patients using a nationwide claims database.

Methods: We performed a retrospective analysis of a national claims database (Symphony) of over 250,000,000 patients in the US to identify an aggregated cohort of CP patients and those CP patients with ≥3 PERT-Rx for 2017. CP patients & PERT-Rx were mapped using physician prescriber zip code and stratified into 3 groups: NPF-C zip codes, zip codes within a 50 mile radius of NPF-C, and zip codes over 50 miles. Rates of PERT-Rx in CP patients in NPF-C zip codes were compared to the surrounding 50 mile radius zip codes and those over 50 miles to assess the effect of NPF-C on PERT-Rx using chi-square and ANOVA analyses.

Results: There were 163,724 CP patients identified nationwide in 2017, of which 25,927 had PERT- Rx (Overall treatment 15.8%). 1458/6376 (22.9%) CP patients treated for EPI in NPF-C zip codes received PERT-Rx, compared to 6733/44,680 (15.1%) in the 50 mile surrounding radius (chi-square P < 0.0001). PERT-Rx rates in NPF-C zip codes were significantly higher than in geographically near & remote locations (ANOVA P < 0.0001). Rates of PERT-Rx within 50 miles were numerically similar to those over 50 miles.

Conclusion: Substantial gaps in treatment of EPI in CP patients exist nationwide. Rates of PERT- Rx in CP patients were significantly higher in NPF-C zip codes compared to surrounding areas and zip codes over 50 miles. The presence of pancreatic specialty centers may improve EPI care in CP.

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The sTRA Glycan Increases Screening and Diagnostic Accuracy of CA 19-9 for Pancreatic Cancer

D. Barnett,1,2 Y. Liu,1 B. Staal,1 K. Partyka,1 Z. He,3 Y. Huang,3 A. Singhi,4 R.R. Drake,5 A. Maitra,6 R.E. Brand,4 B.B. Haab.1 1 The Van Andel Research Institute, Grand Rapids, MI; 2 Michigan State University, East Lansing, MI; 3 Fred Hutchinson Cancer Research Center, Seattle, WA; 4 University of Pittsburgh Medical Center, Pittsburgh, PA; 5 Medical University of South Carolina, Charleston, SC; 6The University of Texas MD Anderson Cancer Center, Houston, TX.

Background: The CA 19-9 biomarker is elevated in a substantial group of patients with pancreatic cancer, but not enough to be reliable for the detection or diagnosis of the disease.

Methods: We hypothesized that a glycan called sTRA complements CA 19-9, and that the two biomarkers in combination provide added information over either biomarker individually. We first asked whether the secreted levels of each marker reflected tissue expression. In cell culture and PDX mouse models of pancreatic ductal adenocarcinoma (PDAC), the amount of CA 19-9 or sTRA in the media or serum, respectively, was proportional to the expression in tissue, with each model expressing primarily one or the other. We observed a similar relationship in matched sera and primary tumors from 52 patients with PDAC. Next, we tested whether a biomarker panel comprising CA 19-9 sandwich immunoassay and sTRA detected with two capture antibodies performed better than either marker individually for distinguishing PDAC from benign pancreatic diseases.

Results: In a training set of 197 subjects (97 cancer from all stages and 100 benign disease), a panel optimized for specificity gave sensitivity/specificity 65%/97% compared to 47%/97% for CA 19-9, and a panel optimized for sensitivity gave 96%/67%, compared to 96%/14% for CA 19-9. The application of the markers to an independent, blinded set of 147 subjects gave 54%/95% and 94%/38% for the panels, relative to 30%/97% and 87%/34% for CA 19-9 at the preset thresholds. Adjustments of the marker thresholds upon unblinding of the data yielded 70%/97% and 96%/37%, relative to 31%/97% and 96%/9% for the equivalently-optimized CA 19-9 thresholds.

Conclusion: Thus, sTRA and CA 19-9 are complementary serological biomarkers that in combination improve performance over CA 19-9 for distinguishing pancreatic cancer from benign diseases of the pancreas.

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Continued Failure to Operate on Early Stage Pancreatic Cancer

K. Baugh,1 H.S. Tran Cao,1,2 G. Van Buren,1 E.J. Silberfein,1 C. Hsu,1 O. Barakat,1 W.E. Fisher,1 N.N. Massarweh.1,2 1 Michael E DeBakey Department of Surgery, Baylor College of Medicine, Houston, TX; 2VA HSR&D Center for Innovations in Quality, Effectiveness and Safety, Michael E DeBakey VA Medical Center, Houston, TX.

Background: Although surgery offers the greatest survival benefit in pancreatic ductal adenocarcinoma (PDAC), between 1995 and 2004 only 29% of patients with stage I PDAC underwent surgery. Race, income status, and education level were associated with whether a patient was offered surgery. We hypothesized over the ensuing decade and after illumination of socioeconomic disparities utilization of surgery would increase.

Methods: Retrospective cohort study of patients with clinical stage 1 PDAC (i.e.: T1N0M0 and T2N0M0) in the National Cancer Database (2004–2012). A non-parametric test of trend was used to evaluate utilization of resection over time. Multivariable logistic and Cox regression were utilized to identify factors associated with not receiving resection and to evaluate the association between receipt of surgery and overall risk of death, respectively.

Results: Among 8084 patients, 41.8% did not have surgery–surgical contraindications were documented in 6.5%; 3.1% refused surgery; 30.8% were classified as “not offered surgery”; 1.4% did not have surgery due to “unknown” reasons. Resection increased (45.4% 2004 to 57.7% 2012; trend test, P < 0.001), non-surgical treatment decreased (27.7% to 18.2%; trend test, P < 0.001) and no treatment remained stable. Patients were less likely to undergo surgery if they were black, on Medicare/Medicaid, older than 80 years, had less education, or lower annual income (P < 0.001). Resection was more likely at academic institutions and comprehensive cancer centers (P < 0.001). Surgery was associated with a median survival of 24.0 months compared to 7.9 without surgery (non-surgical cohort—hazard ratio, 2.39; 95% CI [2.26-2.53]).

Conclusion: Although utilization of resection in early stage PDAC has improved, a large percentage of patients without any identifiable contraindications still fail to undergo surgery. Socioeconomic factors continue to be associated with failure to offer surgery, highlighting the need to re-evaluate and address barriers to care.

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Naltrexone and Methyl Naltrexone Reduce the Severity of Chronic Pancreatitis in Mouse Models

E.P. Bava, A. Nikam, M. Tarique, H. Cheema, U. Hooda, R. Dawra, V. Dudeja, A.K. Saluja. Department of Surgery and Sylvester Comprehensive Cancer Center, University of Miami Miller School of Medicine, Miami, FL.

Background: Chronic pancreatitis (CP) leads to irreversible replacement of pancreatic parenchyma with fibrotic connective tissue. There are reports that naltrexone is an antagonist at TLR4 receptors and has anti- muscarinic action. We examined the effect of naltrexone and methyl-naltrexone which are opioid receptor antagonists on CP using two different mouse models of the disease.

Aim: To evaluate the effect of naltrexone and methyl-Naltrexone on the progression and severity of chronic pancreatitis.

Methods: L-arginine induced CP was initiated by i.p. injections of L-arginine (4.5 g/kg x2 hourly, once a week x4). Animals were randomized after third week and assigned to saline, naltrexone (50 mg/kg/d i.p.) or methyl-naltrexone (2.5 mg/kg/d s.c.) group. All animals were euthanized after 7 weeks from start of model. Cerulein-induced CP was induced by cerulein injections (50 ug/kg x7, i.p., hourly x twice weekly x10 weeks). After 6 weeks, animals were randomized and were assigned to either saline or naltrexone or methyl-naltrexone treatment group for 4 weeks. Pancreatic atrophy, fibrosis markers, acinar cells loss, inflammatory cells infiltration were evaluated.

Results: There was significant increase in pancreas to mouse weight ratio for both the drugs in L-arginine CP model (Naltrexone + CP = 4.51 ± 0.47; Methyl-naltrexone + CP = 4.2 ± 0.29, vs CP only = 2.6 ± 0.26; P = 0.0009). The increase in pancreas to mouse weight ratio was reproduced in cerulein model (Naltrexone + CP = 5.27 ± 0.36, Methyl-naltrexone + CP = 5.29 ± 0.18 vs CP only = 3.64 ± 0.2; P = 0.001). H&E showed reduction in pancreatic atrophy and chronic inflammation in treatment groups. Sirius red staining for collagen, fibrosis markers and pro-inflammatory cytokines’ mRNA showed reduction in treatment groups.

Conclusion: Our study shows that Naltrexone and Methyl-Naltrexone reduce acinar cell loss and fibrosis, and promotes pancreatic regeneration thereby reducing the severity and progression of chronic pancreatitis.

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Pirfenidone Treatment Potentiates Reversal of Well-established Chronic Pancreatitis in Mouse Model

E.P. Bava, A. Nikam, M. Tarique, H. Cheema, U. Hooda, R. Dawra, A.K. Saluja, V. Dudeja. Department of Surgery and Sylvester Comprehensive Cancer Center, University of Miami Miller School of Medicine, Miami, FL.

Background: Chronic pancreatitis (CP) is a fibro-inflammatory disease of the pancreas with no known therapeutic agent available to reduce the progression or reverse it. Pirfenidone is an antifibrotic, analgesic, antipyretic, anti-oxidant and anti-inflammatory agent that has been FDA approved for idiopathic pulmonary fibrosis. Its role in chronic pancreatitis has not been investigated before. We explored the antifibrotic effect of pirfenidone and pancreatic regeneration in well-established cerulein CP.

Aim: To evaluate the effect of pirfenidone on well-established chronic pancreatitis in mouse model.

Methods: Cerulein-induced chronic pancreatitis was induced in male and female mice by cerulein injections (50 ug/kg x7,i.p., hourly x twice weekly x10). After 11 weeks, animals were randomized and assigned to either saline or pirfenidone treatment group (400 mg/kg/d by oral gavage for 5 weeks). Animals were euthanized after 17 weeks of start of the model. Pancreatic atrophy, fibrosis, acinar cells loss, and inflammatory cells infiltration were evaluated.

Results: There was significant increase in pancreas to mouse weight ratio in both female and male mice [(female mice: CP only, 6.0 ± 0.32 vs pirfenidone group, 8.61± 0.44, (P < 0.001); control 9.41 ± 0.32 vs pirfenidone group = 8.61 ± 0.44, P > 0.10 ); (male mice: CP only = 4.11 ± 0.17, vs pirfenidone group = 5.92 ± 0.39; P < 0.0001)]. H&E showed reduction in pancreatic atrophy and chronic inflammation in the pirfenidone treated CP groups as compared to CP only groups. Sirius red staining for collagen and fibrosis markers showed reduced fibrosis in pirfenidone groups.

Conclusion: Pirfenidone treatment potentiated reversal of well established CP related changes to near normal in female mice and significantly reduced its severity in male mice. Compared to 11 weeks, at 17 weeks atrophy was less, which indicates that there is some reversal in this model without treatment and pirfenidone is enhancing that process.

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Correlation of Intra-abdominal Pressure (IAP) With Inflammatory Markers and Outcome in Patients With Acute Pancreatitis (AP)

B.L. Bellam,1 Narendra Dhaka,1 S.K. Sinha,1 V. Gupta,2 T.D. Yadav,2 R. Kochhar.1 Departments of 1 Gastroenterology and 2General Surgery, Post Graduate Institute of Medical Education and Research, Chandigarh, India.

Objectives: To ascertain correlation of IAP with inflammatory markers and outcome in AP.

Methods: Baseline IAP (normal ≤5 mm Hg, intra-abdominal hypertension [IAH] >12 mm Hg and abdominal compartment syndrome; ACS >20 mm Hg along with new-onset organ failure) was measured at admission along with inflammatory markers (C-reactive protein, IL-6 and IL-10) in patients with AP from 2016 to 2017. IAP was correlated with outcome measures including organ failure (OF), persistent OF (POF), CT severity index (CTSI), APACHE II score, infective pancreatic necrosis (IPN), hospitalization duration, intensive care unit (ICU) stay, need for percutaneous catheter drainage (PCD) and surgery and mortality.

Results: In 145 (102 males; age, 42.01 ± 1.16 years) patients, disease severity was mild in 15 (10%), moderate in 34 (23%), and severe in 96 (64.9%). Organ failure was seen in 108 (73%) patients of whom 79 (53.4%) had POF. IAH was seen in 85 (58.6%) patients, out of whom 22 (25.8%) had ACS. The mean baseline IAP was 18.46 ± 4.53 vs 7.33 ± 3.08 mm Hg in patients with and without IAH. Levels of IL-6 (P=0.00) and IL-10 (P = 0.00) were significantly higher in patients with IAH as compared to those without IAH but not CRP (P = 0.45). IAH was significantly associated with higher frequency of OF (P = 0.01), POF (P = 0.00) number of OF (P = 0.00), high APACHE II score (P = 0.00), and CTSI (P = 0.00), IPN (P = 0.00), longer hospital stay (P = 0.00), and ICU stay (P = 0.03), increased requirement of PCD (P = 0.00), and increased mortality (P = 0.01), but not with need for surgery (P = 0.12). ACS was found to be significantly associated with POF (P = 0.00), longer hospital stay (P = 0.02), PCD requirement (P = 0.00), and increased mortality (P = 0.02).

Conclusion: IAH correlated with levels of inflammatory cytokines. Patients with IAH had more severe disease, poor outcome and more need for interventions. Our data suggest routine monitoring of IAP in moderate to severe pancreatitis.

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Developing a Serum Proteomics Pipeline for the Mapping of TPIAT-induced Changes to the Serum Proteome

M.D. Bellin,1 T. Bjerg Bennike,2 S. Ahmed,2 Z. Cruz-Monserrate,3 G.J. Beilman,1 D.L. Conwell,3 H. Steen.2 1 Department of Surgery, University of Minnesota, Minneapolis, MN; 2 Boston Children’s Hospital Harvard Medical School, Boston MA; 3The Ohio State Univerisity Wexner Medical Center, Columbus, OH.

Background: The major challenge of managing chronic pancreatitis (CP) is the lack of accurate radiologic and endoscopic modalities for early detection. Biopsy for “gold standard” histology is high risk and exocrine insufficiency is a late event occurring when >90% of the gland has been scarred. TPIAT affords a unique opportunity to study the molecular ramifications upon removal of the pancreas. We hypothesize that the diseased pancreas modulates the serum proteome, which will allow for a more rational choice of blood-based biomarkers for pancreatic disorders.

Aim: Map the TPIAT-induced changes to the serum proteome from TPIAT.

Methods: Serum samples were collected pre- and post-TPIAT from 48 patients. We optimized our plasma/serum proteomics pipeline to a) efficiently process 96 serum samples within a workday and b) map ∼500 plasma/serum proteins covering >7 orders of magnitude dynamic range requiring less than 1 μl of serum or plasma.

Results: A total of 487 proteins were identified in serum ranging from serum albumin with a concentration of ∼1 mM to fatty acid synthetase at a concentration of ∼10 pM. Sixteen serum proteins were identified as being significantly up-/downregulated upon TPIAT. Fourteen of these sixteen proteins had been described as showing pancreatic disease-induced altered abundance levels, including thrombospondin which was recently described as a potential serum marker for pancreatic adenocarcinoma. We also detected several immunoglobulins with increased levels after TPIAT; we had similar findings when mapping the urinary proteomes pre and post TPIAT. Furthermore, we noticed a significant downregulation of those serum apolipoproteins, which have faster clearance rates.

Conclusion: Most of the statistically significant TPIAT-induced changes in the serum proteome have been described in the context of pancreatic diseases and malignancies, validating our findings. However, the often-unexpected directionality of the observed TPIAT-induced changes in the serum proteome highlights the need to differentiate between pancreatitis dependent and pancreatic function- dependent serum proteome changes as TPIAT will have an opposing effect on such changes.

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Investigating the Urinary Proteome Pre and Post Total Pancreatectomy With Islet Auto- transplantation (TPIAT)

M.D. Bellin,1 T. Bjerg Bennike,2 S. Ahmed,2 Z. Cruz-Monserrate,3 R. Arora,2 G.J. Beilman,1 R. Arnaout,2 D.L. Conwell,3 H. Steen.2 1 Department of Surgery, University of Minnesota, Minneapolis, MN; 2 Boston Children’s Hospital Harvard Medical School, Boston MA; 3The Ohio State University Wexner Medical Center, Columbus, OH.

Background: The major challenge of managing chronic pancreatitis (CP) is the lack of accurate radiologic and endoscopic modalities for early detection. Biopsying the pancreas is not feasible due to the associated high risks and exocrine insufficiency often only occurs when >90% of the gland has been scarred. We hypothesize that samples from TPIAT patients afford the unique opportunity to identify potential biological mechanisms and molecular implications of the presence/absence of the pancreas.

Aim: Determine the TPIAT-induced changes to the urinary proteome.

Methods: Urine samples were collected before the surgery and 12 to 18 months post TPIAT from 22 patients. The urine samples were prepared for proteome analysis using our in-house developed high throughput urine proteomics pipeline suing 96-well plate sample processing and LC/MS analysis on QExactive mass spectrometers.

Results: A total of 2484 proteins were identified in urine. 8% of these proteins showed significant up-/downregulation. We noticed a strong sex difference in the TPIAT-induced changes in the urinary proteome: not a single urinary protein showed significant changes pre-/post-TPIAT for the males, while >25% of the female urinary proteome showing multiple-testing corrected statistically significant abundance changes. The causes and implications of this observation is currently being investigated. Additional notable TPIAT-induced changes in the urinary proteomes included the post TPIAT upregulation of all 45 identified immunoglobulins as well as a striking 30- to 50-fold downregulation of the plaque subcomplex of the desmosome (P = 0.1 to 0.0001 at an FDR of 5%). Finally, we noticed that glomerularly filtered proteins behaved differently pre and post TPIAT than proteins of kidney origin such as uromodulin/Tamm-Horsfall protein.

Conclusion: Using our high throughput urine proteomics pipeline identified numerous TPIAT-induced changes to the urine proteome. Detailed proteome analysis indicates a more substantial crosstalk between the pancreas and the kidney than previously appreciated.

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Updates on the Prospective Observational Study of TPIAT (POST): Approach and Participant Characteristics

M.D. Bellin,1 M. Abu-El-Haija,2 D. Adams,3 S. Ahmad,4 A.N. Balamurugan,5 G.J. Beilman,1 S. Chinnakotla,1 D.L. Conwell,6 T.B. Dunn,1 M.L. Freeman,1 T. Gardner,7 J. Hodges,1 M. Hughes,5 V. Kirchner,1 L.F. Lara,6 L. Long-Simpson,1 K. Morgan,3 J.D. Nathan,2 B. Naziruddin,8 A. Posselt,9 T.L. Pruett,1 V.K. Singh,10 K. Smith,7 M. Wijkstrom,11 P. Witkowski,11 Y. Yang.1 1 University of Minnesota Minneapolis, MN; 2 Cincinnati Children’s Hospital, Cincinnati, OH; 3 The Medical University of South Carolina, Charleston, SC; 4 University of Cincinnati Medical Center, Cincinnati, OH; 5 University of Louisville, Louisville, KY; 6 The Ohio State University Medical Center, Columbus, OH; 7 Dartmouth-Hitchcock Medical Center, Lebanon, NH; 8 Baylor Health, Dallas, TX; 9 University of California San Francisco, San Francisco, CA; 10 John Hopkins Medical Institutions, Baltimore, MD; 11 University of Pittsburgh Medical Center, Pittsburgh, PA; 12University of Chicago, Chicago, IL.

Background: The optimal approach to patient selection for and timing of TPIAT remains unclear. The POST study is collecting detailed assessments on TPIAT recipients at multiple U.S. centers to determine what baseline characteristics predict successful relief of pain, improved quality of life, and minimal diabetes.

Methods: Patients scheduled for TPIAT at participating centers are eligible for recruitment. Baseline assessments include patient characteristics, pancreatitis history, pain burden and medications, and quality of life. Follow up at 6 months, 1 year, and yearly thereafter includes assessment of pain and quality of life (Aim 1) and diabetes outcomes (Aim 2). Health care utilization before and after TPIAT is also collected to determine cost-effectiveness of TPIAT (Aim 3). Serum, plasma, urine, genetic tissue, and a pancreas biopsy are collected at baseline for future studies.

Results: As of June 2018, 128 participants were enrolled. Patient characteristics include: 82% with chronic pancreatitis, 81% with recurrent acute pancreatitis, mean age 32.7 (SD, 16.6) years, 35% male, 91% Non-Hispanic, 93% white, and 16% on medical assistance. The most common risk factors for pancreatitis included genetic mutations associated with pancreatitis risk in 55%, and obstructive factors in 29%. Prior treatments included endoscopic therapies in 87%, cholecystectomy in 65%, pancreatic surgery in 13%. 75% used non-opioid and 69% used opioid analgesics in the 2 weeks before enrollment, with mean opioid dose 89.5 (SD, 128.5) oral morphine mg equivalents per day. Numeric pain score (0-10 scale) was 7.8 (SD, 2.7) during acute exacerbations and 5.2 (SD, 2.3) on average at baseline. 10% used insulin before TPIAT.

Conclusions: Patients undergoing TPIAT in POST are more often young, female, Non-Hispanic white, with genetic or obstructive risk factors for pancreatitis. The majority require chronic opioids before TPIAT. Planned follow-up assessments will facilitate risk modeling for predictors of success for pain, quality-of-life, and diabetes outcomes.

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Pancreatic Acinar Carcinoma Has Distinct Mutation Patterns Than PDAC and Neuroendocrine Tumor

Y. Bi,1 Y. Liu,2 M. Raimondo,1 M. Wallace,1,3 K. Mody,3 H. Asbun,4 J. Stauffer,4 B. Ji.5 1 Department of Gastroenterology, Mayo Clinic, Jacksonville FL; 2 Department of Bioinformatics, Mayo Clinic, Rochester MN; Departments of 3 Medical Oncology, 4General Surgery, and 5Cancer Biology, Mayo Clinic, Jacksonville, FL.

Background: Pancreatic acinar cell carcinoma (ACC) is a rare pancreatic cancer with poor prognosis. Therapeutic approaches for ACC in advanced disease are limited as the molecular mechanism of ACC is poorly understood.

Methods: Whole exome sequencing was performed on 11 ACC surgical samples. Rigorous filtering steps were preformed to remove potential germline variants: 1) Allele frequency >0.01 in multiple polymorphic databases; 2) Minor allele frequency <1% or coverage depth <10; 3) non-functional variants; 4) Variant Quality Score Recalibration predicted score >99.9%; 5) Synonymous variants, non-coding variants. Mutation profiles and signatures were evaluated using Mutational Patterns package.

Results: Average 313 point somatic mutations (range, 121-1118) were identified in each patient. Three novel mutations were identified in majority of ACC patients: NBPF (neuroblastoma breakpoint family) 20 c.130G>C (10/11), E2F4 c.956_958delGCA (9/11) and TBP c.231delG (6/11). BRCA2 (6/11), COL12A1 (4/11), FAT1 (3/11), FAT3 & FAT4 (2/11), SMAD4 (2/11) were also frequently mutated. Interestingly, KRAS (0/11) and TP53 (1/11), frequent mutations of PDAC, were rarely mutated in ACC. MEN1, MUTYH and CHEK2, frequently mutated in pancreatic neuroendocrine tumors, are not found in ACC.

All ACC patients exhibited mutation cosmic signature. Six patients exhibited cosmic signature 6 which is associated with defected DNA mismatch repair. Five patients exhibited signature 3 which is associated with double strand DNA repair failure. Pathway mutation pattern analysis showed most patients had perturbed activities in MAPK, WNT, JAK-STAT, mTOR and NOTCH pathways. Cell cycle and DNA repair related pathways were also affected in most patients (10/11 and 9/11 respectively).

Conclusion: We identified 3 novel mutations in ACC and showed that ACC exhibits distinct mutation signatures with defect cell cycle and DNA repair functions. The data may be useful to identify therapeutic markers and predict therapy response in a subset of ACCs.

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Wnt/β-catenin Signaling Plays Diverse Functions During the Process of Fibrotic Remodeling in the Exocrine Pancreas

M. Bläuer, M. Laaninen, J. Sand, J. Laukkarinen. Tampere Pancreas Laboratory, Department of Gastroenterology and Alimentary Tract Surgery, Tampere University Hospital; Faculty of Medicine and Life Sciences, University of Tampere, Tampere, Finland.

Background: Wnt/β-catenin signaling plays vital roles in early development and tissue homeostasis. Dysregulation of the pathway has been implicated in the pathogenesis of cancer and fibrosis in numerous tissues, including the pancreas. Our aim was to provide further insight in Wnt/β-catenin signaling in the exocrine pancreas in relation to fibrosis and to elucidate in vitro the role of cross-talk between acinar and stellate cells (PSCs) on the cellular expression of selected Wnt/β-catenin pathway-associated proteins.

Methods: Pancreatic specimens from patients undergoing pancreatic resection were classified according to the degree of pancreatic fibrosis. The expression of β-catenin, Wnt2, Wnt5a and SFRP4 were analyzed by immunohistochemical means. A long-term acinar-stellate cell co-culture system was employed to study the effect of humoral interactions of the proteins in both cell types. The cells were maintained for four days in parallel mono- and co-cultures and thereafter subjected to immunocytochemical analyses.

Results: In human pancreatic tissue the fibrotic microenvironment caused redistribution of the studied proteins in and between epithelial and stromal compartments, compared to acinar-rich microenvironment. In non-fibrotic and moderately fibrotic tissue the proteins were expressed exclusively in acinar cells but resided predominantly in stromal fibroblastoid/stellate cells and macrophages in highly fibrotic tissue. Subcellular changes in the expression of immunoreactive β-catenin and Wnt5a were detected. The in vitro data suggest an involvement of acinar cell/PSC cross-talk in mediating the changes observed in tissue specimens.

Conclusion: Wnt/β-catenin pathway-associated proteins are amply expressed in the exocrine pancreas with distinct changes in their cellular and subcellular expression patterns along with increasing levels of fibrosis. Wnt/β-catenin signaling is suggested to play diverse functions during the process of fibrotic remodeling in the exocrine pancreas. The in vitro data renders our co-culture setting a relevant experimental tool to study Wnt/β-catenin signaling in the exocrine pancreas.

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Scoring Systems as Clinical Predictors of Pancreatic Necrosis and Pseudo-Cyst in Montenegrin Population

R. Boricic,1 M. Lukic,2 B. Smolovic.2 1 Emergency Center and 2Department of Gastroenterohepatology, Clinical Center of Montenegro, Podgorica, Montenegro.

Background: Scoring systems are used for a quick orientation in patients with acute pancreatitis (AP). Our prospective study was designed to assess the strength of scoring systems: Acute Physiology and Chronic Health Examination (APACHE) II-24, APACHE II-48, the bedside index for severity (BISAP) and computed tomography severity index (CTSI) in predicting pancreatic necrosis (PNec) and pseudo cyst (Pc) in patients with AP.

Methods: All necessary data was collected from January 2014 to November 2017. We calculated these scores according to valid rules and their respective abilities were evaluated using trend and discrimination analysis.

Results: Out of the 187 patients, 13 (7.1%) developed PNec and 36 (19.8%) developed Pc. Sensitivity of our scoring system (APACHE II-24, APACHE II-48, BISAP, CTSI) for PNec was 72.7%, 77.8%, 55.6%. and 80.6 %, while specificity was 66,2%, 67,8%, 87,7%, 93.8%, respectively. Sensitivity of our scoring system for Pc was 61.5%, 53.8%, 61.5% and 92.3 % while specificity was 60.1%, 66.3%, 63.3%, 63.3%, respectively. Positive predictive values of our scoring systems for PNec were 0.295, 0.354, 0.476, and 0.957 while negative were 0.913, 0.926, 0.890, and 0.912, respectively. Positive predictive values for Pc were 0.095, 0.089, 0.119, and 0.174 while negative were 0.957, 0.944, 0.945, and 0.943, respectively. Area under curves for APACHE II-24, APACHE II-48, BISAP, and CTSI in predicting PNec was 0.726, 0.776, 0.738, and 0.961, and for Pc was 0.583, 0.565, 0.625, and 0.827, respectively. However, we found that CTSI have significantly higher predictive strength as compared to other scores we used in predicting PNec (P = 0.0947, 0.7906, and <0.0001, respectively) and Pc (P = 0.7231, 0.5341, and <0.0001, respectively).

Conclusion: Our study suggests that CTSI is a useful predictor for the PNec and Pc, more than other scoring systems we used.

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Predicting Acute Pancreatitis (AP) Length of Stay (LOS): “Hotspotting” High-cost Admissions

E. Bowns, A. Hinton, L.F. Lara, P.A. Hart, D.L. Conwell. Section of Pancreatic Disorders, Division of Gastroenterology, Hepatology and Nutrition, The Ohio State University Wexner Medical Center Columbus, OH.

Introduction: AP LOS directly impacts costs and resource allocation. We chose to develop a risk score to identify subjects with AP at risk for prolonged hospitalization.

Aims: 1. Determine risk factors that lead to an increased LOS for patients with AP; 2. Derive a risk score to stratify AP LOS.

Methods: The 2013 Nationwide Readmissions Database (NRD), was used to develop the AP risk score. The NRD contains information on 36 million discharges, containing ICD-9/ICD-10 diagnosis codes. Included in the analysis were adults (>18 years of age) with a primary diagnosis of AP. Excluded were patients who were pregnant, transferred during their stay, or died during admission. SAS 9.4 was used to develop a logistic regression model identifying significant independent predictors of LOS ≥7 days. Based on the coefficients and standard errors from this model, a risk score was developed for LOS ≥7 days.

Results: Overall, there were 217,535 patients included in the analysis (52% male). LOS was strongly associated with costs of admission. Predictors of LOS ≥7 days included age, comorbidities, admission day, hospital type and size, morbid obesity, gallstones, and alcohol use. An AP LOS risk score was developed from these predictors. The AP LOS risk scores were normally distributed and ranged from 0-105. The model (OR; 95% CI) was stratified into low (reference), moderate (2.09; 1.97-2.21) or high risk (4.15; 3.93-4.39) with an AUC of 0.644 for increased LOS ≥7 days.

Conclusion: A risk score for LOS was derived from administrative claims data. Further external validation and testing of this AP risk score is planned. Clinical Implication: “Hotspotting” AP can facilitate reallocation of resources to a subset of high-needs, high-cost AP patients.

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Cutting off the Fuel Supply to the Plasma Membrane Ca2+ Pumps in Pancreatic Cancer: Pyruvate Kinase-M2 (PKM2) as a Potential Therapeutic Target

J.I.E. Bruce, A.D. James, P. Sritangos, T. Attard, L. Barrett, I.W. Oh. Division of Cancer Sciences, School of Medical Sciences, Faculty of Biology, Medicine and Health, University of Manchester, United Kingdom.

Background and Aims: Pancreatic ductal adenocarcinoma (PDAC) has poor survival and limited treatment options. PDAC cells undergo a switch from mitochondrial to glycolytic metabolism (Warburg effect) which facilitates numerous cancer hallmarks. Our previous studies show that inhibiting glycolytic ATP supply to the plasma membrane calcium pump (PMCA) causes cytotoxic Ca2+ overload and cell death. The oncogenic pyruvate kinase-M2 (PKM2) is the major ATP generating glycolytic enzyme and is over-expressed in PDAC. We aim to investigate the functional coupling between PKM2 and PMCA and whether this facilitates cancer hallmarks.

Methods: Human PDAC cells (MIA PaCa-2) were cultured in standard DMEM. Cell surface biotinylation assays were used to identify transmembrane protein-associated glycolytic enzymes. Glycolysis (extracellular acidification rate (ECAR)) and mitochondrial respiration (oxygen consumption rate (OCR)) was assessed using the Seahorse XFe96 analyzer. Cell proliferation/viability and cell migration was assessed using a sulforhodamine-B (SRB)/MTT-based absorbance assay and gap closure assay, respectively. Cellular ATP was assessed using either luciferase-based luminescence or recombinant FRET-based fluorescent sensor (GO-ATeam). Fura-2 imaging was used to asses cytosolic Ca2+ an in situ Ca2+ clearance assay was used to assess PMCA activity. PKM2 knockdown was achieved using siRNA. Cell death was assessed by western blotting for cleaved poly-ADP ribose polymerase (PARP1).

Results: Pyruvate kinase-M2 (PKM2) was found to associate with plasma membrane proteins, potentially providing a privileged ATP supply to the PMCA. Specific inhibition of PKM2 (with shikonin) inhibited the cancer hallmarks; cell proliferation, cell viability and cell migration. This was due to inhibition of glycolysis, ATP depletion, inhibition of PMCA and cytotoxic Ca2+ overload (fura-2 fluorescence). PKM2 knockdown reduced PMCA activity and reduced the sensitivity of shikonin-induced cell death.

Conclusions: These data suggest that the functional coupling between PKM2 and the PMCA may represent a novel therapeutic strategy for the treatment of PDAC.

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Contribution of CXCR3/CXCL4 Signaling Axis to Pancreatic Ductal Adenocarcinoma Metastasis

A. Cannon,1 C. Thompson,1 R. Bhatia,1 B. Hall,2 S. Kumar,1 S.K. Batra.1,3 Departments of 1 Biochemistry and Molecular Biology and 2 General Surgery; 3Fred and Pamela Buffet Cancer Center, University of Nebraska Medical Center, Omaha, NE.

Background: Pancreatic ductal adenocarcinoma (PDAC) is among the most lethal human malignancies with a five-year survival rate near 8%. Dissemination of malignant cells early in disease progression underlies the dismal prognosis of PDAC as evidenced by extremely high rates of recurrence following surgical resection. Literature suggests that metastasizing cancer cells rapidly associate with platelets in circulation; however, the nature and functional consequences of this interaction are not fully understood. We investigated the interaction of platelets with PDAC cancer cells and the contribution of platelet-derived CXCL4 to PDAC metastasis to elucidate a potential mechanism by which PDAC cells surmount the intravascular and colonization phases of metastasis.

Methods: Immunohistochemistry (IHC) and q-PCR analysis were performed to characterize the expression of CXCL4 receptor, CXCR3, in 42 Whipple samples and 2 PDAC cell lines, respectively. Western blot was used to investigate the effect of CXCR3 signaling on mucin expression. A tail vein injection model was used to test the effect of inhibition of CXCR3 and MUC4 overexpression on the intravascular phase of PDAC metastasis. In vitro, low attachment survival and endothelial adhesion assays were used to explore the mechanism of CXCR3’s involvement in PDAC metastasis.

Results: 86% of Whipple samples and all PDAC cell lines showed CXCR3 expression in malignant cells. Treatment of PDAC cells with CXCL4 augmented the expression of MUC1, 4, and 16. Inhibition of CXCR3 suppressed the ability of PDAC cells to colonize the lungs in tail vein injection models (P < 0.05) while mini-MUC4 overexpression augmented lung colonization (P < 0.05). Concurrently, treatment with platelets and recombinant CXCL4 augmented the survival of Capan1 grown in low attachment conditions (P < 0.05) and the ability of Capan1 to adhere to endothelium (P < 0.05) respectively.

Conclusion: These results suggest that CXCL4/CXCR3 signaling aids PDAC cells during the intravascular phase of metastasis.

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Surgical and Patient-Centered Outcomes After Pancreatic Resection for Screening-Detected Lesions in High-Risk Individuals for Pancreatic Cancer

M.I. Canto,1 T. Kerdsirichairat,1 M. Ford,1 M.G. Goggins,1 A. Blackford,2 A.A. Javed,3 T. Abe,1 R.H. Hruban,4 A.M.O. Lennon,1 A.P. Klein,2 I.R. Kamel,5 E.K. Fishman,5 M.J. Weiss,3 C.J. Yeo,6 R.D. Schulick,7 C.L. Wolfgang.3 Departments of 1Medicine (Gastroenterology), 2Division of Biostatistics and Bioinformatics, 3Surgery, 4Pathology, and 5Radiology, Johns Hopkins Medical Institutions, Baltimore, MD; 6Department of Surgery, Thomas Jefferson University, Philadelphia, PA; 7Department of Surgery, University of Colorado, Aurora, CO.

Background: Screening HRI for pancreatic ductal adenocarcinoma (PDAC) and its precursors is a potential approach to improving survival. Little is known regarding the outcomes after pancreatic resection in this population. We aim to identify surgical and patient-related outcomes in high-risk individuals (HRI) undergoing a screening for PDAC.

Methods: HRI enrolled in the prospective Cancer of the Pancreas Screening (CAPS) studies during 1998-2017 consisted of relatives from familial pancreatic cancer kindreds, specific syndromes and germline mutations, who underwent pancreatic resection for detected lesions by EUS, MRI, and/or CT with a follow-up time of ≥ 6 months were included. Surgical outcomes, patient- centered outcomes, pre- and post-operative health-related quality of life (HRQoL) were assessed. Matched comparison of survival and stage shift were performed.

Results: Among 358 HRI with continued surveillance, 46 (12.8%) asymptomatic HRI had surgery for suspected pancreatic neoplasms. 10/11 screening-detected PDAC were resectable. 40 HRI had partial resection, then 8 of these underwent completion pancreatectomy at median 3.8 years (IQR, 2.5-7.6) for a new lesion, of which 2/8 (25%) developed PDAC in the remnant pancreas. Post- operative complications developed in 15 HRI (32.6%). New-onset diabetes mellitus developed in 7/26 HRI after partial resections. Mean follow-up was 7.6 years. HRQoL is preserved postoperatively (physical component score, P = 0.44; mental component score, P = 0.81). Survival is improved (median 143 months) compared with our matched surgical non-CAPS cohort (24.1 months, P = 0.002). PDAC size was smaller (2.5 cm vs 4 cm, P < 0.001) with higher rates of stage 1a at diagnosis (14.3% vs 2.49%, P = 0.02) in CAPS cohort.

Conclusion: Screening HRI leads to detection of PDAC with high resectability, survival and stage shift. Surgical treatment is associated with acceptable morbidity and no mortality. Patient satisfaction is high and HRQoL is preserved.

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Surface Characterization of Seromic Oncosomes for the Early Diagnosis of Pancreatic Cancer

J. Carmicheal,1 C. Hayashi,1 X. Huang,2 L. Liu,2 Y. Lu,2 A. Krasnoslobodtsev,3,4 A. Lushnikov,4 P.G. Kshirsagar,1 A. Patel,1 M. Jain,1 Y.L. Lyubchenko,4 Y Lu,2 S.K. Batra,1 S. Kaur.1 1 Department of Biochemistry and Molecular Biology, University of Nebraska Medical Center, Omaha, NE; 2 Department of Electrical and Computer Engineering, University of Nebraska-Lincoln, Lincoln, NE; 3 Department of Physics University of Nebraska at Omaha, Omaha, NE; 4Nanoimaging Core Facility, College of Pharmacy, University of Nebraska Medical Center, Omaha, NE.

Background: Pancreatic ductal adenocarcinoma (PDAC) is a highly lethal malignancy with the sole curative option of surgical resection. Unfortunately it often presents after it has metastasized or is nonresectable. An urgent need exists for novel biomarkers to detect PDAC. Oncosomes, extracellular vesicles (EVs) released by cancer cells, are an integral part of cancer progression and are present at the earliest stages of disease. We hypothesize that seromic oncosomes can be utilized for the early stage detection of PDAC.

Methods: EVs from normal pancreatic ductal epithelial cells (HPDE), and oncosomes from three pancreatic cancer cell lines (MIA PaCa, CD18/HPAF, and T3M4), representing the wide mutational spectrum of PDAC were isolated using gold standard ultracentrifugation technology and sucrose density gradient. Surface enhanced Raman spectroscopy (SERS), in conjunction with principle component-discriminant function analysis (PC-DFA), was used to classify oncosomes originating from cells in vitro, and patient serum samples. Surface glycosylation status and proteomic profiling was assessed via fluerescence lectin array and trypsin surface shaving followed by mass spectrometry, respectively.

Results: Three-pronged approach was used to identify PDAC specific oncosomes. SERS based label-free analysis of the oncosomal surface correctly characterized EVs as originating from PDAC or healthy cell lines with 90% accuracy and from PDAC patient or healthy patient sera with 66.7% accuracy. 40-lectin array elucidated difference in oncosomal glycosylation compared to EVs from HPDE. Surface profiling of cell line oncosomes and literature comparison, yielded six novel PDAC specific oncosomal surface proteins.

Conclusion: SERS results proved serum oncosomes have potential as non-invasive label free biomarkers for the early stage detection of pancreatic cancer. Further investigation is planned for the validation of the PDAC specific surface markers in patient serum via immunogold staining and exosome specific ELISA (ExoELISA). This study provides novel methods for the early detection of pancreatic cancer, through concomitant oncosome surface characterization modalities.

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Alternative Splice Variants of DCLK1 in Pancreatic Tumor Cells Coordinates Macrophage Polarization and an Immunosuppressive Phenotype

P. Chandrakesan,1,2,3,4 N. Weygant,1,2,3 J. Panneerselvam,1,2,4 N. Ali,1,2,4 R. May,1,3 D. Qu,1,2,3 K. Pitts,1 S. Sureban,1,4 M. Bronze,1 M. Li,1,2 C.V. Roa,1,2,4 C. Houchen.1,2,3,4 1 Department of Medicine and 2 Stephenson Cancer Center, OUHSC, Oklahoma City, OK; 3 COARE Biotechnology, Oklahoma City, OK; 4Oklahoma City VA Health Care System, Oklahoma City, OK.

Introduction: PDAC is characterized by dense tumor microenvironment (TME) with increased infiltration of immunosuppressive cells, which create an enormous challenge to the therapeutic regimen. In particular, M2-phenotype macrophages are immunosuppressive and coexist with tumor epithelial cells, promoting tumorigenesis and help the tumor cells to evade the host anti-tumor immunity. These M2-macrophages are activated by IL-13 and IL-4, alternate pathways. Doublecortin-like kinase 1+ (DCLK1+) tuft cells regulate intestinal parasitic infection through IL-25/IL13 axis. We previously reported that DLKC1 a tumor stem cell marker and a functional protein kinase is increased in the PDAC TME and regulates pancreatic tumor progression. Tuft cells in the pancreas express DCLK1, therefore in this study, we investigate, whether DCLK1 overexpressing pancreatic tumor cells can regulate macrophage differentiation and polarization.

Methods: Gain and loss of function of DCLK1 by lenti- virus infection and si-RNA transfection. DCLK1 overexpressing AsPC1 cells were co-cultured with macrophages, and analyzed for M1 and M2 markers. M2-macrophages were co-cultured with AsPC1 parental cells, analyzed for tumor cell migration and invasion and for the expression of EMT factors.

Results: DCLK1 overexpressing pancreatic tumor cells convert macrophages into M2-phenotypes based on reduced expression of IL-1b and CD86 (M1 markers) and increased expression of CD206 and CD163 (M2 Markers). M2-macrophages co-cultured with AsPC1 parental cells resulted in increased tumor cell invasion and migration and increased expression of EMT factors SNAI1 and SNAI2. DCLK1 Knock down in the DCLK1 overexpression AsPC1 cells resulted in the reversion of M2-phenotype into M1- phenotype and reduced tumor cell invasion, migration and the expression of SNAI1 and SNAI2.

Conclusion: Our data strongly suggest that DCLK1-mediated activation of M2 macrophages results in enhanced tumorigenesis. Overall our findings delineate a strong association between DCLK1 and immunosuppressive TME and provides the rationale for targeting DCLK1 as a potential therapeutic approach to re-engage anti-tumor immunity.

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Activation of Free Fatty Acid Receptors 1 and 4 Increases YAP Activity in Pancreatic Cancer Cells

H.H. Chang,1 L. Ayoub,1 E. Wang,1 O.J. Hines,1 E. Rozengurt,2 G. Eibl.1 Departments of 1Surgery and 2Medicine, David Geffen School of Medicine at UCLA, Los Angeles, CA.

Background: Free Fatty Acid Receptors FFAR1 and FFAR4, members of metabolite sensing G protein-coupled receptors (GPCRs), are currently emerging as novel therapeutic targets for metabolic disorders such as type 2 diabetes mellitus and obesity, known risk factors for pancreatic ductal adenocarcinoma (PDAC). The role of FFAR1 and 4 in malignant diseases, however, is still elusive. To further elucidate the role of these receptors in PDAC, we examined the previously unexplored signaling crosstalk between FFARs and Yes-associated protein (YAP), a key transcriptional regulator downstream of oncogenic Kras in PDAC and demonstrated to be an effector of several GPCRs.

Methods: Expression levels of FFAR1 and FFAR4 were measured in a panel of human PDAC lines by qRT-PCR. Stimulation of the downstream Gαq-coupled response by FFAR1 and/or 4 ligation was assessed by monitoring intracellular Ca2+ mobilization upon exposure to synthetic agonists. Activation of YAP in response to FFAR1 and/or 4 agonists was determined by qRT-PCR analysis of target gene expression, and the dependence of YAP was examined by siRNA-mediated knockdown.

Results: In PANC-1 cells, which express higher levels of both FFAR1 and FFAR4 among several human PDAC cell lines, and exhibit detectable intracellular Ca2+ increase in response to agonists, we found that YAP target genes including connective tissue growth factor (CTGF) and cysteine-rich angiogenic inducer 61 (CYR61) are markedly upregulated (2-10 folds, P < 0.05) upon exposure to FFAR1 (TAK-875), FFAR4 (TUG-891), or dual (GW 9508) agonists at 20, 20, and 50 μM, respectively. In addition, siRNA-mediated knockdown of YAP abolished the increase in the expression of these genes.

Conclusion: These data demonstrate, for the first time, that FFAR1 and FFAR4 agonism increases oncogenic YAP activity in human PDAC cells. Our finding will facilitate future studies addressing current research gaps and major controversy regarding the role of FFARs in PDAC.

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Generation of Immortal Pancreatic Acinar Cell Lines In Vitro by hTERT and Transdifferented Pluripotent Acinar Derived Cells

H.K. Charles Jacob, M.R. Kashyap, A. Ferrantella, S. Kurtom, R. Dawra, A.K. Saluja, V. Dudeja. Department of Surgery, University of Miami Miller School of Medicine, Miami, FL.

Background: Culture of acinar cells has been difficult primarily because of the plasticity of acinar cells which tend to lose their secretory abilities and transform into other cells such as ductal and hepatocyte like cells. The composition of the media also plays a very important role in preventing the transdifferentiation of primary cells isolated.

Results: Transduction of cells with Htert alone allows for keeping the cells in culture however, there is no observed proliferative advantage conferred upon the cells. In order to generate proliferative acinar cell cultures, several methods were followed post generation of pluripotency. By constitutively expressing progenitor markers such as Stathmin and Reg1 in iPS induced cells followed by addition of conditioned media from acinar cells, we have been able to maintain cultures of acinar cells for a period of 5 weeks and have investigated the transcriptome and secretome of these cultured cells post cerulein based injury and have found it to be similar to primary acinar cells.

Methods: Acinar cells were transduced with lentiviruses expressing the Yamanaka factors in a doxycycline inducible construct and with hTERT and or the progenitor markers Stmn1 and Reg1 as constitutive contructs. iPS cells were selected and exposed to conditioned media and the secretome and intracellular proteome was analyzed by using an iTRAQ high throughput quantitative proteomics approach while the transcriptome using the HiSeq2500 and compared with freshly isolated acinii. Cerulein treatments in vitro indicate a similar transcriptome and proteome to control cells.

Conclusion: Here we show for the first time the generation of immortalized acinar cell cultures that can be used for downstream experiments as an alternative to mouse models. The cells are also responsive to cerulein treatment indicating that the cells can be used as an in vitro assay system for assessing damage and or responses to external stimuli.

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Absence of B and T Cells Does Not Affect Development and Severity of Chronic Pancreatitis

H. Cheema, M. Tarique, U. Hooda, E.P. Bava, V. Dudeja, R. Dawra, A.K. Saluja. Department of Surgery and Sylvester Comprehensive Cancer Center, University of Miami Miller School of Medicine, Miami, FL.

Background: Chronic pancreatitis (CP) is irreversible fibro inflammatory disease of the pancreas characterized by acinar cell loss and fibrotic replacement. In recent studies, it has been shown that alternatively activated macrophages (AAM) promote pancreatic fibrosis. Interestingly, pancreatic tissues from human and mouse CP has shown elevated T-cell, possibly suggesting role of adaptive immunity in modulating chronic inflammation and fibrosis.

Aim: To evaluate the role of adaptive immunity in cerulein model of chronic pancreatitis.

Methods: CP was induced by repetitive cerulein injections. In brief, 6-8-week-old C57BL6 or RAG1-/- mice (in C57BL6 background) were given seven hourly intraperitoneal (i.p.) injections of 50 μg/kg/h body weight cerulein 2 days per week, for a total of 10 weeks. Mice were sacrificed at week 7 and week 11. Severity of CP was evaluated by measuring changes in pancreas to mouse weight ratio, histology and Sirius red staining. Acinar cell loss and stellate cell activation was evaluated by IHC and WB. Immune infiltrate in the pancreas was analyzed by flow cytometry.

Results: Rag1-/- mice had significantly more pancreatic atrophy as compared to WT mice, as measured by pancreas to mouse weight ratio (WT mice 4.07±0.17 mg/g vs RAG1-/- mice 3.21±0.161 mg/g). There was no difference in fibrosis and stellate cell activation as shown by Sirius red staining and expression of fibrosis related proteins (vimentin, α-SMA, collagen-1). Total macrophages (CD45+ F4/80+), neutrophils and monocytes (CD45+ Gr-1+), were increased in RAG1-/- mice pancreas, although there was no difference was seen in classically activated macrophages or alternatively activated macrophages.

Conclusion: Absence of B and T cells does not affect development and severity of chronic pancreatitis.

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Depletion of Neutrophils After Development of Acute Pancreatitis Does Not Reduce Worsening of Pancreatic Injury Due to Morphine Treatment

H. Cheema,1 M. Tarique,1 U. Barlass,2 U. Hooda,1 M. Kashyap,1 E.P. Bava,1 S. Roy,1 V. Dudeja,1 R. Dawra,1 A.K. Saluja.1 1 Department of Surgery and Sylvester Comprehensive Cancer Center, University of Miami Miller School of Medicine, Miami, FL; 2Department of Gastroenterology & Hepatology, Rush University Medical Center, Chicago, IL.

Background: Opioids such as morphine are widely used for the management of pain associated with acute pancreatitis (AP). We have previously shown morphine treatment in AP leads to worsening of injury and inflammation, there is increase in pancreatic sequestrated neutrophils. However, the underlying mechanism of what leads to increased sequestration of neutrophils has not been studied. It could be either because of increased recruitment of neutrophils or inability to clear out sequestrated neutrophils.

Aims: To investigate the mechanism which leads to increase in pancreatic neutrophil sequestration after morphine treatment in AP.

Methods: Acute pancreatitis was induced by 12 hourly intra-peritoneal injections of cerulein at a dose of 50 μg/kg/h to 6-8-week-old male wild-type mice. Morphine (25 mg) or placebo pellets were implanted one hour after last cerulein injection and mice sacrificed at 48 and 56 hours. Neutrophils were depleted using a Ly6G antibody after induction of AP. Pancreatic injury was evaluated using pancreatic myeloperoxidase activity (MPO), histology and Immunohistochemistry (IHC). Neutrophil population was studied by flow cytometry, IHC and MPO.

Results: Morphine treatment of mice with acute pancreatitis led to increase in neutrophils in the pancreas as measured by MPO activity, as compared to placebo treatment. Ly6G antibody treatment was able to reduce neutrophils in the blood but there was no change in pancreatic neutrophils population as seen by pancreatic MPO assay and IHC. Histology pictures (H&E staining) and quantification of pancreatic injury on histology showed that was no change in pancreatic injury with LY6G antibody treatment.

Conclusion: Our data suggests that morphine treatment after AP, does not cause increase in pancreatic sequestration of neutrophils but impairs clearance of neutrophils from inflamed pancreas. Role of other immune populations especially macrophages required for clearing out neutrophils needs to be studied further in detail.

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The Tumorigenic Roles of Mutant KrasG12D Isoforms in Pancreatic Cancer

J. Chen,1 A. Haddock,1 F. Gui,1 J. Wan,1 X. Zhu,1 Y. Bi,2 B. Ji.1 Departments of 1 Cancer Biology and 2Gastroenterology and Hepatology, Mayo Clinic, Jacksonville, FL.

Backgrounds and Aims: It is well recognized that Kras signaling plays an important role in pancreatic adenocarcinoma (PDAC) initiation, progression and maintenance. However, it is less acknowledged that Kras encodes two isoforms, Kras-4A and Kras-4B. We aim to elucidate the specific roles of oncogenic Kras-4AG12D and Kras-4BG12D in PDAC.

Methods and Results: Using CRISPR-Cas9-mediated genome editing in the fertilized eggs of the well-established LSL-KrasG12D model, we generated the LSL-Kras-4AG12D and LSL-Kras-4BG12D mouse models. To activate the KrasG12D isoforms, these mice were crossed with pancreatic specific Pdx1-Cre mice. Using a KrasG12D specific antibody, robust KrasG12D expression can be detected in the LSL-Kras-4BG12D/Pdx1-Cre (K4B) mice, though the signal was slightly lower than the total KrasG12D in LSL- KrasG12D/Pdx1-Cre (KC) mice, while the Kras-4AG12D protein is undetectable in the LSL-Kras- 4AG12D/Pdx1-Cre (K4A) mouse. PanIN lesions and focal fibrosis spontaneously developed in both K4B and KC mouse but not in the K4A mouse, albeit the number of PanIN lesions and fibrosis in K4B mouse is relatively lower than those in KC mouse. When challenged with cerulein, both K4B and KC mice developed dramatic acinar to ductal metaplasia (ADM), though there is less desmoplasia and PanIN lesion numbers in the K4B mouse. To study the tumorigenesis potential of theses KrasG12D isoforms, these mice were further crossed with conditional p53 deletion mice. Both LSL- KrasG12D/p53f/f/Pdx1-Cre (KPC) and Kras-4BG12D/p53f/f/Pdx1-Cre (K4BPC) mice developed PDAC and died at 8-10 weeks. In contrast, the Kras-4AG12D/p53f/f/Pdx1-Cre (K4APC) mice did not develop PDAC and have survived to at least 1 year of age.

Conclusion: Taken together, Kras-4BG12D is the major KrasG12D isoform expressed and is sufficient to cause PDAC in mice while Kras-4AG12D is expressed at a lower level and may play a minor role in the development of PDAC.

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Neutrophil-lymphocyte Ratio in Acute Pancreatitis: Diagnostic and Prognostic Role

S. Chooklin, S. Chuklin, G. Shershen, P. Popyk. Regional Clinical Hospital, Lviv, Ukraine.

Background: Acute pancreatitis (AP) is characterized by local and systemic inflammation. Hematologic components of systemic inflammatory response can be specifically used for the prediction of disease severity, including the ratio of neutrophils to lymphocytes (NLR).

Methods: We examined 206 patients with acute pancreatitis. According to the international classification in 51 patients we diagnosed the mild AP, in 98 patients the moderately AP, and in 57 patients the severe AP. We determined the NLR, indicators of hemostasis and inflammation.

Results: Significant difference in the NLR in different variants of acute pancreatitis was noted. In mild AP NLR averaged 6.11±5.07, the moderately AP – 7.28±5.11 and severe – 10.32±7.00. Materiality was in the difference between the rates of patients with mild AP and moderately AP (U = 1883.500, P = 0.013798), mild AP and severe APP (U = 802.5000, P = 0.000062), moderately AP and severe AP (U = 2027.500, P = 0.004502). The direct correlation between the NLR and thrombin time (R = 0.279899, P = 0.022836), the concentration of IL-6 (R = 0.469123, P = 0.005887) and TNF- α (R = 0.355912, P = 0.042069), reverse with the activity of AT III (R = -0.255050, P = 0.038756) confirmed in AP patients. At the same time, was noted direct correlation between the NLR and the severity of AP patients, determined by Ranson (R = 0.304941, P = 0.000008), APACHE II (R = 0.250068, P = 0.000289), BISAP (R = 0.166842, P = 0.01653), SOFA (R = 0.300822, P = 0.000011), Balthazar (R = 0.275644, P =0.022897). In addition, NLR is significantly increased in AP patients with verified pulmonary (F = 14.534, P = 0.00018), cardiovascular (F = 5.9542, P = 0.01554), and multiple organ (F = 5.6287, P = 0.01860) dysfunction.

Conclusion: NLR correlated with inflammation and hypercoagulation in patients with acute pancreatitis, as well as with severity of condition of patients.

