Comparison of Surgical Outcome of Radical Antegrade Modular Pancreatosplenectomy With Standard Retrograde Pancreatosplenectomy and Evaluation of the Prognostic Factors in Left Side Pancreatic Cancer
T. Abe, K. Ohuchida, M. Nakamura. Department of Surgery and Oncology, Graduate School of Medical Sciences, Kyushu University, Fukuoka, Japan.
Background: Distal pancreatectomy (DP) with splenectomy is standard technique for left sided pancreatic cancers. Radical antegrade modular pancreatosplenectomy (RAMPS) is a modification of standard retrograde pancreatosplenectomy (SRPS) to achieve N1 LN dissection, early vascular control and negative posterior margins. However, there have been few studies on the clinical outcome to compare the RAMPS procedure with SRPS.
Method: Ninety-nine patients underwent DP for pancreas body and tail adenocarcinoma between January 2000 and October 2014. Clinicopathologic data were retrospectively analyzed in this study. We compared short- and long-term outcomes of RAMPS and SRPS. Furthermore, we identified prognostic factors associated with survival.
Results: Fifty-nine patients underwent RAMPS and forty patients underwent SRPS. RAMPS showed a larger number of retrieved lymph nodes [28.3 ± 11.8 vs 20.7 ± 10.1, p = 0.0018] and more frequent R0 resection [88.4% vs 67.5%, p = 0.0121]. RAMPS showed a tendency of less intraoperative bleeding than SRPS [500.0 ± 59.2 vs 682.3 ± 71.9, p = 0.053]. Perioperative morbidity and mortality rates were comparable. There were no significant differences in 5-year (33.8% vs 35.1%) and median (47 vs 34 mo) overall survival (P = 0.3806) between RAMPS and SRPS. Furthermore, there were no significant differences in 5-year (22.8% vs 21.0%) and median (25 vs 28 mo) disease-free survival (DFS) (P = 0.7908). In the multivariate analysis, R1 resection, histologic grade and vascular invasion for OS, and R1 resection, histologic grade, initial CEA (≥5 ng/ml) and positive cytology for DFS were independent factors.
Conclusions: RAMPS increased number of retrieved lymph nodes and R0 resection rate compared with SRPS although there were no significant differences in OS and DFS between RAMPS and SRPS.
Minimally Invasive Versus Open Pancreaticoduodenectomy for Pancreatic Adenocarcinoma: A National Perspective on Survival
M. Adam, S. Roman, J. Sosa. Department of Surgery, Duke University Medical Center, Durham, NC.
Background: There has been increasing interest in minimally invasive pancreaticoduodenectomy (MIPD). Recent data suggest that MIPD is associated with inferior short-term outcomes compared to open pancreaticoduodenectomy (OPD). Our aim was to compare survival from MIPD vs. OPD at the national level.
Methods: Patients undergoing pancreaticoduodenectomy for stage I and II pancreatic adenocarcinoma were included from the National Cancer Database (2010-2012). Bivariate/multivariate regression and survival analyses were employed to compare short-term outcomes and survival from MIPD vs. OPD.
Results: A total of 8468 patients met inclusion criteria: 1238 (15%) patients had MIPD and 7230 (85%) OPD. Use of MIPD increased from 314 (12%) cases in 2010 to 482 (16%) cases in 2012. After adjustment for patient, clinical, and pathologic characteristics, MIPD vs. OPD was associated with a similar rate of positive surgical margins (OR 1.03, p = 0.71) and shorter hospital length of stay (−0.3 days, p = 0.006). However, 90-day mortality was significantly higher (OR 1.79, p = 0.002) in the MIS vs. OPD group. Increasing hospital MIPD volume was associated with decreasing odds of 90-day mortality (OR 0.94, p = 0.004). In bivariate analysis, MIPD vs. OPD was associated with higher 90-day mortality for patients undergoing surgery at low-volume (<10 cases/yr) centers (11% vs. 8%, p = 0.05) but not at high-volume (≥10 cases/yr) centers (5% vs. 3%, p = 0.10). The majority of MIPD patients (63%) had surgery at low-volume centers that performed <10 cases/yr: 70% in 2010 and 64% in 2012. Median follow-up was 18 mos (range 1-48 mos). After adjustment, MIPD vs. OPD was associated with a similar overall survival (HR 1.05, p = 0.43).
Conclusions: Overall survival is similar for MIPD and OPD; however, MIPD is associated with increased risk for 90-day mortality. While increasing hospital volume is associated with lower 90-day mortality from MIPD, the majority of patients underwent the procedure at low-volume hospitals. Our results suggest the need for guidelines to inform identification of optimal case volumes for the safe implementation of MIPD.