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CD200 Promotes Immunosuppression in the Pancreatic Tumor Microenvironment

F. Choueiry,1,2 R. Shakya,2 X. Zhang,3 M. Dillhoff,4 D.L. Conwell,1 W.E. Carson, III,4 T.A. Mace.1,2 1 Division of Gastroenterology, Hepatology and Nutrition, Department of Internal Medicine, 2 Comprehensive Cancer Center, 3 Center for Biostatistics, 4Division of Surgical Oncology, Department of Surgery, The Arthur G. James Cancer Hospital and Richard J. Solove Research Institute, The Ohio State University, Columbus, OH.

Background: Pancreatic ductal adenocarcinoma (PDAC) is the third leading cause of cancer-related deaths in the United States. Standard cytotoxic and emerging targeted therapies for PDAC are ineffective treatment options. A significant challenge to overcome in PDAC is the profound systemic immunosuppression that renders this disease non-responsive to immunotherapy. Our supporting data provides evidence that expression of CD200, a regulator of myeloid cell activity, is highly elevated on the cell surface of stromal cells as well as on PDAC cells. We hypothesize that CD200 expression in the PDAC microenvironment limits responses to immunotherapy by promoting expansion and activity of myeloid-derived suppressor cells (MDSC).

Methods: In vitro assays testing for the effect of CD200 on MDSC (CD11b+GR1+) functional responses were measured by flow cytometry. In vivo antibody blocking of CD200 were conducted in subcutaneous MT-5 (KPC derived) tumor bearing mice and in a 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: MDSC isolated from patients and tumor-bearing mice with PDAC were discovered to express CD200 receptor (CD200R). MDSC stimulated in vitro with recombinant CD200 resulted in increased expression of indoleamine 2,3-dioxygenase (IDO), a potent molecule responsible for inhibiting T lymphocyte responses. In vivo studies demonstrated that CD200 antibody blockade limited tumor progression in MT-5 subcutaneous tumor-bearing and in KPC-BCRA2 mice (P < 0.05). Histologic 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 reduced intratumoral MDSC (P < 0.05). Additionally, in vivo blockade of CD200 can also significantly enhance the efficacy of PD-1 checkpoint antibodies compared to single antibody therapies (P < 0.05).

Conclusion: These results indicate that CD200 expression in the PDAC microenvironment may regulate MDSC activity and that targeting CD200 may enhance activity of checkpoint immunotherapy.

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The Circulating Survival Factor Renalase is Transported in High- and Low-molecular Weight Complexes With Serum Binding Proteins

S.L. Chung,1,2 T.R. Kolodecik,1,2 K. Date,3 C. Shugrue,1,2 G.V. Desir,1,2 F.S. Gorelick.1,2 1 Yale University School of Medicine, New Haven, CT; 2 VA CT Healthcare System, West Haven, CT; 3Ochanomizu University, Tokyo, Japan.

Background: Renalase (RNLS) is a 37 kDa circulating protein produced by the kidney and other tissues. We find that RNLS also reduces several forms of acute injury, including that induced by cerulein (CER)-hyperstimulation acute pancreatitis (AP). Thus, genetic deletion of RNLS results in more severe disease and administering recombinant RNLS decreases AP severity. We also find that serum levels of RNLS dramatically decreased within an hour of inducing AP and rebound following resolution. We hypothesized that RNLS might interact with proteins in the serum that mediate its activity, disappearance, and reappearance during acute pancreatitis. To examine this, RNLS complexes from murine serum were characterized.

Methods: Murine plasma was separated by size exclusion chromatography using a Sepharose 6B gel column. Co-immunoprecipitation was done from both plasma and column fractions for RNLS and candidate binding proteins.

Results: We found that plasma RNLS elutes from the sizing column in 2 peaks: first, a major high molecular weight complex (∼200-300 kD) and second lower molecular weight complex of 60-80 kD. Following size exclusion chromatography, proteins that interact with RNLS to from the high molecular weight complex were probabilistically determined by mass spectrometry and confirmed by co-immunoprecipitation. Two members of the alpha-2 macroglobulin protein (A2M) family were identified: pregnancy zone protein (PZP) and murinoglobulin (Mug-1). The profile by gel chromatography of RNLS in human serum was similar to that of mouse. Plasma RNLS rebounded to baseline levels or higher within 24 hours of inducing CER AP; this post rebound form of RNLS was predominantly in the lower molecule weight peak.

Conclusion: Our studies indicate that serum RNLS is predominantly in a complex with proteins that belong to the A2M family. This protein family can bind endothelial cell surface receptors, such as GRP78, and could mediate the disappearance of RNLS in early AP.

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Bacterial Resistance in Islet Preparations for Total Pancreatectomy With Islet Cell Auto-transplantation; Selecting the Optimal Antibiotics

K. Colling,1 A. Coughlan,1 J.J. Wilhelm,2 G.J. Beilman.1,2 1 Division of Critical Care & Acute Care Surgery, Department of Surgery, University of Minnesota, Minneapolis, MN; 2The Schulze Diabetes Institute, University of Minnesota, Minneapolis, MN.

Background: Total pancreatectomy with islet auto-transplantation (TPIAT) is a treatment option for chronic pancreatitis. Removing the inflamed pancreas frequently alleviates or reduces the patient’s pain, and replacing their islet cells into their portal system prevents development of brittle diabetes. TPIAT is often performed in patients that have undergone prior pancreatic instrumentation, leading to bacterial contamination of the pancreatic tissue. Currently ciprofloxacin is infused into islets preparations as prophylaxis. The most effective antibiotic in the preparation solution is not clear. We reviewed our internal culture and sensitivity data to identify the optimal antibiotic regimen.

Methods: A retrospective review was performed of TPIATs performed at the University of Minnesota between October 2009 and April 2018. Cultures were obtained from preservation fluid and final islet preparation. Cultured organisms and their antibiotic sensitivities were reviewed.

Results: During our study period, 55% of patients (217 of 394) had positive islet cultures. The most common bacteria were Enterococcus, Streptococcus, E. coli and Klebsiella. Candida species were isolated in seven patients. Extended spectrum β-lactamase producing organisms were identified in 9 patients (4%) and E. coli was the most common ESBL organism. No methicillin-resistant S. aureus were identified and only one vancomycin resistant Enterococcus.

Conclusion: Islet preparations for TPIAT often have positive cultures. The most common bacteria isolated were Enterococcus, Klebsiella, Streptococcus, and E. coli (18%), not surprising given the proximity to the biliary system. In our study, we report relatively high resistance to our current antibiotic, ciprofloxacin, due to the high rate of resistance in Enterococcus and E. coli species. Given our results a more appropriate antibiotic may be gentamicin.

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The Ohio State University Pancreas Disorders Network U-BioCHIP Platform – An Update

D.L. Conwell,1,2 J. Muntel,3 S. Ahmed,3 P.A. Hart,1 Z. Cruz-Monserrate,1,2 S. Appana,3 L. Lee,1 P. Banks,1 H. Steen.3 1 Division of Gastroenterology, Hepatology, and Nutrition, Department of Medicine; 2 Comprehensive Cancer Center, The Ohio State University Wexner Medical Center, Columbus, OH; 3Department of Pathology, Boston Children’s Hospital and Harvard Medical School, Boston, MA.

Background: The major challenge of managing chronic pancreatitis (CP) is the lack of diagnostic modalities for the detection of early CP prior to irreversible organ damages: routine pancreas biopsy carries risk, exocrine insufficiency only develops clinically when >90% of the gland has been scarred, and advanced imaging is expensive, and often ambiguous for early disease. We hypothesize that the diseased pancreas modulates the urine proteome, which can be utilized to complement current diagnostic modalities.

Aim: Identify changes in the urinary proteome which associate with disease state and pancreatic function.

Methods: Urine samples were collected from 10 (5 female, 5 male) healthy controls, 15 (4 female, 11 male), and 25 (9 female, 16 male) patients with equivocal/mild and moderate/severe CP on MRI/MRCP (Cambridge criteria), respectively. The samples were prepared for proteome analysis using our in-house developed high throughput urine proteomics pipeline. Endoscopic pancreas function test- based bicarbonate levels were available for 29 individuals.

Results: About 1500 urinary proteins were identified. A detailed biostatistical analysis across the three different groups as well as various pairwise comparisons to reflect the varying diagnostic needs of e.g. a principal care physician vs. a pancreatologists were carried out, identifying several biomarker candidates. Furthermore, we evaluated the sex dependency of these changes as independent studies on the urinary proteome changes before and after TPIAT provide strong evidence for such sex dependencies. Finally, we identified protein panels that correlate with bicarbonate levels; the resulting sensitivities ranged from >90% to >70% for moderate/severe and mild/equivocal CP, respectively.

Conclusion: Using our high throughput urine proteomics pipeline identified numerous proteins whose urinary abundance levels are modulated by the presence or absence of CP and that correlate with peak bicarbonate levels in PF. These findings highlight the potential of urinary proteins for diagnostic and functional testing in the context of pancreatic diseases.

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Association of Acinar Secretion and Autophagosome Formation via AP3-Derived Clathrin Coated Vesicles

M. Cooley,1 M. Breen,1 N. Ly,1 K. Ly,1 D.D.H. Thomas,1 S.M. Messenger,1 F.S. Gorelick,2 G.E. Groblewski.1 1 Program in Biochemical and Molecular Nutrition, University of Wisconsin, Madison, WI; 2Department of Internal Medicine, Section of Digestive Diseases, Yale University School of Medicine, New Haven, CT.

Background: Clathrin coated vesicles (CCVs) mediate vesicular trafficking between organelles including the ER, Golgi, endosomes, and plasma membrane (PM). Such trafficking is essential to maintain secretory and degradative pathways crucial for pancreatic acinar cell homeostasis. In acinar cells, tumor protein D52 controls an endolysosomal-derived constitutive-like secretory pathway and influences autophagic flux, which are inhibited during pancreatitis. The autophagy protein ATG16L has been shown to recruit membrane to the growing phagophore through CCVs derived from PM endocytosis. Here we demonstrate that D52 enters a complex with ATG16L1, the autophagy protein LC3, and clathrin heavy chain to modulate secretion and autophagy.

Methods: Co-immunoprecipitations were done in rodent and human primary acinar cells as well as cultured cells to probe protein-protein interactions. Immunofluorescent microscopy was performed to determine protein colocalization. CCV purification from pancreas was done by subcellular fractionation. Pearl mice have a genetic deletion of the β3 subunit of adaptor protein 3 (AP3) preventing Golgi and endosomal CCV formation.

Results: D52 co-immunoprecipitated with ATG16L in rodent and human acini and HCT 116 cultured cells; D52 and ATG16L strongly colocalize in Golgi, PM. D52 contains a putative LC3 interacting region in its C-terminus. LC3 antibodies co-immunoprecipitated D52 and clathrin heavy chains from pancreas membrane fractions. D52/ATG16L1/LC3 colocalize extensively along acinar apical membrane and are retrieved into Rab11-positive recycling endosome compartments during CCK-pancreatitis. Furthermore, D52 is enriched in purified acinar CCVs and strongly colocalizes with AP3. Loss of AP3 activity in Pearl mice enhances unregulated endolysosomal exocytosis measured by LAMP1 surface labeling independent of D52 expression.

Conclusion: These data implicate D52 in CCV trafficking pathways through interaction with ATG16L, LC3, and clathrin heavy chain. We propose that D52 acts as a cargo receptor protein to coordinate vesicular trafficking between the secretory and autophagic pathways to maintain acinar cell homeostasis.

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Heparin vs. Lovenox as Thrombosis Prophylaxis After Total Pancreatectomy With Islet Autotransplantation

A. Coughlan, M. Skube, T. Dunn, G.J. Beilman, M.D. Bellin, K.L. Berry. Department of Surgery, University of Minnesota, Minneapolis, MN.

Background: Portal vein thrombotic events (PVTs) are a known complication associated with total pancreatectomy with islet autotransplantation (TPIAT). Despite this risk the optimal postoperative anticoagulation regimen has not been well studied and standardized. The purpose of this study was to compare two cohorts of patients that underwent TPIAT, those who were given postoperative subcutaneous enoxaparin and those who received a heparin drip for thrombosis prophylaxis.

Methods: We performed a retrospective chart review of consecutive TPIAT cases performed between 2015 and 2017 by two surgeons at a single center who utilized different prophylactic anti-coagulation protocols. Using a chi squared analysis we compared the rate of portal venous thrombotic events, bleeding complications, blood transfusion requirements and need for reoperation between the two groups.

Results: Five of the fifty eight patients (8.6%) developed a PVT. Of the patients who received enoxaparin, 2/30 (7%) developed a PVT vs. 3/28 (11%) of those who received heparin (P = 0.7). Of the patients who developed PVTs, 2/5 were occlusive and 3/5 were non-occlusive. Four of the five PVTs had resolved on repeat ultrasound. When comparing bleeding complications, other thrombotic complications, need for blood transfusions and need for reoperation there were no statistically significant differences between the two groups (P = 0.5, 0.7, 0.3).

Conclusion: These findings suggest that a less aggressive regimen of thrombosis prophylaxis doses may be sufficient to prevent PVTs in patients undergoing TPIAT. Further studies are needed to draw correlative conclusions but there was also no statistical difference in other thrombosis or bleeding complications between patients that received lovenox vs heparin for thrombosis prophylaxis.

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A Pilot Study to Assess the Feasibility of Dietary Soy Isoflavones in Chronic Pancreatitis

Z. Cruz-Monserrate, O. Crowe, J. Ahn-Jarvis, N. Badi, A. Hinton, S. Kaul, H. Komar, S. Krishna, L.F. Lara, G. Lesinski, E. Lombardo, T. Mace, M. Ramsey, K. Roberts, K. Stinehart, Y. Vodovitz, D.L. Conwell, P.A. Hart. Division of Gastroenterology, Hepatology, and Nutrition, The Ohio State University Wexner Medical Center, Columbus, OH.

Introduction: Chronic pancreatitis is a chronic inflammatory disease which can progress to fibrosis causing irreversible organ damage. Currently there are no interventions to delay or stop the progression of disease. In this pilot study we assessed the tolerability of a novel dietary intervention using a soy-enriched bread product and feasibility to reduce circulating inflammatory mediators of chronic pancreatitis.

Methods: Subjects with chronic pancreatitis diagnosed using the APA diagnostic guidelines were recruited and provided a one-week intervention using a soy-enriched dietary bread product. One subject who did not complete the study due to undisclosed solid food dysphagia was categorized as a screen fail and was not included in the final analyses. The dosage of bread was determined using a classic 3+3 dose-escalation design, and dose-limiting toxicities were monitored. Blood was collected, centrifuged, and plasma was aliquoted and frozen at baseline and one-week after the soy-based dietary bread intervention. Plasma cytokine levels were measured using a Meso Scale Discovery multiplex assay kit, including IL-6, IFN-gamma, MCP- 1, and TNF-alpha. Cytokine levels from baseline and following the intervention were compared using Wilcoxon signed-rank test.

Results: A total of nine subjects completed one week of intervention without any dose-limiting toxicities up to a maximum dosage of three slices (99 mg of isoflavones) per day. The plasma concentrations of IL-6, IFN-gamma, and MCP-1 were similar prior to and following the intervention. In contrast, there was a decline in the TNF-alpha levels at one week (2.667 vs 2.382 pg/mL, P = 0.039).

Conclusion: In this pilot study we demonstrate the short-term tolerability of a novel dietary intervention for chronic pancreatitis. There is a reduction in at least one key pro-inflammatory cytokine. Larger studies with a longer intervention period are needed to assess long-term tolerability and the effectiveness of dampening the inflammatory mediators of chronic pancreatitis.

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Obesity is Associated With Increased Healthcare Utilization in Hospitalized Patients With Pancreatic Cancer

Z. Cruz-Monserrate,1 O. Crowe,1 A. Lahooti,1 S. Krishna,1 H. Hussan,1 P.A. Hart,1 M.S. Abougergi,2,3 D.L. Conwell.1 1 Division of Gastroenterology, Hepatology, and Nutrition; The Ohio State University Wexner Medical Center, Columbus, OH; 2 Catalyst Medical Consulting, Simpsonville, SC; 3Division of Gastroenterology, Department of Internal Medicine, University of South Carolina School of Medicine, Columbia, SC.

Introduction: Obesity is a known modifiable risk factor for pancreatic cancer (PC); however, few studies have evaluated the influence of obesity on outcomes and inpatient healthcare utilization in PC.

Methods: The Nationwide Inpatient Sample was used to identify all 2014 admissions for adults with PC. Obesity and morbid obesity were defined using ICD-9 codes. The primary outcome was indication for hospitalization, and secondary outcomes were length of stay (LOS), total hospitalization charges and costs, mortality rate, and hospice consults for metastatic PC. Multivariate regression models were used to adjust for confounders.

Results: A total of 60,140 hospitalizations were included. Of those 3740 (6.22%) and 1415 (2.35%) were for obese and morbidly obese subjects, respectively. The most common primary diagnosis for hospitalization was septicemia, regardless of obesity status. The second most common reason for admission among obese and morbidly obese subjects was a complication from metastasis compared to cancer-related pain for non-obese subjects. LOS was longer for obese (adjusted mean difference (amDifference): 1.19 (0.72–1.66) days, P < 0.01) and morbidly obese (amDifference: 1.79 (1.02–2.57) days, P < 0.01) patients compared with non-obese subjects. In addition, both total hospitalization charges and costs were higher for obese (amDifference: $13,432 ($7848–$19,016), P < 0.01 and $3311 ($2022-$4599), P < 0.01 ) and morbidly obese ($20,528 ($10,735-$30,321), P < 0.01 and $3986 ($2197-$5774), P < 0.01) compared with non- obese subjects. However, the in-hospital mortality rate and palliative care utilization were similar for non-obese (reference), obese (adjusted odds ratio (aOR): 1.00 (0.75–1.34), P = 0.98 and 0.89 (0.72–1.11), P = 0.32) and morbidly obese (aOR: 1.16 (0.75–1.79), P = 0.50 and 1.11 (0.82–1.52), P = 0.49) subjects.

Conclusion: Obesity is associated with different patterns of hospitalization and increased healthcare utilization among hospitalized patients with PC in a dose dependent manner. These findings warrant further research to better understand the reasons behind this disparity and improve both outcomes and cost-effectiveness of care.

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The Effects of Pancreas Fluid (PF) Processing on the Quality and Integrity of Proteins, and Nucleic Acids: A Report From the Biospecimen Committee of the Consortium for the Study of Chronic Pancreatitis, Diabetes, and Pancreatic Cancer (CPDPC)

Z. Cruz-Monserrate,1 G.B. Lesinski,2 W.E. Fisher,3 L.F. Lara,1 M. Topazian,4 N. Badi,1 S. Kaul,1 A. McElhany,3 R. Orr,5 D. Yadav,6 T. Mace,1 P.A. Hart,1 H. Steen,7 D.L. Conwell.1 1 Division of Gastroenterology, Hepatology and Nutrition, Department of Internal Medicine, and Comprehensive Cancer Center, The Ohio State University, Columbus, OH; 2 Department of Hematology and Medical Oncology, Winship Cancer Institute, Emory University, Atlanta, GA; 3 The Elkins Pancreas Center, Michael E. DeBakey Department of Surgery, and Dan L. Duncan Comprehensive Cancer Center, Baylor College of Medicine, Houston, TX; 4 Department of Gastroenterology and Hepatology, Mayo Clinic, Rochester, MN; 5 Indiana Clinical and Translational Sciences Institute, Specimen Storage Facility, Indianapolis, IN; 6 Division of Gastroenterology, Hepatology and Nutrition, Department of Medicine, University of Pittsburgh, Pittsburgh, PA; 7Department of Pathology, Harvard Medical School, Boston, MA.

Background: Endoscopic collection of secretin-induced pancreatic fluid (ePFT) has been used to better understand pancreatic physiology, pathophysiology, and improve clinical diagnosis of pancreatic diseases. Molecular components within the PF can help researchers discover novel disease biomarkers for better characterization of pancreatic disorders. However, PF contains active enzymes that may impact degradation and downstream molecular analyses. Herein, we describe a study of the quality and integrity of proteins, and nucleic acids in the PF using various methods of processing.

Methods: We obtained PF from subjects (n = 9) who underwent endoscopic pancreatic function testing (ePFT) for clinical purposes and provided consent for the study. The PF samples were processed and frozen (-80°C) either at the time of collection, after 1, 2, or 4 hours on ice, or after storage overnight at 4°C. The PF was aliquoted into tubes containing either, RNAase inhibitors, protease inhibitors, or no preservative. Proteins and nucleic acids were isolated using standard methods. Protein degradation was determined by SDS-PAGE, and nucleic acid integrity was determined by the DNA integrity number (DIN) and RNA integrity number (RIN).

Results: No endoscopic complications or adverse events occurred during PF collection. Protein degradation was increased after 4 hrs of storage on ice. Addition of protease inhibitors prevented protein and DNA degradation at all times points. DIN from samples stored overnight at 4°C without preservative and with RNAase inhibitor were significantly lower than in snap frozen samples and those in which a protease inhibitor was added. RIN values where below 3 for every condition tested.

Conclusions: PF samples should be processed and frozen within 4 hours to maintain protein and nucleic acid integrity. Addition of protease inhibitors at the time of PF collection decreases DNA and protein degradation. RNA integrity in PF is low even following the addition of RNAase inhibitors.

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Prolactin Receptor, a Novel Target for Pancreatic Ductal Adenocarcinoma

P. Dandawate,1 G. Kaushik,2 D. Subramaniam,1 C. Ghosh,1 S. Choudhury,1 D. Standing,1 P. Ramamoorthy,1 A. Manzardo,3 A. Sayed,1 T. Banerjee,4 S. Santra,4 M. Butler,3 S.B. Padhye,1,5 J. Baranda,6 A. Kasi,6 W. Sun,6 O. Tawfik,7 D. Coppola,8 M. Malafa,8 S. Umar,2 M.J. Soares,7,9-11 S. Saha,12 S.J. Weir,1,13 A. Dhar,1 R.A. Jensen,1,7 S.M. Thomas,1,14 S. Anant.1,2,5 Departments of 1 Cancer Biology, 2 Surgery, and 3 Psychiatry and Behavioral Sciences, University of Kansas Medical Center, Kansas City, KS; 4 Department of Chemistry, Pittsburg State University, Pittsburg, KS; 5 Interdisciplinary Science and Technology Research Academy, Abeda Inamdar College, University of Pune, Pune, India; Departments of 6 Internal Medicine and 7 Pathology and Laboratory Medicine, University of Kansas Medical Center, Kansas City, KS; 8 Department of Gastrointestinal Oncology, H. Lee Moffitt Cancer Center and Research Institute, Tampa, FL; Departments of 9 Obstetrics and Gynecology and 10 Pediatrics, University of Kansas Medical Center, Kansas City, KS; 11 Center for Perinatal Research, Children’s Research Institute, Children’s Mercy-Kansas City, Kansas City, MO; Departments of 12 Radiation Oncology, 13 Pharmacology, Toxicology and Therapeutics, and 14Otolaryngology, University of Kansas Medical Center, Kansas City, KS.

Background: Pancreatic ductal adenocarcinoma (PDAC) has poor patient outcome with current chemotherapy and hence, we need new therapeutic target. In this regard, we have now identified prolactin receptor (PRLR) to be upregulated in PDACs. Prolactin (PRL) activates PRLR and intracellular signaling through the JAK-STAT and JAK-ERK pathways.

Methods: PDAC cells treated with prolactin (PRL) and/or penfluridol were assessed for expression of JAK2, STAT3 and ERK phosphorylation. CRISPR-Cas9 system was used to knockdown PRLR. Penfluridol (5 mg/kg, i.p.) was administered to C57BL/6 mice bearing orthotopically-injected tumors of UNKC-6141 cells.

Results: Interrogation of the Cancer Genome Atlas database demonstrated higher levels of PRLR mRNA in multiple cancers including PDAC compared to normal controls, which we confirmed by immunohistochemical analyses of a tumor microarray. PRLR is also overexpressed in PDAC cell lines. PRL treatment induced a dose- and time-dependent STAT3, and ERK1/2 phosphorylation in cell lines. PRLR knockdown resulted in lower colony and spheroid formation capacity, decreased cell migration in vitro, and reduced tumor-forming capacity in an orthotopic model in C57BL/6 mice. To identify small molecular inhibitors, we generated a homology model of the PRLR intracellular domain, and identified Penfluridol, a highly potent antipsychotic drug in a virtual screening strategy. Penfluridol directly interacts with the JAK2 binding site of PRLR, which we subsequently confirmed in experimental conditions by surface plasmon resonance technique and cellular thermal shift assay (CETSA) techniques. Moreover, Penfluridol decreased PRL-induced STAT3 and ERK phosphorylation in PDAC cells, as well as colony and spheroid formation, and decreased cell migration. Finally, Penfluridol treatment significantly reduced tumor burden in the orthotopic PDAC model. Mechanistically, we observed Penfulridol induced autophagic related protein expression.

Conclusion: These results suggest that PRL-PRLR signaling is crucial for PDAC growth and repurposing penfluridol to target PRL-signaling is an effective therapeutic targeting strategy for treating the disease.

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A Novel Target for Chronic Pancreatitis Therapy: Blocking Gremlin1 in a Mouse Model

J. Davis,1 K. Liu,1 P. Yu,1 M. Younes,2 T.C. Ko,1 Y. Cao.1 Departments of 1Surgery and 2Pathology & Laboratory Medicine, UTHealth, Houston, TX.

Background: We reported that Gremlin1 (Grem1) is pro-fibrogenic and upregulated in human and mouse chronic pancreatitis (CP). To explore the potential of targeting Grem1 for CP therapy, we administered a Grem1 antibody (VelocImmune® Grem1 Ab, Regeneron Pharmaceuticals) to mice with cerulein-induced CP.

Methods: Based on Grem1’s natural ability to inhibit bone morphogenetic protein (BMP) signaling, we first characterized the Grem1 Ab in vitro by assessing its capacity to restore the suppressed BMP/Smad1/5 signaling by Grem1. Human pancreatic fibroblasts and mouse pancreatic stellate cells were treated in 4 groups: 1) vehicle, 2) BMP2 (50 ng/ml), 3) BMP2 (50 ng/ml)+Grem1 (500 ng/ml)+IgG isotype (5000 ng/ml), and 4) BMP2 (50 ng/ml)+Grem1 (500 ng/ml)+Grem1 Ab (5000 ng/mL). Phospho(p)Smad1/5 levels were assessed by western blotting. For in vivo studies, male C57BL/6 mice were randomly assigned to 3 groups: 1) Control mice receiving normal saline, 2) CP mice receiving cerulein (50 μg/kg, 5 ip injections/day, 3days/wk for 4wks)+IgG isotype, and 3) CP mice receiving cerulein+Grem1 Ab (n = 3/group). IgG isotype or Grem1 Ab was given at 10 mg/kg, ip, 2 days/wk, beginning at the 2nd week of cerulein injections and ending at week 4. Mice were weighed weekly and pancreata were harvested at day 4 post treatment. Histopathologic scores were obtained from H&E stained sections. Sirius red staining was performed for fibrosis assessment.

Results: We confirmed in vitro that Grem1 blocked BMP2-induced pSmad1/5 signaling, which was restored by Grem1 Ab. In vivo, compared to the CP mice receiving IgG isotype, the CP mice receiving Grem1 Ab gained more body weight over the course of the study and had 70% reduced fibrosis at 4wks (P < 0.05). However, acinar injury scores were similar in the CP mice regardless of treatment.

Conclusion: Blocking Grem1 activity by Grem1 Ab in vitro restores BMP2-induced pSmad1/5 signaling, and in vivo attenuates pancreatic fibrosis. These results imply that Grem1 Ab may have therapeutic potential for CP.

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Reconsidering Lymphadenectomy for Locoregional Resectable Non-Functioning Pancreatic Neuroendocrine Tumors

S.W.L. de Geus,1 G.G. Kasumova,1 S.C. Ng,1 T.S. Kent,2 D. McAneny,1 M.H. Kulke,3 J.F. Tseng,1 T.E. Sachs.1 1 Department of Surgery, Boston Medical Center, Boston University School of Medicine, Boston, MA; 2 Department of Surgery, Beth Israel Deaconess Medical Center, Boston, MA; 3Department of Medicine, Boston Medical Center, Boston, MA.

Background: The current treatment guideline for locoregional resectable non-functioning pancreatic neuroendocrine tumors (PNETs) suggests that next to resection lymphadenectomy should be considered in tumors of 1 to 2 cm in size, and recommend lymphadenectomy for PNETs > 2 cm. However, the literature has shown ambiguous results. The purpose of this study was to assess the survival impact of lymphadenectomy in PNETs.

Methods: Patients that underwent pancreatectomy between 2004 and 2014 for non-metastatic PNETs 1 to 4 cm in size were identified from the National Cancer Data Base. Propensity score models predicting the odds of undergoing lymphadenectomy (≥1 nodes examined) were created, and patients were matched based on logit of the propensity score. Survival analysis was performed using the Kaplan-Meier method. Subset analysis was performed in patient with positive (cut-off, > 13 nodes examined) and negative (cut- off > 6 nodes examined) nodes.

Results: In total, 2795 patients were identified. 82.8% of patients underwent lymphadenectomy, 76.9% had negative nodes and the median number of nodes examined was 8 (IQR, 2-14 nodes). On multivariable analysis, lymphadenectomy was associated with tumor size >2 cm (vs. ≤2 cm: OR, 2.55; P < 0.0001), academic facility (vs. non-academic: OR, 1.42; P = 0.0009), moderate/poor differentiation (vs. well: OR, 1.48; P = 0.0193), and negative margins (vs. positive: OR, 2.10; P < 0.0001). After matching, lymphadenectomy was not associated with survival benefit (3-year survival: 95% vs. 94%; P = 0.59).

Similarly, extend of lymphadenectomy did not impact survival in patients with positive (3-year survival: 82% vs. 85%; P = 0.10) and negative (3-year survival: 95% vs. 95%; P = 0.10) nodes.

Conclusion: Although positive lymph nodes remain associated with less favorable survival outcomes, the results of this study suggest that lymphadenectomy is not associated with improved survival.

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Endosonographic Changes in the Pancreas are Common in Patients With New-Onset Diabetes Mellitus

J. de la Fuente,1 W.R. Bamlet,2 M.J. Levy,3 N. Takahashi,4 J.G. Fletcher,4 S.T. Chari,3 S. Majumder.3 Departments of 1 Internal Medicine and 2 Biostatistics; 3 Division of Gastroenterology and Hepatology; 4Department of Diagnostic Radiology, Mayo Clinic, Rochester, MN.

Background: Diabetes mellitus (DM) is known to be associated with pancreatic fibrosis; a condition we termed diabetic exocrine pancreatopathy (DEP). Endoscopic ultrasound (EUS) findings in DEP can potentially overlap with chronic pancreatitis (CP). In a population of patients with new-onset DM (NOD) we aimed to assess the prevalence of pancreatic abnormalities on computed tomography (CT) and EUS.

Methods: Consecutive adults above age 50 years with NOD (fasting glucose ≥ 126 mg/dl or HbA1c ≥ 6.5%) prospectively enrolled in the “Examination of the Pancreas in New-onset Diabetes (EXPAND)” trial, who met criteria (new weight loss >5 lbs., CA 19-9>40 U/mL, and negative depression/anxiety screening) for abdominal imaging were included. Patient demographics, CT, and EUS findings were collected using standardized study forms.

Results: A total of 20 patients with NOD met the inclusion criteria for CT/EUS. All patients were Caucasian, 60% (n = 12) were male, median age was 59 years, and median body mass index was 33.3 kg/m2. Seven patients (29%) had history of tobacco exposure with two current smokers (10%). None of the patients had prior history of pancreatitis. DM was non-insulin dependent, with median HbA1c 7.0. Two subjects (10%) had CT evidence of pancreatic calcification suggestive of chronic pancreatitis. Based on Rosemont criteria 60% (n = 12) had EUS findings either consistent or suggestive of CP. The most prevalent EUS finding was hyperechoic foci (100%, n = 20). There was no statistically significant difference in EUS findings between ever smokers and never smokers. None of the subjects older than 60 years (n = 9) had a normal pancreas on EUS.

Conclusion: Incidental pancreatic findings of CP on EUS are common in patients with NOD above the age of 50 years and in the absence of symptoms should not be used to make a diagnosis of CP.

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Engineering the Mouse Cationic Trypsinogen for Selective Activation by Cathepsin B

A. Demcsák, A. Geisz, M. Sahin-Tóth. Department of Molecular and Cell Biology, Boston University, Henry M. Goldman School of Dental Medicine, Boston, MA.

Background: Intra-pancreatic activation of trypsin is an early event in the development of pancreatitis. The mechanism of trypsin activation, however, is still unclear, as trypsinogen can be activated either by trypsin (autoactivation) or by cathepsin B. Experimental separation of trypsin-mediated versus cathepsin B-mediated activation would require engineering special trypsinogen mutants sensitive to one of these proteases but resistant to the other. Our aim was to develop a mouse cationic trypsinogen mutant which can be rapidly activated by cathepsin B but cannot undergo autoactivation.

Methods: Mouse T7 trypsinogens carrying mutations in the activation peptide (D19A; D20A; D21A; D22A; D23A; K24G; D23A,K24G; D22A,D23A,K24G) were expressed recombinantly and purified by affinity-chromatography. Cathepsin B-mediated trypsinogen activation was determined by enzyme activity assays.

Results: Mutation of the activation site Lys24 to Gly (K24G) abolished autoactivation while cathepsin B-mediated activation was preserved. Among mutations of the various Asp residues, mutation D22A induced a 2-fold increase in the cathepsin B-mediated trypsinogen activation, whereas mutations D19A, D20A, D21A, and D23A had no significant effect. The double mutant D23A, K24G was activated by cathepsin B about 1.6-fold faster, while the triple-mutant D22A, D23A, K24G exhibited 4-fold increased activation by cathepsin B.

Conclusion: We successfully engineered a mouse cationic trypsinogen mutant, which is robustly activated by cathepsin B but exhibits complete resistance to activation by trypsin (autoactivation). Based on our in vitro data, we will generate a novel knock-in mouse model carrying this highly cathepsin B-sensitive trypsinogen variant.

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Pancreatic Triglyceride Lipase (PNLIP) Converts Mild Acute Pancreatitis (AP) Into a Lethal Disease

C. de Oliveira, B. Khatua, A. Bag, M. Lowe, V.P. Singh. Department of Medicine, Mayo Clinic, Scottsdale, AZ.

Background: The severity of clinical AP, unlike typical animal models, is variable and unrelated to the etiology. Pharmacologic approaches have previously identified unregulated visceral fat lipolysis to worsen AP severity irrespective of etiology. However, the main lipase responsible for worsening AP is unknown. Since PNLIP is the most highly expressed lipase in the pancreas, we studied the effect of its genetic deletion on AP severity and acinar injury.

Methods: Triglyceride (Glyceryl trioleate) lipolysis and resulting LDH leakage were studied in mouse pancreatic acini from WT (C57BL/6) and PNLIP knockout mice (KO). For in vivo studies, Leptin -/- mice or dual KO (PTL-/-, Leptin-/-) mice of similar weight (56 ± 5 g) were used. Cerulein AP was induced by IP (50 μg/kg/h x 12) injections for 2 days and outcomes of local injury and systemic severity were measured. A P value of <0.05 was regarded as significant

Results: Triglyceride lipolysis was significantly reduced by 62 ± 15% and LDH leakage by 65 ± 8% in the PTL-/- acini. In vivo, while visceral fat amount and composition (76% unsaturated) and parameters for pancreatitis initiation at 24 hours were similar (Sr. Amylase 2913 ± 1416 vs. 3946 ± 2266); dual KO mice had significantly reduced serum lipase (222 ± 208 vs. 8505 ± 5046U/L), fat pad lipase activity (5.4 ± 8 vs. 666 ± 393U/L), PNLIP on fat pad western blot, 5 days mortality (0 vs 100%, median 32h), serum unbound fatty acids (10 ± 4 vs 73 ± 18 nM), BUN (18 ± 7 vs. 71 ± 37 mg/dl), a normalized pulse distention (332 ± 24 vs. 137 ± 18 μm), core temperature (36 ± 0.6 vs. 31 ± 3 °C), prevention of leucopenia (7.1 ± 2.7 vs. 3.4 ± 1.8 x109/L), visceral fat necrosis.

Conclusion: PNLIP is the principal lipase that worsens the severity of AP. Its genetic deletion improves AP outcomes including SIRS, shock, renal failure without affecting AP initiation. PNLIP worsens AP outcomes via excessive lipolysis of visceral fat and consequently increasing unbound fatty acids which mediate systemic complications.

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Regional Variations in Clinical Characteristics and Management of Pediatric Acute Recurrent and Chronic Pancreatitis: Report From the INSPPIRE Study

C.R. Dike,1 B. Zimmerman,1 M. Wilschanski,2 S.L. Werlin,3 D.M. Troendle,4 U. Shah,5 S.J. Schwarzenberg,6 S.J. Rhee,7 J.F. Pohl,8 E.R. Perito,7 C.Y. Ooi,9 J.D. Nathan,10 V.D. Morinville,11 B. McFerron,1 M.R. Mascarenhas,13 A. Maqbool,13 Q. Liu,14 T.K. Lin,10 S.Z. Husain,15 R. Himes,16 M.B. Heyman,7 T. Gonska,17 M.J. Giefer,18 C.E. Gariepy,19 S.D. Freedman,20 D.S. Fishman,16 M.D. Bellin,6 B. Barth,4 M. Abu-El-Haija,10 M.E. Lowe,21 A. Uc.1 1 University of Iowa, Iowa City, IA; 2 Hadassah Hebrew University Hospital, Jerusalem, Israel; 3 Medical College of Wisconsin, Milwaukee, WI; 4 UTSW, Dallas, TX; 5 Massachusetts General Hospital Children's, Boston, MA; 6 University of Minnesota, Minneapolis, MN; 7 University of California, San Francisco, San Francisco, CA; 8 University of Utah, Salt Lake City, UT; 9 UNSW, Sydney, Australia; 10 Cincinnati Children’s Hospital Medical Center, Cincinnati, OH; 11 Montreal Children’s Hospital, Montreal, Canada; 12 Riley Hospital for Children, Indianapolis, IN; 13 CHOP, Philadelphia, PA; 14 Cedars-Sinai Medical Center, Los Angeles, CA; 15 University of Pittsburgh, Pittsburgh, PA; 16 Baylor College of Medicine, Houston, TX; 17 Hospital for Sick Children, Toronto, Canada; 18 Seattle Children’s Hospital, Seattle, WA; 19 Nationwide Children's Hospital, Columbus, OH; 20 Harvard Medical School, Boston, MA; 21Washington University School of Medicine, St Louis, MO.

Background: It is not known whether the clinical characteristics and management of pediatric acute recurrent pancreatitis (ARP) and chronic pancreatitis (CP) differ across various regions.

Methods: Clinical information from children with ARP and CP were obtained at 20 INSPPIRE (INternational Study Group of Pediatric Pancreatitis: In Search for a CuRE) sites. US Centers were grouped by geographical locations. International centers were grouped as “outside US”. Between-group differences were compared by Pearson Chi-Square test and differences in disease burden among regions were compared by Kruskal-Wallis test.

Results: Of 477 children, 121 (25%) were in the West, 151 (32%) Midwest, 45 Northeast (9%), 77 (16%) South and 83 (18%) outside US. The groups were not different in sex. Hispanic ethnicity was more common in West and South (P < 0.0001); white race in Northeast (P = 0.013). CP diagnosis was less common and time from diagnosis of first acute pancreatitis to CP was longer in children outside US (P < 0.0001 and P < 0.01). Genetic variants were the most common risk factors among all groups; PRSS1 mutations predominated in Midwest (P = 0.001).

Obstructive risk factors, particularly gallstones were more common in South (P < 0.01). Tobacco exposure was more common in South or outside US (P < 0.01). ERCP and CT imaging were more commonly done in US (P < 0.0001), but there were no differences in the use of MRI/MRCP. Constant pain, pain medication use (P < 0.01 for both) and missed school days (P < 0.05) were more reported in West and Midwest. All therapies (medical, endoscopic, surgical) were less commonly utilized by sites outside US (P < 0.0001). Eighty percent of total pancreatectomy and islet autotransplantation (TPIAT) were reported by Midwest sites.

Conclusion: There are significant geographical variations in demographics, risk factors and management trends of pediatric ARP and CP, which affects disease outcomes. The lower frequency of pediatric CP and fewer interventions outside the US is intriguing.

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Pancreas-specific SNAP23 Depletion Impairs Acinar Cell Exocytosis and Activates Autophagy

S. Dolai,1 T. Qin,1 T. Liang,1 F. Kang,1 H.Y. Gaisano.1,2 Departments of 1Medicine and 2Physiology, University of Toronto, Toronto, Canada.

Background: Exocytosis of zymogen granules (ZGs) in pancreatic acinar cells requires SNARE proteins, one of which is SNAP-23, postulated to participate in physiologic apical and pathologic basolateral exocytosis. Autophagy, which maintains acinar homeostasis by eliminating cellular debris and damaged organelles, also involves a series of SNARE-mediated vesicular fusion events. Using a pancreas-specific SNAP-23 knockdown (KD) strategy, we investigated the molecular functions of SNAP-23 in ZG exocytosis, acinar cell autophagy and pancreatitis.

Methods: Adeno-SNAP-23-shRNA injected into rat pancreatic ducts depleted acinar SNAP- 23 in a week. Three weeks post-op, these rats were administered suprmaximal cerulein (10 μg/kg/hr, 6 times) to induce pancreatitis. Pancreatitis was assessed by histology, tissue myeloperoxidase activity, serum amylase and lipase activity. Autophagy and ER stress were assessed by biochemical markers. In vitro SNAP-23-KD (24 hrs) acini were used to examine amylase secretion. Basolateral exocytosis Syntaxin-4 SNARE complex formation was assessed by immunoprecipitation.

Results: We observed ∼ 68% and ∼ 48% SNAP-23-KD in the head portion of rat pancreas and dispersed acini, respectively. SNAP-23-KD reduced amylase secretion by 25% and 40% at physiological and supramaximal CCK-8 stimulation, respectively. Reduced formation of basolateral Syntaxin-4 SNARE complex was observed in SNAP-23-KD acini. SNAP23-KD rats were protected against cerulein pancreatitis with less pancreatic tissue neutrophil infiltration and myeloperoxidase activity, and reduced serum amylase and lipase activity. However, increased vacuolization was observed in SNAP23-KD acini, along with increased LC3-I to LC3- II conversion in pancreas lysates, together indicating increased autophagic vacuole accumulation. Biochemical assays demonstrated increased ER stress in SNAP23-KD acini with elevated ER stress markers Bip and phospho-EIF2-α indicating activation of ER stress-mediated autophagy activation, which we attributed to protein overload resulting from the secretory blockade.

Conclusion: SNAP-23 depletion reduces physiologic and pathologic basolateral ZG exocytosis, the latter abrogating pancreatitis. However, this secretory blockade could lead to increased ER stress-mediated autophagy induction

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Targeting IL-1α Processing and Release to Reverse Epithelial-To-Mesenchymal Transition and Inhibit Invasion in Pancreatic Cancer

A. Dosch, X. Dai, F. Messaggio, A. Gaidarski, S. Srinivasan, M. VanSaun, N. Nagathihalli, N.B. Merchant. Department of Surgery, University of Miami Miller School of Medicine, Sylvester Comprehensive Cancer Center, Miami, FL.

Background: Epithelial-to-mesenchymal transition plays a critical role in initiating pancreatic cancer (PDAC) invasion and is induced by numerous inflammatory signals. Interleukin-1α (IL- 1α) is a major cytokine known to mediate host response to tissue injury by activating EMT pathways and stimulating proliferation in neighboring cells. IL-1α requires cleavage by the enzymes calpain I/II in order to be secreted into the extracellular space and maximally exert its biologic effects. We hypothesize that IL-1α activates EMT pathways to promote a metastatic phenotype in PDAC and that pharmacologic blockade of calpain I/II using the inhibitor, calpeptin, will inhibit IL-1α release to reverse EMT, reduce proliferation, and decrease cellular invasion.

Methods: BxPC3, PANC-1, SW1990, and Capan-1 cells were utilized for our study. Effects of IL- 1α and calpeptin on proliferation were assessed using a 3D spheroid model. Secreted levels of IL- 1α were measured by ELISA. Calpain activation was quantified using fluorometric assay kit. Changes in expression of target EMT genes were quantified using qPCR. Modulation of E- cadherin and vimentin expression was determined by immunofluorescence. Apoptosis was measured using Annexin V staining. Cellular invasion was assessed using Boyden chamber assay.

Results: Metastatic cell lines secreted high amounts of IL-1α and had elevated calpain activity compared to primary tumor cell lines. IL-1α stimulated transcription of pro-EMT genes (Zeb1, Snail, Slug), increased vimentin and downregulated E-cadherin expression, increased cellular invasion, and enhanced spheroid growth. Calpeptin drastically reduced secreted levels of IL-1α, inhibited transcription of pro-EMT genes, restored E-cadherin expression, blocked invasion, reduced PDAC spheroid proliferation, and increased cellular apoptosis.

Conclusion: The inflammatory cytokine IL-1α regulates the EMT pathways essential for initiating invasion and metastasis in PDAC. These data show the novel therapeutic potential of targeting IL- 1α processing and release through inhibition of calpain I/II to reverse EMT and decrease metastases in PDAC.

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Pancreatic Stellate Cell Activation is Mediated Through the SIN3a/HDAC Repressive Complex

J.F. Eisses, A. Mukherjee, S.Z. Husain. Division of Pediatric Gastroenterology, UPMC Children’s Hospital of Pittsburgh, University of Pittsburgh School of Medicine, Pittsburgh, PA.

Background: Acute pancreatic injury is marked by acinar cell loss, followed by transient activation of the mesenchymal cell, the pancreatic stellate cell (PSC). PSC activation during pancreatic injury, transforms PSCs from a quiescent state into myofibroblasts which reorganize ECM to provide a scaffold for regeneration. Chronic pancreatic injury leads to fibrotic replacement marked by extracellular matrix (ECM) protein accumulation and sustained PSC activation. The factors that mediate PSC activation in the context of physiologic recovery and pathological fibrosis have not been well studied.When activated, PSCs phenotypically change morphology and transcriptionally switch gene expression which is characterized by expression of αSMA. Activating signals, such as TGF1β, initiate this switch, however the mechanism for this transcriptional switch remains unclear.

Methods: Here we demonstrate in primary and cultured PSCs, by RNAseq and qPCR, that there are dramatic changes in gene expression during PSC activation. We hypothesized that this transcriptional switch is regulated through the epigenetic repressive complex, SIN3a/HDAC.

Results: Our data suggests that the myofibroblast phenotype is actively repressed through SIN3a/HDAC activity. We demonstrate that myofibroblast genes are repressed by the SIN3a complex in non-activated stellate cells by 1) small molecule inhibition of HDAC activity and 2) knockdown of SIN3a through RNAi. These data suggest that the SIN3a/HDAC complex is required to maintain the quiescent phenotype.

Conclusion: We demonstrate that key transcription factors, such as SOX9 and β-catenin, are regulated by SIN3a/HDAC repressive complexes revealing a mechanism for TGFβ1 activation of PSCs. Understanding the mechanism of PSC activation should reveal how fibrosis is initiated and regulated thereby providing new targets to therapeutically treat pancreatic fibrosis.

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Scheduled Outpatient and Inpatient Opioid Use Leads to Long-Term Use in Patients With Early Chronic Pancreatitis

C. Fan,1 M. Faghih,1 N. Parsa,1 V.S. Akshintala,1 A. Kamal,1 F. Garcia Gonzalez,1 M.A. Makary,2 M.A. Khashab,1 A.N. Kalloo,1,3 H. Al-Grain,4 V.K. Singh.1,3 1 Division of Gastroenterology; Department of Medicine, 2 Division of Surgical Oncology, Department of Surgery, 3 Pancreatitis Center, 4Department of Anesthesiology, Johns Hopkins Medical Institutions, Baltimore, MD.

Background: Since acute (AP) and chronic pancreatitis (CP) lie on a disease continuum, early CP often presents with ongoing episodes of AP (ACP) and the management of pain during these episodes may influence long term opioid use.

Methods: Adult patients admitted to Johns Hopkins Hospital with ACP between 2006 to 2016 were evaluated. AP and CP were defined by the revised Atlanta classification and calcification(s) on imaging, respectively. Total opioid used during the first 7 days of admission was converted to oral morphine equivalents (OME) and divided by the numbers of days of administration to calculate the mean OMEs (MOME) per day(s) of treatment. Opioid use prior to admission included scheduled, PRN and none. Long term opioid use was assessed based on a multistate opioid prescription drug monitoring program (PDMP) and outpatient clinic documentation.

Results: There were 69 patients with ACP (mean age 49.8 ± 14 years; 61% male, 64% alcoholic etiology). Median MOME was significantly lower in patients with no opioid use prior to admission (36 [17.5, 88]) when compared to PRN (93.7 [48,236], P = 0.006) or scheduled (245 [36.7,522], P = 0.005) use. There were 38 (55%) patients with no opioid use prior to their initial admission, of whom 29 (76%) developed ongoing and 9 (24%) had no ongoing opioid use over a mean follow-up of 5.5 years. All patients with opioid use prior to their initial admission continued to use opioids. No demographic or clinical parameters predicted long-term opioid use in patients with no opioid use prior to admission.

Conclusion: Scheduled outpatient and inpatient opioid use led to ongoing opioid use over a mean follow-up of years in 76% of initially opioid naïve patients with early CP. Non-opioid treatment strategies need to be evaluated in early CP as a means of averting long term opioid use and dependence.

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The Best Surgical Technique Against Delayed Gastric Emptying

G. Farkas Jr, G. Lazar. Department of Surgery, University of Szeged, Szeged, Hungary.

Background: After pancreaticoduodenectomy (PD) the two main complications are pancreatic fistula and delayed gastric emptying (DGE). Our aim was to compare Billroth II (BII) with double loop and Braun anastomosis vs. Roux-en-Y (RY) after classic PD investigating the incidence rate of DGE. Furthermore we also tested which anatomic reconstruction is superior: retrocolic or antecolic.

Methods: To compare totally similar number of patients – without any post-operative morbidity or adverse event- we selected 8 patients for 4 groups: BII antecolic, BII retrocolic, RY antecolic, RY retrocolic anastomosis. The age, sex, BMI and final hystology were the same in each group.

Results: The incidence of DGE was equal in both BII group showing no difference how the gastro-enteric anastomosis was performed anatomically 25% vs. 25%. We found only grade B DGE in these two groups. Comparing to the RY groups the incidence of DGE was similar to the BII groups (25%).

However in the antecolic sub group we detected grade A DGE, whereas in the retrocolic group we measeured grade B DGE. Regarding to all cases with DGE we performed gastroscopy and gastrographin swallow to make sure no surgical technical problem was responsible for the DGE.

Conclusion: from our results, however the numbers are few we have the suspicion that the antecolic RY anastomosis can be the best result against DGE after classic PD. Although in the literature no difference were found, but after pylorus preserving pancreaticoduodenectomy where surgeons kept the pylorus. As we performed the classic Whipple procedure with antrectomy we think this can be the main reason having different results.

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Mastl Regulates EGFR Signaling to Promote Pancreatic Cancer Progression

I. Fatima,1 S. Chauhan,1 J. Uppada,1 G. Talmon,2 A.B. Singh,1,3,4 S.K. Batra,1,4 P. Dhawan.1,3,4 Departments of 1 Biochemistry and Molecular Biology, 2 Pathology, 3 VA Nebraska-Western Iowa Health Care System, Omaha, NE and 4Buffet Cancer Center, University of Nebraska Medical Center, Omaha, NE.

Background: Anti-cancer treatments act primarily by damaging the DNA of cancer cells. Upon DNA damage, cells stop proliferation at cell cycle checkpoints, which provides them time for DNA repair. Hence, pharmacological inhibition of checkpoint kinases in combination with the DNA damaging anti-cancer therapies (chemotherapy or radiotherapy) is now emerging as promising cancer treatment strategy. In this regard, pancreatic ductal adenocarcinoma (PDAC) remains one of the most lethal malignancies with a frightening resistance to chemotherapeutic and targeted approaches. Here, along with a key role of KRAS-dependent signaling, receptor tyrosine kinases especially the EGF receptor (EGFR) signaling is strongly upregulated in PDAC. Importantly, Greatwall (Mastl in human; Microtubule-associated serine/threonine-protein kinase-like) promotes normal G2-mitosis transition and is highly upregulated in multiple cancer types and demonstrated to associate with therapy resistance. However, status of Mastl expression in PDAC and its potential role in pancreatic cancer progression and/or therapy resistance remains unknown. Our central hypothesis is that MASTL expression is (a) involved in cancer progression and intrinsic drug resistance and (b) constitutes a potential therapeutic target for PDAC.

Methods: We used immunoblotting, immunohistochemistry and TCGA database analysis to examine Mastl expression and its association with pancreatic cancer progression, and patient survival. Normal and pancreatic cancer cells were used. Genetic and pharmacological manipulations for Mastl expression were performed. ERB family members expression, tumoroigenic and invasive activity were determined.

Results: A robust increase in Mastl expression was found in pancreatic cancer cells compared with the non-transformed pancreatic cells. A similar upregulated expression of Mastl was noted in samples originating from KPC mouse model of PDAC (immunoblotting and the IHC). Analysis of the human pancreatic cancer samples and the TCGA database strongly supported these outcome and suggested a positive association of the Mastl expression with cancer progression and patient mortality. Interestingly, genetic inhibition of Mastl expression in pancreatic cancer cells not only inhibited the ability of these cells to proliferate and invade through the matrix but also inhibited EGFR activation. Further studies revealed that overexpressing K-ras/mutant p53 pathway in HPNE cells (untransformed pancreatic cells) modulates Mastl expression, and thus suggested a causal correlation with K-Ras/EGFR signaling in pancreatic cancer cells.

Conclusion: Our results suggest a cross talk of Mastl with K-ras/EGFR signaling pathway in pancreatic cancer progression. We hypothesize that combinatorial therapy targeting Mastl along with Gemcitabine would overcome the drug resistance in the PDAC. Thus, this study identifies a novel approach for the treatment of tumors resistant to traditional EGFR inhibitors.

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Pancreas-specific Secretory Pathway Calcium ATPase 2 Affects Calcium Influx and ER Calcium Stores Through Store-Operated and Store-Independent Calcium Entry

M. Fenech,1,2,3 M. Carter,1,2 C. Pin.1,2,3,4 1 Children’s Health Research Institute; Departments of 2 Pediatrics, 3 Physiology and Pharmacology and 4Oncology; Schulich School of Medicine & Dentistry, Western University, London, Canada.

Introduction: Acinar cell exocytosis requires spatiotemporal accumulation of cytosolic Ca2+. Cytosolic Ca2+ levels are regulated by numerous factors including Ca2+ATPases that pump Ca2+ out of the cytosol, Store-independent Ca2+ entry (SICE), which maintains Ca2+ influx from extracellular spaces, and Store-operated Ca2+ entry (SOCE), which allows Ca2+ uptake directly into the endoplasmic reticulum (ER) upon store depletion. We previously identified a pancreas-specific isoform of secretory pathway Ca2+-ATPase 2 (termed SPCA2C), which consists of the last 136 amino acids of SPCA2, likely disrupting its Ca2+-ATPase function. Other laboratories have identified interactions of the full-length SPCA2 with Orai1, a plasma membrane Ca2+ channel involved in SOCE and SICE. The goals of this study were to determine if SPCA2C regulates cytosolic Ca2+ levels and the mechanisms by which it works.

Hypothesis: SPCA2C affects cytosolic Ca2+ levels through several pathways including SICE and SOCE.

Methods: Epitope-tagged SPCA2C (SPCA2C-FLAG) was expressed in HEK293A cells with/without stable expression of Orai1YFP followed by fura2-ratiometric imaging to examine cytosolic [Ca2+] under normal conditions, or following stimulation of GPCR or SOCE signaling. SPCA2C localization was examined through co-immunofluorescence analysis (IF) for FLAG and organelle-specific markers. Additionally, Orai1-SPCA2C interaction was determined by co-immunoprecipitation (co-IP).

Results: SPCA2C localized to the ER and golgi in the presence and absence of ORA1. Forced SPCA2C expression resulted in increases in (a) resting cytosolic [Ca2+], (b) Ca2+ release in response to carbachol stimulation, (c) Ca2+ in ER stores, and (d) Ca2+ influx through SOCE and SICE, all of which indicates a functional role for SPCA2C in Ca2+ homeostasis. Furthermore, Co-IP showed interaction between Orai1 and SPCA2C, which was altered by co-expression of STIM1.

Conclusion: Our findings suggest SPCA2C plays a role in maintaining Ca2+ homeostasis possibly through multiple mechanisms. Future studies will focus on determining if SPCA2C functions in a similar fashion in acinar cells.