Even Stage IIB Operated Pancreatic Ductal Adenocarcinoma (PDA) can Achieve Over 5 Year Survival: The Finnish Register Study 2000–2013 With Pathological Re-Evaluation
R. Ahola,1 A. Siiki,1 K. Vasama,2 J. Sand,1 J. Laukkarinen.11Dept. of Gastroenterology and Alimentary Tract Surgery, Tampere University Hospital, Tampere, Finland; 2Dept. of Pathology, Fimlab Laboratories, Tampere University Hospital, Tampere, Finland.
Introduction: The survival of patients with operated pancreatic ductal adenocarcinoma (PDA) has been associated with tumour-free resection margins, small tumour size and centralisation. However, no previous reports about long-term survivors of PDA with a confirmed histology covering a whole single nation exist.
Aims: Our aim was to analyse characteristics of PDA long term survivors operated in Finland 2000-2008.
Patients and Methods: Combining the information from the Finnish national cancer register, operation register and patient archives, PDA patients were selected. Histological slides of patients with over 5-year survival were re-analyzed by an expert pancreatic pathologist. Operation, demographic and oncologic data were recorded. The cut point of survival was 31.12.2013.
Results: Out of the 580 operated PDA patients, 75 survived over 5 years. 7 patients were excluded due to missing histological slides and 23 after re-analysis of histology. By the end of May 2015, 35 of the long-term survivors were confirmed as PDA. Median age at the time of operation was 61 years, 45% were female and 51% had no previous medical history. 57% received adjuvant therapy. The range of survival among long-term survivals was 5.0-13 years. 22 patients were still alive at the time of analysis. 60% were operated in the university (teaching) hospitals. 57% of the patients had R0 resections and the tumours were staged as IA/IB/IIA/IIB in 6.5/6.5/35/48%.
Conclusions: This is the largest reported single-nation cohort of long-term survivors with histologically confirmed PDA. Long-term survival can be achieved even in stage IIB and with R1 resection. For the length of survival, we suspect that tumour biology may play a more important role than tumour size or negative resection margin.
Development of a Novel Biocompatible Bond for the Prevention of Pancreatic Fistulas
M. Aikawa, M. Miyazawa, S. Ishida, K. Okada, Y. Watanabe, K. Okamota, I. Koyama, T. Taguchi. Saitama Medical University, International Medical Center, Hidaka, Saitama, Japan.
Pancreatic fistulas (PFs) are often associated with severe complications, such as intra-abdominal abscess and hemorrhage. Although many techniques have been developed for the prevention of PFs, an ideal prevention method is lacking. We have developed a novel biocompatible bond (BCB) for organ repair or adhesion. In the present study, we compared the safety and effectiveness of our BCB to those of conventional fibrin glue (FG) in pigs.
Materials and Methods: The BCB includes a cross-linking agent synthesized from organic acid and a biological polymer confirmed to be of high-strength and low toxicity in clinical use. Six pigs were laparotomized under general anesthesia, and the body and tail of the pancreas were exposed. A defect was prepared on the surface of the pancreatic body (10 × 10 mm), and a stump was prepared on the pancreatic tail. In 3 of the 6 pigs, NVs coated with the BCB were patched and fixed on the pancreatic defect and stump (BCB group), and in the remaining 3 pigs, NVs coated with FG were patched and fixed on the pancreatic defect and stump (FG group).
Results: All of the operated pigs survived until sacrifice. At 2 weeks after the operation, in the BCB group, the defect and stump were coated with NV and membranous tissue, while in the FG group, the defect and stump showed severe adhesion with the surrounding bowel. At 8 weeks after the operation, in the BCB group, the defect and stump were covered with thin connective tissue without adhesion, while in the FG group, the stump had infected pseudocyst and the defect showed severe adhesion with the surrounding bowel. These findings indicate that PFs occurred in the FG group but not in the BCB group.
Conclusion: The BCB coated on an NV is safe and effective for the prevention of PFs resulting from pancreatic injury and may be used in the clinical setting.
Verification of the Utility of International Consensus Guidelines 2012 for the Management of IPMN and MCN of the Pancreas
J. Akao,1 K. Shimizu,1 T. Ajihara,1 K. Nagao,1 J. Tahara,1 Y. Takayama,1 K. Shiratori,1 M. Yamamoto,2 T. Furukawa.31Department of Gastroenterology; 2Gastroenterological Surgery; 3Institute for Integrated Medical Science, Tokyo Women's Medical University, Tokyo, Japan.
Introduction: The international consensus guidelines 2012 are quite useful for suspecting the possibility of malignancy in IPMN patients, but it is still difficult to make a definitive preoperative diagnosis based on the diagnostic imaging findings, especially in BD (branch-duct)-type IPMN.
Aim and Methods: To determine whether the diagnostic algorithm of the guidelines is useful for differentiating malignant IPMNs from benign IPMNs we compared the preoperative diagnostic imaging findings and the postoperative histological grading of the lesions of 50 patients who underwent surgery for IPMNs in our hospital.
Results: Of the 7 MD (main-duct)-type-IPMNs, 1 was graded as invasive carcinoma, 4 as high-grade dysplasia, and the malignancy rate, which included invasive carcinoma and high-grade dysplasia, was 71.4%. The grade of the BD-IPMN was invasive carcinoma in 4 cases, high-grade dysplasia in 12 cases, and the malignancy rate was 39.0%. Comparison between the preoperative classifications and the histological diagnoses showed that 2 of the 12 BD-IPMNs with high-risk stigmata were histologically diagnosed as invasive carcinoma and 6 as high-grade dysplasia, and that 2 of the 15 BD-IPMNs with EUS features in the presence of worrisome features were diagnosed as invasive carcinoma and 5 as high-grade dysplasia, thereby resulting in the malignancy rate of 55.6%. Both of the 2 patients with invasive carcinoma classified as a BD-IPMN with EUS features in the presence of worrisome features had a family history of pancreatic cancer.
Conclusion: The guidelines 2012 were quite useful for differentiating malignant IPMNs from benign IPMNs. We recommend more aggressive surveillance of IPMN patients who have a family history of pancreatic cancer.
Predictors of 30-Day Readmission in Patients Undergoing Pancreatic Resections for Neoplastic and Inflammatory Diseases of the Pancreas in a Single Tertiary Healthcare Institution
L.I. Amodu,1 A. Levy,1 M. Akerman,2 M. Tiwari,1 G.I. Georgiev,1 M. Beltran Del Rio,1 J. Nicastro,1 G.F. Coppa,1 E. Molmenti,1 H.L. Rilo.11Center for Diseases of the Pancreas, Department of General Surgery, Hofstra North Shore-LIJ School of Medicine, Manhasset, NY; 2Department of Biostatistics, Feinstein Institute for Medical Research, Manhasset, NY.
Introduction: Hospital readmissions following surgery have important implications in patient recovery, physician-specific outcome measures and healthcare cost. 30-day readmissions are usually attributed to the index surgical procedure and post-operative complications. We sought to determine predictors of 30-day readmission following pancreatic resections in a single tertiary healthcare institution.
Methods: We retrospectively collected information from the records of 111 patients who underwent pancreatic resections from 2004-2013. We performed a univariate screen of possible factors associated with 30-day readmission using the Fisher’s exact test for categorical variables and the Mann-Whitney test for continuous variables; we performed a multivariate analysis using logistic regression to determine the independent effects of each factor.
Results: 17 (15.3%) patients were readmitted within 30 days of discharge. The most common reasons for readmission were dehydration (18%), and sepsis (12%). Multivariate logistic regression found that the development of any type of intra-abdominal fluid collection (OR = 3.65, p<0.0229), and superficial surgical site infections both occurring within 30 days of resection (OR = 6.36, p<0.0411), were associated with readmissions within 30 days of discharge.
Conclusion: Our data concludes that factors predictive of 30-day readmission are under surgeon and patient control; the risk of 30-day readmission after pancreatic resections can be modified by attention to surgical technique and sterility, pre-operative patient optimization, and environmental and lifestyle modifications.
Statins but Not Aspirin nor Their Combination Have a Chemopreventive Effect on Pancreatic Cancer Occurrence
L. Archibugi, P. Maisonneuve, M. Piciucchi, R. Valente, G. Delle Fave, G. Capurso. Digestive & Liver Disease Unit, Sapienza University, S. Andrea Hospital, Rome, Italy.
Background: Pancreatic Ductal Adenocarcinoma (PDAC) has increasing incidence and poor prognosis, mostly due to delayed diagnosis. Prevention can be a cornerstone in the fight against this deathly cancer. In this context, a chemopreventive action of aspirin and statins might prove interesting. However, published data are conflicting, with effect sometimes limited to subgroups; also, the possible combined effect of the two drugs has never been explored.
Aim: To investigate the possible protective role of aspirin and statin use and their combination on PDAC patients.
Methods: Case-control study with risk factors screened through questionnaires about environmental factors, family and medical history. Cases were matched to controls for age and gender with a 1:2 ratio. Power and required sample size were calculated after the enrollment of the first 200 controls.
Results: 346 patients with PDAC and 692 matched controls (54% males, mean age 69) were enrolled. Aspirin (19.1% vs 23.8%) and statin (19.1% vs 24.9%) use differed among cases and controls. At multivariate logistic regression analysis adjusted for possible confounding factors, statin use was shown to be a protective factor (OR 0.66; 95% CI: 0.46-0.96; p=0.03) while aspirin use only showed a trend towards being protective. >5-year use of aspirin (7.2% vs. 10.8%) or aspirin and statins combined use (10.1% vs. 11.3%) showed no protective effect. All known risk factors for PDAC were confirmed in our study.