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LAT2 Regulates Glutamine-dependent mTOR Activation to Promote Glycolysis and Chemoresistance in Pancreatic Cancer

M. Feng,1 G. Xiong,1,2 Z. Cao,1 G. Yang,1 S. Zheng,1 J. Qiu,1 L. You,1 L. Zheng,3 T. Zhang,1,4 Y. Zhao.1 Departments of 1 General Surgery and 3 Nuclear Medicine, Peking Union Medical College Hospital, Chinese Academy of Medical Sciences and Peking Union Medical College, Beijing, China; 2 Department of Biliary-Pancreatic Surgery, Affiliated Tongji Hospital, Tongji Medical College, Huazhong University of Science and Technology, Wuhan, China; 4Clinical Immunology Center, Chinese Academy of Medical Sciences and Peking Union Medical College, Beijing, China.

Background: Reprogrammed energy metabolism has become an emerging hallmark of cancer in recent years. Transporters have been reported to be amino acid sensors involved in controlling mTOR recruitment and activation, which is crucial for the growth of both normal and tumor cells. L-type amino acid transporter 2 (LAT2), encoded by the SLC7A8 gene, is a Na+-independent neutral amino acid transporter and is responsible for transporting neutral amino acids, including glutamine, which can activate mTOR. Previous studies have shown that LAT2 was overexpressed in gemcitabine-resistant pancreatic cancer cells. However, the role of LAT2 in chemoresistance in pancreatic cancer remains uncertain and elusive.

Methods: The effects of LAT2 on biological behaviors were analyzed. LAT2 and LDHB levels in tissues were detected, and the clinical value was evaluated.

Results: We demonstrated that LAT2 emerged as an oncogenic protein and could decrease the gemcitabine sensitivity of pancreatic cancer cells in vitro and in vivo. The results of a survival analysis indicated that high expression levels of both LAT2 and LDHB predicted a poor prognosis in patients with pancreatic cancer. Furthermore, we found that LAT2 could promote proliferation, inhibit apoptosis, activate glycolysis and alter glutamine metabolism to activate mTOR in vitro and in vivo. Next, we found that gemcitabine combined with an mTOR inhibitor (RAD001) could reverse the decrease in chemosensitivity caused by LAT2 overexpression in pancreatic cancer cells. Mechanistically, we demonstrated that LAT2 could regulate two glutamine-dependent positive feedback loops (the LAT2/p-mTORSer2448 loop and the glutamine/p-mTORSer2448/glutamine synthetase loop) to promote glycolysis and decrease gemcitabine (GEM) sensitivity in pancreatic cancer.

Conclusion: Taken together, our data reveal that LAT2 functions as an oncogenic protein and could regulate glutamine-dependent mTOR activation to promote glycolysis and decrease GEM sensitivity in pancreatic cancer. The LAT2-mTOR-LDHB pathway might be a promising therapeutic target in pancreatic cancer.

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The Long Non-coding RNA GSTM3TV2 Enhances Drug Resistance in Pancreatic Cancer by Negatively Modulating Let-7 and EGFR

M. Feng,1 G. Xiong,1,2 G. Yang,1 Z. Cao,1 L. You,1 T. Zhang,1 Y. Zhao.1 1 Department of General Surgery, Peking Union Medical College Hospital, Chinese Academy of Medical Sciences and Peking Union Medical College, Beijing, China2, Department of Biliary-Pancreatic Surgery, Affiliated Tongji Hospital, Tongji Medical College, Huazhong University of Science and Technology, Wuhan, China.

Background: Understanding the mechanisms implicated in drug resistance of pancreatic cancer is critical to improving patient outcomes. Recent studies indicated that long non-coding RNAs (lncRNAs) may emerge as key molecules in drug resistance. However, the functional contribution of lncRNAs to drug resistance in pancreatic cancer remains elusive.

Methods: The effects of lncRNAs on biological behaviors were analyzed. lncRNAs levels in tissues were detected, and the clinical value was evaluated.

Results: Here, we demonstrated that the lncRNA Homo sapiens glutathione S-transferase mu 3, transcript variant 2, non-coding RNA (GSTM3TV2) enhances drug resistance in pancreatic cancer by negatively modulating let-7 and EGFR. Firstly, we screened lncRNAs associated with drug resistance in pancreatic cancer and identified a panel of lncRNAs dysregulated in gemcitabine-resistant pancreatic cancer cells. We then identified GSTM3TV2 as a key regulator enhancing the gemcitabine resistance of pancreatic cancer cells in vitro and in vivo. Further studies demonstrated that GSTM3TV2 functioned as a competing endogenous RNA (ceRNA) by competitively sponging let-7 to promote gemcitabine resistance. In addition, we observed that GSTM3TV2 could directly bind epidermal growth factor receptor (EGFR) mRNA and decrease EGFR expression at the protein level, resulting in erlotinib resistance in vitro and in vivo. Meanwhile, we demonstrated that the expression levels of GSTM3TV2 were significantly increased in pancreatic cancer tissues and were associated with poor prognosis.

Conclusion: Together, these results indicated that GSTM3TV2 could be a new therapeutic target and prognostic marker in pancreatic cancer.

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Stromal HSF1 Promotes Tumor Progression in a Mouse Model of Pancreatic Cancer

A. Ferrantella, B. Garg, S. Kurtom, B. Giri, V. Sethi, H.K.C. Jacob, S. Lavania, S. Banerjee, R. Dawra, A.K. Saluja, V. Dudeja. Department of Surgery, University of Miami Miller School of Medicine, Miami, FL.

Introduction: Stromal cells within the tumor microenvironment promote cancer progression in pancreatic ductal adenocarcinoma (PDAC), but the mechanism by which this occurs is not well understood. Heat shock factor-1 (HSF1), a transcription factor that mediates responses to proteotoxic stress, is known to play a pro-survival role in cancer cells, but its role in the stroma of pancreatic cancer is unknown. Here, we aim to delineate the role of HSF1, specifically in the stroma, in a mouse model of PDAC.

Methods: Pancreatic cancer cells were isolated from KrasG12D, Trp53R172H, Pdx-1-Cre (KPC) mice, a genetically engineered mouse model for PDAC. Pancreatic stellate cells (PSCs) were extracted from wild-type C57BL/6 (WT) mice or HSF1 knockout mice. KPC cells were injected alone or co-injected with either WT PSCs or HSF1-/- PSCs into the pancreata of 8-week-old WT female mice to induce tumors. This simulated a stromal compartment with or without HSF1, respectively. Tumor size was measured at endpoint.

Results: Mice co-injected with KPC cancer cells and WT PSCs developed larger tumors than those injected with KPC cells alone. However, this increased tumor burden was abrogated when HSF1 was depleted in the stroma, suggesting that stromal HSF1 promotes tumor progression.

Conclusion: Our findings indicate that HSF1 expression by stromal cells promotes tumor growth in pancreatic cancer. Targeting HSF1 activity may be an effective therapeutic strategy to abrogate this stroma-mediated pathogenicity.

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Lipopolysaccharide Reduces Cancer Recurrence and Metastasis in an Mouse Model of Pancreatic Cancer

A. Ferrantella, B. Giri, S. Kurtom, H.K.C. Jacob, P. Sharma, P. Roy, B. Garg, V. Sethi, S. Lavania, A.K. Saluja, V. Dudeja. Department of Surgery, University of Miami Miller School of Medicine, Miami, FL.

Introduction: Lipopolysaccharide (LPS) is an integral component of the outer membrane of gram-negative bacteria. Preclinical studies have demonstrated the anti-tumor effects of LPS in a few cancers, but these studies have been limited by the use of immunodeficient animal models. Here, we show that treatment with LPS reduces cancer recurrence and metastasis using an immunocompetent mouse model of pancreatic cancer.

Methods: Pancreatic cancer cells were isolated from KrasG12D, Trp53R172H, Pdx-1-Cre (KPC) mice, a genetically engineered mouse model for pancreatic ductal adenocarcinoma. KPC cancer cells were injected into the pancreata of C57BL/6 mice to induce tumors, and the pancreatic tumors were resected after 25 days. Following resection, the mice were randomized to receive treatment with LPS (5 mg/kg) or vehicle twice weekly by intraperitoneal injection. The mice were followed for cancer recurrence. In a separate experiment, KPC cancer cells were injected into the spleens of C57BL/6 mice to induce liver metastases. Following intra-splenic injection, the mice were randomized to receive treatment with LPS (5mg/kg) or vehicle twice weekly by intraperitoneal injection. Liver metastases were measured at the endpoint.

Results: Treatment with LPS resulted in less cancer recurrence following resection of pancreatic tumors, and the median survival for the LPS-treated group was more than double that of the vehicle-treated control group. Additionally, liver metastases were drastically reduced in LPS-treated mice.

Conclusion: Our findings indicate that LPS inhibits cancer progression in pancreatic cancer. Understanding the mechanism by which this occurs may reveal new therapeutic targets against cancer.

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Exocrine Pancreatic Function Determines Major Shifts in Intestinal Microbiota Composition and Diversity

F. Frost,1 T. Kacprowski,2,3,4 M. Rühlemann,5 R. Bülow,6 J.P. Kühn,6,7 A. Franke,5 F.A. Heinsen,5 M. Pietzner,8 M. Nauck,8,4 U. Völker,2 H. Völzke,9 A.A. Aghdassi,1 M. Sendler,1 J. Mayerle,1,10 F.U. Weiss,1 G. Homuth,2 M.M. Lerch.1 Departments of 1 Medicine, 2 Functional Genomics, Interfaculty Institute for Genetics and Functional Genomics, University Medicine Greifswald, Greifswald, Germany; 3 Chair of Experimental Bioinformatics, TUM School of Life Sciences Weihenstephan, Technical University of Munich, Freising, Germany; 4 DZHK (German Centre for Cardiovascular Research), Partner Site Greifswald, University Medicine Greifswald, Greifswald, Germany; 5 Institute of Clinical Molecular Biology, Christian Albrechts University of Kiel, Kiel, Germany; 6 Department of Diagnostic Radiology and Neuroradiology, University Medicine Greifswald, Greifswald, Germany; 7 Institute of Radiology, University Medicine, Carl-Gustav-Carus University, Dresden, Germany; 8 Institute of Clinical Chemistry and Laboratory Medicine, University Medicine Greifswald, Greifswald, Germany; 9 Institute for Community Medicine, University Medicine Greifswald, Greifswald, Germany; 10Department of Medicine II, University Hospital, LMU Munich, Germany.

Background: The intestinal microbiome is an important determinant of health and changes in its composition have been linked to inflammatory, metabolic, and malignant disorders.

Currently known regulators of the microbial community structure only explain small parts of its variation. We investigated how variation in exocrine pancreatic function affects the intestinal microbiota.

Methods: Intestinal microbiota were determined by 16S rRNA gene sequencing from stool samples in 1795 individuals of the population-based Study-of-Health-in-Pomerania (SHIP). Fecal pancreatic elastase ELISA measurements, and in a subset of 435 participants secretin- stimulated pancreatic fluid secretion, were performed for determination of the exocrine pancreatic function.

Results: Changes in fecal pancreatic elastase levels explained larger parts of microbiota variation than age, sex, body-mass-index, smoking, alcohol consumption, or dietary factors. Reduction of exocrine pancreatic function was accompanied by reduction of Bacteroides and a concomitant increase of Prevotella, indicating a shift from a so-called type-1 to a type-2 enterotype. Variation in pancreatic fluid secretion alone exhibited a much smaller association to microbial diversity.

Conclusion: Exocrine pancreatic function appears to be one of the most important host factors regulating the intestinal microbiota composition and diversity. The results from secretin-stimulated pancreatic fluid secretion investigations indicate that products secreted by acinar cells, and not ductal cells, are mainly responsible for shaping the intestinal microbiota.

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Retrospective Comparision Analysis Between Pathology and the Fukuoka Consensus Guidelines in Resected IPMNs in a Single Center

W. Gao, H. Li, Q. Xu, H. Shi, Z. Lu, M. Tu, C. Xi, K. Jiang, J. Wu, F. Guo, J. Chen, J. Wei, C. Lu, C. Dai, Y. Miao. Pancreas Center, First Affiliated Hospital of Nanjing Medical University, Nanjing, China.

Background: The management of intraductal papillary mucinous neoplasm (IPMN) improves constantly. According to the Fukuoka consensus guidelines (FCG), IPMN with worrrisome features should be closely followed up and with high-risk stigmata should be considered for surgical resection. However, the FCG is highly dependent on imaging features. There are uncertainties in the interpretation of imaging features in practice.

Method: A retrospective analysis of 130 cases of surgical resection IPMN confirmed by postoperative pathology in the First Affiliated Hospital of Nanjing Medical University from 2010 to 2017. Fifty-five of the patients with this hospital image (CT or MRI or MRCP) were re-read by two radiologists in a blind and blind manner to determine whether the imaging findings are consistent with the worrrisome features and high-risk stigmata of the FCG.

Results: The age of 130 patients with IPMN was 65.68±10.62 years-old, male: female was about 2:1, and 36.9% of patients were asymptomatic. The mean postoperative hospital stay was 19.2±14.6 days. Preoperative imaging showed IPMN only with worrrisome features, and 10.0% of patients postoperative pathology was canceration; Preoperative imaging showed IPMN with high-risk stigmata, and 31.3% was canceration. By telephone follow-up, the overall survival (OS) of patients with IPMN canceration was significantly lower than that of patients with non-cancerous IPMN (P = 0.0002).

Conclusion: The image characteristics specified in the FCG are uncertain in practical applications. Different radiologists have different interpretation for the imaging indicators in the FCG. The main factor affecting the OS of patients with IPMN is whether it is cancerous. Some patients with IPMN who have only worrrisome features have been cancerous and should be followed closely or considered for surgical resection. However, the practical safety needs to be further study prospectively because of outpatient follow-up cases and invasive carcinoma cases not included.

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Diffusion-Weighted Magnetic Resonance Imaging of Pancreatic Ductal Adenocarcinoma: Correlation With Metastatic Disease Potential and Overall Survival

A. Garces-Descovich,1 T.C. Morrison,2 K. Beker,1 A. Jaramillo-Cardoso,1 A.J. Moser,3 K.J. Mortele.1 1 Division of Abdominal Imaging, Department of Radiology, Beth Israel Deaconess Medical Center, Harvard Medical School, Boston, MA; 2 Division of Abdominal Radiology, Department of Radiology, Boston University Medical Center, Boston University, Boston, MA; 3Pancreas and Liver Institute, Beth Israel Deaconess Medical Center, Harvard Medical School, Boston, MA.

Background: To analyze the relationship between the apparent diffusion coefficient (ADC) of pancreatic ductal adenocarcinoma (PDAC) and the presence or development of metastasis, and overall survival.

Methods: A total of 65 patients with histopathologically proven, treatment naive PDAC with staging DWI- MRI between January 2012 and December 2014 were evaluated; 17/65 patients were excluded. Data for the remaining 48 patients (24 men; median, 65.5 years; IQR, 56 – 77 years) was obtained during a 4-year follow-up period (mean: 397 days ± 415.1). Overall correlation between ADC and presence or development of metastatic disease was assessed using descriptive statistics. Overall survival and mortality analysis was performed using Pearson correlation and Kaplan-Meier curves.

Results: Of 48 patients, 10 either had metastases at the time of staging MRI or went on to develop metastatic disease (n = 12). Among the latter, mean time from staging MRI to metastasis was 258 ± 274.1 days. Majority (86%) of metastases were observed in the liver (n = 19). During the follow-up period, the remaining 26 (54 %) patients never developed metastases. Patients with metastases (n = 22) had significantly lower mean pretreatment ADC (1.27 mm2/s) than those without metastases (1.43 mm2/s) (P = 0.047). ADC of PDAC had a positive correlation with survival: patients with PDAC that showed lower ADCs (< 1.36 mm2/s) had significantly worse 4-year overall survival rates than patients with PDAC that showed higher ADCs (P = 0.036).

Conclusion: Pretreatment ADC values of PDAC are significantly lower in patients who have or will develop metastatic disease and correlate with a worse overall survival.

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Nationwide Trends in Hospital Readmissions, Predictors, and Healthcare Utilization in Patients With Pancreatic Cancer

S.K. Garg,1 S. Sarvepalli,2 H. Goyal,3 H. Kandlakunta,1 M. Sanaka.2 1 Division of Gastroenterology & Hepatology, Mayo Clinic, Rochester, MN; 2 Department Of Gastroenterology, Cleveland Clinic, Cleveland, OH; 3Mercer University School of Medicine, Macon, GA.

Background: National estimates of 30-day readmissions in pancreatic cancer patients in US are unknown. Objective of our study was to identify 30-day readmission rates, predictors of readmissions and economic impact of readmissions.

Methods: We used National Readmission Database (NRD-year 2010–2014), an all payer dataset designed to generate national estimates for readmissions. Pancreatic Cancer patients were identified by ICD-9 codes. All-cause 30-day readmission rates were calculated. The independent predictors of unplanned 30-day readmissions were identified by logistic regression adjusting for stratified cluster design of NRD.

Results: There were 387,453 patients admitted for pancreatic cancer between 2010 and 2014 (48.7% females). Most patients (52%) were between 66 to 84 years of age. Readmission rates improved from 30.2% in 2010 to 28.7% in 2014. Of these, 21% were readmitted for primary diagnosis of pancreatic cancer and 10% admitted for sepsis. Admission during a weekend (OR, 1.101; 95% CI, 1.064-1.139), private insurance (OR, 1.128; 95% CI, 1.063–1.198), being diagnosed with venous thromboembolism (OR, 1.119; 95% CI, 1.075, 1.164), having chemoradiation (OR, 4.268; 95% CI, 3.626–5.023), or ERCP (OR, 1.124; 95% CI, 1.084–1.166) were all associated with increased odds of any-cause readmission. Compared to patients between 18–44 years of age, those between- 45–64 (OR, 0.842; 95% CI, 0.77–0.922), 65–84 (OR, 0.685; 95% CI, 0.622–0.753), or were >85 (OR, 0.442; 95% CI, 0.397–0.491) had decreased odds of readmission. Compared to admission in a metropolitan teaching hospital those admitted to a non- metropolitan hospital (OR, 0.965; 95% CI, 0.931-0.999), or metropolitan, non-teaching hospital (OR, 0.851; 95% CI, 0.796-0.91) had reduced odds of readmission. Additionally, patients with lower quartile median incomes or discharged to home had increased odds of readmission.

Conclusion: Readmissions related to pancreatic cancer are substantial. In this study we have demonstrated factors associated with readmission in patients with pancreatic cancer. This information can be used to inform and modify healthcare policy.

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Incidence, Admission Rates, Economic Burden, and Predictors of Adult Emergency Visits for Pancreatic Cancer, Data From the National Emergency Department Sample, 2006 to 2014

S.K. Garg,1 S. Sarvepalli,2 H. Goyal,3 H. Kandlakunta,1 M. Sanaka.2 1 Division of Gastroenterology & Hepatology, Mayo Clinic, Rochester, MN; 2 Department of Gastroenterology, Cleveland Clinic, Cleveland, OH; 3Mercer University School of Medicine, Macon, GA.

Background: Pancreatic cancer (PC) is a relatively uncommon cancer usually presenting at advanced age with dismal prognosis and often leads to unplanned emergency department (ED) visits. However, to date, the healthcare burden of PC-related ED visits has not been assessed. Using the largest national cohort of ED visits, we aimed to investigate trends in ED utilization, and assess factors associated with hospital admission from ED.

Methods: The Nationwide Emergency Department Sample (NEDS; 2006–2014) was interrogated to identify patients with primary diagnosis of PC (ICD-9 CM code: 157.0-157.9). The yearly incidence of ED visits, rates of hospitalization, length of stay (LOS), and total charges associated with ED or subsequent inpatient stays were assessed. A survey logistic regression model was used to determine predictors of hospitalization.

Results: A total of 175,833 PC-related ED visits were identified. Interestingly, overall incidence of these visits gradually increased from 17,974 in 2006 to a peak of 22,479 in 2014. The mean ED charges per visit, adjusted to inflation increased significantly from $1274.45 in 2006 to $3145.56 in 2014. However, a slight decrease in hospitalization rates was noted from 88.6% in 2006 to 82.1% in 2014. Also, in-hospital deaths decreased from 11.5 % to 7.1%. The average LOS in the hospitals decreased from 7.4 to 6.6 days. Using multivariate analysis, the most significant factors associated with hospitalization were female gender, older age groups, ED in a metropolitan teaching hospital, presence of liver metastasis, biliary duct obstruction, lung metastasis, and higher Charleson comorbidity score.

Conclusion: Increasing ED visits and charges associated with PC are concerning. However, decreasing trend in the hospitalizations and mortality are encouraging. The results of our study can be used to identify the PC patients who tend to visit ED more frequently and are at higer risk of hospitalization.

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Nationwide Trends in Opiate-related Hospitalizations in Patients With Chronic Pancreatitis

S.K. Garg,1 S. Sarvepalli,2 H. Goyal,3 H. Kandlakunta,1 M. Sanaka.2 1 Division of Gastroenterology & Hepatology, Mayo Clinic, Rochester, MN; 2 Department of Gastroenterology, Cleveland Clinic, Cleveland, OH; 3Mercer University School of Medicine, Macon, GA.

Introduction: Opiates are commonly prescribed in chronic pancreatitis (CP) patients. However, burden of inpatient stays for opioid dependence and overdoses in CP has not been examined. In this study, using a large national inpatient database, we examined burden, trends and associated factors in opiate-related hospital stays (ORHS) in CP patients.

Methods: National inpatient sample (NIS) was interrogated to identify discharges with primary or secondary diagnosis of CP (ICD-9-CM: 577.1) between 2007 to 2014. ORHS were defined as all-listed diagnoses associated with opioids or illicit substances such as heroin. Survey logistic regression was performed to identify demographic and hospital factors associated with ORHS in CP

Results: Among 1,173,703 hospitalizations with CP, 52,457 (4.5%) were opiate-related. During the study period, ORHS increased by 58%, from 3.4% to 5.3%. Although, 18-44 age group had highest rates of ORHS at 5.9%, surprisingly a 148% increase in ORHS was seen in 65-84 age group. Females had a slightly greater rate of ORHS (female, 4.6%; male, 4.4%) with slightly sharper rise (female, 60%; male, 56%). With regard to etiology of CP, patients with alcohol use (5.8%) had highest ORHS rate, followed by smoking (5.4%), and biliary disease (2.6%). With regards to trends an opposite pattern was noted. CP patients with biliary disease (132.6%) experienced highest rise in ORHS, followed by smoking (46.6%), and alcohol-related diseases (33.3%). In multivariate analysis female sex, younger age, non-weekend admission, staying in wealthier zipcode, white race, medicare insurance, fewer comorbidities, alcohol use, smoking, and normal weight were associated with greater risk of ORHS.

Conclusion: According to a previous HCUP analysis, ORHS accounted for 0.2% of all hospitalizations. However, strikingly patients with CP have >20 times increase in incidence of ORHS with upward trends. Clearly, burden and trends of ORHS in CP are alarming, and highlight a present and urgent need for intervention. Further, patient and hospital-related factors associated with ORHS can help make informed decisions for opiate prescriptions in CP patients.

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Incidence of Pancreatitis With the Use of Immune Checkpoint Inhibitors (ICI) In Advanced Cancers: A Systematic Review and Meta-Analysis

J. George, J.W. Yoo, N. Joshi, J.J. Farrell. Department of Internal Medicine, Bridgeport Hospital-Yale New Haven Health, Bridgeport, CT; Section of Digestive Diseases, Yale University School of Medicine, New Haven CT.

Background: Systemic immune side effects including pancreatitis have been reported with the use of Immune checkpoint inhibitors (CTLA-4, PD-1 and PDL-1). However the true incidence, risk, causes (tumor or drug specific) of pancreatitis and relation to other immune side effects, especially diabetes mellitus are unknown.

Methods: We performed a systematic review of all clinical trials using ICI for the incidence of any grade or grade 3&4 pancreatitis or IDDM. In addition to the grade of pancreatitis or IDDM, the drugs used including combinations, study design and tumor treated were recorded. Statistical analysis was done by Chi Square test. P < 0.05 was considered statistically significant.

Results: A total of 37 clinical trials involving 7387 patients are included in the study. The incidence of any grade pancreatitis after ICI use is 2.7% (211/7702) and grade 3-5 is 1.9%(150/7702). Patients treated with CTLA-4 inhibitors have increased incidence of pancreatitis when compared to patients treated with PD1 inhibitors (3.6% vs1.8% P <0.05). Patients treated with ICI for melanoma have increased incidence of pancreatitis when compared to non-melanoma cancers (3.7% vs 1.2% P <0.05). We also noted an additive increase in incidence of AP with combination ICI of PDI and CTLA4 inhibitors. There is limited information about the response to steroids for cases of pancreatitis.

Conclusion: Our study provides precise data for the incidence of pancreatitis among patients using ICI based on tumor types and ICI regimens. ICI use for solid tumors is associated with increased incidence of all grades of pancreatitis, especially for CTLA-4 agents and ICI combination. Although it does not appear to be associated with mortality, ICI related pancreatitis should be recognized early for appropriate treatment and to reduce complications.

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KDM3A, an Epigenetic Mark and DCLK1, Pancreatic Cancer Stem Cell Marker, Interactions Promotes Stemness and Tumorigenesis

C. Ghosh,1 K. Palaniyandi,1 S. Paul,1 P. Dandawate,2 S. Rawal,2 D. Subramaniam,2 S. Padhye,1 S. Gunewardena,3 S. Thomas,1 R. Jensen,1 S. Maliski,4 S. Weir,5 T. Iwakuma,1 S. Anant,1 A. Dhar.1 Departments of 1 Cancer Biology, 2 Surgery, 3 Molecular and Integrative Physiology, 4 Nursing, 5Pharmacology and Toxicology, University of Kansas Medical Center, Kansas City, KS.

Background: Pancreatic ductal adenocarcinoma (PDAC) is the major leading cause of cancer related human death in the US. 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.

Methods: KDM3A influences tumor growth and regulates stemness. Therefore, our goal is to find out the role of KDM3A in tumor progression in PDAC through interactions with DCLK1. 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’s 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: Co-expression of DCLK1 with KDM3A influenced stemness and enhance tumor progression in PDAC.

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Preoperative Biomarkers as Predictors of Non-Resectability and Early Recurrence in Pancreatic and Periampullary Tumors

M. Gil,1 A. Gomes,2 R.V. Martins,2 A. João,2 V. Nunes.2 1 Faculty of Medicine, University of Lisbon, Lisbon, Portugal; 2HBP Surgery, Hospital Prof. Doutor Fernando Fonseca, Amadora, Portugal.

Background: Pancreatic cancer has a dismal prognosis. Surgical resection is the only option for curative intent. Some patients who are offered surgical resection are found to have unresectable disease or early recurrence. There are no efficient biomarkers available for accurate prediction of resectable disease or long term survival. This study aims to evaluate the predictive value of systemic inflammatory and nutritional biomarkers with regards to disease staging at diagnosis, surgical resectability and early recurrence.

Methods: Data of patients with pancreatic or peri-ampullary carcinoma diagnosed between January 2005 and August 2017 in Hospital Prof. Doutor Fernando Fonseca was retrospectively analysed. The neuthrophil-to-lymphocyte ratio (NLR), lymphocyte-to- monocyte ratio (LMR), platelet-to-lymphocyte ratio (PLR), C-reactive protein (CRP), serum albumin (ALB), CRP-to-ALB ratio, Prognostic Nutritional Index (PNI), Hounsfield Unit Average Calculation (HUAC), modified Glasgow Prognostic Score (mGPS) and CA 19-9 were compared according to disease staging at diagnosis, intra- operative staging and early recurrence (<6 months). Non parametric statistics were used. Multivariate logistic regression and ROC curve analysis were performed.

Results: 391 patients were included. NLR, LMR, CRP, ALB, CRP/ALB, PNI, CA 19-9 and mGPS were associated with metastatic disease at diagnosis. The multivariate analysis showed that NLR, mGPS, CRP/ALB and CA 19-9 were independent predictors of disease staging at diagnosis. ROC curve analysis showed low to moderate AUC values. 108 patients underwent surgery, of which 23.8% were found to have unresectable disease at intra- operative staging. 26.9% had early disease recurrence. When adjusted for histology, none of the biochemical and radiological biomarkers were independent predictors of unresectable disease or early recurrence.

Conclusion: Biochemical and radiological inflammatory and nutritional biomarkers at diagnosis were predictors of disease staging with low performance. Pre-operative markers were not predictors of unresectable disease or early recurrence. Variables, other than the ones measured, modulate the disease prognosis assessed by the selected outcomes.

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Coexistence of CPA1, SPINK1, CFTR, CTRC, PRSS1 Gene Mutations in Acute Pancreatitis and Pancreatic Cancer

S. Głuszek,1,2 M. Wawszczak,1 W. Adamus-Białek,1 M. Majchrzak,1 J. Klusek,1 D. Kozieł.1 1 Department of Surgery and Surgical Nursing with the Scientific Research and Genetic Laboratory, Faculty of Medicine and Health Sciences, Jan Kochanowski University, Kielce, Poland; 2Department of General, Oncological and Endocrinological Surgery, Voivodeship Hospital, Kielce, Poland.

Background: Environmental factors significantly increase the risk of acute pancreatitis (AP) and pancreatic cancer (PC) as well as mutations in genes related with premature activation of pancreatic enzymes or hereditary predispositions. The aim of the study was to analyze the coexistence of CPA1, SPINK1, CFTR, CTRC and PRSS1 gene mutations in AC and PC patients.

Methods: The analysis included DNA isolated from blood of 148 patients with AP, 20 patients with PC and 48 controls. The mutations in CPA1, SPINK1, CFTR, CTRC and PRSS1 were analyzed using PCR and sequencing method.* The coexistence of studied mutations was evaluated by the statistical analysis with the Bonferroni correction (P < 0.05/28=0.00179).

Results: There were no statistically significant differences in coexistence of mutations in CPA1, SPINK1, CTRC, CFTR between investigated groups. However, within CPA1 10 characteristic mutations were identified. Seven mutations were specific for PC patients, and three of them occurred at a frequency greater than 50%. Coexistence of minimum two mutations were identified in 12 cases (60%). All three mutations were identified in 5 cases (25%). There were no cases of coexistence in controls and AP patients.

Conclusion: The lack of the correlation between the mutations in the CPA1 gene and other tested genes may results from different phenotypic consequences of these mutations. It was found that there is a correlation between the number of CPA1 mutations and the persistence of pancreatic cancer. The accumulation of CPA1 mutations may lead to dissfunction of caboxypeptidase 1 and increase the stress in the endoplasmic reticulum (ERS), which further can causes the generation of new mutations and intensify the pancreas pathology.

*SPINK1, CFTR, CTRC, PRSS1 variants were identified by Artur Kowalik from Department of Molecular Diagnostics, Holycross Cancer Centre, Kielce, Poland

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Temporal Relationship Between SIRS, Organ Failure, and Death in Acute Pancreatitis: Data From a Large, Multicenter, International Study (APPRENTICE Study Group)

A. Gougol,1 P. Paragomi,1 I. Pothoulakis,1 R. Talukdar,2 R. Kochar,3 M.K. Goenka,4 A. Gulla,5,6 J.A. Gonzalez,7 V.K. Singh,8 M. Ferreira,9 T. Stevens,10 S.T. Barbu,11 H. Nawaz,12 S.C. Gutierrez,13 N.O. Zarnescu,14 G. Capurso,15 J. Easler,16 K. Triantafyllou,17 M. Pelaez-Luna,18 S. Thakkar,19 C. Ocampo,20 E. de-Madaria,21 G.A. Coté,22 B.U. Wu,23 A. Kamal,8 L. Archibugi,15 S. Tiwari,4 X. Gao,1 G. Tang,1 G.I. Papachristou.1 1 University of Pittsburgh Medical Center, Pittsburgh, PA; 2 Asian Gastroenterology Institute, Hyderabad, India; 3 Postgraduate Institute of Medical Education and Research, Chandigarh, India; 4 Apollo Gleneagles Hospitals Kolkata, Kolkata, India; 5 Georgetown University Hospital, Washington, DC; 6 Lithuanian University of Health Sciences, Kaunas, Lithuania; 7 Universidad Autónoma de Nueva León, Monterrey, Mexico; 8 Johns Hopkins Medical Institutions, Baltimore, MD; 9 Hospital Nacional de Itauguá, Itaugua, Paraguay; 10 Cleveland Clinic Foundation, Cleveland, OH; 11 University of Medicine and Pharmacy "Iuliu Hatieganu", Cluj-Napoca, Romania; 12 Eastern Maine Medical Center, Bangor, ME; 13 Hospital Nacional "Profesor Alejandro Posadas", Buenos Aires, Argentina; 14 University of Medicine and Pharmacy, Bucharest, Romania; 15 Digestive and Liver Disease Unit, S. Andrea Hospital, Sapienza University of Rome, Rome, Italy; 16 Indiana University School of Medicine, Indianapolis, ID; 17 Attikon University General Hospital, Athens, Greece; 18 Instituto Nacional de Ciencias Médicas y Nutrición Salvador Zubirán-Universidad Autónoma de Mexico, Mexico City, Mexico; 19 Allegheny General Hospital, Pittsburgh, PA; 20 Hospital General de Argudos "Dr. Cosme Argerich", Buenos Aires, Argentina; 21 Investigación Sanitaria y Biomédica de Alicante (ISABIAL - Fundación FISABIO), Alicante, Spain; 22 Medical University of South Carolina, Charleston, SC; 23Kaiser Permanente Los Angeles Medical Center, Los Angeles, CA.

Background: Although systemic inflammatory response syndrome (SIRS) has been associated with worse outcomes in acute pancreatitis (AP); its time relationship with persistent organ failure (POF) and death has been poorly described. Our aim was to investigate the dynamic relationship between SIRS, POF and death in AP.

Methods: APPRENTICE is a multi-national, prospective study in AP, initiated in 2015. Twenty-two international centers participated across 4 continents. SIRS was defined as of ≥2/4 criteria. Organ failure (OF) was defined by modified Marshall score ≥2 for respiratory, renal, or cardiovascular. POF referred to OF lasting ≥ 48 hours. Death was defined as in-hospital mortality.

Results: 1544 patients were prospectively enrolled (age 50, 52% male), of whom 166 (11%) developed POF and 39 (2.5%) died. SIRS on admission was found in 41%, with 22% developing POF at a median of 40 (18-85) hours. The incidence of POF peaked within 24 hours of admission, with 78% developing POF within first 96 hours of hospitalization. Among patients with POF, 47% progressed to multi-organ failure (MOF) and renal was the first organ to fail in 45%, two concurrent organs failed in 30%, and respiratory in 23%. Among patients with POF, 20% died at a median of 9 (3-24) days after OF onset. In regards with the organ failing first in patients with MOF, renal was associated with lower mortality (20% in renal vs. 65% in all others, P < 0.01).

Conclusion: The findings of this multinational study demonstrate that SIRS is common, develops early in AP, progresses to OF within 2 days, and about a fifth of patients with POF die at a median of 10 days. The above results from APPRENTICE, will provide knowledge to clinicians in regards with the natural history of AP and also guide investigators with the design of clinical trials.

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Deletion and Insertion Mutations in the Gene Encoding the Digestive Enzyme Carboxyl Ester Lipase (CEL): A Role in Pancreatic Disease?

A. Gravdal,1,2,3 R.S. Brekke,1,3 K. El Jellas,1,3,4 X. Xiao,5 M.E. Lowe,5 D. Lombardo,6 E. Mas,6 B. Johansson,1,7 K. Fjeld,1,2 A. Molven.1,3,4 1 KG Jebsen Center for Diabetes Research, Department of Clinical Science, University of Bergen, Bergen, Norway; 2 Department of Medical Genetics, Haukeland University Hospital, Bergen, Norway; 3 Gade Laboratory for Pathology, Department of Clinical Medicine, University of Bergen, Bergen, Norway; 4 Department of Pathology, Haukeland University Hospital, Bergen, Norway; 5 Department of Pediatrics, Division of Gastroenterology, Washington University School of Medicine, St. Louis, MO; 6 Aix-Marseille University, INSERM, CRO2, Center for Research in Biological Oncology and Oncopharmacology, Marseille, France; 7Department of Pediatrics, Haukeland University Hospital, Bergen, Norway.

Background: The last exon of the CEL gene is highly polymorphic because of a variable number of tandem repeats (VNTR) region. Rare single-base deletions (DEL) within the VNTR lead to frameshifts and a new C-terminal sequence of the protein. Some DEL carriers suffer from MODY8, an inherited disorder characterized by exocrine pancreatic dysfunction and diabetes. In contrast, single-base insertions (INS) are common within the VNTR and result in a truncated protein with only five new C-terminal amino acids. The aim of this study was to gain more knowledge about the role of the CEL VNTR in pancreatic disease.

Methods: Plasmid constructs encoding different INS and DEL variants of CEL were transiently transfected into HEK293 cells. We studied CEL expression by Western blotting, immunofluorescent staining, confocal imaging and immunohistochemistry. We employed antibodies specific for the tail of normal CEL (anti-PAVIRF) and for the INS variant tail (anti- PRAAHG).

Results: MODY8-causing DEL variants showed elevated intracellular accumulation compared with DEL variants not known to associate with disease. DEL variants that all had the same theoretical molecular weight migrated with different band sizes. This observation suggested different patterns of post-translational modification in the repeat region. Varying levels of the DEL variants in the insoluble cell fraction suggested that expanding the aberrant CEL tail makes the protein more prone to aggregation.

The tested INS variants behaved similarly to the normal CEL protein with regard to secretion and intracellular accumulation. Intriguingly, by using the anti-PRAAHG antibody, we could detect specific expression of INS variants in germ-line INS-negative human pancreatic tissue sections, both in cancer and non-cancer specimens. This observation possibly reflects somatic mutational events.

Conclusion: The level of expression and secretion of CEL varied according to VNTR length and composition. CEL variants appear most pathogenic when the mutational event has taken place in the more proximal VNTR repeats. Naturally occurring INS variants are most likely benign.

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Effective Experimental Therapeutics in a Novel Humanized Animal Model of Hereditary Pancreatitis

F. Gui,1 Y. Zhang,1 X. Zhan,1 J. Wan,1 J. Chen,1 X. Zhu,1 A. Haddock,1 Y. Li,1 L. Zhang,2 S.J. Pandol,3 C.D. Logsdon,4 Y. Bi,5 B. Ji.1 1 Department of Cancer Biology, Mayo Clinic, Jacksonville, FL; 2 Department of Pathology, Mayo Clinic, Rochester, MN; 3 Department of Medicine, Cedars-Sinai Medical Center, Los Angeles, CA; 4 Department of Cancer Biology, MD Anderson Cancer Center, Houston, TX; 5Division of Gastroenterology and Hepatology, Mayo Clinic, Jacksonville, FL.

Background: Hereditary pancreatitis (HP) is an inherited form of pancreatitis characterized by recurrent acute pancreatitis (AP) which progresses to chronic pancreatitis (CP). The cumulative risk of pancreatic cancer in HP patients is 40%. In this study, we aimed to generate a HP mouse model for studying its pathogenesis and develop therapeutics.

Methods: We used a human Bacterial Artificial Chromosome harboring the full-length human PRSS1 gene with intact PRSS1 promoter, exons, and introns. An R122H point mutation was introduced using Galk-mediated recombineering technology. The human PRSS1 expression level was detected by Western blot and RT-PCR. Pancreatitis was initiated by cerulein (50 ug/kg). An FDA approved DrugD was used to prevent and treat the disease.

Results: The expression level of PRSS1R122H was at ∼50% of the endogenous human PRSS1 protein level. In humans with a heterozygous PRSS1 mutation, it is expected that 50% of the total PRSS1 protein is mutant. Therefore, transgenic expression of mutant PRSS1 equivalent to 50% of total human PRSS1 protein is physiologically relevant. These mice did not develop spontaneous AP. However, when challenged with cerulein, PRSS1R122H mice presented with more severe AP. Ten days after AP induction, pancreatic histology of control WT mice showed complete recovery. However, pancreata of the PRSS1R122H mice developed chronic inflammation with acinar atrophy, stellate cell activation, collagen deposition and adipocyte replacement. Pretreatment with DrugD completely prevented the development of AP. Treatment with DrugD starting at 24 hours after cerulein induction significantly ameliorated its progression to CP.

Conclusions: We have successfully developed an animal model that faithfully recapitulates human HP and identified a drug that is effective for its prevention and therapy. This novel clinically relevant model will provide a powerful tool for elucidating the molecular mechanisms of pancreatitis and testing new preventive and therapeutic interventions.

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Activity of Heat Shock Protein 70 Gene Promoter Polymorphism and Acute Pancreatitis

A. Gulla,1,2,3 K. Stuopelyte,4 A. Kasarinaite,4 S. Jarmalaite,4 K. Strupas.1,2 1 Department of Gastroenterology, Surgery, Nephro-Urology, Vilnius University Hospital “Santaros Klinikos”, Vilnius, Lithuania; 2 Faculty of Medicine, Vilnius University, Vilnius, Lithuania; 3 Department of Surgery, Georgetown University Hospital, Washington DC; 4Division of Human Genome Research Centre, Institute of Biosciences, Life Sciences Center, Vilnius University, Vilnius, Lithuania.

Introduction: Acute pancreatitis is a severe and life-threatening disease, which can lead to pancreatic necrosis, acute lung injury, SIRS and MODS. The inducible heat shock protein- 70 is anti- oxidative, anti-inflammatory, and cytoprotective enzyme that is induced in response to cellular stress. HSP-70 promoter contains (GT)n dinucleotide repeats and is highly polymorphic in the population. In this study, we hypothesized that different SNPs in HSP-70 promoter can influence the occurrence of acute pancreatitis due to its protective function.

Methods: Acute pancreatitis (n = 28) patients and age- and sex-matched healthy controls (n = 6) were studied. Peripheral blood samples form pancreatitis patients were collected on admission. Genomic DNA was extracted from the blood samples of patients and control groups. Four single nucleotide polymorphisms (SNPs) of HSP70-gene family were selected: rs1061581 A>G (HSPA1B), rs1008438 A>C (HSPA1A), rs2227956 A>G (HSPA1L) and rs1043618 G>C (HSPA1A). DNA was extracted from the whole blood, and polymorphism frequencies determined by TaqMan allelic discrimination assays were compared between patients with AP and controls.

Results: The subjects were categorized into 4 groups based on the genotype results: A/G (rs1061581), A/C (rs1008438), A/G (rs2227956), C/G (rs1043618). The presence of A/A was similar between the patient group (71.4%) and the controls (77.7%). Interestingly, 46.6 % of patients were carries of A/G vs 83.3% A/A control subjects.

Conclusion: Our data demonstrate a strong bias toward A/G (rs2227956). Among patients with acute pancreatitis. Further studies are now underway to analyze the pancreatic levels of HSP-70 protein in acute pancreatitis patients and controls and to determine whether the presence of HSP- 70 upregulation and consequently promoter its protective anti-inflammatory function in acute pancreatitis.

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Hypoxia Mediated L-2-Hydroxyglutarate Accumulation Regulate Self-Renewal in Pancreatic Cancer

V.K. Gupta, N. Sharma, K. Kesh, R. Hadad, B. Durden, V. Dudeja, A.K. Saluja, S. Banerjee. Department of Surgery, University of Miami Miller School of Medicine, Miami, FL.

Introduction: Pancreatic cancer remains a major health issue in United States along with a 5- year survival rate of only 7%. Pancreatic cancer cells have a high demand for metabolic nutrients to fuel their rapid proliferation and therefore undergo extensive metabolic reprogramming, which is driven by oncogene-mediated cell-autonomous pathways, the unique physiology of the tumor microenvironment, and interactions with non-cancer cells. This results in abnormal accumulation of various metabolites which causes both metabolic and nonmetabolic dysregulation and potential transformation to malignancy and therefore termed as “oncometabolites.” Hypoxia is a major hallmark of pancreatic tumors which associated with increase in self-renewal.

Aim: The present study investigates the role of Hypoxia mediated altered oncometabolite in regulating self-renewal in Pancreatic Cancer.

Methods: Pancreatic Cancer cells (MIA PaCa-2 and S2-VP10) cultured in hypoxic environment (1.0% Oxygen) for 24 hrs. Cell lysates and culture supernatant analyzed for metabolites using gas chromatography-mass spectrometry (GC-MS). Chiral derivatization by R(-)-2-butantol and acetic anhydride was done to separate L-2HG and D-2HG enantiomers by GC-MS. Gene expression were analyzed in different cell lines and conditions by QPCR.

Results: Metabolic mass spectrometric analysis of hypoxic pancreatic cancer cells showed significant accumulation of 2-hydroxyglutarate (2-HG) both in cells and culture supernatant. Further analysis showed Hypoxic cells selectively accumulate L- enantiomer form. Self-renewal requires critical balance between stemness and differentiation. Current study showed for the first time that L-2-HG regulates self-renewal by increasing expression of genes associated with stemness (Sox-2, CD133) and by decreasing expression of differentiation genes (PdX-1, HB9, NKX6.1). Similar genes expression was observed in cells under hypoxic environment. Further analysis showed that L-2-HG mediated epigenetic changes regulates these self-renewal gens in pancreatic cancer.

Conclusion: Our results indicated that hypoxia mediated accumulation of L-2-HG upregulate self-renewal by shifting critical balance of gene expression towards stemness in Pancreatic cancer. This increase in stemness contribute to chemoresistance and highly metastatic tumors associated with Pancreatic cancer.

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Sonic Hedgehog Signaling Pathway Promotes Pancreatic Cancer Pain via Nerve Growth Factor

L. Han, L. Cheng, Q. Ma. Department of Hepatobiliary Surgery, First Affiliated Hospital, Xi’an Jiaotong University, Xi’an, China.

Background: Many pancreatic cancer (PC) patients suffer from abdominal pain and back pain. However, the cause of pain associated with PC is largely unclear. In this study, we tested the potential influence of the sonic hedgehog (sHH) signaling pathway on PC pain.

Methods: Substance P (SP) and calcitonin gene-related peptide (CGRP) expression was measured in cultured PC cells and dorsal root ganglions (DRGs) by real-time PCR, western blotting analysis and ELISA. Small interfering RNA transfection and plasmid constructs were used to regulate the expression of sHH in the AsPc-1 and Panc-1 cell lines. Pain-related behavior was observed in an orthotopic tumor model in nude mice.

Results: In this study, the results showed that sHH increased the expression of SP and CGRP in DRGs in a concentration- and time-dependent manner. Additionally, sHH secretion from PC cells could activate the sHH signaling pathway and, in turn, increase the expression of nerve growth factor (NGF), P75 and TrkA in DRGs. Furthermore, the sHH signaling pathway and NGF/NGF receptor contributed to pain factors and pain behavior.

Conclusion: Our results demonstrate that PC pain originates from the sHH signaling pathway, and NGF mediates the pain mechanism via regulating SP and CGRP

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Pancreatic ERCP Training is Inadequate and Infrequently Performed in Independent Practice: Results From a Prospective Multicenter Study Evaluating Learning Curves and Competence Among Advanced Endoscopy Trainees

S. Han,1 R. Keswani,2 M. Hall,3 D. Early,4 E. Aagaard,4 V. Simon,1 L. Carlin,1 S. Ellert,1 W. Abidi,5 T. Baron,6 B. Brauer,1 H. Chatrath,7 G.A. Coté,8 K. Das,9 C. DiMaio,10 S. Edmundowicz,1 I. El Hajj,11 H. Hammad,1 S. Jalaj,6 M. Kochman,9 S. Komanduri,2 L. Lee,5 D. Mullady,4 R. Muthusamy,7 A. Nett,12 M. Olyaee,13 K. Pakseresht,13 P. Perera,12 P. Pfau,14 C. Piraka,15 A. Rastogi,16 R. Shah,1 R. Sharma,17 J. Scheiman,12 J. Tabibian,9 D. Uppal,18 S. Urayama,17 A. Wang,18 T. Zuchelli,15 E. Bowman,14 G. Lang,4 D. Loren,19 A.H. El Chafic,19 P. Yachimski,20 A. Gamboa,20 J. Poneros,21 W. Wassef,22 M. Sohail,22 T. Stevens,23 B. Confer,23 N. Guda,24 S.I. Gan,25 A. Razzak,25 S. Gordon,26 J. Ferriera,26 S. Banerjee,27 A. Choudhary,27 A. Dries,28 J. Buscaglia,29 D. Tzimas,29 S. Zepeda-Gomez,30 S. Kenshil,30 F. Lukens,31 M. Barte,l31 P. Jowell,32 J. Spaete,32 B. Riff,10 C. Harris,33 S. Saligram,33 B. Gannavarapu,2 A. Chak,34 A. Singh,34 S. Wani.1 1 University of Colorado, Aurora, CO; 2 Northwestern University, Chicago, IL; 3 Children’s Hospital Associates, Cincinnatti, OH; 4 Washington University in St. Louis, St. Louis, MO; 5 Brigham and Women’s Hospital, Boston, MA; 6 University of North Carolina, Chapel Hill, NC; 7 University of California, Los Angeles, Los Angeles, CA; 8 Medical University of South Carolina, Charleston, SC; 9 University of Pennsylvania, Philadelphia, PA; 10 Icahn School of Medicine at Mount Sinai, New York City, NY; 11 Indiana University, Indianapolis, IL; 12 University of Michigan, Ann Arbor, MI; 13 Kansas University Medical Center, Kansas City, KS; 14 University of Wisconsin, Madison, WI; 15 Henry Ford Hospital, Detroit, MI; 16 Kansas City VA Medical Center, Kansas City, KS; 17 University of California, Davis, Davis, CA; 18 University of Virginia School of Medicine, Charlottesville, VA; 19 Thomas Jefferson University, Philadelphia, PA; 20 Vanderbilt University, Nashville, TN; 21 Columbia University, New York City, NY; 22 University of Massachusetts Medical Center, Worcester, MA; 23 Cleveland Clinic, Cleveland, OH; 24 Aurora Health Care, Milwaukee, WI; 25 Virginia Mason Medical Center, Seattle, WA; 26 Dartmouth-Hitchcock Medical Center, Lebanon, NH; 27 Stanford University, Stanford, CA; 28 Carolinas Medical Center, Charlotte, NC; 29 Stony Brook University, Stony Brook, NY; 30 University of Alberta, Edmonton, Edmonton, Canada; 31 Mayo Clinic School of Graduate Medical Education, Jacksonville, Jacksonville, FL; 32 Duke University, Durham, NC; 33 Moffitt Cancer Center, Tampa, FL; 34University Hospitals Case Medical Center, Cleveland, OH.

Background: Pancreatic ERCP remains a cornerstone in the management of pancreatic disease but is technically complex. Limited data exist regarding current pancreatic ERCP training in advanced endoscopy training programs (AETPs).

Aims: Establish learning curves in pancreatic ERCP.Report practice patterns in pancreatic ERCP among advanced endoscopy trainees (AETs) during the 1st year of independent practice.

Methods: In Phase I of this prospective multicenter study, AETs were graded on every 5th ERCP using the TEESAT; a validated tool that grades both technical and cognitive aspects. Learning curves were created using cumulative sum analysis for overall, technical and cognitive components. AETs with at least 20 evaluations were included for final analysis. In Phase II, AETs logged performance data on all ERCPs completed during independent practice.

Results: 24 AETs were included in the final analysis (Phase I). Overall, 1339 ERCP exams received grading. A minority (4.6%) were pancreatic ERCPs, with indications including pancreatic stricture (43.3%), stones (22.9%) and recurrent acute pancreatitis (17.9%). 45% of pancreatic ERCPs were ASGE grade of difficulty 3, compared to 7% in biliary ERCPs (P < 0.01). Only 4 AETs had enough data to generate meaningful learning curves. No AETs achieved overall technical competence or competence in individual endpoints such as cannulation or sphincterotomy. Aggregate learning curves demonstrated that the “average” trainee would achieve cognitive, but not technical, competence in pancreatic ERCP at the completion of training. 22 AETs participated in Phase II, performing 3620 ERCPs of which only 7.8% were performed for pancreatic indications. The overall cannulation rate was 92.2% and 85.7% in native papilla cases.

Conclusion: AETs receive minimal training in pancreatic ERCP during their AETP and continue to perform low volume pancreatic ERCP in independent practice, which have significant implications for AETs and AETPs as novel strategies are warranted to increase AET exposure to pancreatic ERCPs.

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Neoplastic and Stromal Cell Cross-Talk Promotes a Collagen Signaling Complex in Pancreatic Cancer

T. Hank, K.C. Honselmann, A. Li, D. Birnbaum, S.B. Begg, K.D. Lillemoe, A.L. Warshaw, C. Fernández-Del Castillo, A.S. Liss. Department of Surgery, Massachusetts General Hospital and Harvard Medical School, Boston, MA.

Background: Pancreatic ductal adenocarcinoma (PDAC) is characterized by an extensive desmoplastic reaction formed by extracellular matrix (ECM) components and cancer-associated fibroblasts (CAFs). The ECM represents a heterogenous group of proteins that support soluble and mechanical signaling in the tumor. Despite its importance, little is known about the regulation of ECM expression in PDAC. Here we investigate the regulation of collagen XVII and collagen VII that are key components of an epithelial cell signaling complex.

Methods: Expression patterns of COL17A1 and COL7A1 were investigated by qPCR in mono- and co- cultures of PDAC cell lines and immortalized cultures of CAFs. Gene expression of laser capture microdissected (LCM) human PDAC samples were analyzed by RNA-sequencing. Immunohistochemistry was performed in patient-derived xenograft tumors and murine pancreas to determine the expression of collagen XVII.

Results: COL17A1was highly expressed in PDAC cells in comparison to COL7A1, which was almost absent. Conversely, CAFs did not express COL17A1 but expressed high levels of COL7A1. Upregulation of COL17A1 and robust expression of COL7A1 was observed in PDAC cells upon co-culture with CAFs. Additionally, COL7A1 expression increased in CAFs after co-culture with PDAC cells; however, CAFs still failed to express COL17A1. Importantly, these co-culture models recapitulate the expression of COL17A1 and COL7A1 observed in tumors. Analysis of LCM human PDAC samples revealed COL17A1 was predominantly expressed in the cancer cells, whereas COL7A1 was more highly expressed in the stroma. The expression of collagen XVII appears to be unique to pancreatic cancer as immunohistochemistry analyses showed a strong expression of collagen XVII, which was absent in normal and chronic inflammatory pancreatic tissue.

Conclusion: These findings highlight the differential contribution of PDAC cells and CAFs to the production of a collagen XVII and VII signaling complex. Moreover, PDAC-CAF cross-talk regulates ECM expression and is required for the neoplastic expression of collagen XVII.

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Validating an Ex Vivo Model of Human Acute Pancreatitis

B. Hasdemir,1 G. Szot,2 A. Bhargava.1,3 Departments of 1 Ob-Gyn, 2 Transplant Surgery, and 3The Osher Center, University of California San Francisco, San Francisco, CA.

Background: The pathophysiological mechanisms underlying acute pancreatitis (AP) have largely been identified and characterized in animal models, because biopsy samples are not collected from patients with AP diagnosis. While the initiating insults for human AP and animal models are different, several key characteristics of AP are thought to be common in rodents and humans, such as alterations in intracellular signaling (Ca2+ signaling) and secretory response of the acinar cells (increase in amylase and lipase secretion). We have previously shown that Urocortin 1 (Ucn1), a neuropeptide hormone, ameliorates cerulein-induced AP symptoms in mice, including decreasing serum amylase.

Aim: To characterized transcriptome in human acinar cells subjected to insults that result in AP in animal models.

Methods: Human acinar cells were treated with cerulein (AP) and pretreated with Ucn1 (Ucn1+Cerulein) to evaluate rescue pathways activated by Ucn1. Vehicle-treated acinar cells served as controls. Amylase secretion was measured. RNASeq was performed to determine changes in transcriptome profile of genes involved in metabolism, stress responses, and Ca2+ homeostasis from mouse AP samples.

NanoString analysis was used to confirm changes in expression levels of specific common genes identified in human acinar cells with ex-vivo AP.

Results: Transcriptome profiling from mouse AP pancreatic tissue using RNASeq identified several genes involved in cell metabolism, cellular stress responses, and calcium homeostasis, pathways that are also known to be activated in human pancreatitis. These included several genes involved in fibrosis and vimentin, a protein expressed in stellate cells.

Conclusion: Before potential prophylactic therapy or therapeutic interventions in humans can be designed, it is instrumental that the commonalities between the responses of animal and human pancreatic acinar cells during AP are established.

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Eschericha coli Bacterobilia is Associated With Severe Postoperative Pancreatic Fistula After Pancreaticoduodenectomy

M. Heckler, C.W. Michalski, D. Winter, Z. Zhou, L. Bing L, M.W. Büchler, T. Hackert. Heidelberg University, Heidelberg, Germany.