Conclusions: A chemopreventive effect for statin, but not for aspirin use, was observed in our study. The described OR is similar to that recently reported in another large C-C study in the US. The possible combined chemopreventive effect of aspirin and statins was hereby analyzed for the first time with null results. All known factors associated with increased risk for PDAC were confirmed, supporting the genuineness of our population.
Pancreatic Cancer in Women: Late Onset of Menopause, Use of Hormone Replacement Therapy and Two-parity are Protective Factors
L. Archibugi, M. Piciucchi, R. Valente, G. Zerboni, S. Stigliano, M. Signoretti, G. Delle Fave, G. Capurso. Digestive & Liver Disease Unit, Sapienza University, S. Andrea Hospital, Rome, Italy.
Background: The incidence of Pancreatic Ductal Adenocarcinoma (PDAC) is slightly higher in men than in women, although the difference in smoking and alcohol consumption between the two genders does not explain this disparity completely. Reproductive and hormonal factors might have an influence, as estrogen inhibits growth of pancreatic cancer in rat models and lowers the incidence of diabetes, but published data are inconsistent.
Aim: To investigate the role of reproductive and hormonal factors on PDAC occurrence in women.
Methods: This is a case-control study on women; risk factors were screened through questionnaires about gynecologic and medical history. Cases were matched to controls for age, with a 1:2 ratio.
Results: 160 PDAC and 320 matched controls (mean age 70 in both groups) were enrolled. Age of onset of menopause was significantly higher in controls (48.9 vs. 50; p=0.02) and at multivariate logistic regression analysis adjusted for possible confounding factors, older age at menopause (OR:0.96 per year; 95% CI:0.92-1; p=0.05), use of hormonal replacement therapy (HRT) (OR:0.14; 95% CI:0.04-0.49; p=0.002) and having given birth to two children (OR:0.62; 95% CI:0.39-0.98; p=0.04) were significant, independent protective factors. No difference among cases and controls was found on age of menarche, nulliparity, use of oral contraceptive or number of abortions. Having given birth to more than 4 children showed a trend towards being a risk factor.
Conclusions: In our study, late onset of menopause, use of HRT and having given birth to two children are protective factors for the occurrence of PDAC, with a trend towards being a risk factor for having given birth to more than four children. These data confirm some previous findings on menopause age and number of births while, to our knowledge, this is the first study to show a protective effect of HRT.
Preoperative Independent Prognostic Factors in Patients With “Resectable” Pancreatic Ductal Adenocarcinoma Following Curative Resection: Neutrophil-lymphocyte Ratio and Platelet-Lymphocyte Ratio
S. Asari, H. Toyama, T. Goto, S. Terai, T. Ajiki, T. Matsumoto, K. Shinozaki, M. Kido, A. Takebe, M. Tanaka, K. Kuramitsu, H. Kinoshita, T. Fukumoto, Y. Ku. Division of Hepato-Biliary-Pancreatic Surgery, Department of Surgery, Kobe University Graduate School of Medicine, Kobe, Japan.
Background: We have reported that preoperative higher neutrophil-lymphocyte ratio (NLR) and platelet-lymphocyte ratio (PLR) can be independent predictive risk factors in borderline resectable pancreatic ductal adenocarcinoma (BR-PDAC) patients following curative resection (S. Asari et al. Surgery Today 2015: In press). The aim of this study was to examine whether NLR and/or PLR could be preoperative independent prognostic factor(s) in patients with “resectable (R)”-PDAC.
Method: Between January 2003 and June 2013 at Kobe University Hospital, 190 consecutive PDAC patients who underwent surgical curative resection were retrospectively studied. Six PDAC patients who underwent neoadjuvant chemotherapy or chemoradiation therapy were excluded. The 184 PDAC patients were stratified into R- and BR-PAC patients according to the National Comprehensive Cancer Network guidelines published in 2013.
Results: In the R-PDAC patients, 5-year overall survival rates (OS) and median survival time (MST) after surgical resection were 21% and 24.3 months, respectively. In the BR-PDAC patients, 5-year OS and MST were 6% and 22.1 months, respectively (P = 0.04). In the R-PDAC patients with NLR > 3 (n = 50) and NLR ≤ 3 (n = 97), 5-year OS were 0% and 25.7%, respectively (MST: 17.5 months vs. 28.4 months), indicating a significant difference in OS between the two groups (P = 0.04). In the R-PDAC patients with PLR > 225 (n = 32) and PLR ≤ 225 (n = 115), 5-year OS were 11.7% and 23.0%, respectively (MST: 21.1 months vs. 24.6 months). There was no significant difference in OS between the two groups (P = 0.32). Univariate analysis revealed that preoperative albumin (P = 0.01), preoperative mGPS (P = 0.02), preoperative NLR (P = 0.05), intraoperative blood transfusion (P = 0.01), UICC-stage (P < 0.01), residual tumor status (P = 0.02), and adjuvant chemotherapy (P < 0.01) were significantly associated with OS. Multivariate analysis demonstrated that neither preoperative NLR nor PLR could be predictive factor in R-PDAC patients.
Conclusion: Neither preoperative NLR nor PLR can offer independent prognostic information regarding overall survival in “R-PDAC” patients following curative resection.
Small Molecule Inhibitors of Cyclophilin D to Protect Mitochondrial Function as a Possible Treatment for Acute Pancreatitis
M. Awais,1 E. Shore,2 R. Gibson,3 M.A. Javed,1 L. Wen,1 D. Latawiec,1 N. Kershaw,2 S. Pandalaneni,3 D.N. Criddle,1,4 A. Tepikin,1,4 N. Berry,2 L-Y. Lian,3 P. O’Neill,2,5 R. Sutton.11NIHR Liverpool Pancreas Biomedical Research Unit, Royal Liverpool University Hospital; 2Department of Chemistry; 3NMR Centre for Structural Biology; 4Department of Cellular and Molecular Physiology; 5MRC Centre for Drug Safety Science; University of Liverpool, United Kingdom.
Introduction: The mitochondrial permeability transition pore (MPTP) is a major contributor to acute pancreatitis, causing loss of mitochondrial membrane potential (Δψm), diminished ATP production and pancreatic acinar cell (PAC) necrosis. Cyclophilin D (CypD) is a mitochondrial matrix peptidyl prolyl cis-trans isomerase (PPIase) that promotes formation of the MPTP. We aimed to develop small molecule (SM) specific inhibitors of CypD as a possible treatment for AP and other conditions where MPTP plays a role.
Methods: Molecular modelling was used to identify SMs predicted to bind with human CypD and to inform chemical synthesis of compounds targeting the enzymatic site on CypD. Binding affinities (Kd) were determined by isothermal titration calorimetry and the inhibitory effect on enzymatic activity of CypD (Ki) measured by PPIase assay. Structures of CypD/SMs complexes were determined by X-ray crystallography. Confocal microscopy was used to evaluate the ability of SMs to protect Δψm and inhibit necrosis of PACs.
Results: SMs (coded 3C28, 3C34, 3C41 and 3C53) showed affinity for CypD with Kd values of 4.9, 0.5, 1.1 and 0.06 μM, respectively. The Ki values of 3C28, 3C34, 3C41 and 3C53 were 9.1, 0.1, 0.95 and 0.032 μM, respectively. All the four compounds (10 μM) protected Δψm and reduced necrosis in freshly isolated PACs in the presence of taurolithocholate acid sulphate (500 μM, p<0.01 3C28, 3C53 vs controls; p<0.05 3C34, 3C41 vs controls).
Conclusion: 3C28, 3C34, 3C41 and 3C53 bind with and modulate the PPIase activity of CypD, preserve Δψm and inhibit necrosis in PACs, at Kd/Ki below 100 nM for 3C53. Further optimization of our SMs is underway.
Applying Healthcare Utilization Data to Predict Pancreatic Cancer
A. Baecker,1,2 S.J. Pandol,1 A. Hendifar,1 N. Nissen,1 B. Wu,3 C.Y. Jeon.1,21Cedars-Sinai Medical Center, Los Angeles, CA; 2UCLA Fielding School of Public Health, Los Angeles, CA; 3Kaiser Permanente Los Angeles Medical Center, Los Angeles, CA.
Background: Patients with pancreatic cancer present with a constellation of non-specific symptoms and conditions prior to diagnosis that could identify a high risk population who may benefit from screening.
Objective: To examine the pattern of healthcare utilization for conditions, symptoms and medications associated with pancreatic adenocarcinoma within 2 years prior to cancer diagnosis.
Method: We employed the linked Surveillance, Epidemiology and End Results tumor registries program and Medicare data files to describe the healthcare utilization patterns of 15,290 patients diagnosed with primary pancreatic adenocarcinoma. We extracted Medicare inpatient and outpatient claims that listed ICD-9 diagnoses of acute pancreatitis, chronic pancreatitis, cachexia or anorexia, poorly controlled type 2 diabetes mellitus, abdominal pain, or nausea or vomiting. In patients who also enrolled in the prescription drug coverage plan (N=2,198), we examined prescription filling patterns for proton-pump inhibitors. Diagnosis or prescription trends were then assessed over the 7 quarters leading up to the diagnosis of pancreatic cancer, excluding the quarter prior to cancer diagnosis.
Results: The proportion of patients with a claim for each condition increased from the 8th quarter to the 2nd quarter prior to the diagnosis of pancreatic adenocarcinoma: chronic pancreatitis, 0.16% to 0.67%; acute pancreatitis, 0.34% to 1.31%; cachexia/anorexia, 1.1% to 3.2%; poorly-controlled diabetes, 4.3% to 6.4%; abdominal pain, 4.8% to 8.7%; nausea/vomiting, 1.5% to 2.3%. The proportion of patients with a filled proton-pump inhibitor prescription also increased from 15.0% to 20.6% in the same period.