Introduction: The benefit of preoperative biliary stenting in the treatment of PDAC is controversially debated. Data from recent meta analyses favor primary surgery for the majority of resectable pancreatic cancers. Regardless of this evidence, preoperative biliary drainage/stenting via endoscopy (EBS) is commonly performed, often before involvement of a surgeon. Though stenting itself has been shown to increase specific postoperative complications, there are only little data on the impact of different microbial stent colonization patterns. This study investigated the association of bile duct stenting, microbiological dislocation of gut flora to the biliary compartment and postoperative complications.

Methods: Patient data was derived from a prospectively maintained database. Patients receiving pancreaticoduodenectomy (PD) for malignant disease in the head of the pancreas with prior EBS were included. Microbiological data was obtained through conventional culture.

Results: 298 PD patients with preoperative EBS were enrolled in this study. Severe postoperative complications were associated with stent colonization: Postoperative pancreatic fistula type C (POPF C) occurred more frequently in E. coli colonized patients (sample estimated odds ratio [OR], 4.07), the rate of lymphatic fistula was elevated in Enterococcus colonized patients (OR, 3.25). Longer stenting duration was associated with the prevalence of these specific microbial pathogens.

Conclusion: Preoperative EBS should be avoided before PD due to a high risk of bacterial stent colonization associated with a significant risk for severe postoperative morbidity, especially when E. coli or Enterococcus species are present. Upfront resection can minimize microbial translocation and associated complications. Antibiotic prophylaxis and treatment in stented patients should be tailored to the individual patients´ profile.

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Diagnostic and Prognostic Value of Circulating KRAS Mutations in Resectable Pancreatic Ductal Adenocarcinoma

J. Hipp,1 S. Hussung,2 J. Duyster,2 S. Fichtner-Feigl,1 R. Fritsch,2 U.A. Wittel.1 Departments of 1 General and Visceral Surgery and 2Oncology, University of Freiburg Medical Center, Freiburg, Germany.

Introduction: Histopathological diagnosis and determination of the prognosis may be difficult in patients with resectable pancreatic cancer. This problem may be overcome by liquid biopsy where mutated DNA is detected in the blood of cancer patients. We evaluated the diagnostic and prognostic value of detection of circulating KRAS-mutations (ctDNA) for patients with resectable pancreatic ductal adenocarcinoma (PDAC).

Methods: KRAS-mutations (KRAS G12D, V, R and C) were quantified in circulating cell free-DNA from 57 patients with PDAC and 38 controls by droplet digital PCR. The correlation of survival, detection of cell free tumor DNA and CA 19-9 was analyzed.

Results: ctDNA was detected in 10/44 (23%) patients with resectable PDAC, 7/13 (54%) with locally advanced and metastatic resectable PDAC and in 4/38 (11%) without malignant pancreatic tumor. Sensitivity, specificity, positive and negative predictive value for ctDNA was comparable to CA 19-9 (ctDNA 30% (17/57), 89% (34/38), 81% (17/21) and 44% (34/38); CA 19-9 >40 U/l 66% (35/53), 83% (29/35), 85% (35/41) and 62% (29/47). Overall (OS) and disease free-survival (DFS) for primary resectable PDAC did not differ dependent on the detection of ctDNA (DFS: ctDNA+: 8.8 months, ctDNA-: 8.5 months; P = 0.841). Disappearance of ctDNA after tumor resection (n = 4) was associated with a substantial improvement of disease free survival (DFS: ctDNA+→ctDNA-: 22.5 months; Postoperative ctDNA+: 7.0 months; P = 0.043).

Conclusion: ctDNA is a promising diagnostic tool for non-invasive diagnosis of PDAC. Patients with sustained ctDNA after tumor resection are at risk for early recurrence while the perioperative drop of ctDNA is a positive prognostic marker after curative resection of PDAC.

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A Novel Matrisome Signature Predicts Overall Survival in PDAC

K. Honselmann,1,2 D. Birnbaum,1,3 S. Begg,1 F. Pascal,3 D. Birnbaum,3 F. Bertucci,3 D. Ting,4 E. Tai,4 C. Monsalve,1 M. Mino-Kenudson,5 K.D. Lillemoe,1 C. Fernández-Del Castillo,1 A.L. Warshaw,1 A. Liss.1 1 Department of Surgery, Massachusetts General Hospital and Harvard Medical School, Boston, MA; 2 Department of Surgery, University Medical Center Schleswig-Holstein, Campus Luebeck, Luebeck, Germany; 3 Department of Surgery and Molecular Oncology, Marseille Cancer Research Center, Paoli-Calmettes Institute, Aix-Marseille University, Marseille, France; 4 Massachusetts General Hospital Cancer Research Center, Harvard Medical School, Boston, MA; 5Department of Pathology, Massachusetts General Hospital and Harvard Medical School, Boston, MA.

Background: PDAC is characterized by a strong desmoplastic reaction, contributing to its dismal prognosis. The extracellular matrix and its affiliated proteins are composed of as many as 1029 proteins which collectively are referred to as the matrisome. The aim of this study was to define the PDAC matrisome.

Methods: We performed RNA-seq from laser capture microdissected (LCM) stromal and epithelial compartments of 24 resected human PDAC samples. Clinicopathological and gene expression data from 14 available datasets from whole tissue gene expression Omnibus and TCGA databases of 573 resected PDAC patients were collected and hierarchical clustering as well as multivariate analysis were performed.

Results: In LCM PDAC samples, a total of 654 matrisome genes were expressed. Interestingly, 80% of these genes were commonly expressed in stromal and cancer cells. Matrisome genes uniquely expressed in cancer and stromal compartments encoded soluble factors involved in signaling pathways, including sonic hedgehog and chemokines, respectively. To determine whether differences in the matrisome are associated with patient outcome, supervised analysis of TCGA long-term (>36 months) (n = 13) and short-term (2-6 months) (n = 28) survivors was performed. This identified 65 differentially expressed matrisome genes, which predicted these two subgroups with 80% accuracy (P = 3.44E-04). In an independent cohort (n = 532), this 65- gene signature classified patients into “short-term survivor-like” (n = 370) and “long-term survivor-like” (n = 162) groups with a 2-year survival of 34% (95% CI, 29-40) and 51% (95% CI, 43-60), respectively (P < 0.001). This matrisome signature was independently associated with overall survival in multivariate analysis, even when known molecular subtype signatures were included.

Conclusion: This study demonstrates a substantial contribution of cancer cells to the expression of the matrisome, revealing an uncharacterized epithelial role in regulation of the PDAC stroma. In addition, differences in the expression of matrisome components robustly predict PDAC patient outcome, indicative of the importance of the matrisome to PDAC biology.

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Role of NLRP3 Inflammasome on the Severity of Chronic Pancreatitis in Mouse Models

U. Hooda, M. Tarique, V. Ramakrishnan, M. Kashyap, H. Cheema, E.P. Bava, R. Dawra, V. Dudeja, A.K. Saluja. Department of Surgery and Sylvester Comprehensive Cancer Center, University of Miami Miller School of Medicine, Miami, FL.

Background: Chronic pancreatitis (CP) is characterized by presence of inflammatory infiltrates and fibrosis of the pancreas. Growth factors and inflammatory cytokines activate the Pancreatic Stellate Cells which are responsible for the fibrotic changes in pancreas. Role of NLRP3, the chief inflammasome involved in sterile inflammation, has not been studied in CP.

Aim: To evaluate the effect of MCC950, a small molecular inhibitor of NLRP3 inflammasome, on progression and severity of CP.

Methods: Cerulein-induced CP was induced by intraperitoneal injections (50 ug/kg x7, hourly, twice weekly x10 weeks). Animals were randomized and were assigned to either saline or MCC950 (40 mg/kg/d intraperitoneally) at 6 weeks and were euthanized at 11 weeks. L-arginine induced CP was initiated by intraperitoneal injections of L-arginine (4.5 g/kg x2, weekly x4).

Treatment was continued from 3rd to 7th week. Animals were euthanized and tissue samples were harvested.

Results: H&E showed minimal reduction in pancreatic atrophy and no changes in inflammatory infiltrate in treatment groups compared to CP only group. Sirius red staining, IHC for Vimentin, Coronin-1 and CK-19 did not show any difference between the two groups. Western blots showed reduction in α-SMA but no change in Desmin and Amylase levels. mRNA of fibrosis makers and pancreatic regeneration markers were similar in both groups. Flow cytometry from pancreatic tissue showed a non-significant change in inflammatory cell population with treatment.

Conclusion: Our study shows that selective NLRP3 inflammasome inhibition in animal models of CP does not reverse the damage. Other inflammasome complexes might have a significant role and should be investigated.

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The Predictive Factor for the Mortality of Acute Pancreatitis on Admission

Y. Hori,1 N. Jinno,1 I. Naitoh,1 Y. Tsuji.2 1 Department of Gastroenterology and Metabolism, Nagoya City University Graduate School of Medical Sciences, Nagoya, Japan; 2Department of Clinical Education, Shiga University of Medical Science, Shiga, Japan.

Background: The Revised Atlanta Classification is widely used for the evaluation of the severity of acute pancreatitis (AP). However, we cannot use this classification within 48 hours after the onset of AP. The aim of this study was to investigate the predictive factors of mortality in patients with AP on admission.

Methods: We evaluated the association between mortality of AP and clinical parameters at the time of admission in patients with AP from April 2013 to December 2017 at one university hospital and one tertiary-care referral center.

Results: A total of 203 consecutive patients were enrolled. Nine patients (4.4%) were deceased despite multidisciplinary treatment. In multivariate analysis, hematocrit ≥ 40 % (odds ratio [OR], 1.07; 95% confidence interval [CI], 1.01-1.13; P = 0.027), BUN ≥ 40 mg/dL (OR, 1.25; 95% CI, 1.10-1.42; P = 0.001), base excess <3.0 mmol/L (OR, 1.15; 95% CI, 1.05-1.26; P = 0.004), and inflammation extend to rectovesical excavation (OR, 1.11; 95% CI, 1.01-1.23; P = 0.029) on admission were significantly associated with mortality.

Conclusion: Among image findings, inflammation extend to rectovesical excavation was the only independent predictive factor for the mortality in AP. This simple CT finding on admission is promising prognostic factor for AP.

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Invasive Candidiasis in Patients With Severe Acute Necrotizing Pancreatitis was Associated With the Antimicrobial Prophylaxis: A Large Retrospective Multicenter Cohort Study

M. Horibe,1 M. Sanui,2 M. Sasaki,3 Y. Ogura,1 H. Sawano,4 T. Goto,5 T. Ikeura,6 T. Takeda,7 T. Oda,8 H. Yasuda,9 E. Iwasaki,10 T. Kanai,10 T. Mayumi.11 1 Tokyo Metropolitan Tama Medical Center, Fuchu, Japan; 2 Jichi Medical University Saitama Medical Center, Saitama, Japan; 3 National Cancer Center Hospital, Tsukiji, Japan; 4 Osaka Saiseikai Senri Hospital, Suita, Japan; 5 Hiroshima City Hiroshima Citizens Hospital, Hiroshima, Japan; 6 Kansai Medical University, Hirakata, Japan; 7 Graduate School of Medicine, The University of Tokyo, Tokyo, Japan; 8 Iizuka Hospital, Iizuka, Japan; 9 Japanese Red Cross Musashino Hospital, Musashino, Japan; 10 Keio University School of Medicine, Tokyo, Japan; 11School of Medicine University of Occupational and Environmental Health, Fukuoka, Japan.

Background: The patients with acute necrotizing pancreatitis (ANP) have a high risk of mortality. Although antimicrobial prophylaxis was expected to improve the outcome of these patients, no benefit was observed. However, antimicrobial prophylaxis continues to be used widely in actual practice, and no report of its negative effects on patient outcomes exists. Therefore we evaluated the association between invasive candidiasis as a negative outcome and antimicrobial prophylaxis.

Methods: We extracted data on patients with severe ANP from a large retrospective cohort database of all consecutive patients with severe acute pancreatitis admitted to any of 44 institutions between 2009 and 2013 in Japan. We compared the patients who received antimicrobial prophylaxis to those who did not.

Results: Of 1159 patients with severe acute pancreatitis, we abstracted 101 patients with pancreatic necrosis greater than 50%. Of these patients, 72 (71.3%) received antimicrobial prophylaxis and 29 (28.7%) did not. Patients with antimicrobial prophylaxis showed a significantly higher incidence of invasive pancreatic candidiasis than did those with no antimicrobial prophylaxis. (9 vs 0 patients, P = 0.04) Multivariable logistic regression analysis was performed after adjusting for the need for surgical interventions. Both antimicrobial prophylaxis (P = 0.006) and the need for surgical interventions (P < 0.001) were independently associated with invasive pancreatic candidiasis. Meanwhile, antimicrobial prophylaxis was not found to be associated with mortality (P = 0.66).

Conclusion: Antimicrobial prophylaxis in patients with severe ANP can lead to the development of invasive pancreatic candidiasis and is not recommend for routine use in these patients.

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A CDKN2A (p16) Mutation Associated With Familial Pancreatic Cancer Reduces Protein Stability and Functionality

I.P. Horn,1 D.L. Marks,1 T.L. Hogenson,1 L.L. Almada,1 L.E. Goldstein,1 P.A. Romecin Duran,1 A.M. Vrabel,1 G. Cui,2 K.G. Chaffee,3 W.R. Bamlet,3 G. Mer,2 H. Li,4 G.M. Petersen,5 M.E. Fernandez-Zapico.1 1 Schulze Center for Novel Therapeutics, Division of Oncology Research; Divisions of 2 Biochemistry and Molecular Biology, 3 Biomedical Statistics and Informatics, 4 Molecular Pharmacology and Experimental Therapeutics, and 5Epidemiology, Mayo Clinic, Rochester, MN.

Background: Among the major cancers, pancreatic cancer has the worst survival and historically has been the least studied. While pancreatic cancer was long thought to be a sporadic disease, genetic susceptibility is now clearly implicated in subsets. Our group previously identified familial pancreatic cancer kindreds that segregate a missense mutation, L16R(47T>G), in the cell cycle regulator gene, CDKN2A(p16INK4A).

Methods: We sought to identify the molecular mechanism and biological significance of this mutation contributing to families’ increased susceptibility to pancreatic cancer using a combination of biochemical and cell biological methods.

Results: p16 L16R showed less protein expression than the wild type (WT), although L16R expression could be restored completely by treatment with the proteasome inhibitor bortezomib and partially by the knock down of REG-gamma. In silico molecular modeling suggested that the L16R mutation prevents the protein from folding properly. p16 L16R was also less efficient in arresting cells in G1 phase than similar expression levels of WT p16, and reduced binding of L16R to p16 targets, CDK4 and CDK6, was also observed. Of note, the same effects were not observed in other p16 missense variants presented in familial and sporadic pancreatic cancer.

Conclusion: We conclude that the L16R mutation of CDKN2A results in an improperly folded protein with reduced functionality. These findings in part explain the increased risk of pancreatic cancer in families carrying this mutation.

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Prevalence of Abnormal Glucose Metabolism in Pediatric Acute, Acute Recurrent, and Chronic Pancreatitis

L. Hornung,1 L. Denson,2,3 A. Husami,4 T.K. Lin,2,3 K. Matlock,3,5 J.D. Nathan,6 J.J. Palermo,2,3 T. Thompson,2 C.A. Valencia,4,7 X. Wang,4 J. Woo,1,3 K. Zhang,4 D. Elder,3,5 M. Abu-El-Haija.2,3 Divisions of 1 Biostatistics and Epidemiology; 2 Gastroenterology, Hepatology and Nutrition, Cincinnati Children’s Hospital Medical Center, Cincinnati, OH; 3 Department of Pediatrics, University of Cincinnati College of Medicine, Cincinnati, OH; Divisions of 4 Human Genetics, 5 Pediatric Endocrinology, 6 Pediatric General and Thoracic Surgery, Cincinnati Children’s Hospital Medical Center, Cincinnati, OH; 7PerkinElmer Genomics, Branford, CT.

Background: Type 3C Diabetes, or diseases of the exocrine pancreas, have been reported in~30-50% of adults with pancreatitis. The incidence of glucose abnormalities or risk factors that predict development of glucose derangements in the pediatric pancreatitis population are not known.

Methods: Retrospective chart review from 1998-2016 yielded glucose-related testing for patients diagnosed with acute pancreatitis (AP), acute recurrent pancreatitis (ARP) and chronic pancreatitis (CP). We compiled glucose testing (GT), defined as glucose values, hemoglobin A1c and oral glucose tolerance and mixed meal testing. Patient and clinical characteristics were analyzed. American Diabetes Association guidelines were utilized for diagnosing prediabetes/diabetes based on GT.

Results: Fifty-two children with pancreatitis had available GT; 58% (30/52) developed ARP and 37% (19/52) developed CP. Twenty-five percent (13/52) had abnormal GT. Of the 13 patients with abnormalities, 15% (2/13) occurred after 1st AP, 46% (6/13) on or after the 2nd AP attack, and 38% (5/13) after CP. A significantly higher proportion of abnormal GT was seen in patients (85%, 11/13) with a BMI >85th percentile compared to patients with normal glucose (28%, 11/39) (P = 0.0007). A significantly higher proportion of patients with abnormal GT (77%, 10/13) had a severe acute pancreatitis (SAP) episode prior to testing compared to only 10% (4/39) with normal GT (P < 0.0001). Presence of exocrine pancreatic insufficiency prior to GT or positive genetic testing did not differ between groups.

Conclusion: In our sample, abnormal GT was identified as early as after the 1st AP attack, and a higher proportion of glucose abnormalities were identified after the 2nd AP attack even in the absence of CP. We found being overweight/obese and having SAP during the prior AP episode were associated with abnormal GT. We propose systematic screening for abnormal glucose after the first AP episode to better establish the timing of diabetes progression.

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Serum Tissue Factor/Tissue Factor Pathway Inhibitor Ratio Predicts Non-Mild Acute Pancreatitis

W. Hu, J. Yang. Department of General Surgery, Affiliated Hangzhou First People’s Hospital, Zhejiang University School of Medicine, Hangzhou, China.

Background: Tissue factor (TF) and tissue factor pathway inhibitor (TFPI) are key regulators of external coagulation pathways. This study aimed to investigate the potential of serum TF, TFPI and TF/TFPI ratio to predict non-mild AP.

Methods: Eighty patients were prospectively included in the study, and were divided into mild and non-mild according to the revised Atlanta classification. Venous blood was collected within 24 hours when the patient was first diagnosed with AP. Serum TF and TFPI concentrations were measured by ELISA method. Receiver operating curves was employed to evaluate the capacity of serum TF, TFPI, and TF/TFPI for the prediction of non-mild AP.

Results: Serum TF and TFPI levels in patients with non- mild AP were higher than that in mild AP (P < 0.05), whereas serum TF/TFPI was lower in patients with non-mild AP than mild AP. High serum TF and TFPI were not available in distinguishing mild AP from non-mild AP, with an AUC of 0.516 (95% CI, 0.651-0.923) and 0.541 (95% CI, 0.621-0.932), respectively. Surprisingly, low TF/TFPI ratio was available in distinguishing mild AP from non-mild AP, with an AUC of 0.816 (95% CI, 0.654-0.856). The cut-off value of TF/TFPI was 1.8 with sensitivity 83%, specificity 74%, respectively. The combination of serum TF/TFPI and C-reactive protein, the AUC value was significantly higher than each of them individually (AUC, 0.912; 95% CI, 0.853-0.972), suggesting TF/TFPI combined with C-reactive protein increase the ability to predict non-mild AP.

Conclusion: We conclude that serum TF/TFPI has a clinical ability to distinguish mild from non-mild AP in the early stages of AP.

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M2-like Macrophage is Essential for Acinar Cell Regeneration After Acute Pancreatitis

W. Hua, J. Xue. State Key Laboratory of Oncogenes and Related Genes, Stem Cell Research Center, Ren Ji Hospital, School of Medicine, Shanghai Jiao Tong University, Shanghai, China.

Background: Acinar cell regeneration, facilitating by acinar-to-ductal metaplasia (ADM) and proliferation, is a critical event after pancreas injury. Macrophages are a determinant during AP pathogenesis, here we wonder the phenotype and role of macrophages in pancreas regeneration after AP.

Method: Using cerulein-induced acute pancreatitis (AP) model, we examined the dynamic change of macrophages in pancreas during AP recovery. Macrophage depletion by Clodronate Liposomal or using IL-4R1 knockout mice to examine the role of macrophage after acute phase of AP.

Results: We found that the M1 macrophage in acute phase of AP were switched to M2-like macrophages (F4/80+CD206+) in the phase of ADM. Depletion of macrophage by Clodronate Liposomal after acute phase of AP could dramatically block the ADM and delay the pancreas recovery. M2-like macrophages are dependent on IL-4R1 signaling, and we found that mice lacking IL-4Ra, showed less difference during AP, but exhibited impaired pancreas recovery after AP. Furthermore, RNA-Seq analysis of pancreatic macrophages isolated from different phases during AP recovery provide cues for understanding these macrophage phenotype and function.

Conclusion: Macrophage switches to M2-like phenotype after acute phase of AP, which is essential for acinar cell regeneration after acute pancreatitis.

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Radiofrequency Ablation Plus Stenting vs. Stenting Alone in the Treatment of Pancreatic Cancer and Cholangiocarcinoma

T. Hucl,1 P. Macinga,1 D. Gogova,1 J. Fronek,2 J. Spicak.1 Departments of 1 Gastoenterology and Hepatology and 2Surgery, Institute for Clinical and Experimental Medicine, Prague, Czech Republic.

Background: Radiofrequency ablation (RFA) uses high frequency current to induce heat induced necrosis of the surrounding tissue. Retrospective data show that endoscopically delivered endoluminal RFA can be used to treat malignant bile duct obstruction, however concerns have been raised regarding its safety.

Methods: A prospective randomized controlled study (protocol NCT03166436) of endoluminal biliary RFA plus stenting (self expendable metal stent, SEMS) versus stenting only in the treatment of malignant biliary obstruction (pancreatic cancer and cholangiocarcinoma).

Results: A total of 31 patients were included in the study (18 males, 13 females, mean age 73 years). Fourteen patients had pancreatic cancer (8 males, 6 females, mean age 73 years), 17 patients had cholangiocarcinoma (10 males, 7 females, mean age 73 years). Eighteen patients were ramdomized to RFA plus stenting and 13 patients to stenting only. Acute cholecystitis in a patient with pancreatic cancer and intact gallbladder treated with RFA and SEMS requiring antibiotic administration and gallbladder drainage occurred within a week after the procedure, the patient fully recovered and is still alive. Fourteen patients (45%) died (9 in RFA plus stenting and 5 in stenting only, NS). Stent obstruction occurred in 17% in RFA plus stenting group and in 22% in stenting only group (NS) in a mean follow-up time of 8 months and was always successfully treated endoscopically.

Conclusion: Preliminary data of our randomized study show that endoluminal radiofrequency ablation is a safe technique with a limited risk of complications. Its efficacy in terms of survival and stent patency can be truly evaluated only when more patients are included in the study.

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Diagnostic Yield of a Conventional Endoscopic Ultrasound-Fine Needle Aspiration (EUS-FNA) and Two Endoscopic Fine Needle Biopsy (EUS-FNB) Needles in Solid Lesions and Pancreatic Cancer: A Single Center, Retrospective Study

F. Iqbal,1 J. Miranda,1 A. Aditi,3 B. Huang,1 A. Ballonado,1 M. Hassan,2 M. Arain.1 1 Gastroenterology Division, University of California, San Francisco, San Francisco, CA; 2 Stanford University, Stanford, CA; 3InSite Digestive Health Care, Daly City, CA.

Background: To evaluate and compare the diagnostic yield of the solid lesions sampled using an FNA (Echotip Ultra® (EC) and two FNB (Acquire ® (AC) and SharkCore (SC) needles.

Methods: A retrospective review of consecutive patients undergoing an index EUS procedure for evaluation of a solid lesions was performed. Data collected included location, lesion size, anesthesia type, needle type number of passes, presence of rapid onsite cytologic evaluation (ROSE), diagnostic yield, adverse events and final diagnosis.

Results: A total of 329 lesions were sampled between July 2016 and May 2018. The overall diagnostic yield was 93.6%. The diagnostic yield of the EC needle was lower than both the SC and AC needles (86.2 vs 96 and 94.9% respectively, P = 0.014). The odds of obtaining a higher yield using an EUS-FNB needle were also higher for pancreatic solid lesions (SC vs EC, OR 8.98, P = 0.05 and AC vs EC, OR 7.07, P < 0.05). The diagnostic yield for lesions assessed using more than one needle in the same procedure (due to a non-diagnostic yield with the first needle using ROSE) was 100% (n = 33). There were 76 patients diagnosed with pancreatic cancer of whom 63 patients (82%) needed a single needle and 13 (17%) needed two needles (mean of 4.5 vs 2.7 passes, P < 0.01).

Conclusion: EUS-FNB needles had a better diagnostic yield than the EUS-FNA needle overall and in pancreatic solid lesions. An approach of switching to a second needle was associated with achieving a diagnosis in all lesions when the first needle did not yield a diagnosis. All pancreatic cancers were diagnosed at the index EUS procedure with 17% requiring two needles to reach an onsite diagnosis. In conclusion, FNB needles and switching to a second needle are both associated with a higher diagnostic yield using EUS sampling.

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Clinical Characteristics, Prognosis, and CFTR Mutations of Cystic Fibrosis in Japan

H. Ishiguro,1 A. Yamamoto,1 K. Fujiki,2 M. Nakakuki,1 I. Taniguchi,1 Y. Kozawa,1 Y. Sohma,3 S. Naruse,4 Y. Takeyama,5 the study group for pediatric rare and intractable hepato-biliary-pancreatic diseases, the Ministry of Health, Labor, and Welfare of Japan. 1 Department of Human Nutrition, Nagoya University Graduate School of Medicine, Nagoya, Japan; 2 Department of Nutritional Sciences, Nagoya University of Arts and Sciences, Aichi, Japan; 3 Department of Pharmaceutical Sciences, International University of Health and Welfare, Tochigi, Japan; 4 Miyoshi Municipal Hospital, Miyoshi, Japan; 5Department of Surgery, Kindai University, Osaka, Japan.

Background: Cystic fibrosis (CF) is rare in Asian populations including Japanese. More than 2,000 different mutations have been reported in CFTR and there are considerable regional and ethnic variations. We have tried to characterize clinical courses of CF and CF-causing CFTR mutations in Japanese.

Methods: Nationwide surveys were conducted every 5 years since 1994 and the registry was established in 2012. All exons, their boundaries, and promoter region (up to ∼1,000 bp upstream) of the CFTR gene were directly sequenced and genomic rearrangements were examined by multiplex ligation-dependent probe amplification. RT-PCR analysis of CFTR transcripts from nasal swab was performed in 2 Japanese patients who had CFTR mutations only in one allele.

Results: Since 1994, clinical data of 117 CF patients (male 60, female 57) have been collected and 40 patients are now registered. The estimated prevalence rate is about 1 per 590,000 live births. The median survival time is about 20 years. Their clinical courses are similar to those of European CF patients and most of them die of respiratory failure. Twenty-nine consecutive patients with definitive CF since 2004 were analyzed for CFTR mutations. Among 41 alleles inherited from Japanese/Asian ancestry, 16 different mutations including 6 novel mutations were found, but European-type mutations were not found. A large genomic deletion spanning exons 16, 17a and 17b (c.2908+1085_3367+260del7201) was detected in 16 alleles and most frequent in Japanese CF alleles. Analysis of nasal CFTR transcripts revealed deletion/skipping of exon1 in one patient and reduction of transcript amount in another patient. European type mutations including F508del and R1066C were found in 17 alleles inherited from European ancestry.

Conclusion: Nationwide survey for 25 years and registry demonstrate very low incidence and poor prognosis of CF in Japanese. The spectrum of CFTR mutations of Japanese is different from that of Europeans.

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Histopathological Changes of Pancreatic Cancer Stroma Induced by nab-Paclitaxel

K. Ishido, D. Kudo, N. Kimura, T. Wakiya, K. Hakamada. Department of Gastroenterological Surgery, Hirosaki University Graduate School of Medicine, Hirosaki, Japan.

Background: The aim of this study was to clarify the histopathological changes in pancreatic duct adenocarcinoma (PDAC) induced by nab-Paclitaxel (NP).

Methods: This was a retrospective study of 25 patients with PDAC who underwent curative resection after chemotherapy between 2010 and 2017 at our institution. Ten of the patients received NP plus gemcitabine (GnP group) and 15 received S-1 plus gemcitabine (GS group) as preoperative treatment. Pathological evaluation of nuclear, cytoplasmic and stromal features was conducted, and scored from -1 to +3. The clinical and pathological features in the two groups were then compared.

Results: The serum CA 19-9 levels in the GnP and GS groups were reduced to 10.1% and 44.7% after chemotherapy, respectively (P = 0.01). With regard to changes in tumor size, the median tumor reduction rates in the GnP and GS groups were 28.6% and 68.2%, respectively (P = 0.005). With regard to pathological changes, the cytoplasmic vacuole score and the intensity of Masson trichrome (MT) staining were significantly higher in the GnP group than in the GS group (2.0 vs 0.93, P = 0.001; 1.57 vs -0.44, P < 0.001, respectively). Additionally, the inflammatory infiltration score and the degree of fibroblast immaturity were significantly lower in the GnP group than in the GS group (-0.21 vs 1.88, P < 0.001; 0.29 vs 1.88, P < 0.001, respectively).

Conclusion: GnP/GEM had a strong anticancer effect on PDAC. It was considered that NP markedly enhanced the degree of stromal fibrosis in PDAC, suggesting a novel anticancer mechanism that reduces the number of cancer-associated fibroblasts.

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Serum CA 19-9 as Predictor in Preoperative Parameters for Resectable Pancreatic Adenocarcinoma

J. Itakura, M. Watanabe, N. Hosomura, H. Amemiya, H. Kawaida, H. Okamoto, H. Kohno, D. Ichikawa. Department of Surgery, University of Yamanashi, Yamanashi, Japan.

Background: To evaluate the preoperative factors predict postoperative recurrence of pancreatic cancer (PDAC) we examined the correlation between the preoperative factors and the recurrence type and Relapse-Free Survival (RFS) from the resected cases of PDAC in our institute.

Methods: Hundred forty-two PDAC patients that enforced R0-1 resection were enrolled in this study. The recurrence cases of these were analyzed for the site of the recurrence and RFS. For the preoperative factors the highest CA 19-9 value (CA 19-9), reduction rate of CA 19-9 after treatment (RR) and neutrophil lymphocyte rate (NLR) were estimated.

Results: Eighty-nine of these 142 cases showed recurrence. They consist of 5/35/38/11(stage IA/IIA/IIB/III) cases. As for the surgery, 55 PD, 32 DP, and 2 TP were performed. In regard to the first recurrence form, 32 cases had liver metastasis (H), 29 cases had local recurrence (L), 17 cases had pulmonary metastasis (P), and 11 cases had peritoneum dissemination (D). The average RFS of each recurrence group were 8.7/11.4/22.3/21.9 (H/D/L/P) month, and these were significantly different. The CA 19-9 was 508 U/ml in the recurrence cases and 171 U/ml in the non-recurrence cases with significant difference. According to the recurrence form, CA 19-9 was 187/244/739/845 (P/L/D/H) U/ml with significant difference. While RR and NLR had no significant correlation with RFS. To distinguish the early and the late recurrence cases we developed ROC curve and based on this analysis 512 U/ml was suggested for the best cut-off value.

Conclusion: Serum CA 19-9 value was significant pre-operative parameter predicts recurrence and the type of recurrence for the resected PDAC. Even if in the macroscopically resectable cases, the high value of preoperative CA 19-9 may indicate potential distant metastasis and early recurrence, and neo-adjuvant treatment should be in consideration for these cases.

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Changes in Composition of Plasma Free Amino Acids (PFAAs) in Progression of Chronic Pancreatitis (CP)

T. Ito,1 M. Taniguchi,1 T. Nakamura,1 K. Nishiguchi,1 A. Asakura,1 M. Mori,1 M. Sawamura,1 Y. Tokuda,1 A. Ishimi,1 Y. Onishi,1 T. Kanno,1 S. Hiyama,1 M. Hamano,1 M. Chiba,1 K. Yamamoto,1 N. Tatsumi,1 S. Kikuchi,2 H. Yamamoto.2 1 Department of Gastroenterology and Hepatology, Japan Community Healthcare Organization, Osaka Hospital, Osaka, Japan; 2Institute of Bio-Fine, Ajinomoto Co., Inc., Kawasaki, Japan.

Background and Aim: It is well-known that digestive organs like liver and pancreas are playing important roles in AA metabolism and homeostasis. Recently we reported that the PFAA profile of pancreatic cancers (PC) was significantly different from that of healthy group (HG) and that the PFAA index is a promising biomarker for screening and diagnosis of PC. However, little is known alteration of PFAA in progression of chronic pancreatitis. In this study, we tried to clarify specific aminogram profiles in CP.

Methods: Sixty-six patients with CP were enrolled consisting of 31, clinically possible or early CP(A); 26, probable CP(B); and 9, definite CP(C). 132 cases of HG were as a control. Concentrations of 19 PFAAs were measured by liquid chromatography–mass spectrometry, using fasting plasma samples from those enrolled cases.

Results: PFAAs analyses indicated that glutamine, histidine, and alanine concentration were increased in CP patients significantly comparing these AA in HG (P < 0.05). Both in CP(A) and CP(B), concentration of several PFAAs were detected to be elevated compared with CP(C). Interestingly, these concentrations were decreasing with good relation during progression of CP.

Conclusion: These findings suggested that the PFAA aminogram in CP was unique with significant difference from that of HG. There would be changes in AA metabolism and homeostasis in progression of CP inducing digestive system disorders. Thus, the PFAA profiles might be one of important biomarkers for assessment of stages in CP, especially for detection of early staged CP.

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Mechanistic Implications of Trefoil Factor 1 (TFF1) in Pancreatic Cancer Chemoresistance

R. Jahan,1 K. Ganguly,1 L.M. Smith,2 I.A. Sheikh,3 Y. Sheinin,4 M.A. Macha,1,5 M. Polland,6 S. Kaur,1 S.K. Batra.1,7,8 Departments of 1 Biochemistry and Molecular Biology, 2 Biostatistics, University of Nebraska Medical Center, Omaha, NE; 3 King Fahd Medical Research Center, King Abdulaziz University, Jeddah, Saudi Arabia; Departments of 4 Pathology and Microbiology, 5 Otolaryngology/Head and Neck Surgery, University of Nebraska Medical Center, Omaha, NE; 6 Nebraska Wesleyan University, Lincoln, NE; 7 Buffett Cancer Center, 8Eppley Institute for Research in Cancer and Allied Diseases, University of Nebraska Medical Center, Omaha, NE.

Background: Chemoresistance is a major cause for high incidence of mortality in Pancreatic Cancer (PC). Thus, identifying molecules facilitating chemoresistance can improve therapeutic efficacy and patient survival. TFF1 is a mucin-associated small secretory molecule and overexpressed in classical subtype of PC, a subtype which showed resistance to gemcitabine. Multipronged approach suggests significant upregulation of TFF1 in preneoplastic lesion and PC. Interestingly, continuous gemcitabine treatment in PC cells significantly upregulated TFF1. Based on these evidence, we hypothesize that TFF1 plays a critical role in PC gemcitabine resistance (GR).

Methods: TCGA database was used to correlate between TFF1 and GR predictor (ratio of gemcitabine metabolic genes). To understand the effect of TFF1 in GR, in vitro studies with control and TFF1-knocked-down in SW1990 cells were performed. Confocal microscopy in human and mouse tissues, immunoprecipitation, and chromatin immunoprecipitation were utilized to identify interacting partner and regulator of TFF1. Protein-protein docking using BioLuminate module was performed to predict CXCR4 as a possible signaling receptor for TFF1.

Results: TCGA database analysis revealed a significant positive correlation between TFF1 and GR predictor in PC (P = 0.0001). Repression of TFF1 in SW1990 cells induced apoptosis, reduced colonies, and modulated apoptotic regulators such as Bax, Bcl-2, caspases in the presence of gemcitabine. Furthermore, TFF1 colocalized and interacted with MUC5AC suggesting their partnering is critical for PC pathogenesis. Interestingly, 16-fold enrichment of GATA-6, an overexpressed transcription factor in classical subtype of PC, was observed on two distinct TFF1 promoter sites and KD of GATA-6 repressed TFF1 expression in PC cells. Moreover, protein- protein docking studies revealed the interaction of TFF1 with CXCR4 at Phe-172, Ser-122 and Glu-1 and recombinant TFF1 protein treatment in SW1990 cells increased CXCR4 mediated downstream signaling.

Conclusion: TFF1 imparts GR in PC by interacting with MUC5AC and modulating apoptotic molecules possibly through CXCR4 which needs further validation.

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Chymotrypsin Protects Against Cerulein-induced Pancreatitis in Mice

Z. Jancso M. Sahin-Tóth. Department of Molecular and Cell Biology, Boston University Medical Center, Boston, MA.

Background: Intra-pancreatic activation of the digestive proteases trypsin and chymotrypsin is an early event in experimental pancreatitis in rodents. Human genetic and biochemical studies demonstrated that chymotrypsin regulates trypsin activity through degradation but evidence from animal models has been lacking.

Aims: The aim of the present study was to evaluate the effect of chymotrypsin deletion on secretagogue-induced pancreatitis in mice.

Methods: We used CRISPR-Cas9 to disrupt the Ctrb1 gene in C57BL/6N mice. Acute pancreatitis was induced by 10 hourly injections of cerulein at a supramaximal stimulatory dose. Pancreatitis severity was determined by histology scoring, measurement of pancreas edema, plasma amylase, and pancreas and lung myeloperoxidase (MPO).

Results: Compared to C57BL/6N controls, CTRB1-deficient mice given cerulein exhibited significant elevations in intra-pancreatic trypsin activity, plasma amylase, pancreas edema, pancreas and lung MPO content, and acinar cell necrosis.

Conclusion: The observations demonstrate that intra-pancreatic chymotrypsin protects against cerulein-induced pancreatitis by reducing harmful trypsin activity.

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Glycosylation Profiles and ABO Blood Group Antigens of the Carboxyl-ester Lipase (CEL) Protein Associated With Chronic Pancreatitis and MODY8 Syndrome

K. El Jellas,1,2 S.M. Haslam,3 M.H. Choi,1,2 A. Dell,3 B.B. Johansson,4 K. Fjeld,5 A. Molven.1,2 1 Gade Laboratory for Pathology, Department of Clinical Science, University of Bergen, Bergen, Norway; 2 Departments of Pathology, Haukeland University Hospital, Bergen, Norway; 3 Department of Life Sciences, Imperial College London, London, United Kingdom; Departments of 4 Pediatrics and Adolescent Medicine, and 5Medical Genetics, Haukeland University Hospital, Bergen, Norway.

Background: CEL is a digestive enzyme secreted by the pancreas. The protein is highly polymorphic because of its O-glycosylated C-terminal region that consists of a mucin-like sequence repeated 3-23 times. Genetic variants (CEL-MODY, CEL-HYB) that alter the mucinous region of CEL have been implicated in pancreatic disease. We examined the glycosylation pattern of the normal CEL protein and assessed whether the pathogenic variants undergo changes in glycosylation.

Methods: CEL was immunoprecipitated from juice obtained from the distal pancreatic duct during surgery. O-glycans were determined by high-sensitivity MALDI-TOF mass spectrometry. We expressed four different CEL variants in HEK293 cells and characterized their O- and N-glycosylation patterns. We changed the N-glycosylation site of the variants by in vitro mutagenesis and evaluated the effect on subcellular localization.

Results: The O-glycome of CEL consisted of core 1 and 2 structures in similar proportions, with a composition that depended on ABO gene polymorphisms. The O-glycans of CEL-MODY were 90% of the core 1 type with a high sialic acid content. CEL-HYB, which has only seven putative sites for O-glycosylation distributed over its three mucin-like repeats, contained only mono- and di-sialylated core 1 structures. The N-linked-glycans of CEL-HYB showed a very distinct profile characterized by unprocessed oligomannoses and an increase in fucosylated LacNAc and LacdiNAc structures. When disrupting the N-glycosylation site by introducing a N210Q mutation in all constructs, the subcellular localization of normal CEL and CEL-MODY, but not of CEL- HYB, was altered.

Conclusion: Among digestive enzymes secreted by the pancreas, CEL is unique by being a glycoprotein which reflects the individual’s ABO blood group. Substantial changes in the glycosylation pattern were observed when modelling pathogenic CEL variants in HEK293 cells. The biochemical alterations on CEL-MODY and CEL-HYB may also occur in human acinar tissue and could be involved in initiation/progression of pancreatic disease.

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Arterial Resection in Radical Pancreatectomy for Pancreatic Cancer

K. Jiang, P. Wu, Z. Lu, K. Zhang, C. Dai, J. Wu, W. Gao, J. Chen, J. Wei, F. Guo, J. Yin, Y. Miao. Pancreas Center, The First Affiliated Hospital of Nanjing Medical University, Nanjing, China.

Background: Tumor-induced arterial abutment/encasement is commonly presented in patients with locally advanced pancreatic cancer. However, arterial resection, to achieve curative resection (R0 resection) is a controversial approach, due to the potential complications. In this study, we aimed to analyze whether pancreatectomy with simultaneous resection of tumor-involved arteries is a safe approach with acceptable patient survival.

Method: We reviewed all patients with arterial tumor infiltration pancreatic cancer (T4NxM0, stage III) between Sept 2009 and Feb 2017. No patient received neoadjuvant therapy before surgical treatment.

Results: We reported 16 patients underwent arterial resection during pancreatectomy in our center. Major surgical approach was Appleby procedure (12/16), Pancreaticoduodenectomy (PD) with resection and reconstruction of replaced right hepatic artery (3/16) and PD with resection of common hepatic artery (1/16). Median age was 63 (53-78) years. Median operative time was 310 (195-530) min with median blood loss 400 (50-1800) ml. Postoperative morbidity included 7 cases of grade B postoperative pancreatic fistula (POPF), 1 cases of grade C Postpancreatectomy hemorrhage and grade C POPF with no perioperative mortality. Median postoperative hospital stay was 13 (8-36) d. R0 resection was achieved in 7 patients (43.75%, 7/16). Until the last time of follow-up (Apr 2017), 9 patients (56.25%, 9/16) are dead, 5 patients are alive without local recurrence or distant metastases, with Median Survival Time (MST) was 13 months and the longest survival time was 36 months. Compared with other T4NxM0 patients (stage III, n = 34) underwent palliative treatment, radical pancreatectomy combined with arterial resection could prolong the overall survival time in stage III pancreatic cancer patients (One-year survival rate: 54.27% vs 19.61%, P = 0.049; Two-year survival rate: 33.92% vs 0%, P = 0.028; MST: 13.0 months vs 9.0 months, P = 0.1167).

Conclusion: Planned arterial resection at the time of pancreatectomy can be performed with acceptable morbidity and mortality, which may increase the possibility of R0 resection and improve survival in patients with pancreatic cancer.

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Protective Effect of Urocortin 2 in Cerulein-Induced Acute Pancreatitis

J. Yuan,1 B. Hasdemir,2 S.J. Pandol,1 A. Bhargava.2,3 1 Cedars-Sinai Medical Center, VA West Los Angeles Medical Center, and University of California Los Angeles, Los Angeles, CA; 2 Department of Ob-Gyn and 3The Osher Center, University of California, San Francisco, San Francisco, CA.

Background: Dysregulation of secretory function is seen in acute pancreatitis (AP). Corticotropin Releasing Factor (CRF) and urocortin (UCN) are emerging as key regulators of pancreatic function through CRF receptors (CRFRs). UCN was shown to be de novo expressed in acinar cell in murine model of AP. UCN mediates cellular signaling via activation of the subtype 2 CRF receptor (CRFR2). Increased ubiquitination and change in vimentin expression were noted in CRFR2 null mice with AP compared with WT mice suggesting a role for the CRFR2 in regulating signals of pancreatitis.

Aim: The present study aimed to evaluate the protective effects of a novel CRFR2 ligand, urocortin 2 (UCN2) on cerulein-induced AP.

Methods: Male Sprague-Dawley rats were randomized into groups to receive an intraperitoneal (IP) injection of mUCN2, CRFR2-specific antagonist astressin2B or saline (10 μg/kg body weight). After 30 min, the pre-treated rats were again divided in to 2 groups to either receive 4 hourly IP injections of cerulein (CR, 20 μg/kg body weight, n = 3/group) or saline. Rats were euthanized and pancreatic tissue and blood collected for further analyses.

Results: UCN2 attenuated the severity of AP in rats as reflected by reduced serum amylase and lipase activity, histology (reduced cell necrosis and inflammatory cell infiltration), and reduced nuclear factor NF-κB activity.

Conclusions: The present findings show that UCN2 has a protective effect in AP model via modulation of the NF-κB pathway.

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Disruption of Endosomal Trafficking by Knockout of Tumor Protein D52 Results in Acinar Damage and Proliferation Independent of an Immune Response

E.K. Jones, D.D.H. Thomas, E.J. Phelan, M.M. Cooley, N Ly, G.E. Groblewski. Program in Biochemical and Molecular Nutrition, University of Wisconsin, Madison, WI.

Background: Acinar cell damage is traditionally associated with pancreatitis; however, the mechanisms regulating this pathological process remain unclear. Acute pancreatitis (AP) results in a rapid loss of the endosomal regulatory proteins, D52, Rab5 and EEA1 resulting in loss of VAMP8 mediated zymogen granule (ZG) secretion and intracellular trypsin accumulation. We hypothesize that the rapid loss of D52 during induction of AP represents a key pathological event in disease progression.

Methods: D52-floxed mice were crossed with tamoxifen-inducible elastase cre mice to produce acinar specific D52 deletion (D52-/-). Tamoxifen was administered by oral gavage at 60-100 d of age and pancreases were analyzed 21 d later.

Results: Loss of D52 caused disruption of organelles within the acinar cell, ER dilation, vacuolization, loss of zymogen granules, expanded apical membrane, increases in expression of endosomal regulatory proteins (Rab5, EEA1) and a loss of Golgin-98 staining. This cellular damage was concurrent with a transcriptional profile shift towards a proliferative program consistent with acinar to ductal metaplasia. D52-/- pancreases displayed a 3-fold increase in cyclin D and increases in ductal and progenitor markers: Sox9 (2.5x), β-catenin (4.5x), cytokeratin 19 (2x), E-cadherin (3x), Nestin (2.5x), and Hes1 (1.5x). Acinar proliferation was confirmed by increases in KI-67 positive nuclei. Interestingly, preliminary data suggest that acinar cell damage and regeneration did not induce an immune response as no changes were seen in expression of TNFα, Tgfβ, IL-6, F4/80, CCL2, CD5, and CD19.

Conclusion: These results indicate that loss of D52 during AP exacerbates acinar damage and enhances acinar proliferation. D52-/- may provide an in vivo model to study acinar cell damage and proliferation separated from the immune response allowing for better mechanistic insight into the early stages of pancreatitis.

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The Effect of Pancreatic Cancer Patient Derived Serum on Macrophage M1/M2 Polarization

M. Juusola,1 H. Mustonen,1 S. Vainionpää,1 M. Vähä-Koskela,2 P. Puolakkainen,1 H. Seppänen.1 1 Department of Surgery, Helsinki University Hospital and University of Helsinki, Helsinki, Finland; 2Institute for Molecular Medicine Finland (FIMM), University of Helsinki, Helsinki, Finland.

Background: Monocytes differentiate into inflammatory M1 or anti-inflammatory M2 macrophages in tissue. We set out to explore whether serum from pancreas cancer patients and healthy controls could alter the differentiation of monocytes into M1 or M2 macrophages.

Methods: Monocytes were left to mature into macrophages in media supplemented with pancreatic cancer patient or control serum (15%). Two different cancer cell line cells (MIA PaCa-1 and HPAF) were added to the cultures. After two days of co-culture, the macrophages were harvested and their expression of CD markers was measured by flow cytometry. Cytokine levels in serum were assessed by Q-Plex (Biosciences). Cancer cell migration rate was measured by microscopy.

Results: The pancreatic cancer patients (n = 14) and control serums from healthy individuals (n = 6) differed in levels of cytokines. Patient derived serum was significantly richer in IL-1b (P = 0.041), IL-6 (P = 0.041), IL-10 (P = 0.02), TNFα (P = 0.02) and had higher levels of Rantes (P = 0.008).

The expression of CD markers connected with macrophage differentiation changed depending on the culture conditions. Co-culture with MIA PaCa-1 and HPAF significantly increased CD209 (P = 0.004) and CD86 (P < 0.001) expression. Interestingly, CD86 (M1 marker) expression increased more in the presence of control than patient serum when co-cultured with cancer cells (P = 0.029). No difference was found in the initial expression of CD markers in patient derived monocytes and monocytes drawn from healthy controls (P = 0.537).

The presence of macrophages increased the migration of cancer cells in serum supplemented media (P < 0.001). No difference was found between patient and control derived serum with respect to migration rate.

Conclusion: M1 polarization may be reduced in pancreatic cancer patient macrophages compared to healthy controls when cultured in autologous serum. Further studies need to be conducted to examine whether this effect is due to the altered cytokines in patient sera or due to intrinsic differences in monocytes from patients and healthy individuals.

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Portal Vein Complications After PV/SMV Resection and Reconstruction in Pancreatic Surgery

S. Kagawa, H. Yoshitomi, K. Furukawa, T. Takayashiki, S. Kuboki, S. Takano, D. Suzuki, N. Sakai, H. Nojima, T. Mishima, E. Nakadai, M. Miyazaki, M. Ohtsuka. Department of General Surgery, Graduate School of Medicine, Chiba University, Chiba, Japan.

Background: PV/SMV resection and reconstruction has become standard procedure for experts in pancreatic surgery. The more we do such surgeries the more reports of complications are increasing. Postoperative complications for PV/SMV resection include narrowing and twisting of the anastomosis, accompanying thrombosis. In such cases, portal hypertension causes hepatic failure, intestinal congestion and bleeding. (e.g. Sinistral portal hypertension)

Methods: Among 919 cases of pancreatectomy for pancreatic diseases in Chiba University Hospital, 242 patients who underwent PV/SMV resection and reconstruction were analyzed retrospectively.

Results: The surgical procedures were 196 cases of PD, 31 cases of DP, 15 cases of TP. There was no significant difference in postoperative portal vein thrombus formation rate among 16 PD cases (8.2%), 3 DP cases (9.7%), and TP 1 cases (6.2%). The average length of infiltrate was 15.3 mm in Venorrhaphy, 22.9 mm in End-to-end, 13.5 mm in Patch graft, and 32.9 mm in Segmental graft. Thrombus formation rate were 4.9% in Venorrhaphy, 10.5% in End-to-end, 0% in Patch graft, and 6.5% in Segmental graft. Among who received End-to-end PV reconstruction, four patients were required reoperation due to the PV thrombus. In 2 cases, thrombus was removed by reconstruction with left renal vein graft within 48 hours after first surgery and PV blood flow was obtained.For the other two cases, one patient received metallic stent placement on 27 days after surgery and the other one patients required reoperation on 8 days after first surgery. Unfortunately, in both cases PV blood flow was in sufficient. Of these series of PV/SMV resection and reconstruction cases, 5 patients were not due to postoperative complications but received metallic stenting for cancer recurrence.

Conclusion: For postoperative portal vein thrombosis due to the tension for end-to-end anastomosis, early re-operation with auto-vein graft is important.

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New Onset Pre-Diabetes: Clue to Pre-Diagnostic Pancreatic Cancer

H. Kandlakunta, A. Sharma, S.H. Patlolla, S.J. Singh Nagpal, S.T. Chari. Division of Gastroenterology and Hepatology, Mayo Clinic, Rochester, MN.

Background: Subjects aged ≥50 years with new-onset diabetes mellitus (NOD) are a high-risk group for pancreatic cancer (PC), with ∼1% being diagnosed <3 years of meeting NOD criteria. The mean lead time between onset of NOD and PC diagnosis is ∼6 months. Hyperglycemia precedes PC diagnosis by ∼36 months. Therefore, we aimed to look for clues that may provide earlier diagnosis of PC by using new-onset of pre-diabetes (NOPD) with varying blood glucose (BG) cutoff values for pre-diabetes.

Methods: Using Rochester Epidemiology Project (REP) data, we abstracted all BG values measured on subjects aged ≥50 years in Olmsted County (OC), MN. We assembled a cohort of subjects between 2000 and 2015 who for the first time met NOPD criteria using following BG cutoff values: NOPD-1 (FBG>110/A1C >5.6); NOPD-2 (FBG>115/A1C>5.8) and NOPD-3

(FBG>120/A1C >6.0). To determine the risk of PC in each NOPD category, we identified all PC subjects (n = 219) within same OC population and time period. We also applied enriching new- onset diabetes for pancreatic cancer (ENDPAC) score to each NOPD category. The lead time was calculated between glycemic onset of NOPD and PC diagnosis.

Results: The PC prevalence in NOPD-1 was 0.2% (n = 23/8065) with a mean lead time of 10.9 months (range, 0.03-34). The PC prevalence in NOPD-2 was 0.42 (n = 29/6915) with a mean lead time of 11 months (range, 0.03-34). The PC prevalence in NOPD-3 was 0.60 (26/4312); with a mean lead time of 13 months (range, 0-33). An ENDPAC score of ≥3 in NOPD-1, NOPD-2 and NOPD-3 enriched the PC prevalence to 0.75, 1.5 and 2.5 respectively.

Conclusion: Incidence of PC in NOPD ranges from 0.3% to 0.6% depending on the glycemic cutoff criteria used for pre-diabetes. ENDPAC score of ≥3 enriches NOPD-3 for PC to ∼2.5% warranting need for clinical work-up.

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Does Prompt Work Up for Acute Pancreatitis Interrupt the Natural History of Pancreatic Cancer?

H. Kandlakunta, A. Sharma, S. Jiv S. Nagpal, S.T. Chari. Division of Gastroenterology and Hepatology, Mayo Clinic, Rochester, MN

Background: Pancreatic cancer (PC) subjects are known to present with acute pancreatitis (AP). However, whether immediate work-up for AP interrupts the natural history of PC is unknown. In current study, we aimed to estimate the 3-year PC risk following an episode of AP in subjects ≥50 years and determine the proportion and characteristics of PC subjects presenting with AP prior cancer diagnosis.

Methods: The Rochester Epidemiology Project (REP) index codes were used to assemble a population-based cohort of all subjects ≥50 years who were hospitalized in Olmsted County (OC), MN (n = 1147) for AP between 2000 and 2015 (AP cohort). An established cohort of PC subjects ≥50 years (PC-cohort, n = 219) within same time period from OC was manually reviewed to identify those who developed AP ≥36 months prior to PC diagnosis using ACG criteria. We abstracted demographic, clinical, radiological and survival data on all PC-AP patients.

Results: The 3-year incidence of PC in AP cohort was 1.1% (13/1147). The median time between clinical work-up from AP episode to PC diagnosis was 0.1 months. In PC cohort, 9 subjects (4.1%) were identified with AP prior to cancer diagnosis; of these, 8 (89%) had no cachexia symptoms (fatigue/anorexia), 5 (56%) had no weight loss, and 6 (67%) had no jaundice at the time of PDAC diagnosis. Four (45%) had no mass on imaging at the time of AP diagnosis and 6 patients (67%) underwent surgical resection for curative purpose. The mean survival of the PC-AP patients was 18 months.

Conclusion: The 3-year risk of PC in AP subjects ≥50 years is ∼1.2%; with ∼5% PCs presenting with prior AP diagnosis. Lower rates of cachexia, weight loss, jaundice and mass on imaging along with higher surgical rates and longer survival may suggests interruption in the natural history of PC due to prompt work-up for AP.

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Tff2+ Cells in the PDG Compartment Regenerate the Main Duct Epithelia After Inflammatory Injury

V. Kansal, P. Mondal, D. Maroni, K.L. McAndrews, J. D. Price, S.P. Thayer. Surgical Oncology, University of Nebraska Medical Center, Fred & Pamella Buffett Cancer Center, Omaha, NE.

Background: Epithelial cell maintenance and repair is regulated through stem cell compartments capable of cell division, self-renewal, migration and differentiation. Under normal physiological conditions, the turnover rate of pancreatic epithelium is very low, whereas in response to inflammatory injury, rapid regeneration takes place. Our knowledge is very limited in terms of understanding the role of these stem cell niches in pancreatic epithelial repair and regeneration. Our laboratory has identified a novel stem cell compartment termed Pancreatic Duct Glands (PDGs), which are the compartment-of-origin for epithelial cells. Somatic progenitor cells proliferate and migrate out of the PDGs to regenerate the epithelium of the main pancreatic duct (MD) after inflammatory injury. PDGs also have a distinct molecular profile (uniquely expressing Shh, Tff2, and Lrig1) from the main duct epithelium.

Methods: To investigate the role of Tff2+ PDG cells as a progenitor stem cell compartment responsible for epithelial renewal and regeneration, we used a newly created Tff2-CreERT2 mouse and performed an in-vivo lineage tracing of Tff2+ cell progeny within the PDGs and main duct epithelium over the course of 5 days.

Results: After inducing inflammatory injury using cerulein, Tff2+ cells expanded within the PDGs and migrated to the main duct epithelium. We saw maximum expansion of Tff2-lineage cells within the MD epithelium at day 1 post-inflammation. The numbers of Tff2-lineage cells observed in the MD decreased by day 2 and was near baseline by day 5. The cells within the PDG were observed to migrate and repopulate the MD epithelium in its proximity. The percent colony size of tagged MD epithelia was relative to the percent tagging of the parent PDG compartment.

Conclusion: These results suggest that Tff2+ cells within the PDGs mark an epithelial progenitor cells that gives rise to mature differentiated progeny that migrates to the pancreatic duct.

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The Effect of Stent Assisted Percutaneous Endoscopic Necrosectomy for Infected Pancreatic Necrosis: A Propensity Score Matched Cohort Study

L. Ke, W. Mao, J. Zhou, B. Ye, G. Li, J. Zhang, P. Wang, Z. Tong, W. Li. Surgical Intensive Care Unit (SICU), Department of General Surgery, Jinling Hospital, Medical School of Nanjing University, Nanjing, China.

Background: In the past 10 years, a very wide range of minimally-invasive techniques were proposed for debridement of infected pancreatic necrosis (IPN). In the present study, we aimed to assess the efficacy of using a relatively new procedure called stent assisted percutaneous endoscopic necrosectomy (SAPEN) instead of conventional endoscopic debridement (ED) in our previously reported 4-step approach in managing IPN.

Methods: From Jan 2015 to May 2018, consecutive patients with IPN receiving either SAPEN or conventional ED were put into two time intervals to conduct a before-and-after comparison according to the treatment strategy. In the SAPEN procedure, a fully covered self-expanding esophageal metal stent was applied to construct a tough and reusable sinus tract to access the necrosis for further necrosectomy. Due to the imbalance in patient numbers, propensity score match was used to generate a 1:1 conventional group for analysis. Matching was based on the admission APACHE II score, demographics (age and sex), time interval from AP onset to admission and etiology.

Results: A total of 263 eligible patients with IPN were identified including 33 ones who underwent the SAPEN procedure (12.5%). One-to-one propensity score matching generated 32 pairs with well-balanced baseline characteristics as no significant difference was detected for organ function at admission and comorbidities. The mortality (21.8% vs 15.6%, P = 0.522) and other important outcome measures like new-onset organ failure (46.9% vs 40.6%, P = 0.614) and bleeding requiring intervention (21.9% vs 25.0%, P = 0.768) did not differ between the two groups. The SAPEN group showed significant shortened hospital stay (33.6 ± 20.8 days vs 51.1 ± 29.7 days, P = 0.01), although both groups needed a median of 3 times of ED procedures.

Conclusion: Comparing with the conventional ED, the SAPEN procedure could reduce the length of hospital stay in IPN patients without adding additional clinical risks. A larger study is warranted to verify the results.

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Outcomes of Endoscopic Therapy in Patients With Acute Recurrent Idiopathic Pancreatitis Associated With and Without Pathogenic Gene Variants

T. Kerdsirichairat,1 M. Faghih,1 N.Y. Jalaly,1 O. Brewer Gutierrez,1 C. Fan,1 A. Kamal,1 V.S. Akshintala,1 C. Walsh,2 M.A. Makary,2 A.N. Kalloo,1 V. Kumbhari,1 M.A. Khashab,1 V.K. Singh.1 Divisions of 1 Gastroenterology and Hepatology and 2Surgical Oncology, Department of Surgery, Johns Hopkins Medical Institutions, Baltimore, MD.

Background: Data on outcomes of endoscopic therapies in acute recurrent idiopathic pancreatitis associated with and without pathogenic gene variants are limited.

Aim: To compare rates of recurrent acute pancreatitis or abdominal pain after ERCP in patients with acute recurrent idiopathic pancreatitis (ARIP) with and without pathogenic gene mutation(s).

Methods: A retrospective database of patients with ARIP from 2010-2018 was evaluated. We compared those with gene-positive and gene-negative variants including PRSS1, SPINK1, CFTR, and CTRC mutations. Exclusion criteria were follow-up time < 6 months, chronic pancreatitis (CP) by definite M-ANNHEIM criteria, lack of at least 2 episodes of AP, or other identifiable cause(s) of acute recurrent pancreatitis. Index ERCP was defined as the last ERCP with interventions. In those without ERCP, the index was tabulated from the time of genetic testing results. Primary outcomes included documented episodes of AP and episodes of pancreatic-type abdominal pain per 6 months.

Results: Among 588 patients with ARP and/or CP, 70 had ARIP. There were 48.6 % females, median age was 36 years. Pathogenic gene variants were found in 34 patients and included CFTR (n = 22, 64.7%), SPINK1 (n = 7, 20.6%), and heterozygous CFTR/SPINK1 (n = 5, 14.7%). 87.1% of patients underwent ERCP for RAP, of whom 19.7% were found to have pancreatic ductal stricture. Over a median follow-up time of 293 weeks, 90% had recurrence of AP and/or pain within 6 months after index ERCP and/or genetic test (90.2% vs 88.9% in those with vs without ERCP, P = 0.91), and 15.7% underwent total pancreatectomy with islet autotransplant (no difference among ERCP vs non-ERCP groups 16.4% vs 11.1%, P = 0.67).

Conclusion: Regardless of the presence of pathogenic genetic variants, the vast majority of ARIP patients have recurrence of AP or abdominal pain within 6 months of index ERCP.

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Visibility, Artifact, and Migrations Using Three Types of Fiducial Marker Placement With a 22-Gauge Needle for Pancreatic Ductal Adenocarcinoma Patients Receiving Stereotactic Body Radiation Therapy

T. Kerdsirichairat,1 S.H. Kim,1,2 A. Narang,3 M. Villafania,3 M.A. Khahsab,1 A.M. Lennon,1 V. Singh,1 M.I. Canto,1 E.J. Shin.1 1 Division of Gastroenterology and Hepatology, Department of Medicine, Johns Hopkins Medical Institutions, Baltimore, MD; 2 Division of Gastroenterology and Hepatology, Chonbuk National University, Jeonju, Republic of Korea; 3Radiation Oncology and Molecular Radiation Sciences, Johns Hopkins Medical Institutions, Baltimore, MD.

Background and Aims: EUS-guided fiducial placement is critical for stereotactic body radiation therapy (SBRT) for borderline resectable and locally advanced pancreatic ductal adenocarcinoma (BR/LA PDAC). We aim to identify visibility, artifact and migration rate of three types of fiducials.

Methods: A retrospective study was conducted using data from patients with BR/LA PDAC who underwent EUS-guided fiducial placement between 8/2014-2/2017 with follow-up until 2/2018. Three types of fiducials were compared: 1. Beacon (BC, 0.43-mm diameter), 2. Visicoil (VS, 0.35- mm diameter), and 3. Gold Anchor (GA, 0.28-mm diameter). A subjective visualization scoring system was used to assess the visibility and artifact on the scale 0-2: 0 = not visible/no artifact, and 2 = clearly visible/significant artifact. The average fiducial migration was calculated.

Results: Fifty-five patients underwent EUS-guided placement of 167 fiducials (36 BC, 27 VS and 104 GA). The median number of fiducials placed were 3. Technical success was 100% with no peri- procedural adverse events. 24.3% of patients received GA had only 2 fiducials (P = 0.04 compared to other fiducials). At simulation CT, three types of fiducials had comparable performance. However, at cone-beam CT during SBRT, GA and VS outperformed BC given better visibility (median 2 versus 1, P = 0.04) and less migration (median 0.12 versus .28 mm, P = 0.04).

Conclusion: GA and VS had comparable visibility, artifact and migration rate. Compared to GA, BC had less visibility, less artifact and worse migration rate. GA implantation perhaps poses increased technical difficulty based on lower number of fiducials deployed.

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Stroma Drive Metabolic Reprogramming and Induces Stemness Properties in Pancreatic Cancer Cells

K. Kesh, V. Gupta, P. Dauer, N. Sharma, R. Hadad, V. Dudeja, A.K. Saluja, S. Banerjee. Division of Surgical Oncology, Department of Surgery, University of Miami Miller School of Medicine, Miami, FL.

Introduction: Pancreatic Ductal Adenocarcinoma (PDAC) is one of the most devastating human malignancies with poor survival rate owing to chemoresistance and relapse. Small population of Tumor Initiating Cells (TIC) are considered major players driving these traits. Recent literature has demonstrated the importance of microenvironment “niche” in enrichment of these TIC populations. The desmoplastic stroma in pancreatic cancer comprising of stromal cells and immune cells are instrumental in pathogenesis and progression of PDAC. Among many cytokines, IL-6 a pro-inflammatory cytokine plays a major role in it.In this study, we show that stromal cell secreted IL6 altered metabolic reprogramming and increased cancer stemness in PDAC cells.

Methods: PDAC cells cultured with pancreatic stellate cells condition media (PSC- CM) or recombinant IL6 and stem ness markers expression were investigated.Cytometry Bead Array (CBA) were used for cytokine profiling. Metabolic alteration along with TIC gene expression were measured using flow cytometry and Seahorse.

Results: PDAC cells when cultured with PSC-CM exhibited increase glucose uptake along withan enrichment of CD133+ population. An analysis of the conditioned media using CBA reveled that PSC-CM is enriched with cytokine IL-6 and TNF-α. Treatment of PDAC cells with recombinant IL-6 increased expression of self-renewal genesSOX2, OCT4, Nanog along with biochemical marker ALDH. In addition, TIC marker CD133 and invasion marker gene MMP7 expression also increased upon IL-6 induction. Moreover, IL6 treatment increased glucose uptake and lactate secretion. Small molecule inhibitor Stattic (inhibitor of STAT3) inhibit glucose uptake and stemness gene expression.

Conclusion: Our study shows that stromal IL-6 altered metabolic reprogramming in cancer cells as well as increased cancer stemness and invasion. In future, the underlying mechanisms of IL-6 driven metabolic alteration and its impact on stemness and metastasis will be investigated.

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Economic Impact of Pancreatic Enzyme Replacement Therapy (PERT) in Pancreatic Cancer (PC) Patients Who May Have Exocrine Pancreatic Insufficiency (EPI) Following Pancreatic Surgery

N. Khandelwal,1 D. Harb,1 P. Reilly,1 S. Manthena,1 J. Vora,1 G. Morris-Stiff.2 1 AbbVie Inc., North Chicago, IL; 2Section of HPB Surgery, Digestive Disease Institute, Cleveland Clinic, Cleveland, OH.

Background: EPI is a known consequence of pancreatic head resection. Following surgery, patients with EPI should be commenced on PERT as soon as their diet is resumed. This study evaluates the economic impact of PERT use among PC patients following pancreatoduodenectomy (PD).

Methods: Retrospective longitudinal analysis was conducted using Truven Health Analytics® claims data. Patients age ≥18 with PC diagnosis, who had undergone PD (January 2008-June 2016), and who were continuously eligible in a health plan for at least 3-months prior and 6- months post-index date, were selected. Patients in whom PERT was initiated within 3 weeks from surgery (PERT group) were compared to matched controls who did not receive PERT (non- PERT group). PERT group was matched 1:2 to non-PERT group based on age, sex, health plan type, and baseline co-morbidities. Health care resource use and costs were compared across the two groups during the three-month follow-up period.

Results: A total of 819 patients were included in the analysis (PERT group = 273; non-PERT group = 546). At baseline, patient characteristics, including healthcare resources and costs were shown to be comparable across both cohorts. During the 3-month follow-up period, patients in the PERT group had lower resource use (i.e. lower hospital, ER, and outpatient visits) vs non- PERT group. Additionally, medical costs ($93,553 vs. $105,405; P = 0.0166) and total healthcare costs ($96,334 vs. $106,820; P = 0.0348) were significantly lower in the PERT group vs non- PERT group.

Conclusion: This study shows that, at a 3-month follow-up period, pancreatic cancer patients who had PD and in whom PERT was initiated within the first 3 weeks following surgery incurred significantly lower healthcare costs than those who did not receive PERT.

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Highly Exothermic Fatty Acid-membrane Phospholipid Interaction Results in Immune Cell Death Which Underlies Development of Infected Pancreatic Fluid Collections (Pfcs)

B. Khatua, A. Bag, C. de Oliveira, R. Pannala, R. Cartin-Ceba, V.P. Singh. Department of Medicine, Mayo Clinic, Scottsdale, AZ.

Background: PFCs developing during clinical acute pancreatitis (AP), which starts sterile, can get infected. The large interval (>4 week) between AP onset and PFC drainage prevents systematic studies of this progression from sterile to infected PFCs. Since post-operative PFCs also get infected, but can be drained earlier, we studied mechanisms common to the progression of sterile to infected PFCs.

Methods: Clinically, etiology of PFC, demographics, fatty acids (GC-MS), lipase, microbiology, and WBC counts were studied. Macrophage membrane phospholipids were analyzed using thin layer chromatography and their interaction with fatty acids was studied using isothermal titration calorimetry (ITC). Cerulein pancreatitis was induced ± the triglyceride of oleic acid [glyceryl tri-oleate (GTO)] or GTO+ the lipase inhibitor orlistat.

Results: Of the 9 post-operative PFCs (8 distal-pancreatectomy, 1 Whipple) 4 were early (< 2 weeks). These were sterile, unlike PFCs drained later. All 9 post AP collections were infected. Early PFCs had higher unsaturated fatty acid (86 ± 2 vs. 66 ± 2%), oleic acid (51 ± 4 vs. 34 ± 2%), and pancreatic lipase activity (30 ± 9 vs. 12 ± 4 KU/L). Infected AP patients had a larger WBC count drop (-3.7 ± 2.4x109/L vs. 1.4 ± 3.3 x 109/L) by day 3 vs. admission. 5/9 infected AP patients developed sepsis (P < 0.03). Sub-micellar oleic acid interacted with the membrane phospholipid dioleyl phosphatidyl choline exothermically. The enthalpy change (-31.4 ± 2.4 Kcal/mol) exceeded common protein-protein interactions by 3-10 fold. In vivo cerulein+ GTO significantly increased serum oleic acid (0.79 ± 0.6 vs. 0.06 ± 0.01 mM in controls), caused peripheral WBC apoptosis (8.5 ± 6.1% vs. 1.4 ± 0.8% in control) and leucopenia (2.3 ± 1.0 x 109/L vs. 12.5 ± 4.1x109/L in controls), which orlistat prevented. A similar pattern of PBMC, macrophage injury was noted in vitro.

Conclusion: Lipolytically generated UFAs like oleic acid integrate into macrophage membrane phospholipids via a strong exothermic reaction and cause Immune cell death. This converts early sterile PFCs into infected ones.

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ADM Are Classified Three Distinct Phenotypical Profiles Depending on its Different Microenvironment

S. Kibe,1 K. Ohuchida,1 Y. Ando,1 S. Takesue,1 M. Nakayama,1 T. Abe,1 S. Endo,1 K. Koikawa,1 T. Okumura,1 C. Iwamoto,2 K. Shindo,1 T. Moriyama,3 K. Nakata,1 S. Nagai,1 Y. Miyasaka,1 M. Shimamoto,4 T. Ohtsuka,1 K. Mizumoto,4 M. Nakamura.1 Departments of 1Surgery and Oncology, 2 Advanced Medical Initiatives and 3 Endoscopic Diagnostics and Therapeutics, Graduate School of Medical Sciences, Kyushu University, Fukuoka, Japan; 4Kyushu University Hospital Cancer Center, Fukuoka, Japan.

Background and Aim: The pancreas is a specific organ easily to induce inflammation, fibrosis, and atrophy because most of the pancreatic parenchyma composed of acini that produce digestive enzymes. However, the role of acini in tumor-surrounding microenvironment affect to the development and progression of pancreatic cancer is not understood. We focused on the pancreatic acinar cell plasticity which we reported as acinar-to-ductal metaplasia (ADM) in the invasive front of pancreatic cancer. We investigated the relation between cancer-associated ADM and local invasion of pancreatic cancer and clarified the distinct phenotype of cancer-associated ADM in the comparison with the pancreatitis-associated ADM or sporadic ADM, which is not associated with cancer or pancreatitis.

Methods: We analyzed pancreatic tissues from patients and KPC (KrasLSL-G12D/+;Trp53LSL-R172H/+;Cre) mice with pancreatic cancer. In vitro, we isolated pancreatic acinar cells from KPC mice and performed ADM assay in three-dimensional culture model. In vivo, we analyzed the correlation between pancreatic tumor progression and acinar cell plasticity around tumor in orthotopic transplantation model. Furthermore, we performed gene expression analysis to clarify the feature of each type of microenvironment-dependent ADM.

Results: Based on immunohistochemical analyses, ADM was significantly observed in the invasive front of pancreatic cancer (P < 0.01). In vitro, when pancreatic acinar cells expressed KrasG12D or stimulated with transforming growth factor-α (TGFα), the cells formed ADM-like structures (P < 0.01). In vivo, orthotopic KC(KrasLSLG12D/+;Cre) mouse models represented cancer-associated ADM around tumor, induced desmoplasia in the invasive front and the accelerated tumor progression compared with the control mouse models (P < 0.01). Gene expression analysis revealed that cancer-associated ADM, chronic pancreatitis-associated ADM, and sporadic ADM show distinct phenotypical gene expression profiles.

Conclusions: The present data suggest the possibility that mechanisms of induction of ADM are different depending on its microenvironment, and acinar cell plasticity via cancer-associated ADM contributes to local invasion to pancreatic parenchyma of cancer cells.

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Use of Periodic-acid Schiff and Alcian Blue (PAS-AB) Stains for Diagnosis of Pancreatic Cancer

Y. Kinowaki,1 Y. Matsuda.2 1 Division of Surgical Pathology, Tokyo Medical and Dental University Hospital, Tokyo, Japan; 2Department of Pathology, Tokyo Metropolitan Geriatric Hospital, Tokyo, Japan.

Background: Accurate diagnosis of malignant and benign pancreatic lesions by ultrasound-guided fine-needle aspiration (EUS-FNA) can be challenging, because the amount of sample and cost of sample preparing are limiting. Periodic-acid Schiff and Alcian blue (PAS-AB) staining is a simple staining method to detect mucin which has been routinely performed in many institutes. In the present study, we focused on the expression pattern of PAS-AB for pancreatic cancer.

Methods: Pancreatic tissue samples were obtained from 114 Japanese patients who underwent surgical treatment at Tokyo Metropolitan Geriatric Hospital, Tokyo, Japan. PAS-AB stained slides were evaluated for intensity and staining pattern. Intensity scores were rated according to the cytoplasmic or membrane staining from 0 to 3 (0: none, 1: weak, 2: moderate, 3: strong), proportional scores were defined by percentage of cytoplasm- or membrane-stained cells and staining pattern were classified as pink (P), blue (B), and pink and blue (P+B). “Strong reactivity” was defined as over 5% of tumor cells were stained with 2 or 3 intensity scores. Immunohistochemical detection was performed with MUC1, MUC2 and MUC5AC.

Results: Pancreatic carcinoma that were composed of invasive ductal carcinoma and intraductal papillary mucinous carcinoma showed strong expression for PAS-AB in 65.5% and the other lesions that included intraductal papillary mucinous adenoma, autoimmune pancreatitis, serous cystic neoplasm and neuroendocrine tumor showed 60.0%. B or P+B pattern were observed in 93.8% of pancreatic carcinoma and in 58.3% of other lesions (P < 0.01). In pancreatic carcinoma, MUC1, MUC2, and MUC5AC were positive in 60.5%, 3.9%, and 70.6%. B or P+B pattern in PAS-AB stains were highly detected in pancreatic carcinoma than MUC1 or MUC5AC.

Conclusion: Pancreatic carcinoma showed blue or pink and blue pattern, indicating acidic mucin. PAS-AB staining might be helpful to detect pancreatic carcinoma.

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Radical Surgery for Locally Advanced Pancreatic Cancer After Neoadjuvant Treatment: Resection Margin Status and Its Prognostic Impact

U. Klaiber,1 E.S. Schnaidt,1 U. Hinz,1 U. Heger,1 M. Gaida,2 M.W. Büchler,1 T. Hackert.1 1 Department of General, Visceral and Transplantation Surgery, University of Heidelberg, Heidelberg, Germany; 2Institute of Pathology, University of Heidelberg, Heidelberg, Germany.

Background: In patients undergoing upfront surgery for primary resectable pancreatic cancer resection margin (R) status was shown to be a relevant determinant for survival. So far, the prognostic impact of R status after neoadjuvant treatment for locally advanced pancreatic cancer (LAPC) is poorly investigated. The aim of this study was to evaluate the impact of R status on survival in patients undergoing pancreatic surgery following neoadjuvant treatment for LAPC.

Methods: Prospectively collected data of all consecutive patients undergoing pancreatic resection following neoadjuvant treatment for LAPC were analyzed. R status was categorized as R0 (tumor-free margin ≥1mm), R0 CRM+ (circumferential margin positive), and R1 (microscopic tumor infiltration). Clinicopathological characteristics and outcomes were compared between the R groups. R status and other parameters were tested for survival prediction.

Results: A total of 280 patients were included. R status was R0 in 82 (29.3%) patients, R0 CRM+ in 99 (35.4%) patients, and R1 in 99 (35.4%) patients. Median overall survival was 25.1 months (R0) vs. 15.3 months (R0 CRM+) vs. 18.6 months (R1) with a 3-year survival rate of 35.0%, 20.7%, and 16.1%, respectively (P = 0.0076). Median disease-free survival was months (R0) vs. 8.9 months (R0 CRM+) vs. 8.3 months (R1) with a 3-year survival rate of 19.6%, 9.8%, and 5.9%, respectively (P = 0.0250). Considering the neoadjuvant treatment period, 5.1 months (median) are to be added. In multivariable analysis, R status was not confirmed a significant prognostic factor for overall and disease-free survival.

Conclusion: In patients undergoing neoadjuvant treatment for LAPC survival rates after R0, R0 CRM+, and R1 resections are much better than those observed after exploration alone. From the present data, radical pancreatic resection following neoadjuvant treatment for LAPC should be offered to all patients fit for surgery provided that complete macroscopic tumor clearance can be achieved.

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KIAA1199 is Induced by Inflammation and Enhances Malignant Phenotype in Pancreatic Cancer

S. Kohi, N. Sato, N. Matayoshi, A. Koga, T. Amaike, Y. Kudo, Y. Adachi, K. Hirata. Department of Surgery, University of Occupational and Environmental Health, Fukuoka, Japan.

Background: Recent evidence suggests a critical role of hyaluronan (HA), especially low-molecular-weight (LMW)-HA, in the aggressive tumor phenotype. Increased expression of KIAA1199, a newly identified protein involved in HA degradation, has been reported in various cancers. However, little is known about the functional significance of KIAA1199 in pancreatic ductal adenocarcinoma (PDAC).

Methods: Using siRNA knockdown and forced expression models, we investigated the effects of KIAA1199 expression on malignant behaviors of PDAC cells. We also examined the effect of inflammation on the transcriptional regulation of KIAA1199.

Results: Knockdown of KIAA1199 expression using siRNA resulted in decreased cell migration and proliferation. On the other hand, forced expression of KIAA1199 significantly enhanced the migration and invasion. Importantly, increased KIAA1199 expression was associated with an increased level of LMW-HA. Exposure to IL-1ß and LPS increased the KIAA1199 transcription. In contrast, treatment with NS-398, a cyclooxygenase-2 inhibitor, decreased the KIAA1199 expression.

Conclusion: These findings suggest that increased KIAA1199 expression may contribute to the aggressive phenotype partly through increasing the LMW-HA concentration. Our present results also suggest a possible link between inflammation and induced KIAA1199 expression during PDAC progression.

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Endo180 Expression and Histologic Categorization in Cancer Stroma is an Independent Prognostic Index in Pancreatic Cancer

K. Koikawa,1 K. Ohuchida,1 A. Yonenaga,1 A. Sagara,1 Y. Ando,1 S. Kibe,1 S. Takesue,1 H. Nakayama,1 C. Iwamoto,2 K. Shindo,1 T. Moriyama,1 K. Nakata,1 Y. Miyasaka,1 T. Ohtsuka,1 K. Mizumoto,1 M. Nakamura.1 Departments of 1 Surgery and Oncology and 2Advanced Medical Initiatives, Graduate School of Medical Sciences, Kyushu University, Fukuoka, Japan.

Background: Abundant stroma is a characteristic of pancreatic ductal adenocarcinoma (PDAC) and contribute to the promotion and suppression of pancreatic cancer cells. We previously reported that Endo180 of PSCs is involved in remodeling of pancreatic stroma.

Methods: We evaluated the expression of Endo 180 in 114 invasive PDAC patients who underwent surgery in our department by examined the relationship with each factor reflecting cancer malignancy. Furthermore, we evaluated stroma volume by Sirius red Staining.

Results: In all 114 cases, PSCs were stained with Endo 180 and we classified into 3 groups of weak (n = 29), moderate (n = 43), strong (n = 42) according to the expression intensity. Endo 180 high expression in PSCs correlated to T factor (P = 0.0137) and tumor grade (P = 0.0218), with a significant decrease in overall survival (P < 0.001) and disease free survival (P < 0.001). On the other hand, pancreatic cancer cells were not stained with Endo180 about in half of cases, and we classified into 2 groups of negative (n = 54), positive (n = 60). There was no correlation with clinicopathological factors and prognosis. Next, we classified the tumor stroma volume into low (n = 43), moderate (n = 35) and high (n = 36) depending Sirius red staining positive area. Low stroma correlated to Tumor grade (P = 0.0130) and lymphatic vessel infiltration (P = 0.0216), and decreased the overall survival period (P < 0.001) and the disease-free survival period (P < 0.001) significantly. Endo 180 expression intensity correlated with stroma volume, and the stroma volume was significantly smaller (P < 0.001) as the Endo 180 high expression group.

Conclusion: High expression of Endo 180 in PSCs is a poor prognostic factor, and by targeting Endo 180 in PSCs, it is expected to suppress infiltration of pancreatic cancer and further improve prognosis.

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βIII-Tubulin is a Brake for External Cell-Death Signaling and Regulates TRAIL Sensitivity in Pancreatic Cancer

J. Kokkinos,1 G. Sharbeen,1 J. McCarroll,2,3 C. Boyer,3 A. Akerman,1 J. Youkhana,1 T.P. Davis,4 D. Goldstein,1,5 P.A. Phillips.1,2 1 Pancreatic Cancer Translational Research Group, Lowy Cancer Research Centre, School of Medical Sciences and Prince of Wales Clinical School, UNSW Sydney, Sydney, Australia; 2 Children’s Cancer Institute, Lowy Cancer Research Centre, UNSW Sydney, Sydney, Australia; 3 Australian Centre for Nanomedicine (ACN), ARC Centre of Excellence in Convergent Bio- Nano Science and Technology, UNSW Sydney, Sydney, Australia; 4 ARC Centre of Excellence in Convergent Bio-Nano Science and Technology, Monash Institute of Pharmaceutical Sciences, Monash University, Melbourne, Australia; 5Prince of Wales Hospital, Prince of Wales Clinical School, UNSW, Sydney, Australia.

Background: Pancreatic cancer (PC) has a dismal prognosis requiring new therapeutic strategies. We identified that inhibition of βIII-tubulin (shRNA) in PC cells decreased tumor growth and metastases in mice (Oncotarget, 6;2235-49, 2015). However, since there are no inhibitors of βIII-tubulin, we developed a nanoparticle to deliver siRNA and silence βIII-tubulin in mouse pancreatic tumors (Biomacromolecules, 17; 2337-51, 2016). Aims: (1) To assess the effect of therapeutic inhibition of βIII-tubulin using nanoparticle-siRNA on pancreatic tumor growth in mice. (2) To investigate the pro-apoptotic pathways controlled by βIII-tubulin in PC cells.

Methods: In vivo: Mice with orthotopic MIA PaCa-2 tumors (n = 10/group) were treated (5 weeks post- implantation) with nanoparticle+βIII-tubulin siRNA or control siRNA for 3.5 weeks (i.v x2 weekly). Tumor volume was measured and cleaved caspase-8 assessed in tumors. In vitro: PC cells (MIA PaCa-2, PANC1) treated ± βIII-tubulin-siRNA ± Caspase-8 or 9 inhibitors and Caspase-8/9 activities and apoptosis measured. Sensitivity to TNF-related apoptosis inducing ligand (TRAIL; extrinsic apoptosis inducer) was tested (proliferation, apoptosis).

Results: Nanoparticle+βIII-tubulin siRNA reduced PC tumor growth (103 ± 17.5 mm3; P < 0.05) versus controls (194.9 ± 31.2 mm3). Silencing βIII-tubulin in PC cells significantly increased the activity of caspase-8 (MIA PaCa-2: 53±13% increase vs control, *P < 0.05, n = 4) and caspase-9 (45±9% increase vs control, *P < 0.01, n = 5). However, βIII-tubulin knockdown-induced apoptosis was only blocked by inhibiting caspase-8. Furthermore, knockdown of βIII-tubulin combined with TRAIL increased apoptosis (75 ± 8% increase vs TRAIL alone, *P < 0.01, n = 5), and reduced cell proliferation. Inhibition of βIII-tubulin in vivo also increased caspase-8 activity in PC tumors.

Conclusion: Nanomedicine/siRNA inhibition of βIII-tubulin represents a new class of therapeutic with potential to improve PC patient outcome. Our novel results demonstrated βIII-tubulin silencing in PC cells activates extrinsic apoptosis and sensitises to an inducer of extrinsic apoptosis (TRAIL). βIII-tubulin inhibition in the clinic may improve the efficacy of extrinsic apoptosis inducers, opening an unexplored avenue for PC treatment.

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Female Sex and Renalase Deletion Both Result in More Severe and Prolonged Pancreatitis

T. Kolodecik,1,2 G. Desir,1,3 F. Gorelick.1,2,4 1 Section of Digestive Diseases, Department of Internal Medicine, Yale University School of Medicine, New Haven, CT; 2 Veterans Administration Connecticut Healthcare, West Haven, CT; Departments of 3 Nephrology and 4Cell Biology, Yale University School of Medicine, New Haven, CT.

Background: Few studies have systematically examined the differences in acute pancreatitis (AP) severity between males and females. However, it is generally accepted that young females have a 2-4 fold increased risk of developing severe pancreatitis after endoscopic retrograde cholangiopancreatography (ERCP). Renalase (RNLS), a cytoprotective factor released by the kidney, can reduce the severity of acute pancreatitis. It can also directly regulate immune cell (macrophage) function. In this study we examined in vivo AP responses in young male and female mice and the impact of genetically deleting RNLS on this response.

Methods: AP was induced in 2-3 month-old male and female mice [C57BL/6 (WT) and RNLS-/- (RNLS-KO)] by six hourly intraperitoneal injections of cerulein (CER, 50 ug/kg). Mice were sacrificed at various times after initiating AP. Pancreatic tissue and blood samples were analyzed for AP responses.

Results: We found that female WT mice had more rapid onset, more severe disease and slower recovery than WT males as indicated by changes in serum amylase, edema and markers of histologic damage (edema, vacuolization and pyknotic nuclei). Histologic damage was greater and recovery was delayed in the RNLS KO vs WT. However, the difference observed between WT males and females were largely absent in RNLS KO animals.

Conclusion: Our studies indicate that AP severity and progression in mice vary by sex. Females appear to have a more rapid onset of disease and slower resolution. RNLS deletion leads to a more severe acute response, and delayed resolution regardless of sex. The mechanism by which female mice have more severe disease is unclear but may be relevant to the increased risk of females developing post-ERCP pancreatitis, which tends to be more severe.

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Fully Covered Self-expanding Metal Stents for Benign Refractory Pancreatic Duct Strictures in Chronic Pancreatitis: Long-term Outcomes

T. Korpela, M. Udd, O. Lindström, L. Kylänpää. Gastroenterological Surgery, Abdominal Center, Helsinki University Hospital and University of Helsinki, Helsinki, Finland.

Background: Pancreatic duct (PD) strictures are common adverse event in chronic pancreatitis (CP). Primary treatment for refractory PD strictures is endotherapy (ET), including insertion of multiple plastic stents. In addition, fully covered self-expandable metal stents (FC-SEMSs) have also been successfully used, however, more long-term studies is needed to clarify the complications rate and efficiency.

Methods: This retrospective study comprised 17 patients (16 men and one woman) with symptomatic CP and refractory fibrotic main pancreatic duct (MPD) stricture treated with FC– SEMSs between 2010-2018 at Helsinki University Hospital. Treated strictures were located in pancreatic head in all patients. Technical success was defined as accurate positioning of the stent and resolution of the MPD stricture at ERCP. The clinical result was defined as successful when relief of pain was achieved at the end of follow-up.

Results: In 10 patients (59%), stricture resolution was accomplished. Clinical success was achieved in 11 patients (65%). Median duration of stenting was 169 days (range, 15-804). All FC–SEMSs [13 Niti-S (bumpy type) stents, five Hanaro stents and two Viabil stents] were easily removed without any complications. Long-term technical and clinical result (≥24 months) was achieved in ten patients (59%). Early complications (≤7 days) was reported in two patients (12%): one pancreatitis and one cholestasis, both was managed conservatively. Late complications (≤7 days) were intolerable abdominal pain in 5 patients (29%), pancreatitis in 3 patients (18%), cholestasis in one patient (6%) and migration in 7 patients (35%) (all Niti- S stents). There was significant difference in stricture resolution (P = 0.004) and pain improvement (P = 0.022) in stent placement episodes without stent migration compared to stent placement episodes with migration.

Conclusion: FC-SEMS placement is effective and safe treatment for this advanced group of patients, however, migration appears to affect the clinical and technical outcome.

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Confocal Laser Endomicroscopy Detection of Advanced Neoplasia in IPMNs: An International External Interobserver and Derivation Study

S.G. Krishna,1 J.M. Dewitt,2 C.J. DiMaio,3 P. Kongkam,4 B. Napoleon,5 M.O. Othman,6 D. Tan,7 S. El-Dika,1 S. McCarthy,1 J. Walker,1 M.E. Dillhoff,8 A. Manilchuk,9 P.A. Hart,1 Z. Cruz-Monserrate,1 D.L. Conwell.1 1 Division of Gastroenterology, Hepatology and Nutrition, The Ohio State University Wexner Medical Center, Columbus, OH; 2 Division of Gastroenterology, Department of Medicine, Indiana University Hospital, Indianapolis, IN; 3 Division of Gastroenterology, Icahn School of Medicine at Mount Sinai, New York City, NY; 4 Department of Medicine, Faculty of Medicine, King Chulalongkorn Memorial Hospital, Chulalongkorn University, Bangkok, Thailand; 5 Department of Gastroenterology, Ramsay Générale de Santé, Hôpital privé Jean Mermoz, Lyon, France; 6 Section of Gastroenterology and Hepatology, Department of Medicine, Baylor College of Medicine, Houston, TX; 7 Department of Gastroenterology and Hepatology, Singapore General Hospital, Bukit Merah, Singapore; 8 Division of Surgical Oncology, The Ohio State University Wexner Medical Center, Columbus, OH; 9Department of General Surgery, The Ohio State University Wexner Medical Center, Columbus, OH.

Background: Prior studies have validated EUS-guided needle-based confocal laser endomicroscopy (nCLE) diagnosis of IPMNs by detection of characteristic “papillae”. We sought to further assess the performance and interobserver agreement (IOA) of EUS-nCLE for differentiating IPMNs with high grade dysplasia (HGD) or adenocarcinoma (HGD-Ca) from those with low or intermediate-grade dysplasia (LGD).

Methods: In a prospective single center study evaluating EUS-nCLE guided management of pancreatic cysts, 26 patients with IPMNs (surgery = 24; confirmatory cytology/metastatic adenocarcinoma = 2) were identified. EUS-nCLE imaging variables predictive of HGD-Ca were internally derived amongst 3 EUS-MDs with experience in nCLE. Six external endosonographers (nCLE experience>30 cases) blinded to all clinical data, reviewed all nCLE videos. After 2 weeks, the assessors reviewed the same images in a different sequence. A self- study tutorial of nCLE variables predictive of HGD-Ca was provided before each review. Prior to the second review, an interactive web-based discussion was also conducted. The IOAs (κ statistic) of diagnostic nCLE image patterns to detect HGD-Ca were calculated.

Results: Among the subjects with IPMNs (16 mixed, 10 branch-duct; mean size 3.4±1.7 cm), there were 9 with LGD and 17 with HGD-Ca. The observers identified papillary structures diagnostic of IPMN in all videos. The overall sensitivity, specificity, accuracy, and IOA of EUS-nCLE to diagnose HGD-Ca was 90%, 73%, 84%, and κ = 0.54 (moderate), respectively.

Correlating with histopathological progression of dysplasia in IPMNs, nCLE imaging characteristics of papillary “thickness” (indicative of cellular and nuclear stratification) and “darkness” (increased nuclear/cytoplasmic ratio) were the most sensitive (90% and 91%) and accurate (86% and 84%) with substantial (κ = 0.61) and moderate (κ = 0.55) IOAs for detecting HGD-Ca, respectively.

Conclusion: In this derivation study, blinded external endosonographers identified several EUS-nCLE imaging features of IPMNs that are associated with HGD-Ca. Further studies are needed to refine and validate these features for applying EUS-nCLE to identify advanced dysplasia.

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EUS-Guided Needle Based Confocal Laser Endomicroscopy for the Management of Pancreatic Cystic Lesions: A Single Center Study

S.G. Krishna, P.A. Hart, A. Malli, D. Kruger, S.E. El-Dika, S.T. McCarthy, J.P. Walker, M. Dillhoff, C. Schmidt, A. Manilchuk, T. Pawlik, K. Porter, Z. Cruz-Monserrate, D.L. Conwell. The Ohio State University Wexner Medical Center, Columbus, OH.

Background: Endoscopic ultrasonography (EUS)-guided needle based confocal laser endomicroscopy (nCLE) image patterns to identify mucinous pancreatic cystic lesions (PCLs) have been validated among independent external observers and in resected ex vivo PCLs. We sought to evaluate the diagnostic accuracy of EUS-nCLE for differentiating mucinous from non-mucinous PCLs.

Methods: In a prospective, single center study, 134 of 186 consecutive subjects referred for EUS with a suspected PCL on cross-sectional imaging (size ≥1.8 cm) also met inclusion criteria for nCLE imaging. A definitive diagnosis (histopathology or cyst-fluid molecular analysis) was available in 92 subjects. A clinical diagnosis (cyst resolution or CEA<5ng/ml and absence of mucin or KRAS/GNAS mutations and no multiple cysts, communication/dilation of main pancreatic duct) was available in 12 subjects. Diagnostic indices of different modalities were computed for all subjects.

Results: Among 134 subjects (60.4 ± 14.4 years; 50% female), mean cyst size was 36.6 ± 16 mm, mean duration of nCLE was 7.3 ± 2.9 min. The overall risk of EUS-FNA-nCLE associated acute pancreatitis was 3.7% (5/134; all of mild-severity). There were no statistical differences in sex, smoking, alcohol, diabetes, and preceding acute pancreatitis between mucinous and non-mucinous PCLs.

In both cohorts (n = 92; definitive diagnosis) and (n = 104; addition of clinical diagnosis), the sensitivity, specificity, and accuracy of EUS-nCLE for the diagnosis of mucinous-PCLs was 93%, 100%, and 95% respectively. Comparatively, the accuracy of any combination of CEA and/or cytology was 66%, 67%, and 66% respectively.

In a cohort where results of EUS-nCLE and cyst fluid molecular analysis was available (n = 46), the sensitivity, specificity, and accuracy of EUS-nCLE for mucinous PCLs was 100%, 96%, and 98% respectively.

Conclusion: In this large, prospective cohort of subjects, endomicroscopy of PCLs was more accurate than current ‘standard of care’ for the differentiation of mucinous from non-mucinous PCLs. These single center results warrant further validation in larger, multi-center studies.

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Systemic Anticoagulation is Associated With Decreased Mortality and Morbidity in Acute Pancreatitis

P.T. Kröner, M. Raimondo, M. Wallace, B. Ji, Y. Bi. Department of Gastroenterology and Cancer Biology, Mayo Clinic, Jacksonville, FL.

Background: Acute pancreatitis is an acute inflammatory process of the pancreas with considerable mortality and morbidity. Current treatment is limited to fluid resuscitation, supportive care and complication management. Inflammation and coagulation pathways are interacted and coagulopathy is common in AP patients. The aim of this study was to explore the effect of systemic anticoagulation prior to AP onset on the outcomes of AP.

Methods: A case-control study using the NIS 2014, the largest publically available inpatient database in the US was performed. All patients with a principal diagnosis of AP on ICD9 codes were included. Patients on systemic anticoagulation were identified using ICD9 codes. The primary outcome was the odds of AP in patients on systemic anticoagulation compared to patients not on anticoagulation. Secondary outcomes were mortality, morbidity, length of hospital stay, total hospitalization charges and costs. Propensity score matching was used to create a 1:1 matching population for gender, age and Charlson Comorbidity Index. Multivariate regression was used to adjust for patient zip code, income, hospital region, location, size and teaching status.

Results: Out of 442,535 patients with AP, 12,735 were on systemic anticoagulation prior to AP. The mean age was 66 and 47% were female. After adjusting for confounders, patients on systemic anticoagulation prior to AP onset displayed odds ratio 0.56 of AP compared to patients who were not on anticoagulation. Patients on anticoagulation displayed decreased mortality (OR, 0.65, P < 0.01), shock, AKI, ICU admission, multi-organ failure, hospital costs and charges compared to patients who were not on systemic anticoagulation.

Conclusion: Patients on systemic anticoagulation have decreased odds of AP occurrence, mortality, and improved outcomes compared to patients who are not on systemic anticoagulation. These findings suggest that anticoagulation may play a role in addressing the coagulopathy triggered by AP, which may result in improved gland perfusion, decreased multi-organ failure and hence better outcomes.

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Acute Pancreatitis in the United States: A Decade of Outcomes

P.T. Kröner, A.C. Rodríguez, J.E. Corral, M. Raimondo. Department of Gastroenterology, Mayo Clinic, Jacksonville, FL.

Background: Despite changes in management guidelines and medical advances, some studies show mortality of acute pancreatitis (AP) as unchanged. The aim of this study was to explore the changes in occurrence, mortality and resource utilization of patients hospitalized for AP throughout the past decade using a national database.

Methods: Retrospective cohort study using the NIS 2005-2014, the largest publically available inpatient database in the US. All principal ICD-9 diagnosis codes for AP were included. The primary outcome was overall inpatient mortality. Secondary outcomes mortality trends of co-existing acute kidney injury (AKI), use and timing of ERCP, economic burden (adjusted for inflation) and length of stay (LOS). Multivariate analysis yielded adjusted odds ratios (aOR) and means to control for confounders, comparing outcomes in the year 2014 against 2005.

Results: 2,716,400 patients with AP were included. Mean age was 53 years and 50% were female. In the study period, inpatient mortality decreased from 1.19% to 0.66% (P < 0.01), aOR:0.48 (P < 0.01). ERCP use proportional to cases did not change. Early ERCP (within 48 hours) use increased from 53.2% to 78.1% (P < 0.01), aOR, 1.33 (P < 0.01). Mortality early ERCP cases was lower than the non-early ERCP cohort (0.68% vs. 1.01%, P < 0.01), aOR, 0.68 (P < 0.01). AKI cases increased from 3.7% to 9.6% (P < 0.01), aOR, 2.51 (P < 0.01). Mortality in patients with AKI and AP decreased from 14.4% to 4.6% (P < 0.01) aOR, 0.44 (P < 0.01). Costs and LOS decreased, while charges increased significantly.

Conclusion: Mortality in patients with AP has decreased in the past decade. Mortality in patients with associated AKI has decreased even more dramatically, likely evidencing adherence to fluid resuscitation guidelines. Although ERCP use has not changed, the shift in tendency towards early ERCP is evident, along with its reflected benefits in decreased mortality. Although LOS and hence costs decreased, charges increased likely due to evolving insurance models

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End-Stage Renal Disease in Acute Pancreatitis: A Nationwide Analysis of Outcomes

P.T. Kröner, A.C. Rodríguez, J.E. Corral, M. Raimondo. Department of Gastroenterology, Mayo Clinic, Jacksonville, FL.

Introduction: Management of acute pancreatitis (AP) in end stage renal disease (ESRD) is challenging as fluid resuscitation is limited by volume overload. Limited data exist regarding the effects of ESRD on AP. Aim of this study was evaluating occurrence of AP and its outcomes on ESRD.

Methods: Case-control study using the NIS 2014, largest inpatient database in US. Included all patients with ICD9CM diagnosis of AP and associated ESRD. No patients were excluded. Primary outcome: prevalence of AP in ESRD compared to non-ESRD. Secondary outcome: inpatient mortality, morbidity, resource utilization, length of hospital stay (LOS), hospital charges and costs. Propensity score matching used to create a matching population; regressed against age and Charlson Comorbidity Index (CCI). Multivariate analysis used to adjust for income in patients’ zip code, hospital region, location, size and teaching status.

Results: 382,595 patients with AP, 15,430 cases were propensity-matched, of which 7380 had ESRD. Mean age 56, 49% females. Inpatient prevalence of AP in ESRD was 20.4/1000 admissions compared to 10.6/1000 in non-ESRD. Patients with ESRD had higher adjusted propensity- matched odds of 1.51 (P < 0.01) of hypercalcemia-related AP and of miscellaneous causes of AP (aOR, 1.29, P < 0.01) compared to non-ESRD. Adjusted mortality was increased in ESRD group (aOR, 2.11, P < 0.01). No differences observed in inpatient morbidity, LOS or economic burden.

Conclusion: Inpatient prevalence of AP is higher in patients with ESRD contrast to non-ESRD. ESRD patients had higher odds of hypercalcemia-related AP, as well as miscellaneous causes of AP such as medication use, which is understandable given that they are inherent factors to ESRD. Mortality was also increased, which may be related to the challenges of treating AP in ESRD or from the coexistence of other comorbidities. Further studies are warranted to study disease burden from these coexisting diseases.

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Acute Pancreatitis Outcomes in Advanced Chronic Kidney Disease and Kidney Transplant History: A Nationwide Analysis

P.T. Kröner,1 H. Wadei,2 A.C. Rodríguez,1 M. Raimondo.1 Department of 1 Gastroenterology and 2Transplant Nephrology, Mayo Clinic, Jacksonville, FL.

Background: The prevalence of acute pancreatitis (AP) in stage 5 chronic kidney disease (CKD5) has not been clearly defined. The aim of this study was to compare the prevalence, etiology and outcomes of AP in CKD5 and kidney transplant recipients with those without CKD using a large national database.

Methods: Using the 2014 Nationwide Inpatient Sample (NIS), patients with ICD-9 principal diagnosis code for AP were identified. All patients were >18 yrs old and had no history of pancreas transplant. Patients with AP and CKD5 on and off dialysis, as well as kidney transplant recipients were identified and were propensity-matched in a 1:1 fashion and regressed against gender, age, ethnicity and Charlson Comorbidity Index to patients with AP and no CKD. Multivariate logistic regression was used to adjust for patients' median income, hospital region, hospital size and teaching status.

Results: 433,805 patients with AP were included, of which 690 CKD5 were not on dialysis, 11,415 were on dialysis, and 1,320 had history of kidney transplant. Inpatient prevalence of AP was comparable between the non-CKD, CKD5, and kidney transplant populations. Crude and adjusted mortality was higher in CKD5 (OR, 2.72; P < 0.01) and kidney transplant (2.29, P = 0.02) patients. CKD5 patients were more likely to develop shock (OR, 1.53; P < 0.01) and require ICU (OR, 1.32; P < 0.01) than the non-CKD group. Alcoholic and gallstone AP were more common in non-CKD group while hypercalcemia-related AP was more common (OR, 1.52; P = 0.01) in the CKD5 group.

Conclusion: The adjusted prevalence of AP is comparable between non-CKD patients, CKD5 and kidney transplant recipients. Adjusted mortality is more than double in CKD5 and kidney transplant recipients compared to non-CKD patients. Dialysis-dependent CKD5 patients hospitalized with AP had highest rate of shock and ICU stay compared to non-CKD patients. Hypercalcemia is the main association with AP in the CKD5 population irrespective of dialysis need.

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Genome Profiling and Development of Personalized Therapy for Esophagogastric Adenocarcinoma

H. Kuasne,1 H. Zhao,1 D. Zuo,1 M. Souleimanova,1 V. Pilon,1 A. Monast,1 A.M. Fortier,1 Y. Stern,1,2 C. Martínez Ramírez,1,3 M. Issac,4,5 N. Bertos,4,5 S. Bailey,4,5 J. Cools-Lartigue,4,5 A. Bass,6 L. Ferri,4,5,7 M. Park,1,2,3,5,7 V. Sangwan.4,5 1 Rosalind and Morris Goodman Cancer Research Center, Departments of 2 Biochemistry, 3 Pathology, 4 Surgery, McGill University, Montreal, Canada; 5 Research Institute, McGill University Health Center, Montreal, Canada; 6 Dana Farber Cancer Institute, Harvard University, Boston, MA; 7 Department of 3Medicine, McGill University, Montreal, Canada.

Background: Adenocarcinomas of the distal esophagus and proximal stomach are the fastest rising malignancies in North America. The five-year survival rate for Esophago-Gastric Adenocarcinoma (EGA) is about 20%, since a majority of patients presents with advanced disease. Molecular profiling of individual tumors shows co-occurring oncogenic alterations. These genetic vulnerabilities can be exploited to selectively target the tumor with one or more therapeutic strategies.

Results: Co-occurrence of alterations in multiple genes leads to suboptimal response to monotherapies and rapid development of resistance to targeted agents, as observed in EGA, ultimately driving poor outcome. We have developed a unique resource of matched patient-derived xenografts (PDXs), organoids and cell lines from 31 EGA patients. Based on the genetic profiling of this resource, we are currently testing FDA-approved treatment combinations directed against multiple genetic perturbations to effectively target EGA with a goal of selecting targeted mono- or combination therapies that will provide maximal therapeutic benefits for EGA patients and improve response to treatment by preventing the development of a resistant cell population. We have identified FDA-approved targeted therapies in vitro using organoids derived from PDXs; subsequent in vivo validation of identified treatments was performed using PDX models.

Conclusion: EGA is usually diagnosed at an advanced stage, due to the lack of specific symptoms in early-stage disease and the absence of effective screening tools. Thus, most tumors are disseminated by the time of treatment. Local resection followed by adjuvant therapy is only curative in a minority of cases. We have identified novel approaches to enhance the efficacy of systemic therapy. This is predicted to have a significant impact on clinical care.

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Gut Microbiota Depletion Attenuates Pancreatic Cancer Metastases

S. Kurtom, V. Sethi, A. Ferrantella, B. Giri, B. Garg, H.K.C. Jacob, S. Ramakrishna, A.K. Saluja, V. Dudeja. Department of Surgery, University of Miami Miller School of Medicine, Miami, FL.

Background: Trillions of bacteria reside in the gut and play an important role in intestinal homeostasis. There is growing evidence demonstrating the gut microbiome’s role in modifying the immune system. Our study aimed to evaluate microbiome-driven immunological mechanisms, specifically toll- like receptor (TLR) activation, in metastatic murine pancreatic ductal adenocarcinoma (PDAC). TLRs mediate the recognition of microbial ligands to facilitate immune-mediated elimination of pathogens. TLR2 and TLR4 are important mediators of the inflammatory response in cancer. We hypothesize that gut microbiome depletion decreases liver metastases, via abrogation of the TLR-induced inflammatory response.

Methods: C57BL/6J mice received either oral saline or a gut sterilizing cocktail of poorly absorbable broad spectrum antibiotics (Vancomycin, Ampicillin, Amphotericin B, Metronidazole, and Neomycin). These mice then received intrasplenic injection of pancreatic cancer cells derived from KPC (Kras LSL.G12D/+; p53 R172H/+;Pdx::Cre) and PKT (Ptf1acre/+ ;LSL-KrasG12D/+ ;Tgfbr2flox/flox) mice. To evaluate the role of TLR activation, intrasplenic KPC injection was subsequently repeated in TLR2-/- or TLR4-/- mice. Mice were sacrificed and the tumors were immunophenotyped and immunostained for multiple antigens.

Results: Gut microbiome depletion decreased hepatic metastases in KPC injected wild type and TLR4-/- mice and PKT injected WT mice. This tumor inhibitory effect of gut microbial depletion disappeared in the TLR2-/- KPC group. Antibiotic-treated mice had a decrease in intrametastatic IL-17+ CD4+ cells.

Conclusion: Gut microbiota promote liver metastases in pancreatic cancer, potentially via modulating IL-17+ CD4 cells mediated through the TLR2 pathway.

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Portal Vein Hypertension Induces Lymphangiogenesis in the Pancreas Through Macrophage-Derived VEGF-C Production

S. Lai,1 M. Tanaka,1 T. Utsumi,1,2 J. Mao,1 F. Gorelick,1,2 Y. Iwakiri.1 1 Section of Digestive Diseases, Yale School of Medicine, New Haven, CT; 2VA Connecticut Healthcare System, West Haven, CT.

Background: Lymphatic vessels are a primary pathway for drainage of excessive interstitial fluid and infiltrating immune cells in pathological conditions. Little is known about the formation of new lymphatic vessels (lymphangiogenesis) in the pancreas. The aim of this study was to determine 3D structure of lymphatic vessels and the mechanism of lymphangiogenesis in the pancreas of rats with increased portal vein pressures.

Methods: Partial portal vein ligation (PPVL) surgery was performed in rats to induce portal vein hypertension. Pancreases were collected 3 and 10 days after PPVL as well as from rats with sham operation. Lymphatic vessels (LVs) and lymphangiogenesis were determined by LYVE-1/podoplanin and podoplanin/PCNA co-immunolabeling, respectively. 3D images of LVs were visualized using scanning confocal microscopy.

Results: Severe pancreatic edema was observed at 3 days after PPVL, but was cleared by 10 days. Lyve-1+/podoplanin+ LVs significantly increased at 3 days (3.6-folds, P < 0.01) and continued to increase at 10 days after PPVL (4.5 folds, P < 0.01) compared to those of sham rats. The number of CD68-positive macrophages increased significantly at 3 days (3.3-fold, P < 0.01) but decreased by 10 days (1.6-fold, P < 0.05) compared to that of sham rats.3D imaging showed newly developed LVs along with pancreatic blood vessels. Macrophage depletion by clodronate liposomes significantly decreased LVs (2.5-fold, P < 0.01) in the pancreas of rats with 10-day PPVL.Treatment of 10-day PPVL rats with a neutralizing antibody to VEGF-C, the most potent lymphangiogenesis inducer, significantly decreased pancreatic lymphangiogenesis (2.0-fold, P < 0.05) compared to 10-day PPVL rats given control IgG. Given that macrophages are known as a source of VEGF-C, macrophages contribute to pancreatic lymphangiogenesis induced by portal vein hypertension through VEGF-C production.

Conclusion: Portal vein hypertension by PPVL induces edema and lymphangiogenesis in the pancreas, suggesting a link between edema formation and lymphangiogenesis. Macrophage-derived VEGF-C mediates pancreatic lymphangiogenesis.

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TLR1 Predicts Favorable Prognosis in Pancreatic Cancer

M. Lanki,1,2 J. Hagström,2,3 H. Mustonen,1 H. Seppänen,1 C. Haglund.1,2 1 Department of Surgery, University of Helsinki and Helsinki University Hospital, Helsinki, Finland; 2 Research Programs Unit, Translational Cancer Biology, University of Helsinki, Helsinki, Finland; 3Department of Pathology and Oral Pathology, University of Helsinki and Helsinki University Hospital, Helsinki, Finland.

Background: The link between inflammation and carcinogenesis is irrefutable. Trying to pinpoint key factors at play, cancer research has found interest in Toll-like receptors (TLRs), through which pathological molecular patterns trigger immune cell response. TLRs appear to show prognostic value in adenocarcinomas of the mouth, colon and ovaries. We set out to investigate whether the expression of Toll-like receptors 1, 3, 5, 7 and 9 could be used for prognostic evaluation in pancreatic ductal adenocarcinoma (PDAC) patients.