Conclusion: The increasing frequency of visits for unspecific symptoms and conditions related to pancreatic cancer could be utilized to identify patients who may have undiagnosed pancreatic cancer.
Evaluation of Human Pancreatic Cancer Cell Viability Following Administration of SBP-101 in the Presence and Absence of Gemcitabine and Nab-Paclitaxel
C. Baker,1 S. Arora,2 S. Gagnon,2 M. Cullen,2 A. Shah.21Biocurity, Inc., New Smyrna Beach, FL; 2Sun BioPharma, Inc., Gainesville, FL.
Introduction: SBP-101 is an analogue of the native polyamine (PA) spermine. The PA uptake transport system is up-regulated in a number of neoplasms including pancreatic ductal adenocarcinoma (PDA) suggesting a promising therapeutic target. This study evaluated the anti-proliferative effect of SBP-101 in the presence and absence of gemcitabine (GEM) and nab-paclitaxel (NAB) in six human PDA cell lines.
Methods: AsPC-1, BxPC-3, Capan-1, HPAF-II, MIA PaCa-2 and PANC-1 cells were seeded in 96-well plates and allowed to adhere overnight. After 24 h fresh media was added containing 0.5-10 μM concentrations of SBP alone or + 0.5 μM GEM, + 5 nM NAB, or + both, or containing GEM, NAB or GEM+NAB alone. Cell proliferation was measured in triplicate 24, 48, 72 and 96 h post-treatment and IC50 values were calculated.
Results: SBP-101 produced an anti-proliferative effect in all cell lines; maximal inhibition most often occurred with 10 μM. At 96 h, maximum mean inhibition with 10 μM SBP-101+GEM+NAB compared with GEM+NAB was 97.3% vs. 38.5% (BxPC-3), 90.1% vs. 47.1% (Capan-1), 89.7% vs. 38.3% (ASPC-1) and 39.4% vs. 8.0% (MIA PaCa-2) (p<0.005). SBP-101 alone produced greater inhibition than GEM+NAB in ASPC-1, BxPC-3 and Capan-1 cells (p<0.005). In most cell lines IC50 decreased with SBP-101 as treatment duration increased and combination treatments with SBP-101 resulted in a further decrease in IC50, indicating an additive or synergistic effect with GEM and NAB on the decrease in cell viability.
Conclusion: SBP-101, both alone and in combinations with GEM and NAB, exhibited a marked anti-proliferative effect against PDA. SBP-101 alone and SBP-101+GEM+NAB were more effective than GEM+NAB, the current standard of care. These results confirm the anti-neoplastic potential of SBP-101 and offer a rationale for its further investigation as a treatment for human pancreatic cancer.
Antibiotic use Early in Acute Pancreatitis: International Overview of Compliance With Guidelines
M. Baltatzis, S. Jegatheeswaran, A.K. Siriwardena. Regional Hepato-Pancreato-Biliary Surgery Unit, Manchester Royal Infirmary, Manchester, United Kingdom.
Introduction: Current International Association of Pancreatology/American Pancreatic Association guidelines state that “Intravenous antibiotic prophylaxis is not recommended for the prevention of infectious complications in acute pancreatitis (AP)”. This recommendation, based on meta-analyses of randomized trials is reflected worldwide in national guidelines. The aim of the present study is to obtain a global overview of compliance in antibiotic use in acute pancreatitis.
Methods: A computerized search was carried out of Pubmed using the keywords “antibiotic prophylaxis” and “acute pancreatitis”. Articles were retrieved if they included original data on the use of antibiotic prophylaxis in acute pancreatitis, either obtained from physician surveys or from national database reports. Searches for the period 1992-2015 resulted in 228 unique citations of which 5 reports met inclusion criteria.
Results: One national administrative patient database report from Japan included 7,193 patients of whom 5,242 (80%) had antibiotic prophylaxis for mild AP and 613 (91%) for severe AP. Median (sem) duration of treatment was 8.1 ± 13.6 days in mild disease and 17.9 ± 18.2 days in severe. Questionnaire surveys from France, USA and the UK reported on the practice of 1,118 respondents. Overall 52% of respondents used antibiotic prophylaxis in acute pancreatitis with individual survey response ranges of 8% - 88%. Only one study provided antibiotic-related adverse event reporting.
Conclusion: Summation of questionnaire survey results may exaggerate inherent biases. Accepting this, the present study is the first global overview of the practice of antibiotic prophylaxis in acute pancreatitis. Results show that antibiotic prophylaxis in AP is widespread in all healthcare systems sampled despite consistent negative recommendations from national and international guidelines.
Microenvironment Mediated Altered Metabolic Pathways Confer Increased Chemoresistance in CD133+ Tumor Initiating Cells
S. Banerjee, A. Nomura, P. Dauer, B. Garg, V. Dudeja, S. Ramakrishnan, A. Saluja. Division of Basic and Translational Research, Department of Surgery, University of Minnesota, Minneapolis, MN.