Methods: We collected tumor biopsies from 154 stage I-III PDAC patients who were surgically treated at Helsinki University Hospital between 2000 and 2011. We used tissue microarray and immunohistochemistry to assess the expression of TLRs 1, 3, 5, 7, and 9 in PDAC tissue.

Differences in staining results against clinicopathological parameters were tested with Fisher’s test. For survival analysis we used the Kaplan-Meier method and log-rank test, and the Cox regression proportional hazard model for univariate analyses. Patients receiving neoadjuvant therapy were excluded from the study.

Results: High TLR1 expression was observed in 60 (39%), high TLR3 in 48 (31%), high TLR5 in 58 (38%), high TLR7 in 14 (9%), and high TLR9 in 22 (14%) patients. Univariate analysis showed high TLR1 expression to associate with slightly better survival in pancreatic cancer patients (Mean survival time 4.3 (95% CI, 3.2-5.5) years and 2.1 (95% CI, 1.3-3.0) years; Log rank with Sidak adjustment for multiple comparisons, P = 0.0439). Also, we found noteworthy how poorly those few patients with negative in TLR1, TLR3, TLR7 and TLR9 expression fared.

Conclusion: We found high TLR1 expression to be of positive prognosis in PDAC patients.

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Secretory Unit of Islet Transplant Objects (SUITO) Index and Homeostasis Model Assessment (HOMA) Beta Score Are Ineffective at Predicting Outcomes in Patients Undergoing Total Pancreatectomy and Islet Autotransplantation

L.F. Lara, S. Meng, J. Buss, A. Rajab, P.A. Hart, D.L. Conwell, S. Black, K. Washburn. The Ohio State University Wexner Medical Center, Columbus, OH.

Background: Total pancreatectomy and islet autotransplantation (TPIAT) is used to treat highly selected patients suffering from chronic pancreatitis. However, prediction of exogenous insulin requirements in patients post-surgery has been difficult. The Secretory Unit of Islet Transplant Objects (SUITO) is an index that has been previously used to predict insulin independence in patients undergoing islet transplantation (where SUITO > 26 is predictive of insulin independence). Homeostasis model assessment of beta cell function (HOMA beta score) has also been used to predict beta cell function.

Methods: Baseline data from six patients who underwent TPIAT was analyzed using both the SUITO and HOMA-beta score parameters. These indices were compared to subsequent islet yields in these patients as well as exogenous insulin requirements at 6 months post-TPIAT.

Results: All patients studied had a SUITO > 26 prior to TPIAT, however, only 2 patients were insulin independent at 6 months post-TPIAT. Comparing SUITO to exogenous insulin requirements showed no correlation between the SUITO measurement and the daily insulin requirements (R = 0.49). In fact, there was shown to be an inverse relationship between the SUITO index and islet yield in these patients (R = -0.35). HOMA-beta scores were similarly ineffective at predicting exogenous insulin requirements in these patients (R = 0.39). However, there did appear to be an inverse correlation between islet dose (IEq/kg) and daily insulin use (R = -0.87).

Conclusion: Based on these results, it appears that neither SUITO nor HOMA-beta score are effective at predicting insulin independence, daily insulin use or islet yield in patients undergoing TPIAT. Islet dose is inversely relate to insulin dependence, and may help predict insulin independence. We understand that, due to our small sample size, these results may be subject to change, and we will reassess with a larger sample size in the future.

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TFEB, the Master Regulator of Autophagy and Lysosomal Biogenesis, Plays Critical Role in Pancreatic Cancer Cell Growth

C. Lawson,1 B. Marchand,2 D. Arsenault,2 M. Groleau,2 L.H. Tai,1 M.J. Boucher.2 1 Department of Anatomy and Cell Biology, University of Sherbrooke, Sherbrooke, Canada; 2Gastroenterology Unit, Department of Medicine, University of Sherbrooke, Sherbrooke, Canada.

Background: It is now well established that autophagy plays a critical role in pancreatic tumorigenesis. The key players underpinning autophagy as well as their contribution to the pancreatic cancer phenotype remain elusive. Recently, we have observed in pancreatic cancer cells and not in non-tumoral pancreatic epithelial cells, an aberrant nuclear localization of the transcription factor TFEB, known as a master regulator of autophagy and lysosomal biogenesis. The aim of this study was to define the contribution of TFEB in pancreatic cancer cell growth.

Methods: We established stable populations of pancreatic cancer cells with reduced expression of TFEB through shRNA strategies. In vitro and in vivo experiments were performed to characterize the growth capacity of control and TFEB-depleted cells.

Results: TFEB depletion did not impair 2D anchorage-dependent growth of pancreatic cancer cells. However, TFEB depletion sensitized cells to apoptotic stimuli such as treatment with DNA damaging agents (etoposide, doxorubicin). When grown in an anchorage-independent manner, shTFEB cells displayed a reduced capacity (by 50%) to form colonies. Furthermore, TFEB depletion in pancreatic cancer cells interfered with primary tumor growth and metastasis when tested in orthotopic xenograft mouse models. As compared to the encapsulated primary tumor formed by control cells, shTFEB tumors displayed much less surrounding desmoplasia as visualized by Trichrome de Masson and Sirius red staining.

Conclusion: Altogether, our findings support a functional role for TFEB in sustaining pancreatic cancer cell growth. Interestingly, our results suggest that TFEB might influence cell growth intrinsically but could also do so by reshaping the microenvironment.

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Tumor Suppressor Long Non-coding RNA AK311005 Coupled With dREAM/Myb-MuvB Complex to Inhibit Pancreatic Ductal Adenocarcinoma Development

C.H. Li,1 C.H. Wong,1 J. H.M. Tong,2 K.F. To,2 Y. Chen.1,3 1 School of Biomedical Sciences, Faculty of Medicine, The Chinese University of Hong Kong, Shatin, Hong Kong, China; 2 Department of Anatomical and Cellular Pathology, Prince of Wales Hospital, The Chinese University of Hong Kong, Shatin, Hong Kong, China; 3Shenzhen Research Institute, The Chinese University of Hong Kong, Shenzhen, China.

Background: LncRNAs contributed to tumorigenic properties by regulating gene expression in cancer cells. However, the role of lncRNAs in pancreatic ductal adenocarcinoma (PDAC) remains largely unexplored. Here, we attempted to identify lncRNAs that were downregulated in PDAC that exhibited tumor suppressor functions.

Methods: LncRNA microarray was conducted to identify lncRNA downregulated in PDAC. SiRNAs were transfected into non-tumor human pancreatic ductal epithelial (HPDE) cells to inhibit the target lncRNA and study the effect to cell proliferation and cell cycle. Gene expression microarray using total RNA extracted from AK311005-inhibited HPDE cells to identify the target genes regulated by AK311005. RNA immunoprecipitation was conducted to identify the protein complex interacted with AK311005.

Results: We identified a novel lncRNA annotated as Ak311005 that was frequently downregulated in PDAC tumor tissues. Loss of AK311005 was critical in PDAC development as downregulation of AK311005 was detected in precursor lesion of PDAC. We further showed that AK311005 played tumor suppressor roles because depletion of AK311005 in HPDE cells significantly promoted cell growth and induced cell cycle progression. We then investigated the role of AK311005 in the development of PDAC by identifying its regulating targets. Depletion of AK311005 in HPDE cells could induce the expression of genes including ANLN, AURKA, DLGAP5 and KIF23. Knockdown of these genes significantly inhibited the PDAC cells growth. Bioinformatics analysis suggested that AK311005 interacted with a gene silencing protein complex dREAM/Myb-MuvB. Analysis of promoter elements of AK311005 targets showed that majority of them harbored dREAM/Myb-MuvB complex binding elements. Knockdown of dREAM/Myb-MuvB complex core members could increase the level of AK311005 targets. By RNA immunoprecipitation, we showed the physical interaction between AK311005 and the core members of dREAM/Myb-MuvB complex.

Conclusion: AK311005 exhibited tumor suppressor functions by silencing its target genes via the recruitment of dREAM/Myb-MuvB complex.

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A Zinc Transporter ZIP4 Promotes Pancreatic Cancer Cell Adhesion, Proliferation, and Spheroid Formation Through Activating Zinc Dependent Transcription Factor (ZEB1)-Integrin-EGFR Signaling Axis

M. Liu, Y. Zhang, 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: Cell adhesion plays a critical role in the development of pancreatic cancer. Integrins have been reported as a mediator of adhesion dependent growth of tumor and spheroid formation. In this study, we aim to investigate whether dysregulated zinc transporter ZIP4 impacts cell adhesion, spheroid formation and proliferation of pancreatic cancer through regulating transcription factor ZEB1, and whether targeting the zinc transport pathway has therapeutic potential strategy of pancreatic cancer.

Methods: Human pancreatic cancer cells AsPC-1, PANC-1, MIA PaCa-2, and KPC mouse model derived cell lines were selected. MTT assay was performed to measure the cell adhesion and proliferation. The morphological characteristics of pancreatic tumor spheroids were studied through spheroid formation assay. Correlations between ZIP4, integrins and downstream targets EGFR were investigated with Western blot. Orthotopic xenograft model was used for in vivo studies.

Results: We found that ZIP4 could promote pancreatic cancer cell adhesion and spheroid formation. Integrin-α3 and integrin-β1 were upregulated by ZIP4. Knocking down integrin-α3 and integrin-β1 diminished cell adhesion, spheroid formation and tumor growth even in the presence of high ZIP4 level cells. We also noticed knocking down integrin-α3 and integrin-β1 inhibited EGFR which are downstream target of integrin. ZEB1 is required for integrin-α3 and integrin-β1 upregulation both in pancreatic cancer and KPC cell lines. And knocking down ZEB1 rescued the promoter activity of integrin-α3 and integrin-β1 enhanced by ZIP4.

Conclusion: It suggested that zinc is the crucial regulator of pancreatic cancer adhesion and ZIP4-ZEB1-integrin-EGFR pathway and may serve as a novel therapeutic strategy of pancreatic cancer.

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Mucin-1 is Required for the Coxsackie Virus B3-induced Inflammation in Pancreatitis

X. Liu,1 D.L. Clemens,2 J.A. Grunkemeyer,3 K. O'Connell,1 J.D. Price,1 M.A. Hollingsworth,1 S.P. Thayer.1 1 Surgical Oncology, University of Nebraska Medical Center, Fred & Pamella Buffett Cancer Center, Omaha, NE; 2 Department of Internal Medicine, University of Nebraska Medical Center, Omaha, NE; 3Midwest Laboratories, Omaha, NE.

Background: Group B Coxsackie viruses (CVB), common and medically important human pathogens, are a frequent cause of acute and chronic inflammatory disease of the human pancreas. Infections with Coxsackie virus cause acute inflammatory pancreatitis in murine models accurately reflecting pathophysiological pancreatitis in humans. We use this virally-induced pancreatitis model to study the role of mucin 1 (MUC-1) during inflammation.

Methods: Wildtype and MUC-1 knockout (MUC-1KO) mice were infected with CVB3 virus and followed for the course of disease. Flow cytometry was used to analyze the amount of infiltrated immune cells in pancreatic tissue. Migration of bone marrow derived macrophages was assessed to compare the functional capability of these cells with/without MUC-1.

Results: We report that MUC-1 deficiency reduced the degree of pancreatic inflammation that resulted from infection with Coxsackievirus B 3 (CVB3). CVB3-infected MUC-1KO mice had significantly reduced infiltration of macrophages into CVB3 infected murine pancreas. Our results support previously reported evidence linking MUC-1 to inflammation through the NF-κB pathway, as we found that MUC-1 signaling through NF-κB increased expression of ICAM-1, a proinflammatory mediator that recruits macrophages. Further investigation revealed that bone marrow derived macrophages (BMDM) from the MUC-1KO mice exhibited defective migration properties, at least in part because of low expression of the chemokine receptor CCR2 and the integrin Very Late Antigen 4 (VLA-4).

Conclusion: The results presented here provide novel insight into the role of MUC-1 in regulating the cellular microenvironment during progression of chronic inflammatory diseases.

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Risk Factors and Nomogram for Pancreatic Stone Formation in Chronic Pancreatitis Over a Long-Term Course: A Cohort of 2153 Patients

Y. Liu,1 L. Hao,1,2 Z.L. Xu,3 T. Wang,1,2 H.L. Guo,1 J. Pan,1 D. Wang,1 Y.W. Bi,1 J.T. Ji,2 L. Xin,1,2 T.T. Du,1 J.H. Lin,1 D. Zhang,1 X.P. Zeng,1 W.B. Zou,1 H. Chen,1,2 T. Xie,4 B.R. Li,5 Z. Liao,1,2 Z.J. Cong,6 L.S. Wang,3 L.H. Hu,1,2 Z.S. Li.1,2 1 Department of Gastroenterology, 2 Digestive Endoscopy Center, Changhai Hospital, The Second Military Medical University, Shanghai, China; 3 Department of Gastroenterology, The Second Clinical Medical College (Shenzhen People’s Hospital), Jinan University, Guangdong, China; 4 Department of Gastroenterology, Zhongda Hospital, Southeast University, Nanjing, China; 5 Department of Gastroenterology, Air Force General Hospital, Beijing, China; 6Department of General Surgery, Renji Hospital, Shanghai Jiaotong University, Shanghai, China.

Background: Pancreatic stones are pathognomonic of chronic pancreatitis (CP). This study aimed to determine the incidence, identify the risk factors, and develop a nomogram for pancreatic stones in CP patients.

Methods: Patients with CP admitted to our center from January 2000 to December 2013 were enrolled. Cumulative rates of pancreatic stones after the onset of CP and after the diagnosis of CP were calculated. Patients were randomly assigned, in a 2:1 ratio, to the training and validation cohort. Based on the training cohort, risk factors were identified through Cox proportional hazards regression model, and nomogram was developed. Internal and external validations were performed based on the training and validation cohort, respectively.

Results: With a total of 2153 CP patients, pancreatic stones were detected in 1626 (75.5%) patients, with a median follow-up of 7.8 years. Age at the onset of CP (HR, 1.022), BMI (HR, 0.965), smoking (HR, 1.169), DM (HR, 0.686), pancreatic pseudocyst (HR, 0.607), biliary stricture (HR, 0.586), severe acute pancreatitis (HR, 0.459), and type of pain were identified risk factors for pancreatic stones development. The nomogram with these 8 factors achieved good accuracy (concordance indexes of 0.781 and 0.559 in the training and validation cohorts, with well-fitted calibration curves.

Conclusion: The nomogram achieved an individualized prediction of pancreatic stones development in CP. It may help the early diagnosis and management of pancreatic stones.

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Risk Factors and Nomogram for Steatorrhea in Idiopathic Chronic Pancreatitis

Y. Liu,1,2 L. Hao,1,2 T. Wang,1,2 D. Zhang,1 H.L. Guo,1 J. Pan,1 D. Wang,1 Y.W. Bi,1 J.T. Ji,2 L. Xin,1,2 T.T. Du,1 J.H. Lin,1 X.P. Zeng,1 W.B. Zou,1 Hui Chen,1,2 T. Xie,4 B.R. Li,5 Z. Liao,1,2 Z.J. Cong,6 L.S. Wang,3 L.H. Hu,1,2 Z.S. Li.1,2 1 Department of Gastroenterology, 2 Digestive Endoscopy Center, Changhai Hospital, The Second Military Medical University, Shanghai, China; 3 Department of Gastroenterology, The Second Clinical Medical College (Shenzhen People’s Hospital), Jinan University, Guangdong, China; 4 Department of Gastroenterology, Zhongda Hospital, Southeast University, Nanjing, China; 5 Department of Gastroenterology, Air Force General Hospital, Beijing, China; 6Department of General Surgery, Renji Hospital, Shanghai Jiaotong University, Shanghai, China.

Background: Steatorrhea is a common complication of idiopathic chronic pancreatitis (ICP). Early diagnosis and prediction of steatorrhea is important to decrease the risk of steatorrhea-associated diseases. This study aimed to identify the risk factors and develop nomograms for steatorrhea in ICP.

Methods: ICP patients admitted to our center from January 2000 to December 2013 were included in our study. Detailed information about demographic data, course of disease, medical history, and follow-up evaluations of patients were documented.

Cumulative rate of steatorrhea was calculated by using the Kaplan–Meier method. Patients were randomly assigned, in a 2:1 ratio, to the training and validation cohort. Based on the training cohort, risk factors for steatorrhea were identified through Cox proportional hazards regression model, and nomograms was developed, respectively. Internal and external validations were performed based on the training and validation cohort, respectively.

Results: The median follow-up duration for the whole cohort was 8.6 years. After fully exclusion, 1633 patients with ICP were finally enrolled. Steatorrhea was found in 20.8% (339/1633) of patients after the onset of CP. Cumulative rates of steatorrhea in ICP were 11.57% (95% confidence interval [CI], 10.00%-13.14%), 12.92% (95% CI,

11.16%-14.68%), and 15.13% (95% CI, 13.17%-17.09%), respectively. Male patients (hazard ratio [HR], 1.862; 95% CI, 1.123-3.086), DM (HR, 2.036; 95% CI, 1.064-3.898), pancreatic duct successful drainage (HR, 0.449; 95% CI, 0.258-0.784) and initial manifestations were identified risk factors for steatorrhea development. Both nomograms achieved good concordance indexes with well-fitted calibration curves.

Conclusion: The nomogram achieved an individualized prediction of steatorrhea development in ICP patients. The high-risk populations were suggested to be monitored frequently and replacement therapy of pancreatic enzyme could be started earlier, which might help to avoid severe complications related to steatorrhea.

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SetD2 is a Barrier to Inflammation- and Kras-driven Acinar-to-Ductal Metaplasia and PanIN Formation

P. Lu,1 N. Niu,1 Y. Sun,2 L. Li,3 J. Xue.1 1 State Key Laboratory of Oncogenes and Related Genes, Stem Cell Research Center, Ren Ji Hospital, School of Medicine, Shanghai Jiao Tong University, Shanghai, China; 2 Department of Biliary-Pancreatic Surgery, Renji Hospital, School of Medicine, Shanghai Jiao Tong University, Shanghai, China; 3School of Biomedical Engineering and Med-X Research Institute, Shanghai Jiao Tong University, Shanghai, China.

Background: The histone H3K36 methyltransferase SetD2 is frequently mutated or deficient in a variety of human tumors, including pancreatic ductal adenocarcinoma (PDAC). Nevertheless, nothing is known about the role of SetD2 in PDAC, especially during early pancreatic carcinogenesis.

Method: TCGA(PAAD) public database and PDAC tissue array with SetD2 staining was used to investigate the clinical relevance of SetD2 mutant / expression in PDAC. Furthermore, to define the role of SetD2 in PDAC, we generated conditional SetD2 knockout mice (PDXcreSetD2flox/flox) with or without KrasG12D mutant. To determine the role of SetD2 in pancreas, animal models and in vitro ADM assay were used. RNA-seq and H3K36me3 Chip-seq was performed to uncover the mechanism.

Results: SetD2 mutant and low expression was correlated with poor prognosis in patients with PDAC. Next, we found SetD2 was critical for pancreatic acinar cells maintenance under homeostasis and regeneration/recovery after pancreas injury (like pancreatitis). In addition, SetD2 ablation drastically accelerated the formation of pancreatic intraepithelial neoplasia (PanIN) and PDAC induced by KrasG12D mutant. Moreover, SetD2 deficiency promoted acinar-to-ductal metaplasia (ADM) using in vitro assay. Furthermore, by using RNA-Seq and H3K36me3 Chip-seq, we identified genes involved in MYC signaling was prominently upregulated in SetD2 deficient pancreas.

Conclusion: These results demonstrate that SetD2 deficiency could promote ADM and PDAC progression under inflammation- and Kras mutant, which provides a better understanding and potential target strategy for PDAC patients with SetD2 mutant/ low SetD2 expression.

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New Observations on the Utility of CA 19-9 as a Biomarker in Lewis Negative Patients With Pancreatic Cancer

G. Luo,1 X. Yu,1 A.L. Warshaw.2 1 Department of Pancreatic Surgery, Shanghai Cancer Center, Fudan University, Shanghai, China; 2Department of Surgery and the Warshaw Institute for Pancreatic Cancer Research, Massachusetts General Hospital, Harvard Medical School, Boston, MA.

Background: CA 19-9 is the best-validated biomarker for pancreatic cancer. The NCCN guideline asserts that “CA 19-9 will be undetectable in Lewis antigen-negative individuals”. However, reports of CA 19-9 secretion and its significance in Lewis (-) patients with pancreatic cancer have been inconsistent. This study was to examine serum CA 19-9 levels in patients with pancreatic cancer according to Lewis status.

Methods: Patients with pancreatic cancer (1482 cases) were retrieved from a prospectively maintained database. Patients with benign pancreatic disease (210 cases) and normal subjects (315 cases) were used as controls. Lewis genotypes were examined by fucosyltransferase 3 (FUT3) sequencing.

Results: In patients with pancreatic cancer, 8.4% of subjects were Lewis (-), but only 41.9% of Lewis (-) subjects had CA 19-9 values ≤ 2 U/mL. CA 19-9 was even elevated (> 37 U/mL) in 27.4% of Lewis (-) patients. The area under the ROC curve for CA 19-9 as a diagnostic biomarker was 0.842 in Lewis (-) patients with pancreatic cancer, which is closing to that of CA 19-9 applied in all of patients with pancreatic cancer (0.898). Lewis (-) status was an independent prognostic factor for shorter survival in a multivariable analysis (HR, 1.30, 95% CI, 1.03-1.64; P = 0.028).

Conclusion: Not all Lewis (-) patients with pancreatic cancer are non-secretors of CA 19-9. Contrary to general understanding, CA 19-9 can retain its utility as a biomarker in these patients in spite of Lewis (-) genotype.

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Visceral Artery Pseudoaneurysm in Necrotizing Pancreatitis: Incidence and Outcomes at a High-Volume Center

T.K. Maatman,1 M.A. Heimberger,2 K.A. Lewellen,2 A.M. Roch,1 C.L. Colgate,3 E.P. Ceppa,1 M.G. House,1 A. Nakeeb,1 C.M. Schmidt,1 N.J. Zyromski.1 1 Department of Surgery, Indiana University School of Medicine, Indianapolis, IN; 2 Indiana University School of Medicine, Indianapolis, IN; 3Center for Outcomes Research in Surgery (CORES), Indiana University School of Medicine, Indianapolis, IN.

Background: Visceral artery pseudoaneurysm (PSA) occurs in necrotizing pancreatitis (NP); however, little is known about its natural history. This study aimed to evaluate the incidence, presentation, management, and outcome of PSA at a high-volume pancreatitis center.

Methods: All NP patients treated between 2005 and 2017 were prospectively catalogued into the institution’s NP database. Those diagnosed with visceral artery pseudoaneurysm were reviewed for incidence, time from NP onset to PSA development, presenting sign, PSA location, management, and PSA-specific mortality.

Results: A total of 647 NP patients were treated between 2005 and 2017. Visceral artery pseudoaneurysm was diagnosed in 25 patients (3.9%) (total of 28 pseudoaneurysms) The most common artery involved was splenic (36%) followed by gastroduodenal (25%), gastroepiploic (11%) and pancreaticoduodenal (11%). The most common presenting symptom was bloody drain output (32%) followed by incidental CT scan finding (24%), abdominal pain (20%), and GI bleed (8%). Median time from onset of NP to diagnosis of PSA was 68 days (range 1-957). All patients diagnosed with PSA underwent treatment, regardless of presentation. Patients were sucessfully treated with percutaneous angioembolization in 23/25 cases (92%). Two required operative management (8%). PSA-specific mortality in the setting of NP was 16% (4/25).

Conclusion: Visceral artery pseudoaneurysm occurs in 4% of NP patients. Percutaneous angioembolization effectively treats most, however mortality of PSA is high (16%). Therefore, a high degree of clinical suspicion remains critical for early diagnosis of this potentially fatal problem.

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PMCA Pump Dysfunction Leads to Ca2+ Overload and Pancreatic Ductal Epithelial Cell Damage in Cystic Fibrosis

T. Madacsy,1,2 A. Varga,1,2 A. Schmidt,1 J. Fanczal,1,2 P. Pallagi,3 Z. Rakonczay,4 P. Hegyi,5,6 Z. Razga,7 A. Kleger,8 I. Nemeth,9 M. Gray,10 J. Maleth.1,2 1 First Department of Medicine, University of Szeged, Szeged, Hungary; 2 MTA SZTE Momentum Epithel Cell Signalling and Secretion Research Group, Szeged, Hungary; 3 Department of Pharmacology, University of Szeged, Szeged, Hungary; 4 Department of Pathophysiology, University of Szeged, Szeged, Hungary; 5 Institute for Translational Medicine and First Department of Medicine, University of Pécs, Pécs, Hungary; 6 MTA-SZTE Translational Gastroenterology Research Group, Szeged, Hungary; 7 Department of Pathology, University of Szeged, Szeged, Hungary; 8 Department of Internal Medicine I, University Medical Center Ulm, Ulm, Germany; 9 Department of Dermatology, University of Szeged, Szeged, Hungary; 10Newcastle University, Institute for Cell and Molecular Biosciences, Newcastle, United Kingdom. [10 cited insitutions but no author with 10 superscript]

Introduction: The cystic fibrosis transmembrane conductance regulator (CFTR) has a major role in pancreatic ductal secretion and its genetic defects damage the pancreas. It is known that intracellular Ca2+ homeostasis is disturbed in bronchial epithelial cells in cystic fibrosis (CF), but the connection of CFTR and the intracellular Ca2+ signaling has never been suggested in pancreatic damage in CF before.

Aims: Our aim was to characterize the Ca2+ homeostasis of CFTR-deficient PDEC.

Methods: Wild type (WT) and CFTR knockout (KO) mouse pancreatic ductal (PDEC) and acinar cells (PAC), human CF pancreatic cell line (CFPAC-1) and human pancreatic organoids generated from induced pluripotent stem cells (IPSC) of controls and CF patients were used in the study. Intracellular Ca2+ levels, mitochondrial membrane potential (ΔΨm) and mitochondrial morphology was assessed using fluorescent probes and transmission electron microscopy, respectively. Immunofluorescent staining and quantitative PCR measurements were performed to detect changes of protein expressions. Protein ligation assay (PLA) was performed to detect contact between proteins.

Results: The plateau phase of the agonist-induced Ca2+ signal was significantly elevated in CFTR KO PDEC caused by decreased function of the plasma membrane Ca2+ pump (PMCA). Functional inhibition of CFTR had no effect on the PMCA activity. Whereas native CFPAC-1 cells, CF human organoids and murine PDEC treated with siCFTR showed similarly impaired PMCA activity. On the other hand, different strategies to restore the CFTR expression, such as Sendai virus mediated gene delivery in CFPAC-1, or VX-809 treatment of CF organoids completely restored PMCA function. As a downstream consequence, sustained [Ca2+]i elevation decreased ΔΨm and released cytochrome c in CFTR KO PDEC without significant alteration of mitochondrial morphology. Immunostaining revealed the colocalisation of PMCA4 and CFTR on the apical membrane of polarized, primary PDEC and PLA confirmed the intimate proximity of the proteins. Calmodulin, a possible link among CFTR and other proteins, showed plasma membrane localization in WT PDEC, which was shifted to the cytosol of CFTR KO cells.

Conclusion: Impaired expression of the CFTR leads to disturbed Ca2+ homeostasis and mitochondrial damage in primary PDEC due to the decreased activity of PMCA. These changes can contribute to the pancreatic damage seen in cystic fibrosis.

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Disordering of Endo-lysosomal System in Pancreatitis

O.A. Mareninova, I. Yakubov, I. Gukovsky, A.S. Gukovskaya. VA Greater Los Angeles Healthcare System and University of California Los Angeles, Los Angeles, CA.

Background: Recent studies in genetic and experimental models indicate a key pathogenic role of impaired autophagy in pancreatitis; however, the underlying mechanisms remain poorly understood. Here we hypothesized that endo-lysosomal system, which mediates post-Golgi protein trafficking, endocytosis and autophagy, is disordered in acute pancreatitis (AP). Methods: We analyzed the effects of pancreatitis induced in rats by cerulein (CER-AP) or L- arginine (Arg-AP) on the endo-lysosomal system and autophagy by measuring changes in the lysosomal membrane proteins LAMP1 and LAMP2; Rab proteins, key mediators of protein trafficking; and LC3-II, a marker of autophagic vacuoles. Pancreatic levels of these proteins were measured by immunoblot; their cellular localization, by immunofluorescence; and subcellular distribution, by OptiPrep density gradient fractionation.

Results: Both CER-AP and Arg-AP caused marked reduction in pancreatic levels of LAMPs and membrane-bound (active) Rabs. In control rats, the density of organelles increased in the order: early endosomes (Rab4, Rab5, Rab11) < lysosomes (LAMP1, LAMP2) < late endosomes (Rab7, Rab9) < zymogen granules (trypsinogen). In pancreatitis the lysosomes became lighter, whereas both early and late endosomes shifted to higher-density fractions, the effect being especially pronounced for early endosomes. The results indicate defective early to late endosomes’ maturation and reduced formation of lysosomes in AP models. Correspondingly, maturation/processing of cathepsins is defective in pancreatitis, as demonstrated by accumulation of the intermediate CatB form (partially processed in endosomes) and a decrease in CatB mature form (fully processed in the lysosome). Endosomal sorting was disordered in ex-vivo pancreatitis model, evidenced by transferrin accumulation during its endocytosis in acinar cells. Defective endosomal maturation was associated with impaired autophagy manifested by increased number of autolysosomes. In AP models, trypsin activity co-fractionated with LC3-II and LAMP1, but not with markers of late endosomes.

Conclusion: The results implicate defective endosome maturation in disordered autophagy and endocytosis in pancreatitis.

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Role of NRF2 in the Inflammatory Response of Acute Pancreatitis

O.A. Mareninova,1 S.R. Gretler,1 D.L. Dillon,1 M. Pimienta,2 S.J. Pandol,2 I. Gukovsky,1 A.S. Gukovskaya.1 1 VA Greater Los Angeles Healthcare System and University of California Los Angeles, Los Angeles, CA; 2Cedars-Sinai Medical Center, Los Angeles, CA.

Background: Inflammation is a major determinant of the severity of acute pancreatitis (AP); however, there are no treatments currently available to reduce persistent/uncontrolled inflammation and associated morbidity of pancreatitis. We hypothesized that the interplay between key transcription factor pathways, the proinflammatory NF-κB and the antioxidant NRF2, drives the inflammatory response of acute pancreatitis (AP).

Methods: Wild type (C57BL/6), NRF2 null, and NF-κB deficient (p65+/-;p50-/-) mice were subjected to models of nonalcoholic and alcoholic AP; we also used corresponding ex-vivo models in which acinar cells isolated from these mice were treated with supramaximal CCK or ethanol+palmitoleic acid. NRF2 activator sulforaphane (50 μg/kg i.p.) was given to some mice. We measured inflammatory cell infiltration, NF-κB activation, NRF2 protein level and mRNA expression of its target genes.

Results: Development of AP was associated with NF-κB activation and concomitant inhibition of NRF2, manifest by decreases in NRF2 protein level and mRNA expression of its target NAD(P)H dehydrogenase quinone 1. We also found similar imbalance between excessive NF-κB activation and decreased nuclear level of NRF2 in human disease, as pancreatic tissues from patients with pancreatitis showed a dramatic increase in nuclear p65 and pronounced decrease in NRF2 nuclear localization. NF-κB and NRF2 had opposite effects on inflammation in models of AP: it was suppressed by blockade of NF-κB in p65+/-;p50-/- mice and facilitated by NRF2 genetic ablation. Sulforaphane treatment restored the level and activity of NRF2 and markedly decreased ROS level, dampening NF-κB activation and the inflammatory response in AP.

Conclusion: The results indicate that excessive NF-κB activation suppresses NRF2 activity in pancreas, perpetuating oxidative stress and uncontrolled inflammation in AP. Pharmacologic approaches to stimulate NRF2 (eg, with sulforaphane) could be promising to reduce inflammation and the severity of AP.

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Stromal Fibrosis in Tumor Periphery of Pancreatic Ductal Adenocarcinoma Following Neoadjuvant Chemotherapy Predicts Patient Outcome

Y. Matsuda,1 M. Hiratsuka,2 S. Kawakatsu,3 Y. Inoue,3 K. Matsueda,2 A. Saiura,3 T. Arai,1 Y. Takazawa.4 1 Department of Pathology, Tokyo Metropolitan Geriatric Hospital, Tokyo, Japan; Departments of 2Radiology, 3Digestive and HBP Surgery, and 4Pathology, Cancer Institute Hospital, Japanese Foundation for Cancer Research, Tokyo, Japan.

Background: An increasing number of patients with locally advanced/borderline resectable pancreatic cancer (LA/BR-PC) have been treated with neoadjuvant therapy (NAT). Pathological assessments of the treatment effect is important to predict patient outcomes after pancreatectomy; however, appropriate pathological assessment methods have not been established.

Methods: The study included 82 LA/BR-PC patients, 40 of whom were treated with NAT using 4 courses of gemcitabine plus nab-paclitaxel (GnP) followed by pancreatectomy (GnP group). The remaining 42 patients were treated with upfront pancreatectomy (UP) followed by adjuvant chemotherapy (UP group). We reviewed clinicopathological data of these patients to assess differences between the GnP and UP groups and to evaluate the prognostic impact of residual tumors after GnP.

Results: Compared to the UP group, the GnP group showed lower serum CA 19-9 levels, decreased incidences of lymph node metastasis and lymphovascular invasion, and lower tumor-stroma ratio. Higher incidences of stromal fibrosis with sclerotic changes in the tumor periphery was observed in the GnP group than in the UP group. There were no differences in necrosis and inflammation between the 2 groups. A higher R0 rate tended to be observed in the GnP group (85%) than in the UP group (65%), but this was not statistically significant. As for prognosis, stromal fibrosis in the tumor periphery was correlated to overall survival in the GnP group. However, overall survival did not show any correlation with other clinicopathological factors, including serum CA 19-9 level, CEA, tumor volume, tumor reduction ratio (determined by CT), tumor regression grade (criteria of Evans’ grading system or those of the College of American Pathologists).

Conclusion: The present study revealed that GnP induced stromal fibrosis with sclerosis in tumor periphery, which could predict patients’ outcome. Larger cohort studies to further evaluate the prognostic value of stromal fibrosis, possibly with imaging and biomarkers, are warranted.

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Effect of Recombinant Human Thrombomodulin on Rat Experimental Severe Acute Pancreatitis

M. Matsumoto, K. Kamei, K. Kawaguchi, Y. Takeyama. Division of Hepato-Biliary-Pancreatic Surgery, Department of Surgery, Kindai University Faculty of Medicine, Higashiosaka, Japan.

Background: Early death in severe acute pancreatitis (SAP) is often caused by pancreatic necrosis and multiple organ failure due to microcirculation disorder. The aim of this study is to prove that recombinant human soluble thrombomodulin (rTM) contributes to the prevention of the development of pancreatitis including pancreatic necrosis and remote organ failure.

Methods: Male Wister rats, weighting 240-300 g were used. Cerulein (50 μg/kg) was administered intraperitoneally four times every one hour, and lipopolysaccharide (LPS, 20 mg/kg) was administered intraperitoneally 3 hours after the last injection of cerulein. rTM (3 mg/kg) was injected into the jugular vein 1 hour after administration of LPS (rTM group). As a control, an equal volume of physiological saline was injected instead of rTM (control group). All rats were observed for 24 hours after starting the experiment, and the survival rate was evaluated. All survived rats were sacrificed under general anesthesia, and the blood sample and liver and pancreas were excised. Serum amylase, AST, ALT and HMGB-1 were measured, and the liver and pancreas were examined histologically.

Results: In control group, serum amylase, AST and ALT were 7134 IU/L, 6248 IU/L, and 4074 IU/L, in average respectively. In rTM group, they decreased significantly to 1360 IU/L (P = 0.0008), 145 IU/L (P = 0.0008), and 65 IU/L (P = 0.0012). The overall survival rate in the rTM group was significantly higher than in control group (100% v.s. 40%, P = 0.0017).

There was no death in rTM group within 24 hours. Moreover, serum HMGB1 in rTM group was lower than in control group. Histological analysis revealed that liver injury and pancreatic necrosis were less severe in rTM group than in control group.

Conclusion: Early administration of rTM prevents the development of organ dysfunction and pancreatic necrosis by the maintenance of microcirculation.

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Neoadjuvant Treatments Increase Risk of Postoperative Thrombosis in PDAC

N. Mattila,1,2 R. Lassila,2,3 H. Mustonen,1 C. Haglund,1,4 H. Seppänen.1 1 University of Helsinki and Helsinki University Hospital, Department of Surgery, Helsinki, Finland; 2 Coagulation Disorders Unit, Comprehensive Cancer Center, Department of Hematology, University of Helsinki and Helsinki University Hospital, Helsinki, Finland; 3 HUSLAB Laboratory Services, Clinical Chemistry, Helsinki, Finland; 4Research Programs Unit, Translational Cancer Biology, University of Helsinki, Helsinki, Finland.

Background: Pancreatic ductal adenocarcinoma (PDAC) patients are at increased risk for thrombotic events, especially venous thrombotic events (VTE). VTE in cancer patients increase mortality 4-7-fold. Oncologic treatments, such as surgery and chemotherapy, increase risk of thrombosis, however their association with later thrombotic events needs further research. Patients operated for PDAC in Helsinki, Finland are given one-month prophylactic postoperative anticoagulant treatments (AT) to reduce the risk of immediate thrombotic complications. Patients who would benefit from longer AT need to be identified. We aimed to analyze the association of preoperative clinical and laboratory variables on risk of overall postoperative thrombosis in PDAC.

Methods: Patients operated for PDAC during 2010-2015 at Helsinki University Hospital with preoperative coagulation variables were included (n = 122). Coagulation biomarkers and CA 19-9 were gathered 1-3 days preoperatively. Neoadjuvant treatments (NT), postoperative chemotherapy, and postoperative venous and arterial thrombosis events were gathered from patient records. Patients were followed at least 2.5 years or until death. The outcome variable studied was thrombosis. Differences in outcome were calculated according to the Kaplan-Meier method and the Cox proportional-hazards model was used to analyze risk of thrombosis.

Results: Out of 122 patients, 34 had postoperative venous or arterial thrombotic events. Median time of thrombosis from operation was 258 days. At the end of follow-up, 32 patients (26%) were alive. NT were given to 42 patients. PDAC patients who received NT had an increased risk of postoperative thrombosis when compared to patients without NT (HR, 2.179; P = 0.025). PDAC stage, resection margins, perineural invasion, perivascular invasion, BMI, age at operation, preoperative biomarkers or postoperative chemotherapy was not associated with postoperative thrombotic events in univariate analysis.

Conclusion: NT increase risk of postoperative thrombosis in PDAC patients. As thrombosis appears to worsen survival, further studies are needed to establish whether patients receiving NT would benefit from longer postoperative AT.

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Thrombin Generation is Associated With CA 19-9 In Vitro and in Pancreatic Ductal Adenocarcinoma

N. Mattila,1,2 B. Przybyla,2 H. Seppänen,1 C. Haglund,1,3 R. Lassila.2,4 1 Department of Surgery, University of Helsinki and Helsinki University Hospital, Helsinki, Finland; 2 Coagulation Disorders Unit, Comprehensive Cancer Center, Department of Hematology, University of Helsinki and Helsinki University Hospital, Helsinki, Finland; 3 Research Programs Unit, Translational Cancer Biology, University of Helsinki, Helsinki, Finland; 4HUSLAB Laboratory Services, Clinical Chemistry, Helsinki, Finland.

Background: Pancreatic ductal adenocarcinoma (PDAC), a highly thrombogenic cancer, increases coagulation activity, even without venous thrombotic events (VTE). Reasons behind this thrombogenicity need further exploration. Combining tumor marker CA 19-9 with coagulation variables increases sensitivity and specificity for PDAC diagnosis and prognosis. Our aim was to assess whether CA 19-9 influences coagulation activity and thrombin generation (TG), both in vitro, using variety of CA 19-9 antigens added to healthy plasma, and from PDAC patient samples.

Methods: The dose response of three different commercial CA 19-9 antigens (MyBioSource), was spiked to Standard Human Plasma (SHP, Siemens) at categories of 1000-2000, 100-300, <30 U/mL to assess TG in Calibrated Automated Thrombogram (CAT, Labscan, Thermo Fisher). In addition, we analyzed citrated plasma samples of 33 histopathologically verified treatment-naïve PDAC patients with CA 19-9 categories of <2, 2-30, 90-500 and >700 U/mL against SHP. TG was triggered with 1 pM tissue factor (PPP Low reagent). CAT parameters included lagtime, time to peak (TTP), peak thrombin generation (peak), and endogenous thrombin potential (ETP).

Results: CA 19-9 at 1000-2000 U/mL added to SHP dose-dependently increased TG (P < 0.05), and the highest CA 19-9 (1000-2000 U/mL) increased also TTP and peak versus the lower CA 19-9 (100- 300 U/mL) level (P < 0.05). In accordance, the patients having CA 19-9 >700 U/mL increased ETP, peak and TTP (vs. SHP P < 0.05). ETP and peak were also higher in patients having CA 19-9 at > 700 U/mL versus 90-500 U/mL and <2 U/mL (P < 0.05).

Conclusion: High concentrations (>700 U/mL) of CA 19-9 are associated with increased TG both when spiked into healthy plasma and in PDAC patient plasma. These findings suggest a link between CA 19-9 and increased coagulation activity in PDAC, a hypothesis to be further verified.

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Utility of Arginine Stimulation Testing in the Pre-Operative Assessment of Children Undergoing TPIAT

K.R. McEachron,1 M.E. Skube,1 G.J. Beilman,1 S. Chinnakotla,1 S.J. Schwarzenberg,2 M.D. Bellin.2 Departments of 1 Surgery and 2Pediatrics, University of Minnesota Medical School, Minneapolis, MN.

Background: Islet yield after pancreatic processing is the best predictor of graft function after total pancreatectomy with islet autotransplantation (TPIAT), and the ability to predict islet yield can improve patient selection and preoperative counseling. The first phase insulin and C-peptide responses from arginine stimulation testing (AST) correlate with islet mass in animal models and post-islet transplant studies in humans.

Methods: A retrospective chart review was conducted of pediatric patients who underwent AST prior to TPIAT from 2015 to 2018. Results of preoperative AST, islet yield, and postoperative metabolic outcomes were compiled. Univariate linear regression and logistic regression analysis were used to test the association between AST results and the islet yield, as well as the postoperative graft function (represented by insulin use (Yes/No) and hemoglobin A1C).

Results: Twenty-six patients (81% female, mean age 11.3 ± 4 years) underwent AST prior to TPIAT. The most common indication for TPIAT was hereditary pancreatitis. The C-peptide response to AST (ACRarg) correlated positively with islet yield (r = 0.648, P = 0.0005), while the insulin response (AIRarg) did not (r = 0.262, P = 0.251). C-peptide and insulin responses to AST did not correlate significantly with insulin use or HbA1c at 6 months (n = 22 patients) or 12 months (n = 13).

Conclusion: The acute C-peptide response during AST prior to TPIAT correlated positively with islet yield. Surprisingly, the insulin response during AST did not correlate with islet yield, possibly reflecting technical challenges with insulin assays such as falsely low levels with hemolysis. More investigation is needed to determine the overall clinical significance of AST for TPIAT patients, as baseline tests did not predict graft function in this small cohort.

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Microbial Dysbiosis and Polyamine Metabolism as Predictive Markers for Early Detection of Pancreatic Cancer

R. Mendez,1 K. Kesh,1 N. Arora,2 L. Di Martino,1,4 F. McAllister,5 N. Merchant,1,6 S. Banerjee,1,6 S. Banerjee.1,6 1 Department of Surgery, University of Miami Miller School of Medicine, Miami, FL; Departments of 2 Surgery and 3 Pharmacology, University of Minnesota, MN; 4 Université Grenoble Alpes, Isère, France; 5 Department of Clinical Cancer Prevention, The University of Texas MD Anderson Cancer Center, Houston, TX; 6Sylvester Comprehensive Cancer Center, Miami, FL.

Background: The lack of tools for early detection of pancreatic ductal adenocarcinoma (PDAC) is directly correlated to the abysmal survival rate in patients. In addition to several potential detection tools under active investigation, we tested the gut microbiome and its metabolic complement as one of the earliest detection tools that could be useful in patients at high-risk for PDAC.

Methods: A combination of 16s pyrosequencing and whole-genome sequencing of gut microbiota was used in a spontaneous genetically engineered PDAC murine model (KRASG12DTP53R172HPdxCre or KPC). Metabolic reconstruction of microbiome was done using the HUmanN2 pipeline. Serum polyamine levels were measured from murine and patient samples using standard methods.

Results: Results showed a progressive Proteobacterial and Firmicutes dominance in gut microbiota in early stages of PDAC development. Upon in silico reconstruction of active metabolic pathways within the altered microbial flora, polyamine and nucleotide biosynthetic pathways were found to be significantly elevated. These metabolic products are known to be actively assimilated by the host and eventually utilized by rapidly dividing cells for proliferation validating their importance in the context of tumorigenesis. In KPC mice, as well as PDAC patients, we show significantly elevated serum polyamine concentration. Therefore, at the early stages of tumorigenesis, the gut microbial composition changes in a way to release metabolites that foster host tumorigenesis, thereby fulfilling the ‘vicious cycle hypothesis’ of the role of the microbiome in health and disease states.

Conclusions: Our results provide a potential, precise, non-invasive tool for early detection of PDAC, which will result in improved outcomes.

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SHP2 is Required for Panin Development and PDAC Progression

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.

Background: Pancreatic ductal adenocarcinoma remains one of the deadliest cancers due in part to the inability to effectively suppress oncogenic KRAS. The Src homology region 2 containing protein tyrosine phosphatase (SHP-2) acts as a critical mediator of KRAS activation in response to cytokines, growth factors, and cell-cell interactions. While SHP-2 is ubiquitously expressed in most tissues, it production and activation is upregulated in pancreatic cancer samples and cell lines compared to normal levels. The objective of this study was to determine whether inhibition or ablation of SHP-2 could prevent KRAS activity and prevent PDAC development and progression.

Methods: SHP-2 levels were assessed in PDAC tissue using immunohistochemistry as well as Western blot analysis. For in vitro functional assays, PDAC cell lines were stimulated with pro- tumorigenic factors (Leptin, IL-6, and/or EGF) and monitored for proliferation, migration, and pathway activation alone or in combination with SHP-2 chemical inhibitors. To understand whether SHP-2 was critical for PDAC development and progression, we generated pancreas specific SHP2[INCREMENT]-KC (SHP2fl/fl; KrasG12D+/-; Pdx1cre) mice.

Results: Activated SHP-2 was increased in human and mouse PDAC tumor tissue compared to adjacent normal pancreas tissue. Inhibition of SHP-2 blocked cytokine-induced activation of ERK in PDAC cell lines in a time dependent manner. Additionally, inhibition of SHP-2 blocked cytokine-induced PDAC cell proliferation via EdU analysis and migration via a wound healing assay. In vivo, pancreas specific ablation of SHP-2 prevented the formation of pancreatic intraepithelial neoplasia (PanIN) in SHP2[INCREMENT]-KC mice.

Conclusion: Our studies demonstrate that SHP-2 is essential for PanIN development and that is inhibition suppresses multiple aspects of PDAC. Therapeutic strategies targeting SHP-2 have a high potential to suppress PDAC progression by preventing tumor intrinsic KRAS activity.

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The SNARE Priming Factor Calcium-Dependent Activator Protein for Secretion 2 (CAPS2) Regulates Trypsinogen Trafficking and Exocrine Pancreas Homeostasis

S.W. Messenger, S.E. Maciuba, T.F. Martin. Department of Biochemistry, University of Wisconsin, Madison, WI.

Background: Trypsinogen is important in pathogenesis of pancreatitis however, the exact function remains unclear. A better understanding of the molecular regulators of trypsinogen trafficking will lead to better treatments of the disease. Members of the CAPS family of proteins assemble SNAREs into fusion competent complexes thereby accelerating vesicle fusion. CAPS2 is an endosomal protein expressed in the acinar cells.

Methods: The physiology and pathophysiology of CAPS2 knockout (-/-) pancreas and acini were assessed.

Results: CAPS2-/- mice show a 25% reduction in survival to weaning with those surviving having a 20% decrease in weight. 10-14 week old CAPS2-/- acini have 50% reduced stimulated amylase secretion corresponding with a fourfold increase in intracellular amylase levels. In contrast, CAPS2-/- acini show a 50% reduction in trypsinogen levels with significantly reduced trypsinogen ZGs imaged by confocal microscopy. CAPS2 functions to regulate vesicle acidification as assessed by Lysotracker intensity and CAPS2-/- acini fail to induce intracellular trypsinogen activation during CCK-hyperstimulation even when controlling for reduced levels of trypsinogen. Necrosis as measured by LDH release is significantly reduced 5-fold in the CAPS2-/- acini during CCK-hyperstimulation. These studies show a reduction in features of acute acinar pancreatitis. In contrast, > 6 month old CAPS2-/- pancreas shows loss of acinar cells marked by reduction in amylase staining with the remaining acinar cells showing a loss of cellular polarity when the apically localized tight junction protein, zonula occludin 1, is imaged. An accumulation of fibrotic tissue (fibronectin and alpha-smooth muscle actin) and inflammatory cell infiltration (f4/80, macrophage and CD3, T-cell) is also present. These studies indicate that CAPS2 plays a role in exocrine pancreas homeostasis.

Conclusion: Together, we hypothesize that CAPS2 regulates vesicle acidification that is critical for both trypsinogen activation and acinar cell homeostasis. Future studies will identify the regulatory pathways controlled by CAPS2.

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Lrig1 Positive Epithelial Cells Within PDGs Identify a Unique Mesenchymal Progenitor Compartment

P. Mondal, V. Kansal, D. Maroni, K.L. McAndrews, J.D. Price, S. Thayer. Division of Surgical Oncology, Department of Surgery, Fred and Pamela Buffet Cancer Center, University of Nebraska Medical Center, Omaha, NE.

Background: Leucine-rich repeats and immunoglobulin-like domains containing 1 (Lrig1) is known to mark stem cells in the mammalian epidermis, intestine, colon, and stomach. Upon injury, these stem cells proliferate and repair damages in these tissues. Our laboratory identified Pancreatic Duct Glands (PDGs), a novel stem cell compartment in the pancreas, which repairs the epithelium of the pancreatic main duct upon inflammatory injury. Lrig1 is uniquely expressed in PDGs within the pancreas. Here, we investigated the fate of Lrig1-expressing cells from PDGs in the regenerative response of the pancreatic main duct epithelium after inflammatory injury.

Methods: We used Lrig1-Cre/ERT2 mouse to tag and follow the fate of Lrig1-expressing cells in PDGs after injury and enumerate Lrig1-lineage GFP positive cells in various anatomical regions. Laser capture microscopy-isolated GFP cells were further characterized by transcriptome analysis. Lrig1 knock-out mice were used to identify changes in regenerative response and expression in PDGs due to loss of Lrig1. In vitro studies were used to determine Lrig1 mediated signaling changes under inflammatory condition.

Results: In response to inflammatory injury there was an expansion of GFP positive cells within the PDG compartment but very few GFP positive cells were identified in the main duct over the two weeks course of repair. Instead, the mesenchymal cells around PDGs and main duct epithelium showed an increasing number of GFP positive cells after the injury. These GFP positive mesenchymal cells expressed vimentin, fibronectin and vitronectin. Lrig1-deficient PDGs expressed higher amounts of epithelial-to-mesenchymal transition (EMT)-related genes and transcription factors. Overexpression of Lrig1 in HPDE cells also confirmed its suppression of EMT, observed via a decrease in vimentin and the other EMT regulators.

Conclusion: Lrig1 marks a specific population of stem cells in PDGs that can differentiate into a mesenchymal phenotype after inflammatory injury.

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Tumor Expression of Insulin-like Growth Factor-1 Receptor is Associated With Disease Recurrence and Mortality in Resected Pancreatic Ductal Adenocarcinoma

V. Morales-Oyarvide,1 C. Du,2,3 A. da Silva,2,4 A. Dias Costa,2 M.M. Kozak,5 R.F. Dunne,6 D.A. Rubinson,1 K. Perez,1 Y. Masugi,7 T. Hamada,2 M.W. Welch,1 L.K. Brais,1 C.L. Zellers,1 C. Yuan,1 A. Babic,1 M. Ducar,4,8 A.R. Thorner,1,8 M. Meyerson,1,4,8,9 A. Aguirre,1 M.H. Kulke,10 K. Ng,1 T.E. Clancy,11 J.J. Findeis-Hosey,12 D.T. Chang,5 J. L. Hornick,4 C.S. Fuchs,13 S. Ogino,2,4,9,14,15 A.C. Koong,16 A.F. Heze,l6 J.A. Nowak,2,4 B.M. Wolpin.1 1 Department of Medical Oncology, Dana-Farber Cancer Institute and Harvard Medical School, Boston, MA; 2 Department of Oncologic Pathology, Dana-Farber Cancer Institute, Boston, MA; 3 Department of Medical Oncology, National Cancer Center/Cancer Hospital, Chinese Academy of Medical Sciences and Peking Union Medical College, Beijing, China; 4 Department of Pathology, Brigham and Women’s Hospital and Harvard Medical School, Boston, MA; 5 Department of Radiation Oncology, Stanford Cancer Institute, Stanford, CA; 6 Department of Medicine, Division of Hematology and Oncology, Wilmot Cancer Institute, University of Rochester Medical Center, Rochester, NY; 7 Department of Pathology, Keio University School of Medicine, Tokyo, Japan; 8 Center for Cancer Genome Discovery, Dana-Farber Cancer Institute, Boston, MA; 9 Broad Institute of MIT and Harvard, Cambridge, MA; 10 Section of Hematology/Oncology, Boston University and Boston Medical Center, Boston, MA; 11 Department of Surgery, Brigham and Women’s Hospital and Harvard Medical School, Boston, MA; 12 Department of Pathology, University of Rochester Medical Center, Rochester, NY; 13 Yale Cancer Center, Smilow Cancer Hospital and Yale School of Medicine, New Haven, CT; 14 Department of Epidemiology, Harvard T.H. Chan School of Public Health, Boston, MA; 15 Program in MPE Molecular Pathological Epidemiology, Brigham and Women’s Hospital, Boston, MA; 16Department of Radiation Oncology, The University of Texas MD Anderson Cancer Center, Houston, TX.

Background: Insulin-like growth factor-1 receptor (IGF1R) signaling is important for pancreatic ductal adenocarcinoma (PDAC) development and growth, but little is known regarding IGF1R expression and patient characteristics and outcomes in resected PDAC.

Methods: In 365 patients with resected PDAC from three U.S. centers, we evaluated IGF1R tumor protein expression using immunohistochemistry on whole-slide sections and interrogated IGF1R genomic status using next-generation sequencing. IGF1R expression was measured by H- scores incorporating staining intensity and proportion of positive tumor cells, and its associations with disease-free survival (DFS) and overall survival (OS) were evaluated using Cox regression adjusting for known prognostic factors.

Results: Membranous IGF1R expression was observed in 317 (87%) cases, with substantial intra- and intertumoral heterogeneity. High IGF1R expression was associated with worse DFS comparing the highest vs. lowest expression tertiles (median DFS, 10.8 vs. 16.1 months; adjusted hazard ratio [HR], 1.77; 95% CI, 1.27-2.48; P trend < 0.001) and worse OS (median OS, 17.4 vs. 25.8 months; HR, 1.38; 95% CI, 1.00-1.89; P trend = 0.046). The association between high IGF1R expression and reduced DFS was identified primarily among overweight and obese patients (body mass index ≥25 kg/m2; HR, 3.92; 95% CI, 1.94-7.93, comparing extreme tertiles; P interaction = 0.039). KRAS-mutant tumors had greater IGF1R expression, and IGF1R expression in tumor epithelium versus stroma was inversely correlated. Higher IGF1R expression was associated with higher IGF1R gene copy number (Pearson correlation coefficient = 0.26, P < 0.001). Mutations in IGF1R were infrequent, and no overt loss of function alterations were identified.

Conclusion: Higher IGF1R protein expression was associated with worse patient outcomes in resected PDAC, with particularly poor survival among overweight and obese patients with high tumor IGF1R expression, suggesting a role for IGF1R in obesity-associated PDAC. Our findings suggest IGF1R has important prognostic implications following PDAC resection and underscore the potential value of biomarker-based selection to identify patient subgroups that may benefit from targeted therapies.

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Chymotrypsin Like Protease 1 Knockout Mouse

D. Mosztbacher, Z. Jancso, M. Sahin-Tóth. Department of Molecular and Cell Biology, Boston University, Henry M. Goldman School of Dental Medicine, Boston, MA.