Tumor-initiating cells (TICs), a minor sub-population of tumor cells have been attributed for both chemoresistance and tumor relapse in pancreatic cancer, resulting in very poor survival statistics. However, the mechanism of chemo-resistance in these cells is still unclear.
Our study shows that the hypoxic pancreatic tumor microenvironment contributes to enrichment of CD133+ population in the tumor. This “niche” also promotes increased glycolysis and low mitochondrial activity in CD133+ cells when compared to CD133- population. Our results indicate that this altered metabolism prevents accumulation of Reactive Oxygen Species (ROS) in CD133+ cells, thereby conferring chemo-resistance and evasion of death in this population.
Progression of tumor in the KRasG12D-TP53-PDXcre mice (n=10) indicated that hypoxia developed with tumor progression. Hypoxic regions correlated with increased CD133+ population, having high HIF1A activity. Consistent with this observation, CD133+ cells had increased glucose uptake (765.69 ± 50.43%) and activity of glycolytic pathway enzymes (HK2, LDH) compared to CD133- cells (425.12± 53.98% and 387.96±29.86%). Mass spectrometric analysis (UPLC-TQD) following metabolically labeling CD133+ cells with 13C-6 glucose confirmed this observation. Furthermore, CD133+ cells had low mitochondrial complex I activity (12.3± 0.09%) and complex IV activity (22.77±0.12%), even though both population had functionally active mitochondria. As a result, CD133+ cells resulted in lesser accumulation of ROS compared to CD133- cells in response to standard chemotherapeutic compounds like paclitaxel, 5FU and gemcitabine. Interestingly, CD133+ cells showed increased resistance to all these 3 chemotherapeutic compounds and treatment with Glut1 inhibitor (STF31) or LDH inhibitor (Na-Oxamate) resulted in reversal of this resistance causing cell death in CD133+ cells similar to CD133- cells.
Our study indicates that altered metabolic profile in CD133+ pancreatic TIC protects the cells from apoptosis induced by standard chemotherapeutic agents by reducing accumulation of ROS and decreasing apoptosis, thereby confering chemoresistance to these cells. Since resistance to existing chemotherapy contributes to the poor prognosis in pancreatic cancer, our study paves the way for identifying novel therapeutic targets for managing chemoresistance and tumor recurrence in pancreatic cancer.
Early Imaging in Acute Pancreatitis: Has Utilization Decreased?
P.A. Banks,1 V. Kadiyala,1 S.L. Suleiman,1 J.Y. McNabb-Baltar,1 B.U. Wu,2 R. Khorasani,3 V.K. Singh.41Division of Gastroenterology, Hepatology and Endoscopy, Brigham and Women’s Hospital, Boston, MA; 2Division of Gastroenterology, Pancreatic Disease Center, Kaiser Permanente Los Angeles Medical Center, Los Angeles, CA; 3Department of Radiology, Brigham and Women’s Hospital, Boston, MA; 4Pancreatitis Center, Division of Gastroenterology, Johns Hopkins Medical Institutions, Baltimore, MD.
Background: Early imaging with CT or MRI is common in acute pancreatitis (AP). Available data indicates that routine use is not warranted.
Aim: To compare the frequency of early (within the 1st 24 hours of presentation) radiologic imaging in AP during the period 9/2006-5/2007 (period A) vs 9/2014-5/2015 (period B).
Methods: Analysis of prospective AP database. AP severity was stratified according to the Revised Atlanta Classification (2012). Clinical, CT and MRI imaging data were recorded. Patients who developed AP after admission were excluded. Fisher’s exact test was used to compare imaging frequency (CT, MRI) between time periods.
Results: The total cohort was 188 cases; 84 in period A and 104 in B. There were 123 (65%) mild, 42 (23%) moderate and 23 (12%) severe cases. There was no significant difference in the distribution of severity between periods (p>0.05). 58 cases (21%) were alcohol induced and 96 (34%) biliary. In period A, 46% of mild, 72% of moderate and 64% of severe cases had early imaging. In period B, 63% of mild, 54% of moderate and 92% of severe cases had early imaging. Within each severity category, there was no significant difference in early imaging between the two periods (p>0.05). In mild cases, there was no difference in early imaging in recurrent episodes compared to 1st episodes (A: 50% vs 43%, p=0.752; B: 63% vs 64%, p=1.000).
Conclusion: There was no significant decrease in early imaging in all severity categories in the 2014-2015 period compared to 2006-2007. Even among patients with mild AP, there was no decrease in early imaging in recurrent episodes compared to the 1st episode.