Background: Chymotrypsin is a digestive enzyme, produced by the pancreas as four isoforms (CTRB1, CTRB2, CTRC, CTRL1). Chymotrypsin C (CTRC) degrades trypsinogen, thereby it protects the pancreas against premature trypsinogen activation. Loss of function mutations in the CTRC gene contribute to development of chronic pancreatitis (CP). Mutations in chymotrypsin B (CTRB) may also have an effect on the development of CP while the role of chymotrypsin like protease 1 (CTRL1) in CP is unknown. Our aim was to investigate the effect of Ctrl1 deletion on pancreatitis responses in a mouse model.

Methods: A Ctrl1 knockout (KO) mouse model was created with CRISPR Cas9 technology. Successful deletion of Ctrl1 was verified by reverse transcription (RT) PCR and western blot. Total trypsinogen and chymotrypsinogen content was measured by enzymatic assay. To generate pancreatitis we used 10 hourly intraperitoneal cerulein injections. We measured pancreas mass, pancreatic water content (edema), plasma amylase activity, pancreas myeloperoxidase (MPO) and evaluated histology sections for edema, inflammatory cell infiltration and necrosis. We measured pancreas trypsin and chymotrypsin activity 30 minutes after a single injection of cerulein.

Results: RT-PCR and western blot demonstrated no CTRL1 expression in the KO strain. Total trypsinogen and chymotrypsinogen content was comparable between wild-type and KO mice. In experimental pancreatitis studies, we found no significant difference in pancreas mass, edema, plasma amylase activity and histological scores between wild type and CTRL1 KO strains.

Conclusion: CTRL1 plays no role in the development of acute pancreatitis.

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In the Setting of Asparaginase Exposure in Pancreatic Acinar Cells, Asparagine Synthetase Maintains Acinar Cell Homeostasis at Baseline and Upregulates to Mitigate Cell Injury

A. Mukherjee, N. Ahmed, F. Turay, T.A. Javed, L. Wen, A. Ahmad, S.Z. Husain. Department of Pediatrics, Children’s Hospital of Pittsburgh of UPMC, University of Pittsburgh School of Medicine, Pittsburgh, PA.

Background: The anti-leukemia drug asparaginase causes pancreatitis in 6-10% of patients, and this iatrogenic complication is a major issue for event-free survival. Thus, there is a crucial need to understand the mechanisms underlying asparaginase-associated pancreatitis (AAP). Asparaginase functions primarily by depleting asparagine. The pancreas expresses high amounts of the counter-regulating enzyme asparagine synthetase (ASNS), in order to replenish asparagine stores.

Methods: In the current study, we have evaluated the expression of ASNS by immunoblotting and qPCR. To knockdown ASNS expression or to overexpress exogenous ASNS, in 266-6 cell line, we have used shRNA transfection and ASNS plasmid respectively. We have also characterized the protective role of ASNS in the acinar cell at baseline and with asparaginase exposure using propidium Iodide uptake assay.

Results: Firstly, we demonstrate that, compared to other organs and tissues, ASNS is predominantly expressed in the pancreas. The mechanism for this high level of expression is through greater eIF2α phosphorylation and ATF4 expression. We further show that within the pancreas, ASNS is predominantly expressed in the acinar cells. Asparaginase exposure in mouse (266-6) and rat (AR42J) pancreatic acinar cell lines leads to a profound time- and concentration-dependent increase in ASNS expression. In primary mouse acinar cells ASNS is also upregulated with asparaginase treatment. The induction of ASNS is unique to asparaginase as a trigger for pancreatic injury because other pancreatitis stimuli fail to induce ASNS expression. Next, shRNA knockdown of ASNS leads to acinar cell necrosis and compounds the cell injury with asparaginase exposure. Conversely, overexpression of ASNS protects mouse 266-6 cells at baseline and from necrosis due to asparaginase exposure.

Conclusion: These observations suggest that pancreatic ASNS maintains acinar cell homeostasis and that its upregulation is required to mitigate asparaginase-induced pancreatic cell injury. Therefore, strategies that selectively augment pancreatic ASNS could be used to alleviate AAP.

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Medicaid or Uninsured Status is an Independent Risk Factor for Mortality in Patients With Pancreatic Ductal Adenocarcinoma

D. Mukhija,1 D.P.S. Sohal.2 Departments of 1 Internal Medicine and 2Hematology and Oncology Cleveland Clinic, Cleveland, OH.

Introduction: Pancreatic ductal adenocarcinoma (PDAC) is projected to become the 2nd leading cause of death from cancer in the US by 2030. Outcomes in PDAC continue to remain dismal. While the impact of race, gender, and treatment options on survival in PDAC has been well studied, there are relatively fewer data on the impact of insurance status on survival in these patients.

Methods: Using the Surveillance, Epidemiology and End Results (SEER) database, we identified patients with pancreatic adenocarcinoma. We collected data on patient demographics, year of diagnosis, tumor location, treatment and survival until death or last follow up for the period 1973-2014. Patients ≥ 65 years of age (medicare eligible) and those with other malignancies were excluded. Overall survival was analyzed using Kaplan-Meier method and Log-rank test, and multivariable analyses were performed using the Cox proportional-hazards model.

Results: 15,960 patients were included. The median (IQR) age was 57 (52-61) years; 51.8% were males; 76.4% were white. 66.5% patients had chemotherapy and 14.8% patients had surgery as the first treatment. Three-fourths (75.1%) patients had insurance, 17.3% had Medicaid, and 7.6% did not have any insurance.

The median overall survival was 6 (2-12) months. Patients with no insurance/Medicaid had worse overall survival than patients who had insurance (P < 0.0001). After adjusting for the other statistically significantly associated variables - age, sex, race, marital status, stage of disease, tumor location, surgery, chemotherapy and radiation- patients with Medicaid (HR, 1.18, P < 0.0001) and no insurance (HR, 1.14, P < 0.0001) independently had worse 1-year survival as compared to those with insurance. Survival was similar between patients with Medicaid as compared to no insurance (HR, 0.96, P = 0.37). Results were similar for 5-year survival as well.

Conclusion: Insurance status is independently associated with overall survival in PDAC, with Medicaid and uninsured patients experiencing higher mortality than insured patients.

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Micro-RNAs Effectors of Gastrin: Cholecystokinin Signaling Axis in Pancreatic Cancer (PDAC)

S. Nadella,1 M. Huber,1 M. Zamanis,2 J. Wang,1 N. Shivapurkar,1 R. Tucker,3 H. Cao,1 J. Smith.1,2 1 Department of Medicine, MedStar Georgetown University Hospital, Washington, DC; 2 Department of Oncology, Lombardi Cancer Center, Georgetown University, Washington, DC; 3Department of Comparative Medicine, Georgetown University, Washington, DC.

Background: Gastrin and the cholecystokinin-B receptor (CCKBR) are not found in the normal murine pancreas. However, both become (re)-expressed in PanINs and in PDAC. We previously showed that CCKBR blockade in a KrasG12D genetically engineered murine model of PDAC (KC) delays PanIN progression and decreases fibrosis. When the KC mice were crossed with a gastrin-knockout (GKO) mouse, PanIN progression, fibrosis, and inflammation decreased. We hypothesize that gastrin and CCKBRs interact with micro-RNAs in pancreatic cancer.

Methods: We performed a 376 miRNA array to compare differential miRNA expression between the pancreata of control KC mice and KC mice treated with proglumide for 6 months. We studied miRNA expression between KC mice and GKO/KC mice at age 1 and 8 months. In vitro, using lentivirus-mediated infection, stable murine PDAC clones(mT5) were generated which overexpressed miRNAs-29c, 148a, 375 compared to scrambled controls. CCKBR knockdown (KD) clones from mT5 cells were generated by CRISPR and miRNA expression evaluated by qRT-PCR. mT5 cells were treated for 24 h with gastrin and/or a CCKBR antagonist and miRNA expression examined.

Results: We found significant (>5-fold) changes in 22 miRNAs in the pancreata of KC mice treated with proglumide compared to controls. MiRNAs-29c, 148a, 375 were decreased with PanIN progression but increased with proglumide. mT5 over-expressing miRNAs-29c,148a, 375 showed decreased growth, colony-formation and increased cell death and reduced S-phase by flow cytometry. mT5 CCKBR-KD cells showed decreased growth, colony-formation and increased miRNA-148a expression. Gastrin treatment decreased miRNA-148a expression while proglumide led to increased miRNA-148a expression.

Conclusion: This data implies that several miRNAs mediate the effects of the GAST:CCKBR signaling axis. MiRNAs-29c,375,148a are tumor suppressive miRNAs that are lost during PanIN progression and increased with proglumide. MiRNA-148a levels are inversely correlated with CCKR stimulation. MiRNA-148a along with GAST:CCK might form a coherent feed forward regulatory loop in pancreatic cancer.

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PAD4 Mediated Neutrophil Extracellular Traps (NETs) Promote Platelet Aggregation and Worsen Severity of Acute Pancreatitis

P. Murthy,1 M.T. Lotze,1,2 M.D. Neal,1 D.C. Whitcomb,3 G.I. Papachristou,3 A.H. Zureikat,1 H.J. Zeh,1,4 B.A. Boone.1,5 Departments of 1 Surgery and 2 Immunology and Bioengineering, University of Pittsburgh, Pittsburgh, PA; 3 Division of Gastroenterology, Department of Medicine, University of Pittsburgh, Pittsburgh, PA; 4 Department of Surgery, UT Southwestern, Dallas, TX; 5Department of Surgery, West Virginia University, Morgantown, WV.

Background: Neutrophil extracellular traps (NETs) from activated neutrophils release intracellular contents, including histones, DNA, HMGB1, and other proteins, into the extracellular space. Peptidyl arginine deiminase-4 (PAD4) enables histone/DNA unwinding and expulsion from the neutrophil and is necessary for NET formation. Platelets are critical mediators of the inflammatory response in pancreatitis and NETs activate platelets in sterile inflammation. We evaluated NETs impact on platelet aggregation and pathophysiology of murine and human pancreatitis.

Methods: Acute pancreatitis was induced in C57/Bl6 or PAD4-/- mice following two sequential hourly injections of L-arginine (4 g/kg IP). For survival experiments, mice were injected with L-arginine weekly x 3 weeks. Platelet function was assessed using whole-blood collagen activated impedance aggregometry. Biomarkers of NET formation including cell-free DNA and MPO-DNA conjugates, were measured in serum from patients with mild and severe acute pancreatitis and healthy controls using Quant-It picogreen and ELISA.

Results: L-arginine injection increased serum amylase and trypsin compared with sham controls, consistent with induction of pancreatitis. Platelet aggregation was upregulated in wild type murine pancreatitis (AUC 32.8±1.1 vs. 23.7±2.7, P < 0.001), but diminished in PAD4-/- mice, incapable of forming NETs (AUC 25.3±1.5, P < 0.001). PAD4-/- mice had decreased levels of amylase and trypsin activity, and had improved survival compared with wild-type controls (median survival 15 days vs. >60 days, P < 0.001). In patients with pancreatitis, circulating markers of NET formation including serum cell-free DNA and MPO-DNA conjugates were elevated compared to healthy controls and correlated with disease severity (DNA (pg/mL): Severe:642±193, Mild:281±128, Control:140±33, P < 0.001; MPO-DNA (absorbance): Severe: 109.8±51.5, Mild: 37±39, Control: 0.124±0.02, P < 0.001).

Conclusion: NETs promote platelet aggregation and worsen survival in murine pancreatitis. Biomarkers of NETs are elevated in patients with pancreatitis, suggesting a role for NETs in the pathogenesis of human disease. Further study of NET inhibition as a novel treatment strategy in patients with acute pancreatitis is warranted.

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CCK Receptor Antagonist Therapy Decreases Inflammation and Fibrosis in Chronic Pancreatitis

S. Nadella,1 V. Ciofoaia,1 H. Cao,1 B. Kallakury,2 R. Tucker,3 J.P. Smith.1,4 Departments of 1 Medicine and 2 Pathology, MedStar Georgetown University Hospital, Washington, DC; 3 Department of Comparative Medicine, Georgetown University, Washington, DC; 4Department of Oncology, Lombardi Cancer Center, Georgetown University, Washington, DC.

Background: Chronic pancreatitis is associated with bouts of inflammation, pain, fibrosis, loss of exocrine and endocrine pancreatic function and risk for cancer. Currently there is no treatment to slow or reverse progression of disease. Hypothesis: We believe that activation of the CCK receptor signaling pathway contributes to pancreatitis and blockade of this pathway with CCK receptor antagonists will improve chronic pancreatitis.

Methods: C57BL/6 female mice were treated with the high dose cerulein (50 ug ip x 6 weeks). After chronic pancreatitis was induced, mice entered a ‘recovery phase’ where N = 10 were treated with the CCK receptor antagonist proglumide and N = 10 were treated with proglumide for 2 weeks. Pancreata were dissected, stained with H&E and scored in a blinded fashion for inflammation, fibrosis and edema. Pancreatic fibrosis was analyzed by Masson’s trichrome & anti-SMAα immunoreactivity and scored using a computer assisted program. Blood was collected for pancreatic enzymes. Pancreatic RNA was tested by qRT-PCR for differentially expressed genes.

Results: Chronic pancreatitis was confirmed by histology and by serum lipase. Proglumide therapy significantly reduced fibrosis and inflammation. Lipase blood levels recovered at week 1 with proglumide but not with water. Tissue qRT-PCR showed decrease of amylase, beta-catenin, e-cadherin and collagen 4 gene expression in proglumide treated mice. Edema and fibrosis scores were lower in mice treated with proglumide but not statistically significant. The acinar ductal metaplasia (ADM) scores, fibrosis by morphometry and alpha-SMA staining were significantly lower in proglumide treated mice compared to control mice.

Conclusion: New strategies are needed to decreased inflammation and reduce fibrosis in chronic pancreatitis. CCK receptor antagonist therapy may improve chronic pancreatitis by reversing fibrosis and inflammation. The decrease in ADM may reduce the risk for development of pancreatic cancer.

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Localization of Exosomal miR-21 and Difference in Expression of miR-21 by Treatment

K. Nakamaru, T. Tomiyama, T. Ikeura, K. Uchida, K. Okazaki. The Third Department of Internal Medicine, Kansai Medical University, Hirakata, Japan.

Background: Type 1 autoimmune pancreatitis (AIP) is now recognized in the spectrum of IgG4-related disease characterized by elevated serum level of IgG and IgG4, irregular narrowing of the main pancreatic duct and pancreatic swelling, lymphoplasmacytic infiltration and fibrosis in pancreas. Abnormal immune systems are thought to be involved in pathophysiology of AIP as steroid and immunosuppressants are effective for AIP, its pathophysiology has not been fully elucidated. Exosomes are extracellular vesicles secreted by various cells and play a cell-to-cell communicator by carrying the intracellular components such as miRNAs derived from secretory cells to recipient cells. Exosomes are deeply involved in not only immune homeostasis but also pathophysiology of various disease. We have already demonstrated the significantly higher expression of miR-21-5p in circulating exosomes in patients with type 1 AIP than that in healthy adults. Therefore, we analyzed the source of exosomal miR-21-5p which affect the activation of T-helper 2 cells, cancer-promoting and tissue fibrosis. We also compared the expression of exosomal miR-21-5p in the AIP patients with or without steroid.

Methods: To identify the source of exosomal miR-21, we performed the in situ hybridization (ISH) using the probe for miR-21 on resected specimen of AIP patient. The exosomal miRNAs were extracted from the patient with or without the steroid. Quantitative reverse- transcription polymerase chain reaction (qRT-PCR) was performed in AIP patients with treatment (n = 5) and without treatment (n = 10).

Results: ISH revealed that miR-21 was expressed in various cells in pancreas of AIP patient such as lymph follicle, pancreatic duct epithelium and acinar cell. qRT-PCR revealed that there was no significant difference between with or without steroid (P = 0.513).

Conclusion: Our study suggested that circulating exosomes carrying miR-21-5p were secreted from various pancreatic cells of AIP. However, it seemed that steroid therapy did not affect the expression of circulating exosomal miR-21.

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Surgical Techniques and Complications in Children and Adults Undergoing Total Pancreatectomy With Islet Autotransplantation: Updates From POST

J.D. Nathan,1 Y. Yang,2 P. Witkowski,3 M. Wijkstrom,4 K. Smith,5 V.K. Singh,6 S.J. Schwarzenberg,2 T.L. Pruett,2 A. Posselt,7 B. Naziruddin,8 K. Morgan,9 L. Long-Simpson,2 L.F. Lara,10 V. Kirchner,2 M. Hughes,11 T.B. Gardner,5 M.L. Freeman,2 T.B. Dunn,2 D.L. Conwell,10 S. Chinnakotla,2 G.J. Beilman,2 A.N. Balamurugan,11 S. Ahmad,12 D. Adams,9 M. Abu-El-Haija,1 J. Hodges,2 M.D. Bellin.2 1 Cincinnati Children’s Hospital Medical Center, Cincinnati, OH; 2 University of Minnesota, Minneapolis, MN; 3 University of Chicago, Chicago, IL; 4 University of Pittsburgh Medical Center, Pittsburgh, PA; 5 Dartmouth-Hitchcock Medical Center, Lebanon, NH; 6 Johns Hopkins Medical Institutions, Baltimore, MD; 7 University of California San Francisco, San Francisco, CA; 8 Baylor Health, Dallas, TX; 9 The Medical University of South Carolina, Charleston, SC; 10 The Ohio State University Medical Center, Columbus, OH; 11 University of Louisville, Louisville, KY; 12University of Cincinnati Medical Center, Cincinnati, OH.

Background: Total pancreatectomy with islet autotransplantation (TPIAT) is a viable option for treating debilitating acute recurrent pancreatitis (ARP) and chronic pancreatitis (CP). Literature regarding surgical approaches, complications, and outcomes has been limited to single-center reports.

Methods: Consenting patients undergoing TPIAT at 9 centers in the United States were enrolled in the Prospective Observational Study of TPIAT (POST). We evaluated surgical techniques including alimentary tract and biliary reconstruction, islet isolation and infusion approaches, outcomes including hospital length of stay, reoperation rates and complications, comparing children with adults.

Results: Data from 78 adults and 29 children were abstracted. Open operation was performed in 77 patients (72%), with minimally invasive techniques utilized in 30 (28%). Pancreatectomy was performed in continuity versus by in situ split technique more commonly in adults (65%) than children (34%; P = 0.006). Compared to adults, children more commonly underwent pylorus preservation (93% versus 64%; P = 0.003), Roux-en-Y duodenojejunostomy reconstruction (93% versus 36%; P = 0.0005), and enteric feeding tube placement (97% versus 65%; P = 0.001).

Choledochojejunostomy or hepaticojejunostomy was performed in 93%. Median islet equivalents/kg transplanted was higher in children (5958; IQR, 3014-7325) than adults (2702; IQR, 1487-4381; P = 0.0003), with COBE purification more common in adults (17% versus 0%; P = 0.019). Median length of hospital stay was higher in children (22 days; IQR, 14-27 versus 11 days; IQR, 9-14; P < 0.0001). Readmission rate (21% versus 19%) and reoperation rate (12% versus 15%) within 30 days of TPIAT did not differ between adults and children. Overall complication rate was higher in children (52% versus 31%; P = 0.055), with bleeding requiring transfusion (17.8%), infection requiring radiologic drainage (6.5%), and portal vein thrombosis (6.5%) being most common overall complications.

Conclusion: Pancreatectomy techniques and alimentary tract reconstruction differ between children and adults, with islet yields higher in children. Surgical complication rates trended higher in children, who had a longer hospitalization post-TPIAT.

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Matrix Metalloproteinases as Markers of Severe Acute Pancreatitis

Ł. Nawacki,1,2 U. Grabowska,1 S. Głuszek.1,2 1 Faculty of Medicine and Health Sciences, Jan Kochanowski University, Kielce, Poland; 2Department of General, Oncological and Endocrinological Surgery, Voivodeship Hospital, Kielce, Poland.

Background: Many prognostic scales have been created to predict the development of severe acute pancreatitis (SAP) and death. We tried to compare their clinical usefulness. Moreover, it has been discovered that a certain group of proteases- matrix metalloproteinases (MMP-2 and MMP-9)- can lead to progression of the inflammatory process. We checked if we can use their concentration as a predictor of SAP.

Methods: We enrolled 72 patients with acute pancreatitis treated in our facility. We excluded patients with any chronic disease as well as patients who suffered from AP in the past or with any history of cancer. Patients were divided into three groups according to The Atlanta 2012 Classification. Data on etiological factor, demographics, clinical, radiological and laboratory variables, outcomes was prospectively collected. We also took blood samples to determine MMP concentration. In the last point we made a statistical analysis of the usefulness of available prognostic scales and factors and compared them with the usefulness of MMP concentration as a predictor of SAP.

Results: Among the cohort study there were 42 (58.34%) mild cases, 16 (22.22%) moderately severe- and 14 (19.44%) severe cases. In the group of SAP 6 patients (42.9%) had more than 8 points in APACHE II scale, 2 (14.3%) patients had more than 3 points in Ranson scale, 2 (14.3%) patients had more than 3 points in BISAP scale. Considering the activity of MMP-2 over 50 ng/ml as indicating a severe form of AP, we get sensitivity = 35.7% and specificity = 98.3%. Considering the activity of MMP-9 over 25 ng/ml as indicating a severe form of AP, we get sensitivity = 28.6% and specificity = 100%.

Conclusion: MMP-2 and MMP-9 have a high specificity in predicting the development of SAP.

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Novel Chymotrypsin C (CTRC) Variant c.173C>T (p.T58M) in a Case of Late Onset Recurrent Acute Pancreatitis

B.C. Németh,1,3 T. Takács,1 P. Hegyi,2 M. Sahin-Tóth.3 1 First Department of Medicine, University of Szeged, Szeged, Hungary; 2 Department of Translational Medicine, Medical School, University of Pécs, Pécs, Hungary; 3Boston University Medical Center, Boston, MA.

Background: Chymotrypsin C (CTRC) is a digestive enzyme produced by the pancreas. Active CTRC inhibits early, intrapancreatic activation of trypsinogen and protects against the development of pancreatitis. Loss-of-function mutations that reduce secretion or impair catalytic activity of CTRC are associated with chronic pancreatitis. Some mutations of the CTRC gene induce endoplasmic reticulum (ER) stress that is a trypsin-independent pathway leading to pancreatitis. The Hungarian Pancreatic Study Group (HPSG) recruits patients with pancreatitis. Genetic testing of a patient with late-onset recurrent acute pancreatitis revealed a novel missense variant p.T58M in the CTRC gene. We aimed to study the clinical and biochemical characteristics of the novel p.T58M variant in the CTRC gene.

Methods: Sanger sequencing was performed using genomic DNA sample of patients with pancreatitis after informed consent form was signed. CTRC was expressed recombinantly, and its biochemical characteristics were studied using enzymatic assays and SDS-PAGE. Secretion of the novel variant was studied in transiently transfected HEK293T cells.

Results: The CTRC p.T58M mutation was identified in the heterozygous state in a 49 year old male patient with recurrent acute pancreatitis. Except for smoking no other environmental or genetic risk factors predisposing to pancreatitis were identified in his history. We found that catalytic efficiency of the novel variant on a small peptide substrate was 2 times higher than the wild type CTRC. However, the mutant enzyme cleaved trypsinogen and β-casein somewhat slower compared to the wild type enzyme. CTRC p.T58M did not significantly change the rate of autoactivation and N-terminal processing of trypsinogen and had no effect on secretion from transfected HEK293T cells.

Conclusion: The novel p.T58M mutation of the CTRC gene did not change the biochemical characteristics or cellular secretion of chymotrypsin C. Therefore, it is unlikely that the p.T58M mutation has a significant role in the development of pancreatitis.

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P2 Receptor Blocking Reduces the Severity of Chronic Pancreatitis in Mouse Models

A. Nikam, E. Bava, S. Lavania, R. Dawra, V. Dudeja, A.K. Saluja. Department of Surgery and Sylvester Comprehensive Cancer Center, University of Miami Miller School of Medicine, Miami, FL.

Background: Chronic pancreatitis (CP) is characterized by chronic inflammation, acinar cell loss, and progressive fibrosis of the pancreas. These changes are considered irreversible and no targeted therapy exists for the disease.

Aim: To investigate the role of P2-purinoceptor (P2R) antagonist suramin and inflammasome activation in the progression of pancreatic fibrosis in a mouse model of chronic pancreatitis (CP).

Methods: Chronic pancreatitis was induced in mice by repeated intra-peritoneal injections of cerulein (50 μg/kg x7 hourly, twice a week x 10). After 6 weeks of initiation of the model, animals were randomized and were assigned to either saline or suramin (10 mg/kg per day) treatment groups. The treatment continued for next 5 weeks and animals were euthanized at week 11. Pancreatic chronic inflammation and fibrosis were evaluated by histological score, Sirius red staining, and alpha-smooth muscle actin immunohistochemical staining and compared treatment and vehicle groups. Similarly, fibrotic changes in the pancreas and P2-purinoceptor and inflammosome expression were investigated by mRNA and protein levels along with immunohistological evaluation.

Results: There was significant increase in pancreas to mouse weight ratio between 10 weeks CP + veh vs 10 weeks CP + Suramin. The pancreatic chronic inflammation and the fibrosis indices, such as H&E, fibrosis score, sirius red staining and α-SMA immunohistochemical staining, were noticeably ameliorated.

Conclusion: The results indicate that the extracellular ATP released from the chronically injured pancreatic cells might be a potent damage-associated molecular pattern molecule. Inhibition of P2-purinoceptor by suramin could represent a potential therapeutic target in management of CP.

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Pancreatic Secretory Trypsin Inhibitor (SPINK1) Gene Mutation in Patients With Acute Alcohol Pancreatitis (AAP) Compared to Healthy Controls and Heavy Alcohol Users Without Pancreatitis

A. Nikkola,1,2 K.H. Herzig,3 K. Mäkelä,3 S.J. Mutt,3 T. Lehtimäki,2,4 M. Kähönen,2,5 O. Raitakari,6 I. Seppälä,4 P. Paakkanen,2 H. Seppänen,7 N. Isto,2 J. Sand,1,8 J. Laukkarinen.1,2 1 Department of Gastroenterology and Alimentary Tract Surgery, Tampere University Hospital, Tampere, Finland; 2 Faculty of Medicine and Life Sciences, University of Tampere, Finland; 3 Research Unit of Biomedicine and Biocenter of Oulu, University of Oulu, Oulu, Finland; Departments of 4 Clinical Chemistry and 5 Clinical Physiology, Tampere University Hospital, Tampere, Finland; 6 Department of Clinical Physiology and Nuclear Medicine, Turku University Hospital, Turku, Finland; 7 Department of Surgery, Helsinki University Hospital, Helsinki, Finland; 8Päijät-Häme Central Hospital, Lahti, Finland.

Introduction: Only approximately 5–10% of heavy alcohol users develop AAP. This suggests that additional triggers are required to initiate the inflammatory process. Genetic susceptibility contributes to the development of acute pancreatitis (AP) and for example SPINK1 mutation is a documented risk factor for the disease. However, only few genetic studies have been conducted comparing AP patients and heavy alcohol users without pancreatitis. Our aim was to study the prevalence of SPINK1 (N34S) mutation in patients with acute alcohol pancreatitis (AAP) compared to heavy alcohol users who had never suffered an episode of pancreatitis.

Methods: For the mutational analysis, we obtained blood samples from patients with the first (n = 60) and recurrent AAP (n = 43) and from heavy alcohol users who had never suffered an episode of AP (n = 98). We compared these findings to a Finnish control population (n = 1914). After DNA isolation the prevalence of SPINK1 mutation was determined using suitable primers in PCR.

Results: The N34S mutation was found in 7.8% of the patients with AAP. The prevalence did not differ between first and recurrent AAP and was not associated with the severity of the disease. The prevalence was significantly lower in healthy controls (3.4 %; OR, 2.43; 1.40–5.24) and very low in alcoholics without pancreatitis (1.0 %, 8.20; 1.01–66.67).

Conclusion: Our study demonstrates that the prevalence of the SPINK1 mutation is overrepresented in AAP patients as previously studied, but interestingly, very low in alcoholics without pancreatitis. This finding may play a role in the variable susceptibility to AAP found in heavy alcohol users.

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Imaging Prediction of Islet Yield in Children Undergoing Total Pancreatectomy With Islet Autotransplantation

H. Nolan,1 A.T. Trout,2 M. Abu-El-Haija,3 L. Fei,4 T.J. Lin,3 D. Elder,5 J.D. Nathan.1 1 Division of Pediatric General and Thoracic Surgery, 2 Department of Radiology and Medical Imaging, 3 Division of Gastroenterology, Hepatology and Nutrition, 4 Divison of Biostatistics and Epidemiology, 5Division of Endocrinology, Cincinnati Children’s Hospital Medical Center, Cincinnati, OH.

Background: Total pancreatectomy with islet autotransplantation (TPIAT) can be a definitive surgical therapy for debilitating chronic pancreatitis (CP) in children. Accurate prediction of islet yield, function and ultimately, glycemic control remains elusive. Adult data suggest that preoperative pancreas volumes measured by CT or MRI are predictive of islet yield but similar pediatric data do not exist.

Methods: A retrospective review of clinical and imaging data from children who underwent TPIAT April 2015–May 2017 was performed. Electronic medical records and operative notes were manually reviewed, and clinical data were abstracted. Most recent cross-sectional imaging (CT or MRI) prior to TPIAT was reviewed by a single board-certified pediatric radiologist who manually segmented the pancreas using MIM (MIM Software). Total islet counts (TIC) and total islet equivalents (TIE) were determined following isolation. Univariate correlations were assessed using Pearson’s correlation; logistic and linear regressions were used for modeling.

Results: 17 patients (11 female) with median age 12 years (IQR, 8.5-14.5) were included. Median interval between imaging and TPIAT was 70 days (IQR, 19.5-96). Median segmented pancreas volume was 31.9 mL (IQR, 16.1-50.5). Segmented pancreas volume correlated very strongly with gross pancreatic weight (r = 0.9, P < 0.0001) and correlated strongly with TIE (r = 0.67, P = 0.0034), but only weakly, and not statistically significantly, with TIC (r = 0.34). TIE was strongly associated with height, weight, body mass index and body surface area (r = 0.65-0.76, all P < 0.005).

Regarding glycemic control, TIE/kg body weight was moderately inversely correlated with total daily dose of exogenous insulin at 3 months (r = 0.49, P = 0.048) and 6 months (r = 0.5, P = 0.04) post-TPIAT and was the only predictor of insulin use in regression modeling.

Conclusion: In children, preoperative pancreas volume as measured by CT or MRI reflects gross pancreatic weight at TPIAT and is strongly correlated with TIE. Insulin use post-TPIAT is best predicted by TIE/kg body weight.

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Hs-CRP, Albumin, and CA 19-9 After Neoadjuvant Therapy Predict Postoperative Survival in Borderline Resectable Pancreatic Ductal Adenocarcinoma

A. Nurmi,1 H. Mustonen,1 U.H. Stenman,2 C. Haglund,1,3 H. Seppänen.1 Departments of 1Surgery and 2 Clinical Chemistry, University of Helsinki, Helsinki University Hospital, Helsinki, Finland; 3Research Programs Unit, Translational Cancer Biology, University of Helsinki, Helsinki, Finland.

Background: Systemic inflammation and tumor markers have shown prognostic value in resectable pancreatic ductal adenocarcinoma (PDAC). This study aimed at assessing preoperative laboratory values as prognostic factors in borderline resectable patients treated with neoadjuvant therapy (NAT).

Methods: Between 2000 and 2015 399 consecutive PDAC patients underwent surgery at Helsinki University Hospital. Borderline resectable patients (n = 71) received NAT. Resectable propensity scored patients (n = 172) underwent upfront surgery (US). NAT included folfirinox, single gemcitabine or combined with cisplatin, capecitabine or nab-paclitaxel with or without radiation. Preoperative high-sensitivity CRP (hs-CRP, mg/l), leukocytes (E9/l), albumin (g/l), bilirubin (g/l), platelets (E9/l), CA 19-9 (kU/l) and CEA (μg/l) were assessed. Survival was estimated with Kaplan- Meier and Cox Regression.

Results: In both NAT and US patients, hs-CRP <3 presented with longer survival than hs-CRP ≥3 (NAT 49 vs. 25 months, P = 0.003; US 30 vs. 22 months, P = 0.034). Additionally, in Kaplan-Meier analysis in NAT patients, albumin ≥35 (33 vs. 16 months, P = 0.033) and CA 19-9 ≤37 (43 vs. 19 months, P = 0.001) associated with survival. In NAT patients, those recurring within six months or one year after surgery had significantly higher hs-CRP (4.8 vs. 3.2, P = 0.003; 4.9 vs. 2.0, P < 0.001) and CA 19-9 (353 vs. 28.5, P < 0.001; 160 vs. 20, P < 0.001) and lower albumin (35.9 vs 38.9, P < 0.001; 36.8 vs. 39.2, P < 0.001) than not recurred patients. Hs-CRP ≥3, CA 19-9 >37 and platelets <150 independently predicted worse survival (HR 3.127, P = 0.014; HR 5.373, P = 0.040; HR 2.171, P = 0.039) and adjuvant therapy better survival (HR 0.402, P = 0.016) in multivariate analysis in NAT patients. Only one NAT patient with hs-CRP ≥3 (total n = 33) survived more than five years (hs- CRP = 3.93).

Conclusion: Hs-CRP, albumin and CA 19-9 after NAT predicted postoperative survival in borderline resectable PDAC patients. Hs-CRP <3 was predictive of long-term survival.

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Minimally Invasive and Endoscopic Methods of Treatment of Postnecrotic Pseudocysts of Pancreas and Separated Focuses of Necrosis

N. Omelchuk. Ivano-Frankivsk National Medical University, Ivano-Frankivsk, Ukraine.

Background: Acute necrotic pancreatitis (ANP) remains complicated problem of urgent surgery because of high frequency of systemic, purulent and septic complications, mortality rate, which is in patients with infected pancreonecrosis 14.7 – 26.4 %.

Aims: The purpose of this study is to evaluate efficiency and establish indications for minimally invasive methods of treatment of postnecrotic pseudocysts of pancreas.

Methods: For diagnostics were used ultrasonography, diagnostic laparoscopy, helical CT with contrast strengthening. Endoscopic interventions were applied by duodenoscopes “Olympus” under control of X-ray machine “Siemens BV 300”. Cystodigestive fistulas were created by prickly papilotoms. For providing of long passability of cystodigestive fistula were used two endoprostheses like “pig tail” sized 10 Fr with length 5–6 cm. For transpapillary drainage were used pancreatic endoprostheses like “pig tail”, sized 5 – 7 Fr with length 5 cm.

Results: In 62 (68.2%) patients were applied minimally invasive methods of treatment. Percutaneous external drainage in 33 (53.2%) patients, endoscopic transmural drainage of postnecrotic pseudocysts in 11 (17.7%) patients. Combined endoscopic interventions were applied in 18 (29.1%) patients. In particular, endoscopic transmural drainage with temporary stenting of pancreatic duct in 11 (61.1%) patients, endobiliary stenting with temporary stenting of pancreatic duct in 3 (16.67%) patients, temporary stenting of pancreatic duct in 3 (16.67%) patients, endoscopic transmural drainage with percutaneous external drainage in 1 (5.56%) patient.

Conclusion: Usage of combined minimally invasive methods of treatment of acute necrotic pancreatitis complicated by postnecrotic pseudocysts help to improve results of treatment, reduction of complications amount, contraction of stationary treatment terms and improving of life quality.

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Is Chronic Pancreatitis in Children a Multifactorial Disease?

G. Oracz, K. Wejnarska, E. Kołodziejczyk, J. Kierkus. Department of Gastroenterology, Hepatology, Feeding Disorders and Pediatrics, The Children’s Memorial Health Institute, Warsaw, Poland.

Background: The etiology of chronic pancreatitis (CP) in children is more diverse than in adults. The major cause of CP are gene mutations and anatomic anomalies. The aim of the study was to analyze the coexistence of risk factors in etiology of CP in children.

Methods: A total of 324 patients with CP, hospitalized from 1988 to 2017 were enrolled into the study. Medical records were reviewed for data on presentation, diagnostic findings and treatment. All children were screened for mutations in major pancreatitis-associated genes (SPINK1, PRSS1, CFTR, CTRC).

Results: Multiple risk factors of CP were found in 85 children (26.2%). 74 patients (87%) had 2 risk factors of CP, in 6 children (7%) more than 2 causes were found. Fourteen patients with PRSS1 mutation had other risk factors of CP. Nine of them had mutation in second gene associated with pancreatitis (5-CTRC, 3-SPINK1, 1-CFTR). Three children had anatomic defect (2-pancreas divisum, 1-ansa pancreatica), 1 patient had a history of choledocholithiasis, 1 had hypertriglyceridemia. 22 children (25.9%) were found with two or more coexisting gene mutations, moreover 4 patients of that group had another risk factor (1 child with anatomical defect, 1 with hypertriglyceridemia, 1 with anatomical defect and cholelithiasis). In 39 children (45.9%) with gene mutation, other gene than PRSS1, we found coexisting anomalies such as: 24-anatomical defect (1 patient had additionally hypertriglyceridemia as a third potential cause of CP), 5-biliary tract diseases, 10-lipid disturbances. In group of 10 children without gene mutations anatomical defects were diagnosed with 4-biliary diseases, 3-lipid disturbances and both n = 1. The coexistence of lipid disturbances and biliary track diseases were found in 2 patients.

Conclusion: CP in children is a multifactorial disease and the “double hit” hypothesis has a strong ground. However, determining which risk factor is crucial in etiopathogenesis of CP is unenforceable.

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Warshaw and Kimura Techniques for Spleen-Preserving Distal Pancreatectomy: Both Viable Strategies to Preserve the Spleen! Multicenter Retrospective Cohort With Long-Term Follow-Up

T.l. Pan,1 S. Paiella,1 M. Korrel,2 M. De Pastena,1 M. Besselink,2 G. Butturini,3 O.R. Busch,2 A. Esposito,1 A. Giardino,3 C. Nessi,1 L. Landoni,1 C. Bassi,1 R. Salvia.1 1 General and Pancreatic Surgery Unit, Pancreas Institute, University of Verona Hospital, Verona, Italy; 2 Department of Surgery, Cancer Center Amsterdam, Amsterdam UMC, University of Amsterdam, Amsterdam, Netherlands; 3Pancreatic Surgery Unit, Ospedale Pederzoli, Peschiera del Garda, Verona, Italy.

Background: The Warshaw (WT) and Kimura (KT) techniques are both used for spleen-preserving distal pancreatectomy (SPDP). We aimed to compare short- and long-term postoperative outcomes including quality of life (QoL) after WT and KT and to compare the open and the minimally-invasive approach (MI).

Methods: Patients who underwent SPDP at three high-volume institutions (2000-2016) were included from prospectively maintained databases. Surviving patients received QLQ-C30 and EQ-5D questionnaires to assess QoL.

Results: In total, 164 patients were included, 55 (33.5%) underwent WT and 109 underwent KT (66.5%). Overall mean age was 51 (±16) years. A MI-SPDP was carried out in 95 cases (57.9%), the open approach was chosen in the remaining 69 (42.1%, P < 0.001). Delayed gastric emptying (DGE) occurred more frequently in the WT group (9.1% vs. 1.8%, P < 0.05). No differences were found in terms of other postoperative complications between the two groups. Mortality was nil. The MI-SPDP group showed a statistically significant lower rate of DGE, splenic infarction and abdominal abscesses. After a mean follow-up of 58 months (±52), 111 patients (67%) with at least 1 postoperative cross-sectional imaging were identified. Eighteen (16.2%) SPDP-related consequences were found; the most frequent was perigastric varices (n = 11, 10%), although none of them had a clinical impact. The time-to-event analysis stratified for MI vs open surgery showed that most of the events developed within 12 months from SPDP and that they were more frequent in the open group (20.8% vs. 10.6%, P < 0.05). QoL after SPDP was satisfactory, with an overall QLQ-C30 global health status of 75 and an overall EQ-5D VAS of 77.

Conclusion: Both WT and KT are viable techniques for SPDP. In terms of postoperative complications, WT showed a higher rate of DGE and the MI approach seemed to be less burdened by postoperative complications. Based on our findings, SPDP (possibly in a MI fashion) should be strongly recommended whenever possible for the treatment of benign or low-malignant diseases.

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Validation of Pancreatitis Activity Scoring System (PASS) in a Large, Prospective, Multicenter US Cohort (APPRENTICE Consortium)

P. Paragomi,1 I. Pothoulakis,1 V.K. Singh,2 T. Stevens,3 H. Nawaz,4 J. Easler,5 S.J. Thakkar,6 G.A. Coté,7 P.J. Greer,1 X. Tang,1 A. Gougol,1 G.I. Papachristou,1 B.U. Wu.8 1 University of Pittsburgh Medical Center, Pittsburgh, PA; 2 Johns Hopkins University, Baltimore, MD; 3 Cleveland Clinic, Cleveland, OH; 4 Eastern Maine Medical Center, Bangor, ME; 5 Indiana University School of Medicine, Indianapolis, IN; 6 Allegheny Health Network, Pittsburgh, PA; 7 Medical University of South Carolina, Charleston, SC; 8Kaiser Permanente, Pasadena, CA.

Background: The Pancreatitis Activity Scoring System (PASS) was designed to quantify acute pancreatitis (AP) dynamic activity. We aimed to examine application of PASS in a prospective, multicenter cohort.

Methods: PASS was calculated as the summation of organ failure (×100/organ), oral intolerance (×40), SIRS (×25/criterion), morphine equivalent dose (×5) and pain score (×5). Enrollment was conducted prospectively in 8 US centers. PASS score was recorded on admission, 24, 48, 72 hours, and 7th day of hospitalization. Patients were divided based on length of stay (LOS) (short: <3 days, intermediate: 3-7 days, and long: >7 days). Discharge Pass was defined as a score calculated ≤24 hours before discharge. Kruskal-Wallis test was applied to compare median PASS scores between subgroups. Area under receiver-operating characteristics (ROC) curve (AUC) was performed to investigate the predictive accuracy of admission PASS score.

Results: 562 patients were enrolled [age: 52 (38-65), 47% female]. The overall median admission PASS was 142 (101-208). Admission PASS was significantly different between short [115 (90-160)], intermediate [145 (105-219)], and long [159 (127-219)] LOS subgroups (P <0.001). At 24 hours, PASS dropped to 81 (45-119) for short, 121 (79-197) for intermediate, and 160 (100-234) for long LOS subgroups. Further decline in PASS ensued at 48 hours in short [25 (7-47)], intermediate [94 (40-150)], and long LOS [150 (90-225)]. At discharge, PASS did not show significant difference between LOS subgroups: [40 (10-67) in short, 48 (20-100) in intermediate, and 56 (25-102) in long LOS; P = 0.07]. Overall, median discharge PASS score was 45 (15-90). Admission score of 140 predicted moderate/severe AP with a sensitivity of 68%, specificity of 52%, and an AUC of 0.7.

Conclusion: In this multicenter cohort study, admission PASS was strongly predictive of LOS, while discharge PASS was similar regardless of LOS.

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Using Patient-derived PDAC Cells to Understand c-MYC Involvement in the Drug Resistance Mechanism of Pancreatic Cancer

E.M. Parasido,1 G. Avetian,1 J. Brody,2 J. Winter,2 C.J. Yeo,2 E. London,2 M. Pishvaian,1 E. Glasgow,1 S. Byers,1 C. Albanese.1,3 1 Lombardi Comprehensive Cancer Center, Department of Oncology, Georgetown University Medical Center, Washington, DC; 2 Department of Surgery, Thomas Jefferson University, Philadelphia, PA; 3Preclinical Imaging Research Laboratory, Georgetown University Medical Center, Washington, DC.

Background: Pancreatic ductal adenocarcinoma (PDAC) is typically incurable due to acquired resistance to systemic chemotherapy. A deeper understanding of the mechanisms involving drug resistance is needed to improve patient outcomes. Commercially available PDAC cell lines do not accurately recapitulate a given patient’s tumor. Using the conditional reprogrammed cell (CRC) technique, we established patient-specific cell lines and created nab-paclitaxel (nab- PTX)-resistant clones, which allowed us to better study resistance mechanisms. Recent studies suggest that the c-MYC pathway is involved in PDAC. However, the role of c-MYC in the mechanisms leading to nab-PTX resistance has not been explored.

Methods: Biopsies were collected from treatment-naive PDAC patients, and long-term cultures of PDAC CRCs were established. The PDAC origin of the cell lines was validated. The IC50s for nab-PTX were used to generate drug-resistant clones. Zebrafish and mouse model were used to test the cells’ ability to form tumors and to verify the drug resistance in vivo. RNA Microarray and Western blotting analyses were used to characterize the clones and to identify key pathways involved in the evolution of drug resistance.

Results: Using 2 KRAS-mutant primary cell lines, we generated 3 nab-PTX-resistant clones; subcutaneous injections in nude mice showed the CRCs’ ability to form tumors that recapitulate human PDAC. Resistant profiles were verified in Zebrafish and mice. RNA microarrays identified the involvement of the induction of a pro-inflammatory pathway leading to c-MYC overexpression. Molecular analysis confirmed the role of c-MYC overexpression in the evolution of nab-PTX resistance.

Conclusion: The CRCs methodology addresses the need for a reliable method for generating primary cell lines on a single patient basis. The ability to rapidly model in vitro, and verify in vivo, that the overexpression of c-MYC contributes to the development of nab-PTX resistance is a significant advancement in the field and provides a platform for discovery of more effective treatment of refractory PDAC.

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Metastasis-associated Fibroblasts Promote Tumor-angiogenesis in Metastasized Pancreatic Cancer via Il-8 and CCL2 Biological Axes

T. Pausch, E. Aue, A.F. Valls, Y. Shen, M. Schneider, T. Schmidt. Heidelberg University Hospital, Heidelberg, Germany.

Background: Ductal adenocarcinoma of the pancreas (PDAC) as soon as metastasized cannot be cured by systemic therapy yet. Primary tumor and metastases exhibit a complex cancer pathology characterized by deposition of desmoplastic stroma, which contributes to cancer progression and chemoresistance. In this microenvironment, cancer cells engage in complex interactions with cancer-associated fibroblasts (CAFs). Concerning the role of CAFs it is heavily discussed if they support or fight the tumor. Even more the role of metastasis associated fibroblasts (MAFs) remains to be unclear and should be analyzed in this study.

Methods: In vitro we studied interaction of tumor cells of metastatic PDAC and fibroblasts in the context of tumor angiogenesis. We measured alterations of cell proteome via ELISA- proteome assay and proofed specific expression changes via qPCR. In angiogenesis assays we tried to block specifically expressed cytokine. In vivo we studied impact of antiangiogenic tyrosine kinase inhibitor Sunitinib on hepatic metastasis of PDAC and associated desmoplastic tumor microenvironment via immunohistochemistry.

Results: Enhanced proangiogenic effect of co-cultured tumor cells and fibroblasts was seen. Additionally proliferation of fibroblasts was stimulated by tumor cells. Simultaneously cell proteome got augmented. Specifically up-regulation of cytokines interleukin 8 (Il-8/CXCL8) and chemokine (C-C motif) ligand 2 (CCL2/MCP-1) was seen in fibroblasts. Increased proangiogenic effects of co-cultures could be suppressed completely by blockage of IL-8- and CCL2-pathways. As anticipated in vivo Sunitinib led to reduction of metastasis. But beyond that it led to reduction of MAFs and simultaneously to increased proliferation of tumor cells in the field of micro-metastasis and cancer invasion front.

Conclusion: Our results highlight that IL-8 and CCL2 from MAFs play a central role in tumor angiogenesis of metastasized PDAC which can be suppressed specifically. Nonspecific antiangiogenic therapy in fact reduces volume of metastases and number of MAFs yet it enhances aggressiveness of tumor cells.

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Development on an Indicator of Pancreatic-Leakage to Prevent Postoperative Pancreatic Fistula

T. Pausch,1 C. Mitzscherling,1 O. Aubert,1 M. Golriz,1 A. Mehrabi,1 P. Schuisky,2 B. Gesslein,2 M.W. Buechler,1 T. Hackert.1 1 Heidelberg University Hospital, Heidelberg, Germany; 2Magle Chemoswed, Malmö, Sweden.

Background: Postoperative pancreatic fistula (POPF) remains to be a frequent and serious complication of pancreatic surgery. Intraoperative visualization of pancreatic leakage would enable to localize leaks and to refine closure. Our aim was to develop an indicator that localizes pancreatic leaks.

Methods: In vitro developed indicator was tested and optimized in vivo in final porcine animal experiments (n = 10). Local cytotoxicity and systemic side effects were tested in 48 hours observational porcine experiments (n = 10) performing distal pancreatectomy (DP).

Pharmacokinetics was studied using liquid chromatography-mass spectrometry (LC-MS) to scan porcine peritoneal fluid, portal, and central venous blood for indicator traces.

Cytotoxicity was tested in vitro using resazurin-assay with benign and malignant pancreatic cells. Local pancreatic injury was evaluated in treated porcine tissues by applying established histopathological scores for acute pancreatic injury. In 8 days follow up porcine experiments (n = 16) optimized indicator was finally tested on sensitivity, specificity, and effect of targeted closure on POPF-rate.

Results: An indicator of pancreatic leaks was developed to be easily applicable and to visualize leakage rapidly and reversibly during DP. On postoperative day three sensitivity of 100% and specificity of 33% for occurrence of POPF was seen. No systemic side effects, no pancreatitis, and no bleeding occurred. Histology ruled out local cytotoxicity. Pharmacokinetic analysis detected only minimal and temporary traces of the indicator in portal vein but no traces got into central blood circulation. No cytotoxicity on target cells was detected. Visualized droplet volume was <10 μl. Thus targeted leak-closure followed by tightness-control through indicator-reapplication became possible reducing POPF-rate from 75% to 0% during 8 days follow-up.

Conclusion: This brand new non-toxic indicator visualizes pancreatic leaks during operations easily and fast. It allows localization and evaluation of pancreatic leakage followed by targeted closure with consecutively dramatically reduced POPF-rate. First in men studies will follow soon.

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Evidence for Computed Tomographic (CT) Angiography as the Standard-of-Care Test in the Diagnosis of Post-Pancreatoduodenectomy Hemorrhage

M. Pease, M. Baltatzis, V. Nadarajah, A. Sheen, A.K. Siriwardena, S. Jamdar. Regional Hepato-Pancreato-Biliary Unit, Manchester Royal Infirmary, Manchester, United Kingdom.

Introduction: Hemorrhage after pancreaticoduodenectomy remains a feared and potentially lethal complication. As post-pancreaticoduodenectomy hemorrhage (PPH) is rare, evaluation of optimal diagnostic test is difficult. This study evaluates CT angiography for the diagnosis of post-pancreatoduodenectomy hemorrhage.

Methods: In this single-center, clinical cohort study 178 consecutive patients undergoing pancreatoduodenectomy from October 2014 to February 2017 constitute the study population. Post-pancreatectomy hemorrhage was defined as described by the International Study Group of Pancreatic Surgery (ISGPS). Statistical analysis was performed with IBM SPSS Version 23 (SPSS Inc, Chicago, IL). A receiver operating characteristic curve (ROC curve) was used to investigate the accuracy of the diagnostic test. The study was approved by Institutional Review Board.

Results: CT scan with intravenous contrast was performed in 76 (44%) to investigate suspected postoperative complications. Twenty patients (12%) underwent CT angiography to investigate clinical signs and symptoms of PPH. Their median (range) age was 66 (46-79) years and 17 (85%) were male. Fifteen (75%) had undergone classical pancreatoduodenectomy, 5 the pylorus-preserving variant. Two patients (10%) had undergone portal vein resection. For detection of post-pancreatoduodenectomy hemorrhage CT angiography had a sensitivity of 30% (95% confidence interval 12-54%); specificity 98% (95% CI, 91-99%), Positive Predictive Value 86% (95% CI, 42-88%) and Negative Predictive Value 79% (95%CI, 68-88%). The AUC of the ROC curve was 0.64. There were 3 (15%) in-hospital deaths in patients who had post-pancreaticoduodenectomy hemorrhage.

Conclusion: The important findings of this study are that CT angiography has high specificity but low sensitivity for detection of post-pancreatectomy hemorrhage. A positive result mandates further investigation. A negative result does not rule out hemorrhage but the CT provides additional information on intra-abdominal sepsis. A practical management algorithm for investigation of post-pancreaticoduodenectomy hemorrhage will utilize CT angiography as the first diagnostic step in the majority of patients.

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The Common Truncation Variant P.W358X of the PNLIPRP2 Lipase Is Not Associated With Chronic Pancreatitis

Z.G. Pesei,1,2 B.C. Németh,1,2 E. Hegyi,1,3 A. Szentesi,2,3 Á. Vincze,4 D. Kelemen,5 P. Hegyi,2,3,6,7 M. Sahin-Tóth.1 1 Boston University Medical Campus, Boston, MA; 2 First Department of Medicine, University of Szeged, Szeged, Hungary; 3 Institute for Translational Medicine, University of Pecs Medical School, Pecs, Hungary; 4 Divisions of Gastroenterology, First Department of Medicine, University of Pecs Medical School, Pecs, Hungary; 5 Department of Surgery, University of Pecs Medical School, Pecs, Hungary; 6 Division of Translational Medicine, First Department of Medicine, University of Pecs Medical School, Pecs, Hungary; 7Hungarian Academy of Sciences - University of Szeged, Momentum Gastroenterology Multidisciplinary Research Group, Szeged, Hungary.

Background: Pancreatic Lipase-Related Protein 2 (PNLIPRP2) is a lesser lipase isoform which can digest long chain triglycerides, diglycerides and monoglycerides. In vitro experiments showed that the common nonsense variant c.1074G>A (p.W358X) causes the formation of a shorter misfolded protein that is not secreted well but induces endoplasmic reticulum (ER) stress. ER stress is a known susceptibility factor for chronic pancreatitis, suggesting that the p.W358X mutation of the PNLIPRP2 gene might contribute the development of pancreatitis. Our aim was to investigate whether the p.W358X mutation of the PNLIPRP2 gene is associated with idiopathic and alcoholic chronic pancreatitis.

Methods: In our study we enrolled 115 patients with idiopathic and 142 patients with alcoholic chronic pancreatitis and 200 controls recruited by the Hungarian Pancreatic Study Group (HPSG – www.pancreas.hu). Exon 11 and its flanking intronic regions were analyzed by Sanger sequencing. Expression of the PNLIPRP2 gene was studied by RT PCR using human donor pancreas cDNA samples.

Results: When allele frequencies were considered, the p.W358X mutation was not overrepresented either in the idiopathic or the alcoholic chronic pancreatitis groups compared to the controls (54.8% and 50% vs. 52%, respectively). RT-PCR on a homozygous cDNA sample indicated drastically reduced PNLIPRP2 expression at the mRNA level. Additionally, we identified 5 more PNLIPRP2 variants (c.1071-379delG, c.1071-321C>T, c.1084A< G p.I362V, c.1161A >G p.S387= and c.1181+55C>A) but none were associated with chronic pancreatitis.

Conclusion: The p.W358X mutation of the PNLIPRP2 gene is not associated with chronic pancreatitis. Expression of the truncated protein is inhibited at mRNA level likely by “nonsense-mediated mRNA decay,” therefore, this variant does not cause ER stress in the human pancreas.

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ROBO2 is a Stroma Suppressor Gene in the Pancreas Through Regulation of TGF-beta

A.V. Pinho,1,2,3 M.V. Bulck,4 L. Chantrill,1,2,3 M. Arshi,1,3 T. Sklyarova,4 D. Herrmann,1,2 C. Vennin,1 D. Gallego-Ortega,1,2 A. Mawson,3,5 M. Giry-Laterriere,1,3 A. Magenau,1 L. Baeyens,6 A.J. Gill,1,3,7 P. Phillips,5 P. Timpson,1,2 A.V. Biankin,3,8,9 J. Wu,3,10 I. Rooman.3,4 1 Cancer Division, The Garvan Institute of Medical Research, Sydney, Australia; 2 St. Vincent’s Clinical School, UNSW, Sydney, Australia; 3 Australian Pancreatic Cancer Genome Initiative (APGI), Sydney, Australia; 4 Oncology Research Centre, Vrije Universiteit Brussel, Brussels, Belgium; 5 Lowy Cancer Research Centre, UNSW, Sydney, Australia; 6 Beta Cell Neogenesis Lab, Vrije Universiteit Brussel, Brussels, Belgium; 7 University of Sydney, Sydney, Australia; 8 Wolfson Wohl Cancer Research Centre, Institute of Cancer Sciences, University of Glasgow and West of Scotland Pancreatic Unit, Glasgow Royal Infirmary, Glasgow, United Kingdom; 9 South Western Sydney Clinical School, UNSW, Liverpool, Australia; 10Centre for Cancer Bioinformatics, Peking University Cancer Hospital and Institute, Beijing, China.

Background: Whereas the SLIT ligand - ROBO receptor pathway is typically involved in axonal guidance, our previous work showed that genomic aberrations in SLIT and ROBO genes are frequent in pancreatic ductal adenocarcinoma (PDAC). The role of these genes in pancreatic epithelium remains to be deciphered.

Methods: Real time PCR and RNA in situ hybridization was used to analyse murine normal pancreas, pancreatitis and PDAC. Cell cultures and experimental pancreatitis were studied using pancreas-specific Robo2 (Pdx1Cre;Robo2F/F) and whole-body Slit1 (Slit1-/-) knockout mice. RNA sequencing and immunostainings were used to study a cohort of human PDAC specimens.

Results: Robo2 expression is generally lost in mouse pancreatitis and PDAC, while Robo1 expression is maintained in the epithelium and becomes most prominent in the stroma.

Pancreatic cell cultures from Pdx1Cre;Robo2F/F mice showed abundant Robo1+ myofibroblast cells and induction of the TGF-beta and Wnt signaling pathways. Accordingly, pancreatitis in Pdx1Cre;Robo2F/F mice led to increased myofibroblasts, enhanced collagen crosslinking, augmented T-cell infiltration and up-regulation of pro-tumorigenic immune markers. A TGF-beta inhibitor (galunisertib), promising in clinical trials for PDAC, suppressed these effects. Slit1-/- mice presented a similar phenotype to Pdx1Cre;Robo2F/F animals.

In patient specimens, ROBO2 expression is frequently low while ROBO1 is variably expressed in tumor epithelium and high in stroma. Low ROBO2 expression indicates poor prognosis and within the ROBO2low population, ROBO1high patients present the poorest survival. ROBO1 correlates with markers of activated stroma, Wnt and TGF-beta pathways.

Conclusion: Epithelial Robo2 acts as a stroma suppressor gene by restraining the activation of myofibroblasts and inhibiting the inflammatory response. ROBO1/2 expression in PDAC patients is prognostic and may guide therapy with TGF-beta inhibitors, stroma or immune modulating agents.

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Simvastatin Induces Mitophagy and Restores Cerulein Impaired Phagosome-lysosome Fusion to Reduce the Severity of Acute Pancreatitis

H. Piplani,1,2 S.M. Iannucci,1 J. Germano,1 J. Hou,3 J. Sin,1 Y. Sang,1 A. Gulla,4,5 B.U. Wu,6 R.T. Waldron,2 A. Lugea,2 A.M. Andres,1 R.A. Gottlieb,1 S.J. Pandol.2 1 Smidt Heart Institute, Cedars-Sinai Medical Center, Los Angeles, CA; Departments of 2 Medicine and 3 Pathology, Cedars-Sinai Medical Center, Los Angeles, CA; 4 Department of Surgery, MedStar Georgetown University Hospital, Washington, DC; 5 Vilnius University Hospital Santaros Klinikos, Vilnius, Lithuania; 6Center for Pancreatic Care, Kaiser Permanente Los Angeles Medical Center, Los Angeles, CA.

Background: During pancreatitis, autophagy is activated but the resolution to degrade dysfunctional organelles is impaired triggering accumulation of dysfunctional mitochondria resulting in acinar cell death and pancreatitis responses. However, there is no comprehensive knowledge about the role of mitophagy in pancreatic physiology. Retrospective cohort analyses demonstrated an association between simvastatin use and decreased pancreatitis incidence.

Methods: We investigated the effect of simvastatin in reducing the pancreatic injury during cerulein induced acute pancreatitis. Mice were pretreated with simvastatin (20 mg/kg) for 24 hours followed by 7 hourly cerulein injections and sacrificed after 1 hour of last injection.

Results: Histopathological analysis of pancreas revealed that simvastatin pretreatment reduced necrotic cell death, inflammatory cell infiltration and edema. We found that cerulein induced mitophagy via translocation of Parkin and p62 at mitochondria with autophagosome formation. However, the fusion of autophagosomes with lysosomes was impaired due to defects in LAMP-1, AMPK and ULK-1. Simvastatin abrogated these effects by activating AMPK which further phosphorylated ULK-1 and induced the expression of LAMP-1. EM images showed the increased formation of autolysosomes with simvastatin as compared to autophagosomes accumulation in control group during pancreatitis. Our findings confirmed that simvastatin induces mitophagy and promotes autophagic flux by showing that chloroquine inhibits the process; and that the process involves Parkin, p62 and LC3. Mitochondria from simvastatin treated mice were resistant to calcium overload compared to vehicle control indicative of simvastatin induced mitochondria quality control. Furthermore, Parkin deletion exaggerated the pancreatic injury in mice and simvastatin induced benefit was lost. Clinical specimens from acute pancreatitis patients showed a significant increase in mtDNA release in plasma compared to normal controls. Simvastatin reduced pancreatitis induced mtDNA release in plasma samples of mice.

Conclusion: Our findings reveal the novel role of a mitophagy inducing drug, simvastatin, in preventing the pancreatic cell injury and pancreatitis.

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Characterization of a Mouse Model of Early Chronic Pancreatitis

K. Priyam,1 M.K. Sharma,1 T.G. Jacob,1 P.K. Garg,2 T.S. Roy.1 Departments of 1 Anatomy and 2Gastroenterology, All India Institute of Medical Sciences, New Delhi, India.

Background: Recurrent pancreatic acinar cell injury, with the necrosis and ensuing fibrosis, results in Chronic Pancreatitis (CP). An intermediate stage of the disease could represent ‘Early CP.’ We wanted to determine early changes indicative of ‘Early CP’ in experimental CP.

Methods: CP was induced in 36 male, adult Swiss-albino mice by intraperitoneal injections of L-Arginine (Group-1: Two doses -4.5g/Kg, one-hour apart, days 0, 7, 14, and 21; Group-2:equivalent doses of normal saline). Histo-pathological changes were evaluated every week till day-28.The inflammatory and Pancreatic Stellate Cells (PSCs) were quantified. Fibrosis was assessed by Masson’s trichrome staining. Western blotting was used to study the expression of TGF-β. Fecal fat and oral glucose tolerance test (OGTT) were measured to assess exocrine and endocrine insufficiency, respectively.

Results: Histopathologically, the pancreas showed features of acinar cell injury and inflammatory cell infiltration on day-7, which increased over the next 3-weeks. Fibrosis appeared on day-14. The severity of histopathological changes was more on day-14 than 7 (23.083 ± 0.19 vs 1.6 ± 0.4; P = 0.001) and progressed (Day-28: 30.625 ± 0.072; P = 0.001). The fibrosis score increased from day-14 to day-28 (9.6 ± 1.02 vs 34.68 ± 1.4; P = 0.003). The number of monocytes and macrophages and activated pancreatic stellate cells increased from day-14 to day-28 (74.2 ± 36.8 vs 114.7 ± 88.18; P = 0.02). At each time point, the expression of TGF-β (1.33 ± 0.68 vs 0.5 ± 0.2; P = 0.05) and fecal fat was more in group-1 than controls on day-14 (2.68 ± 0.12 mg/g; P = 0.01) and 28 (5.26 ± 0.54 mg/g; P = 0.01). Blood glucose level was higher in group-1 than controls on day-14(15262 ± 2267 nMol/ml at 120 minutes vs 14460 ± 2264 nMol/ml; at 120 minutes P = 0.01) and on day-28, (6020 ± 451.3314 nMol/ml vs 3715 ± 140.539 nMol/ml; P = 0.04).

Conclusion: We have shown that features of early CP were present at day-14 in the L-arginine model, which progressed till day-28. These findings may have relevance for ‘Early-CP’ in humans for therapeutic interventions to potentially reverse or prevent further progression to advanced-CP.

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Urine Trypsinogen-2 Dipstick Test in Diagnosis of Post-ERCP Pancreatitis

M. Rainio,1 O. Lindström,1 M. Udd,1 P. Puolakkainen,1 U.H. Stenman,2 L. Kylänpää.1 1 Department of Abdominal Surgery, Helsinki University Hospital and University of Helsinki, Helsinki, Finland; 2Department of Clinical Chemistry, University of Helsinki, Helsinki, Finland.

Background: Post-ERCP pancreatitis (PEP) has traditionally been diagnosed with serum or plasma amylase. A rapid dipstick test for urine trypsinogen-2, with a fixed detection limit of 50μg/L, has also been used in diagnosis of pancreatitis. Sample sizes of studies of dipstick test performances has been small and the study designs has been variable. We explored if the urine trypsinogen-2 dipstick test can replace routine amylase controls in the follow up protocol of ERCP patients; urine sample is less invasive and dipstick test can be analyzed without laboratory facilities.

Methods: We analyzed plasma pancreatic amylase, bilirubin, serum and urine trypsinogen-2, and urine trypsinogen-2 dipstick test in 400 patients with native papillae before ERCP, four hours, and 24 hours after ERCP. Urine samples were tested immediately with the urine trypsinogen-2 dipstick test (Actim Pancreatiitis; Medix Biochemica, Kauniainen, Finland). Our criteria for PEP were a new onset of abdominal pain lasting at least 24 hours accompanied with three fold increase of amylase upper reference limit or diagnostic alterations of AP in radiologic examinations.

Results: PEP developed in 15 (3.8%) patients. 14 cases were mild PEPs and one moderately severe PEP according to Atlanta classification. Post-ERCP dipstick test (4 or 24 hours after ERCP) was positive in 12 of 15 PEP patients. When test was evaluated with presence of abdominal pain four hours after ERCP dipstick tests sensitivity was 60%, specificity 99%, positive likelihood ratio value 44.8, negative likelihood ratio value 0.41, and diagnostic odds ratio 110, whereas 24 hours after ERCP the test strip sensitivity was 100%, specificity 98%, positive likelihood ratio value 52.2, and negative likelihood ratio 0.

Conclusion: When dipstick results are evaluated with abdominal pain symptoms it seems to find PEP cases with good accuracy.

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Total Pancreatectomy and Islet Autotransplantation for Treatment of Chronic Pancreatitis Even in Patients With Prior Whipple Procedure

A. Rajab,1 J. Buss,3 P.A. Hart,2 D.L. Conwell,2 S. Meng,3 L. Lara,2 S. Black,1 K. Washburn.1 1 Department of Surgery Division of Transplantation; 2 Division of Gastroenterology, Hepatology and Nutrition; 3Division of Endocrinology, Diabetes, and Metabolism, The Ohio State University Wexner Medical Center, Columbus, OH.

Background: Chronic pancreatitis (CP) is defined by persistent inflammation of the exocrine tissue of the pancreas. Patients suffering from this debilitating disease often require long-term narcotic therapy to manage the chronic pain associated with CP. For patients who have exhausted other endoscopic or surgical therapies, total pancreatectomy with islet autotransplantation (TPIAT) may be an option for treatment.

Methods: Patients underwent a completion pancreatectomy, and the freshly isolated islets were then transplanted back into the patient’s liver as part of the same surgical procedure. Since 2008, data from n = 11 patients who underwent TPIAT at our institution was analyzed. Patients ranged from 22-59 years of age at the time of surgery.

Results: An average islet dose of 7440.2 IEq ± 6230.3 was given (range 957-21,865 IEq). The average c- peptide at 1 year was 2.0 ng/mL ± 1.5 (range, 0.6-4.4 ng/mL). The average hemoglobin A1c level at 1 year post-TPIAT was 6.6 % ± 1.1 (range, 5.4-8.3 %). Daily insulin doses varied from 0-40 U/day. Fifty percent of patients were insulin independent at 1 year post-TPIAT, with the average daily insulin requirement at 1 year post-TPIAT being 10.8 U/day ± 14.1. Of the 11 patients at our institution who have undergone a TPIAT, two patients had previously undergone a Whipple procedure. The average islet dose for these patients was 7238.5 IEq/kg, which was comparable to that of patients who had not undergone previous Whipple (7485.0 IEq/kg). Both patients maintained positive c-peptide post-TPIAT (average of 0.75 ng/ml at 6 months post-TPIAT) with an average insulin requirement of 15 units per day at 6 months. A majority of patients stated improvement in their quality of life post-TPIAT.

Conclusion: Based on these results, we feel that TPIAT is a viable treatment for patients suffering from CP, including patients who have already undergone a Whipple procedure.

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Cholinergic Signaling via Muscarinic Receptors Directly and Indirectly Suppresses Pancreatic Tumorigenesis and Cancer Stemness

B.W. Renz,1 T. Tanaka,2 M. Sunagawa,2 R. Takahasi,2 M. Macchini,2 Z. Dantes,3 G. Valenti,2 M. Ilmer,1 C.B. Westphalen,4 M. Reichert,3 P.E. Oberstein,2 A.C. Iuga,5 J. Werner,1 K.P. Olive,2 T.C. Wang.2 1 Klinikum der Universität München, Klinik für Allgemeine, Viszeral-, Gefäß-, und Transplantationschirurgie, Munich, Germany; 2 Division of Digestive and Liver Diseases and Herbert Irving Comprehensive Cancer Center, Columbia University Medical Center, New York, NY; 3 Department of Medicine II, Klinikum Rechts der Isar, Technische Universität München, Munich, Germany; 4 Department of Internal Medicine III, Hospital of the University of Munich, Germany; 5Department of Pathology and Cell Biology, Columbia University Medical Center, New York, NY.

Background: The nervous system is increasingly recognized as a central regulator of both normal stem cell function and cancer growth across a range of tissues. Whether parasympathetic signaling directly regulates progression of pancreatic cancer (PDAC) has not been defined.

Methods: KC mice were vagotomized and kept on regular water or water supplemented with a muscarinic agonist. Pancreatic tissue was collected and analyzed by IHC and RT-PCR; cells were isolated and assayed for colony and sphere forming assays. Different human and murine PDAC cell lines and primary organoids were subjected to cholinergic and anti-cholinergic drugs and assayed by RT-PCR, Western blot and flow cytometry. CHRM1 was deleted in Pdx1-Cre/KRasG12D (KC) and Pdx1-Cre/KRasG12D /Trp53R172H (KPC) mice. A syngeneic model of metastatic PDAC was also utilized.

Results: In organoid cultures, cholinergic agonists suppressed sphere formation. Pharmacological inhibition or KO of the CHRM1 abolished this effect in vitro. When PANC-1 cells pretreated with direct parasympathetic agonists were assayed in a xenograft model, they formed less tumors. Vagotomy accelerated tumor development in KC, while treatment with the muscarinic agonist bethanechol rescued the phenotype. An effect also seen in KC-CHRM1 KO mice. In KPC mice, bethanechol significantly extended survival. While KO of the CHRM1 (KPM) shortened overall survival. These effects were mediated in a large part through the CHRM1, which inhibited downstream MAPK/EGFR and PI3K/AKT pathways. Enhanced cholinergic signaling led to a suppression of CSCs and CD11b+ myeloid cells in the pancreas, and metastatic growth in the liver.

Conclusion: Taken together, these data suggest that muscarinic signaling directly and indirectly suppresses the growth of PDAC cells and therapies directed at stimulating muscarinic receptors may be useful in inhibiting the progression of PDAC.

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Is Obesity Associated With Main-duct or Mixed-type Intraductal Papillary Mucinous Neoplasm?

A. Rodriguez,1 P.T. Kröner,1 L.K. Mejia,2 P. Kandel,1 M.B. Wallace,1 T.A. Woodward,1 V. Gomez,1 J. Stauffer,3 H. Asbun,3 M. Raimondo.1 1 Department of Gastroenterology & Hepatology, Mayo Clinic, Jacksonville, FL; 2 Department of Internal Medicine, Cleveland Clinic, Cleveland, OH; 3Department of General Surgery, Mayo Clinic, Jacksonville, FL.

Background: Intraductal papillary mucinous neoplasms (IPMN) are mucin-producing exocrine neoplasms. Main duct (MD-IPMN) & mixed-type are associated with a pancreatic ductal adenocarcinoma (PDAC). Obesity is a PDAC risk factor but a correlation with MD-IPMN has not been described. Aim of this study was to determine if obesity was associated with main/mixed duct IPMN.

Methods: Retrospective study of 182 consecutive patients with IPMN at Mayo Clinic Florida from 1997-2017. Two groups: Main/mixed-duct and branch duct (BD-IPMN). Main/mixed-duct IPMN grouped together given risk of progression to PDAC. Sociodemographic, anthropometric, co-morbidities, histology, imaging, and surgical reports were recorded. Obesity classified according to world health organization. Malignancy defined as high-grade dysplasia (HGD) or invasive carcinoma. Continuous variables reported as mean difference (MD) and standard deviation (SD). Logistic regression used to assess for an association between obesity and main/mixed-duct IPMN. P values <0.05 indicated statistically significant difference.

Results: Mean age 68.7+/-9.9 in main/mixed-duct IPMN vs 70.8 +/-9.8 in BD-IPMN, majority females. Ethnicity, race, family and social history did not differ. BMI was 27 kg/m2 in both groups. Patients with obesity did not have higher odds of MD-IPMN OR, 0.49 (95% CI, 0.21-1.14; P = 0.082). Patients with main/mixed-duct IPMN had greater odds of weight loss but was not statistically significant OR, 2.64 (1.22-5.73, P = 0.083). Of 46 patients with MD-IPMN, 42 (91%) had surgery & 4 (9%) were observed; 25 (60%) had malignancy & 17 (40%) had IPMN only.

Conclusion: Study did not demonstrate an association between obesity & main/mixed-duct IPMN but did reveal higher odds of weight loss in this group although it did not reach statistical significance. This could be potentially be explained by small sample size. Studying a larger cohort should be considered, recognition of risk factors may result in earlier diagnosis of MD- IPMN that could potentially lower the incidence of PDAC.

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Colloid vs Tubular Carcinoma Arising From IPMN: The Better Survival is Independent of Stage

C. Rodrigues,1 V. Morales-Oyarvide,1 M. Mino-Kenudson,2 T. Hank,1 M. Qadan,1 C. Ferrone,1 A.L. Warshaw,1 K.D. Lillemoe,1 C. Fernández-Del Castillo.1 Departments of 1 Surgery and 2Pathology, Massachusetts General Hospital, Boston, MA.

Background: Invasive intraductal papillary mucinous neoplasia (IPMN) of the pancreas is a heterogenous entity with tubular and colloid carcinoma being the two most common subtypes. Colloid carcinoma is thought to have a better survival compared to the tubular variant following resection although it remains unclear if this is due to presentation at a different tumor stage. Our aim was to compare the clinicopathological characteristics and survival after adjusting for stage between the colloid and tubular subgroups.

Methods: From a prospective database of resected IPMNs, all cases with a final pathological diagnosis of an invasive IPMN with colloid or tubular histology were analyzed. We compared the clinicopathological features and evaluated progression-free survival (PFS) and overall survival (OS) using multivariate Cox regression adjusting for age, sex, pT and pN stage, tumor grade, resection margins, perineural and vascular invasion and adjuvant therapy.

Results: Of the 100 patients identified, 36 had colloid carcinoma and 64 had tubular carcinoma. There were no differences in clinicopathological features (including TNM-stage) between the two groups except for significantly higher perineural (P = 0.042) and vascular invasion (P = 0.045) in the tubular subgroup. Median PFS for the colloid subgroup was 108.4 months vs 20.8 months in the tubular subgroup with 5-year PFS of 62.9% vs 35.6%, respectively (P = 0.013). Median OS was 155.7 months in the colloid subgroup vs 35.9 months in the tubular subgroup with 5-year OS of 67.3% vs 45.8%, respectively (P = 0.009). In multivariate analyses, colloid carcinoma was associated with significantly longer PFS (HR, 0.43; 95% CI, 0.21-0.86, P = 0.018) and OS (HR, 0.47; 95% CI, 0.22-0.98, P = 0.047) compared with the tubular subgroup.

Conclusion: Colloid carcinoma arising from IPMN has a markedly better PFS and OS compared to tubular carcinoma. This is independent of tumor stage and other clinicopathological features, and suggests a different underlying tumor biology between these cancer subtypes.

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Portal Hypertension in Pancreatic Cancer is Associated With Radiation Therapy

V. Roy,1 G. Cook-Wiens,1 M. Guan,2 V. Placencio,1 A. Torosian,2 J. Gong,3 I. He,1 A. Osipov,4 R. Tuli,1 A. Hendifar.1 1 Cedars-Sinai Medical Center, Los Angeles, CA; 2 University of California Los Angeles, Los Angeles, CA; 3 City of Hope National Medical Center, Los Angeles, CA; 4Johns Hopkins Hospital, Baltimore, MD.

Background: Ascites is a complication in 20% of pancreatic cancer patients but there is limited data on risk factors and treatment. Our hypothesis is that ascites in pancreatic cancer is a complication of portal hypertension and factors such as prior radiation therapy and SMAD4 genetic mutation are associated with an increased risk of developing ascites. Radiation may lead to physiologic changes such as lymphatic injury that can predispose patients to ascites. SMAD4 mutation has been linked with an aggressive subset of cancer and may be associated with the development of advanced complications including ascites.

Methods: We conducted an IRB approved review of a prospectively collected database of 565 consecutive pancreatic cancer patients at a single institution. We analyzed variables including presence of ascites, serum ascites albumin gradient, treatment modalities for ascites, SMAD4 alterations by targeted next generation sequencing, and administration of radiation therapy for patients’ pancreatic cancer.

Results: 302 of 565 patients (53%) developed ascites in their pancreatic cancer course. 164 of these patients were on diuretics, 119 underwent paracentesis, and 32 had placement of drainage catheters. 83 out of 85 patients (98%) who had ascitic fluid analysis had SAAG >1.1 g/dL. 65% of patients without SMAD4 mutation and 67% of patients with the mutation developed ascites. 67% of patients who received radiation therapy developed ascites compared to 47% of those who had not (Fisher’s exact test P < 0.01).

Conclusion: Ascites due to portal hypertension is a significant complication of pancreatic cancer. Radiation therapy appears to be a risk factor for the development of ascites. SMAD4 mutations were not found to have an association with the development of ascites. A variety of methods are employed empirically for palliation of ascites and further investigation is warranted to develop an evidence-based approach to management of ascites in pancreatic cancer.

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Insulin Protects Against Acinar Cell Injury During Experimental Pancreatitis by Inducing the Phosphorylation of AKT and its Downstream Target PFKFB2

R. Sánchez-Alvarez,1 M.D. Sans,2 B. Nelson,2 J.A. Williams,2 J.I.E. Bruce.1 1 Division of Cancer Sciences, School of Medical Sciences, Faculty of Biology, Medicine and Health, The University of Manchester, Manchester, United Kingdom; 2Department of Molecular and Integrative Physiology, University of Michigan, Ann Arbor, MI.

Background: Several lines of evidence suggest a link between diabetes and severity of acute pancreatitis (AP) and thus a putative protective role of insulin. We investigated the protective effect of insulin using cellular and in vivo models of AP in Pancreatic Acinar cell- specific Conditional Insulin Receptor Knock Out mice (PACIRKO).

Methods: Insulin receptors were specifically deleted in pancreatic acinar cells of PACIRKO mice using a tamoxifen-inducible elastase promoter driven Cre-recombinase system. Both PACIRKO and littermate control mice were fed tamoxifen for 4 days and pancreatitis was induced in all of them on day 8 using a cerulein model or the palmitoleic acid (POA)/ethanol model. Histological features of pancreatic injury and inflammation were assessed by H&E and immunohistochemistry. Fura-2 imaging and an in situ Ca2+ clearance assay were used to assess cytotoxic Ca2+ overload and plasma membrane Ca2+ pump (PMCA) activity in collagenase- digested pancreatic acinar cells. Western blotting, using antibodies for phospho-Akt and phospho-PFKFB2 were used to determine the downstream mechanisms of insulin.

Results: Pancreatitis was exacerbated in PACIRKO mice with pancreatitis compared to control mice, as indicated by the analysis of pancreas wet/dry weight ratio, histological features of oedema, necrosis and inflammation (H&E and CD45 staining) and pancreatic tissue cytokine expression (TNFα, IL1β, and IL6). In isolated control pancreatic acinar cells, insulin attenuated the POA -induced Ca2+ overload and inhibition of the PMCA. Insulin induced phosphorylation of Akt and its downstream glycolytic enzyme 6-phosphofructo-2- kinase/fructose-2,6-biphosphatase 2 (PFKFB2). All these effects were abolished in acini from PACIRKO mice.

Conclusion: These data suggest that insulin directly protects acinar cells during pancreatitis due, at least in part, to up-regulation of glycolytic ATP to fuel the PMCA and thus preventing cytotoxic Ca2+ overload and necrosis. This provides the first mechanistic link between diabetes, and the loss of insulin protection, and the severity of acute pancreatitis.

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Assessment of Perioperative Body Composition at Bioimpedance Vector Analysis (BIVA) Predicts Morbidity Following Pancreatic Resection for Cancer

M. Sandini,1 S. Paiella,2 M. Cereda,1,3 M. Angrisani,1 G. Capretti,3 F. Casciani,2 A. Zerbi,3 C. Bassi,2 L. Gianotti.1 1 School of Medicine and Surgery, San Gerardo Hospital, University of Milano-Bicocca, Monza, Italy; 2 Unit of General and Pancreatic Surgery, The Pancreas Institute, Policlinico GB Rossi, University of Verona Hospital Trust, Verona, Italy; 3Pancreatic Surgery, Istituto Clinico Humanitas, Milano, Italy.

Background: Bioimpedance vector analysis (BIVA) is a reliable tool to assess body composition and hydration status. Perioperative sarcopenia, sarcopenic obesity and hyperhydration have been associated with adverse outcomes after pancreatic surgery. Aim of our study was to evaluate whether perioperative BIVA predicts the occurrence of surgery-related morbidity.

Methods: We prospectively measured BIVA of patients undergoing pancreatic resection for cancer at 3 Italian institutions. BIVA was measured prior to surgery and on postoperative day (POD) 1.

Postoperative morbidity was graded per the Clavien-Dindo Classification (CDC), the Comprehensive Complication Index (CCI) and the ISGPS classification.

Results: We analyzed 249 patients. Overall complication rate was 61% and 16.5% of the cases had a CDC ≥ 3. The median CCI was 24 (IQR, 0.0-24.2), and 24 patients (9.6%) had a CCI of ≥ 40 (severe complications).

Preoperative extracellular water (ECW) was significantly higher in patients who experienced severe morbidity according to the CDC [19.4 L (17.5-22.0) vs. ECW 18.2 L (15.6-20.6), P = 0.009 in complicated and uncomplicated, respectively]; ECW was significantly higher in case of either CCI≥40, or not [ECW 20.3 L (18.5-22.7) vs. 18.3 L (15.6-20.6), P = 0.002, respectively].

Preoperative fat mass (FM) was significantly higher in patients developing pancreatic fistula [FM 20.9 kg/m2 (12.4-30.2) vs. 14.6 kg/m2 (9.8-18.8), P < 0.001, respectively].

Hydration status on POD1 was significantly different in patients who either experienced major complications or not, at the CDC and CCI [ECW 23.9 L (20.6-22.5) vs. 19.7 L (17.3-23.1), P = 0.020 and hydration index HI 80.7% (75.3-85.3) vs. 73.9% (73.6-79.8), P < 0.001, for CDC and ECW 24.8 L (20.3-26.7) vs. 19.8 L (17.5-23.5), P = 0.025 and HI 80.4 L (77.1-87.2) vs. 74.0% (73.6-80.7), P = 0.002, for CCI].

Conclusion: Perioperative hydration status significantly predicts major morbidity after pancreatic surgery. BIVA can be useful for rapid evaluation and monitoring of the hydration status and could be considered in clinical practice.

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Preoperative Biliary Stenting and Major Morbidity After Pancreatoduodenectomy - Does Elapsed Time Matter?

M. Sandini,1 K.C. Honselmann,2 D.J. Birnbaum,3 F. Gavazzi,4 M. Chirica,5 U. Wellner,2 T. Guilbaud,3 L. Bolm,2 M. Angrisani,1 V. Moutardier,3 M. Cereda,1,4 É. Girard,5 M. Montorsi,4 T. Keck,2 A. Zerbi,4 L. Gianotti.1 1 Department of Surgery, School of Medicine and Surgery, Milano Bicocca University, San Gerardo Hospital, Monza, Italy; 2 Department of Surgery, University Medical Center Schleswig-Holstein, Campus Luebeck, Luebeck, Germany; 3 Department of Digestive Surgery, Hôpital Nord, Aix-Marseille University, Marseille, France; 4 Pancreatic Surgery Unit, Department of Surgery, Humanitas Research Hospital and University, Rozzano, Milan, Italy; 5Department of Digestive Surgery and Liver Transplantation, Hôpital Michalon, Grenoble University, Grenoble, France.

Background: The effect of preoperative stent duration on postoperative outcomes after PD has not been investigated. We aimed to analyze possible associations between the duration of stent placement before surgery and the occurrence and severity of postoperative complications following pancreatoduodenectomy (PD).

Methods: From 2013 to 2016 patients who underwent PD for any reasons after biliary stent placement at 5 European academic centers were analyzed from prospectively maintained databases. The primary aim was to investigate the association between the duration of preoperative biliary stenting and postoperative morbidity. Patients were stratified by stent duration into three groups: short (< 4 weeks), intermediate (4-8 weeks) and long (≥ 8 weeks).

Results: 312 patients were analyzed. The median time from stent placement to surgery was 37 days (2-559 days) and most operations were performed for pancreatic cancer (67.6%). Morbidity and mortality rates were 56.0% and 2.6%, respectively. Patients in the short group (n = 106) experienced a higher rate of major morbidity (43.4% vs. 20.0% vs. 24.2%, P < 0.001), biliary fistulae (13.2% vs. 4.3% vs. 5.5%, P = 0.031) and length of hospital stay [16 (10-52) days vs. 12 (8-35) days vs. 12 (8-43) days, P = 0.025]. A multivariate adjusted model identified the short stent duration as an independent risk factor for severe postoperative morbidity (OR, 2.64; 95%CI, 1.23-5.67; P = 0.013).

Conclusion: When jaundice treatment cannot be avoided, delaying surgery up to 1 month after biliary stenting may reduce major morbidity, procedure-related complications, and length of hospital stay.

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Electronic Medical Record or Registry Adaptable Tool to Identify High- utilizers in Pancreatitis

S. Sarvepalli,1,2,3 S.M. Sarvepalli,4 M. Parikh,1 P.N. Thota,2 M.R. Sanaka.2 Departments of 1 Hospital Medicine and 2 Gastroenterology and Hepatology, Cleveland Clinic, Cleveland, OH; 3 Department of Bioinformatics, Vanderbilt University, Nashville, TN; 4College of Liberal Arts and Sciences, University of Michigan, Ann Arbor, MI.

Background: Some patients with pancreatitis (high utilizers) require more frequent readmissions than most— resulting in significant healthcare costs. In this study, in addition to identifying factors associated with being a high utilizer, we aim to create an electronic medical record (EMR) or registry adaptable tool to identify these patients.

Methods: Nationwide readmission database (NRD) was interrogated to identify index hospitalizations for pancreatitis (ICD9: 577.0, 577.1) between January and September of 2014. Patients who had ≥3 readmissions within 90 days were defined as high utilizers. Automated stepwise forward logistic regression was used to identify demographic and clinical factors associated with being a high utilizer. Risk stratification tool was created based on this multivariate model. ROC analysis (internal validation with 1000- bootstrapped resamples) and calibration plots were created to evaluate the predictive ability of the model.

Results: N = 62,697 index admissions were identified (mean age, 52; 47.5% female) and 2937 (4.7%) were identified as high-utilizers. High-utilizers were responsible for 10,856 (36.2%) of readmissions over 90 days. Including the index hospitalization, 90-day aggregate cost for high-utilizers was $532 million, and per-person cost was $181,353. Multiple demographic and clinical factors from index admission were associated with being a high-utilizer. ROC analysis resulted in area under the curve (AUC) 0.748 (95% CI, 0.739-0.756) with similar values with analysis on boot-strapped resamples. Calibration plot demonstrates excellent fit using the multivariate model.

Conclusion: High-utilizer status is associated with significant morbidity and healthcare costs. In this nationally representative sample, we have identified factors associated with being a high utilizer following index admission with acute pancreatitis. Our model can be easily adapted into EMR or a registry to identify patients at highest risk of being a high-utilizer. Targeting healthcare resources specifically to these patients can lead to significant reduction in morbidity and healthcare costs.

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Electronic Medical Record or Registry Adaptable Tool to Stratify Risk of Short- and Long-term Readmissions in Acute Pancreatitis

S. Sarvepalli,1,2,3 S.M. Sarvepalli,4 M. Parikh,1 P.N. Thota,2 M.R. Sanaka.2 Departments of 1 Hospital Medicine and 2 Gastroenterology and Hepatology, Cleveland Clinic, Cleveland, OH; 3 Department of Bioinformatics, Vanderbilt University, Nashville, TN; 4College of Liberal Arts and Sciences, University of Michigan, Ann Arbor, MI.

Background: Acute pancreatitis continues to be one of the leading gastrointestinal causes of hospitalizations and readmissions. In this study we aimed to create an electronic medical record (EMR) based risk stratification tool to risk-stratify short- and long-term risk of readmission for pancreatitis or related complications (PRC) after an index admission for acute pancreatitis.

Methods: National readmission database was interrogated to identify index admissions with primary diagnosis of acute pancreatitis (ICD9-577.0) during the first 3 months of 2014. Demographic, clinical, and readmission (within 270 days for primary diagnosis of PRC: 577.0, 577.1, 577.2) details were obtained. Kaplan-Meier curve was created, followed by forward stepwise cox-regression analysis to identify factors associated with first readmission. ROC analysis was performed to identify predictive ability of the model. Internal validity was confirmed on 1000 boot-strapped resamples.

Results: N = 29,021 index admissions (mean age, 52.1; 47.1% female) resulted in 23.4% PRC related readmissions in 270 days (30-days = 7.9%). Demographic, clinical, and procedural factors were associated with time to readmission for PRC. ROC analysis yielded area under the curve of 0.737 (95% CI, 0.730-0.744). ROC analysis on 1000-bootstrapped samples yielded the exact AUC suggesting excellent internal validity. Risk score was developed based on the multivariate model. Figure-2 demonstrates actual readmission rates in the study cohort based on percentile of risk score.

Conclusion: Although patients are at the highest risk of readmission within 30 days of discharge, this accounts for less than half the readmissions. In addition to identifying factors associated with short- and long-term readmission, we created a generalizable (given national cohort) tool that uses coded data available in EMR to risk-stratify patients within a registry or during a post-hospital office visit. Patients thus identified can be triaged for closer monitoring–preferably by a gastroenterologist as these appointments are related to PRC—or targeted for other interventions.

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Impact of Discharges Against Medical Advice in Hospital Stays for Pancreatitis and Factors Associated With It

S. Sarvepalli,1,2,3 S.M. Sarvepalli,4 M. Parikh,1 P.N. Thota,2 M.R. Sanaka.2 Departments of 1 Hospital Medicine and 2 Gastroenterology and Hepatology, Cleveland Clinic, Cleveland, OH; 3 Department of Bioinformatics, Vanderbilt University, Nashville, TN; 4College of Liberal Arts and Sciences, University of Michigan, Ann Arbor, MI.

Background: Discharging a patient against medical advice (AMA) is associated with poor outcomes, and hospitalizations for pancreatitis are especially prone to these events. In this study, in addition to assessing the burden of readmission rates associated with AMA discharges in pancreatitis, we use modelled data to examine the impact of reducing AMA discharge rate on costs and readmissions. Next we identify factors associated with AMA discharges on index admission.

Methods: Nationwide readmission database was interrogated to identify index hospital stays with a primary diagnosis of pancreatitis (ICD9:577.0, 577.1) between January and November of 2014. Multivariate analyses were used to identify adjusted 30-day readmission rates and total 30-day hospital charges (charges associated with index admission + readmission within 30-days) associated with AMA discharge after adjusting for demographic, clinical, and hospital factors. Next, multivariate logistic regression analysis was used to identify factors associated with AMA discharges based on data from index admission.

Results: Of the N = 107,282 index hospitalizations for pancreatitis, 3.7% were discharged AMA. Adjusted 30-day readmission rate for AMA discharges was 26.3% ±0.7%, compared to 16.1% ±0.1% for non-AMA discharges (P < 0.001). Total 30-day hospital charges associated with AMA discharges ($51,816±$926) was significantly higher than patients who did not leave AMA ($42,507 ±$177; P < 0.001). Male sex, Medicaid or self-pay patients, lower median income, having psychiatric disorders, and tobacco or alcohol/substance use are associated with AMA discharges. Older age, non-urban resident, and certain comorbid conditions are associated with reduced risk of AMA discharge.

Conclusion: AMA discharges in pancreatitis are associated with higher readmission rates and healthcare costs. We have identified factors associated with AMA discharges. Our research using a nationally representative sample can inform local and nation healthcare policy. Future research can focus on strategies to reduce AMA discharges in these high-risk patients.

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Efficient Systemic Treatment With Mesothelin-Targeted Oncolytic Adenovirus in Patient- Derived Xenograft (PDX) Model of Pancreatic Cancer

M. Sato-Dahlman, P. Hajeri, H. Yoshida, K. Jacobsen, C. Yanagiba, M. Yamamoto. Department of Surgery, University of Minnesota, Minneapolis, MN.

Background: Pancreatic ductal adenocarcinoma (PDAC) is an aggressive cancer and frequently found after spread. Oncolytic Adenovirus (OAd) is a novel promising therapeutics. While it is efficient, adenovirus has liver tropism due to high expression of primary adenovirus receptor in hepatocytes and capture of the virus by Kupffer cells. This large sequestration hampers the tumor transduction and efficacy upon systemic administration. Therefore, improvement of vector selectivity for avoidance of liver sequestration and efficient transduction is necessary for realization of systemic treatment of spread and/or metastatic PDAC lesions with OAd.

Methods: To improve the systemic distribution, we have generated the PDAC-targeted OAd by high- throughput screening of Ad-fiber library in mesothelin (MSLN) expressing cells.

Results: The targeted OAd binds to MSLN protein, which is overexpressed on the surface of PDAC. MSLN-targeted OAd showed selective and powerful anti-tumor effect against Panc-1 xenografts in both intratumoral and intravenous injection. Importantly, the liver sequestration of MSLN-targeted OAd after systemic injection was less than 1/10 of OAd without targeting (Ad5WT). At day 7, the virus distribution of MSLN-targeted OAd in the tumor was significantly higher than Ad5WT. These results suggest that MSLN-targeted the OAd significantly lowered liver sequestration and increased tumor accumulation upon systemic injection. Regrown tumors were treated with repeated MSLN-targeted OAd injection, and four out of six tumors were controlled. Next, antitumor effect the OAd was tested in patient-derived xenograft (PDX) model which better resembles clinical features of PDAC in patients. After intravenous administration, the MSLN- targeted OAd showed significant antitumor effect compared to the untreated group (P < 0.05), while Ad5WT showed no effect.

Conclusion: In this study, systemic injection of MSLN-targeted OAd showed remarkable anti-tumor effect in both systemic and intratumoral injections at low dose. Our results indicated that tumor targeted-OAd can realize efficient systemic treatment for PDAC which are mostly found with spread or metastatic lesions.

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The Pancreatitis Activity Scoring System and Timing of Oral Refeeding in Acute Pancreatitis

A. Satoh,1 Y. Tanaka,1 F. Shimoda,1 H. Okata,1 K. Hiramoto,1 O. Kimura,1 S. Asonuma,1 K. Umemura,1 M. Hirota,2 T. Shimosegawa.1,2 1 Department of Gastroenterology, South Miyagi Medical Center, Ohgawara, Japan; 2Division of Gastroenterology, Tohoku University Graduate School of Medicine, Sendai, Japan.

Background: Initiation of oral feeding in patients recovering from acute pancreatitis (AP) may cause relapse of pancreatitis but there is no guidance available for the optimal timing of oral refeeding. The Pancreatitis Activity Scoring System (PASS) has been developed by an international group of experts via a modified Delphi process as a tool for objective measurement of real time disease activity. The aim of the present study was to evaluate the usefulness of the PASS score in timing the initiation of oral feeding. We also assessed the relationship between the PASS score and clinical outcomes.

Methods: We conducted a prospective multicenter cohort study. A total of 91 patients with AP were consecutively enrolled. For assigning the severity of pancreatitis, we used Atlanta criteria (2012). The PASS score, demographic, clinical, biochemical and radiological data were recorded and analyzed. The decision to initiate oral feeding in each patient was based on clinician judgement. Nutrition related complications (relapse of pancreatitis, abdominal pain, nausea and vomiting, diarrhea, aspiration pneumonia) were also noted.

Results: 23 suffered from severe AP and 68 from mild AP. At admission, the mean PASS score was 224 in the severe AP group and 131 in the mild AP group. Patients with mild disease demonstrated a rapid decline in PASS scores, whereas the patients who had persistently high PASS scores developed infected walled-off necrosis in the later clinical course. During the oral feeding period, 16 had abdominal pain, including 2 with relapse of pancreatitis. Notably, PASS score >70 on the day of initiating oral feeding was associated with abdominal pain relapse.

Conclusion: These results suggest that assessment of disease activity by the PASS is a practically useful to determine appropriate timing for initiation of oral refeeding.

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Manipulation of the Gut Microbiota Modulates Pancreatic Cancer Burden in Mice

V. Sethi,1 S. Kurtom,1 I. Fernandez,2 J. Kobinger,2 A. Ferrantella,1 H.K. Charles Jacob,1 B. Garg,1 B. Giri,1 P. Sharma,1 P. Roy,1 R. Dawra,1 M. Abreu,2 S. Roy,1 S. Ramakrishnan,1 A.K. Saluja,1 V. Dudeja.1 Departments of 1 Surgery and 2Gastroenterology, Sylvester Comprehensive Cancer Center, University of Miami, Miami, FL.

Background: Multiple cancers, including pancreatic ductal adenocarcinoma (PDAC), present with a changed ‘dysbiotic’ gut microbiota. Whether this dysbiotic state promotes cancer or is a neutral-bystander is still unclear. We aimed to study as well as investigate the immunotherapeutic implications of this relationship.

Methods: To evaluate if PDAC-bearing hosts have cancer-promoting gut microbiota, freshly-weaned wildtype mice (guests) were co-housed with two types of ‘host’ mice: 3-month old cancer- bearing KPC (KrasG12D/+ ;Trp53R172H/+;Pdx-1-Cre) mice or with 3-month old wildtype cancer- naïve mice. Subsequently, the guest mice were given subcutaneous KPC-PDAC and tumor progression was monitored. In following experiments, wildtype mice were depleted of their entire gut microbiome by oral-antibiotics and growth of orthotopic, subcutaneous and metastatic PDAC was compared between them and controls. Specific antibiotics were also combined with immune checkpoint-inhibitors preclinically. Pancreatic tumors and metastases were probed for bacteria and were immunophenotyped through flowcytometry.

Results: Mice co-housed with KPC cagemates had significantly increased tumor burden than mice co- housed with cancer-naïve mice. Depleting gut microbiota dramatically decreased tumor burden in all models of PDAC. Antibiotics also significantly potentiated the efficacy of conventional immunotherapy in PDAC-bearing mice. Interestingly, pancreatic tumors grew viable bacteria on culture medium and this was prevented by poorly-absorbable oral antibiotics, thereby suggesting that gut bacteria were translocating to remote tumor sites. 16S rRNA gene sequencing of KPC metastases confirmed that PDAC metastases also supported a rich microbial ecosystem similar to the gut. Flowcytometry results suggested that gut bacteria activated an IL17-orchesterated pro-tumorigenic myeloid cell inflammation inside the tumor. Neutralizing various nodes of this inflammatory axis by monoclonal antibodies abrogated the tumor-suppressing effects of antibiotics thereby, revealing a possible mechanism.

Conclusion: Our data presents early evidence on the tumor-enhancing potential of gut dysbiosis in cancer- bearing hosts and on the potential use of oral-antibiotics as novel immunotherapeutic agents against pancreatic cancer.

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Metabolically Reprograming Pancreatic Stellate Cells Using a Novel Nanomedicine for the Treatment of Pancreatic Cancer

G. Sharbeen,1 A. Akerman,1 J. McCarroll,2,3 C. Boyer,3 J. Holst,4 J. Youkhana,1 T.P. Davis,5 D. Goldstein,1,6 M. Erkan,7 P.A. Phillips.1,3 1 Pancreatic Cancer Translational Research Group, Prince of Wales Clinical School and School of Medical Sciences, Lowy Cancer Research Centre, UNSW Sydney, Sydney, Australia; 2 Children's Cancer Institute, Lowy Cancer Research Centre, UNSW Sydney, Sydney, Australia; 3 Australian Centre for Nanomedicine (ACN), ARC Centre of Excellence in Convergent Bio-Nano Science and Technology, UNSW Sydney, Sydney, Australia; 4 Origins of Cancer Program, Centenary Institute, University of Sydney, Sydney, Australia; 5 ARC Centre of Excellence in Convergent Bio-Nano Science and Technology, Monash Institute of Pharmaceutical Sciences, Monash University, Melbourne, Australia; 6 Prince of Wales Hospital, Prince of Wales Clinical School, Sydney, Australia; 7Department of Surgery, Koc University, School of Medicine, Istanbul, Turkey.

Background: Pancreatic cancer (PC) is a lethal disease characterised by extensive fibrosis that distorts tumor vasculature creating hypoxia/oxidative stress (OS). This drives PC tumor progression and chemoresistance, and impedes drug delivery. Cancer-associated pancreatic stellate cells (CA- PSCs) orchestrate fibrosis and promote PC growth/metastases, making them key therapeutic targets. Solute carrier 7A11 (SLC7A11) is part of the xCT transport system that controls cystine uptake, required for glutathione (antioxidant) synthesis. We have shown SLC7A11 is upregulated in CA-PSCs, compared to normal human PSCs. We hypothesised that SLC7A11 inhibition will reduce CA-PSC survival and resistance to oxidative stress.

Aims: To assess the effect of SLC7A11 inhibition on (1) CA-PSC growth and metabolism in vitro and (2) pancreatic tumor growth in vivo using a nanomedicine.

Methods: (1) Human patient-derived CA-PSCs (n = 3-5) were transfected with control or SLC7A11-siRNA ± oxidative stress (t-butyl hydroperoxide; tBHP) and viability (trypan blue exclusion), SLC7A11 function (14C-cystine uptake; glutathione), and intracellular OS (CellROX) were measured. (2) Orthotopic PC tumors (PC+CA-PSCs) in mice were treated with control or SLC7A11-siRNA coupled to our novel nanoparticle (Biomacromolecules, 17;2337-51, 2016). Tumor volume and fibrosis (Sirius red) was measured.

Results: (1) SLC7A11-siRNA reduced CA-PSC proliferation (62.2±7.5% of control-siRNA; P < 0.01) and oxidative stress enhanced this effect (7.6±2.6% of control-siRNA; P < 0.0001). SLC7A11 inhibition reduced CA-PSC cystine uptake (51.8±8.8% of control; P < 0.05) and intracellular glutathione (40.0±9.6% of control; P < 0.01), and increased intracellular OS (166.4±26.8% of control- siRNA+tBHP; P < 0.05). (2) SLC7A11 knockdown in orthotopic PC tumors significantly decreased tumor growth (43.9±23.1% decrease; P < 0.05) and fibrosis (45.6±12.3% decrease; P < 0.05), relative to controls.

Conclusion: (1) SLC7A11 regulates CA-PSC proliferation and sensitivity to oxidant stress. (2) Inhibition of SLC7A11 in PC tumors can reduce their growth and alleviate fibrosis. Therapeutic inhibition of SLC7A11 in PC using our novel nanomedicine could potentially improve PC patient outcomes by reducing tumor growth, and reprogramming CA-PSCs and fibrosis.

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Time Course of Metabolic and Soft Tissue Changes in Pre-diagnostic Pancreatic Cancer

A. Sharma,1 R.P. Sah,1 S. Nagpal,1 S.H. Patlolla,1 A. Sharma,2 H. Kandlakunta,1 R.S. Angom,2 S. Vivekanandan,2 N. Takahashi,3 D. Mukhopadhyay,2 S.T. Chari.1 1 Division of Gastroenterology and Hepatology, Mayo Clinic, Rochester, MN; 2 Department of Biochemistry and Molecular Biology, Mayo Clinic, Jacksonville, FL; 3Division of Radiology, Mayo Clinic, Rochester, MN.

Background: Pancreatic cancer (PC) presents with profound metabolic and soft tissue changes whose pre-diagnostic time course is unknown.

Methods: We generated temporal metabolic and soft tissue profiles for 60 months before PC diagnosis. For metabolic profile we compared annual mean fasting blood glucose, serum lipids, body weight and temperature in population-based PC (n = 219) and controls (n = 657). For soft tissue changes we compared to baseline (>18 months before diagnosis), the area of abdominal subcutaneous adipose tissue (SAT), visceral AT (VAT) and muscle at L2/L3 vertebra on subsequent computerized tomography scans in 68 subjects. Reported changes have P < 0.05. To investigate changes in SAT we studied normal human SAT exposed to PC exosomes and SAT of KRAS+/LSL G12DP53 flox/flox mice.

Results: No metabolic or soft tissue changes were noted before 30 months. The period -30 to - 18 months (Phase I, Hyperglycemia) was characterized by new-onset hyperglycemia without soft tissue changes; -18 to -6 months (Phase II, Pre-cachexia) by worsening hyperglycemia and decrease in serum triglycerides, low-density cholesterol, body weight and SAT with preserved VAT and muscle; and -6 to 0 months (Phase III, Cachexia) by marked hyperglycemia with decrease in serum lipids, SAT, VAT and muscle. That decrease in SAT was due to browning was suggested by i) increasing body temperature starting at -18 months ii) upregulation of genes associated with browning in normal human SAT exposed to PC exosomes, iii) decreased triglyceride content and adipocyte size and increased expression of uncoupling protein 1 (UCP1) in SAT from PC-bearing KRAS+/LSL G12D P53 flox/flox mice.

Conclusion: Pancreatic cancer has three distinct and progressive metabolic phases starting 2·5 years before diagnosis. SAT loss starts at -18 months and is likely due to its browning. UCP1 overexpression in SAT biopsy may be a biomarker of PC in weight-losing subjects with new-onset hyperglycemia.

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Pancreatic Ductal Adenocarcinoma is Associated With Elevated Peripheral Blood Monocyte Counts

A. Sharma, S.T. Chari, S. Majumder. Mayo Clinic, Rochester, MN.

Background: Pancreatic cancer (PDAC) is currently the 3rd leading cause of cancer mortality, with overall 5-year survival <10%. Early detection offers the best chance of prolonged survival. In absence of a reliable biomarker, detecting early stage PDAC continues pose a challenge. In our clinical experience we have observed elevated peripheral blood monocyte (PBM) counts in PDAC patients at diagnosis.

Methods: Using Rochester Epidemiology Project (REP) database, we identified all PDAC patients between January 2000 and January 2015 in Olmsted County, MN. From same population, 2 age- and sex-matched disease free controls per case were identified. For both groups we electronically abstracted all absolute PBM levels and analyzed temporal PBM count profiles collected for 24 months before patients received a PDAC. Monocytosis was defined as PBMC>90x 109/liter. Comparison was done using Fisher’s exact test (categorical variable) and two tailed t test (continuous variables). A P value <0.05 was considered significant.

Results: Mean PBM counts (109/liter) in PDAC patients at diagnosis were higher than controls (0.73 vs 0.59; P < 0.001). A higher proportion of PDAC patients manifested monocytosis at diagnosis compared to controls (23% vs 8%; P < 0.001). Comparing values between the groups 24 months prior to PDAC diagnosis, mean PBM counts were similar between cases and controls in the intervals from months 18 to 24 (0.61 vs 0.62; P = 0.95), 18 to 12 (0.62 vs 0.62; P = 0.91) and 12 to 6 (0.62 vs 0.61; P = 0.74), but significantly different in the interval from 6 months to 1 month prior to diagnosis (0.69 vs 0.61; P = 0.03).

Conclusion: Compared to controls, PBM counts are significantly higher in PDAC patients at diagnosis and PBM elevation precedes cancer diagnosis by 6 months. This novel observation can potentially add value to early detection models for PDAC, serving as an enrichment tool in screening high-risk cohorts such as new-onset diabetes.

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Targeting O GlcNAc Modification of SOX2 Affects Self-renewal in Pancreatic Cancer

N. Sharma, V.K. Gupta, P. Dauer, K.K. Kesh, R. Hadad, V. Dudeja, A.K. Saluja, S. Banerjee. Department of Surgery, University of Miami Miller School of Medicine, Miami, FL.

Background: Pancreatic Cancer is an extremely aggressive disease with a bad outcome owing to its late detection, high rate of metastasis and self-renewal post-surgical and chemotherapeutic intervention. In cancer the balance between differentiation and self-renewal is tightly regulated by SOX2. Over expression of SOX2, as in pancreatic cancer, can tilt the balance towards self-renewal and thus increase the population of these undifferentiated “stem cells”.

Methods: A CRISPR based OGT knockout kit was purchased from Origene and a knockout cell line was generated with S2VP10 cells. For the tumor initiation studies OGTi (OGT knockout cells) were implanted subcutaneously at a limiting dilution from 500,000 cells to 500 cells. Site directed mutagenesis was performed using the Quick-change Lightning from Agilent.

Results: In the current study we show for the first time that SOX2 undergoes a type of post translational modification known as O GlcNAc. We also show for the first time that in humans this modification happens at Serine 246 and mutating this particular site via a side directed mutagenesis leads to a loss of O GlcNAcylation of SOX2.O GlcNAc modification of SOX2 further leads to its activation which then leads to its stabilization in the nucleus. A CRISPR-OGT knockout in pancreatic cancer cell line S2VP10 resulted in a delayed tumor initiation. We further showed that mutation of this site (S246A) prevents the modification of Sox2 and its downstream activity. Our study also demonstrated that targeting OGT in vivo with a small molecule inhibitor OSMI, results in decreased tumor burden, delayed tumor progression and a decreased expression of SOX2 in pancreatic cancer cells.

Conclusion: Our study highlights for the first time that the O-GlcNAc transferase dependent SOX2 glycosylation has a profound effect on the transcriptional activity of SOX2 and is instrumental in determining self-renewal in pancreatic cancer which can be targeted therapeutically.

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GAS5 Induces Quiescence in Cancer Stem Cells by Preventing Anabolic Pathways From Fueling Proliferation

N. Sharma, B. Durden, P. Gnamlin, R. Hadad, V.K. Gupta, K. Kesh, V. Dudeja, A.K. Saluja, S. Banerjee. Department of Surgery, University of Miami Miller School of Medicine, Miami, FL.

Background: Cancer stem cells (CSC’s) are known to be quiescent and non-proliferative. This makes them difficult to treat via chemotherapy. In spite of being non proliferative, cancer stem cells also have been shown to channel glucose metabolites via the pentose phosphate pathway and the hexosamine biosynthesis pathway. These CSC’s have also been shown to have high expression of Growth arrest-specific 5/6 (GAS5/6). GAS5 is a non-protein coding RNA or a long noncoding RNA that accumulates in non-proliferating cells.

Methods: Constitutively over expressing CD133 cell line was made by transfecting MIA PaCa-2 cells with CD133 over expressing lentiviral vector. These cells were called A10’s.CD133 positive cells were sorted using either flow cytometry or magnetic separation. Fluorescence in situ hybridization (FISH) assay was used for the detection of GAS5 long non coding RNA.

Results: Our results show that in cancer stem cells glucose preferentially goes to the pentose phosphate pathway instead of the canonical glycolysis pathway. In spite of this accumulation of the metabolites in the cancer stem cells they do not show an increase in proliferation. Interestingly these CD133 positive cancer stem cells also have an increased expression and accumulation of GAS5 as seen by RT-PCR and FISH. SOX2 has been previously shown to regulate the expression of GAS5, we see a decrease in the expression of GAS5 with SOX2 inhibition. GAS5 inhibition also leads to a loss of self-renewal in these cells.

Conclusion: Unfavorable conditions from the microenvironment exerts selective pressure on the tumor due to which some cells turn off their proliferation by increasing the expression of GAS5/6. These genes inhibit the cell cycle at specific nodes to prevent proliferation. If we target GAS5/6 upstream via SOX2 inhibition, we can turn these breaks off and force the cancer stem cell to proliferate and thus make them sensitive to chemotherapy.

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Delineating Prognostic and Biological Subgroups in Pancreatic Cancer Using Patient Transcriptomic Profiles

S. Sharma,1 S. Kaur,1 J. Iqbal,2 L. Smith,3 S.K. Batra.1,4 1 University of Nebraska Medical Center, 2 Department of Pathology and Microbiology, 3 College of Public Health, 4Fred and Pamela Buffet Cancer Center,