Pancreatic Human Fibroblast Expression Profiling Reveals Novel microRNAs and mRNAs in the Diseased Pancreas
L. Barrera,1 B. Lane,1 S. Brumskill,2 F. Oldfield,1 Q. Nunes,1 F. Campbell,3 T. Andrews,3 P. Phillips,4 C. Halloran,1 W. Greenhalf,1 J. Neoptolemos,1 E. Costello.11NIHR Liverpool Pancreas Biomedical Research Unit, United Kingdom; 2Redx Oncology, Liverpool, United Kingdom; 3Department of Pathology, Royal Liverpool University Hospital, United Kingdom; 4The University of New South Wales, Sydney, Australia.
Background: The molecular pathways responsible for the activation of cancer-associated fibroblasts (CAFs) in pancreatic ductal adenocarcinoma (PDAC) and the role of microRNAs (miRNAs) in this process are incompletely understood. A parallel analysis of miRNA and mRNA in CAFs could help elucidate tumor-promoting signaling pathways.
Aim: To identify novel molecular candidates in the activation of CAFs using integrated microarray-based miRNA and mRNA expression profiling.
Patients & Methods: Activated fibroblasts from chronic pancreatitis (CP), PDAC, periampullary tumor (PT) and adjacent normal pancreas (NP) were obtained from patients undergoing pancreatic surgery (n=5 per group). Total RNA was hybridized onto human Affymetrix arrays. Linear models and an empirical Bayesian method of inference were used to detect differential expression. Ingenuity Pathway Analysis (IPA) was used to identify relevant molecular networks. Validation of miRNA/mRNA levels was undertaken using RTPCR and in situ hybridization. Ongoing work is predicting miRNA-mRNA pairs.
Results: Linear Model Contrasts identified 14, 25 and 36 differentially expressed (DE) genes between PDAC-versus-CP, PDAC-versus-PA and PA-versus-CP, respectively. However, fibroblasts derived from diseased pancreas compared with NP revealed over 300 DE genes. MiRNA data identified 28 DE miRNAs across all groups. IPA demonstrated the enrichment of canonical pathways relevant to cancer-associated fibroblast activation.
Conclusion: Identifying miRNA and mRNA targets responsible for driving pathophysiological mechanisms involved in the activation of normal fibroblasts will shed light on specific mechanisms influencing the tumour-stroma crosstalk.
A High Meat Intake Is Associated With an Increased Risk of Pancreatic Cancer: A Prospective Cohort Study (EPIC-Norfolk) Using Data From Food Diaries
A. Beaney,1 P. Banim,2 R. Luben,3 M. Lentjes,3 K.T. Khaw,3 A. Hart.11Norwich Medical School, Norwich, United Kingdom; 2James Paget Hospital, Norfolk, United Kingdom; 3Institute of Public Health, University of Cambridge, United Kingdom.
Introduction and Aims: Carcinogens in red, processed and white meats may be involved in pancreatic carcinogenesis, although data from epidemiological studies on meat intake are conflicting. We investigated this for the first time using data from 7-day food diaries (7-DFDs).
Methods: 23,658 initially well participants, recruited into EPIC-Norfolk cohort study completed 7-DFDs, donated blood for vitamin C analysis and were followed up over 17 years to identify those developing pancreatic cancer. Meat intakes were compared with a sample of 3970 controls and Hazard ratios (HR) calculated for pancreatic cancer. Analyses were performed accounting for age at intake, temperature of cooking and antioxidant intake.
Results: 86 participants (0.36%) developed pancreatic cancer (56% women). In those younger than 60 years at recruitment, all quintiles of red and processed meat intakes were non-significantly associated with risk (Q1 vs Q5, HR=4.62, 95% CI=0.96-22.30, p=0.06 / Q1 vs Q5, HR=3.73, 95% CI=0.95-14.66, p=0.06) with trends across quintiles for red meat (HRtrend=1.33, 95% CI=1.00-1.77) and processed meat (HRtrend=1.37, 95% CI=1.04-1.82). In the whole cohort, or those older than 60 years at recruitment, there were no associations with these. For white meat, no consistent effects were seen. A non-statistically significant risk was observed for cooking meat at both higher and lower temperatures, in those younger than 60 years. In this age group, participants with low serum vitamin C levels had an increased risk with red meat (HRtrend=1.84, 95% CI=1.09–3.14) and processed meat (HRtrend =1.80, 95% CI=1.10-2.96).
Conclusion: Our findings support the hypothesis that potential carcinogens in red and processed meats may be involved in pancreatic carcinogenesis. The effects may be dependent on age at intake, cooking temperatures and anti-oxidant intake.
Agency for Healthcare Research and Quality (AHRQ) Patient Safety Indicators (PSIs) are Associated With Increased Healthcare Utilization, Costs, and Mortality Among Patients Hospitalized With Acute Pancreatitis
J. Behzadi, S. El-Dika, A. Hinton, S. Krishna, S. Moffatt-Bruce, D. Conwell, P. Hart. The Ohio State University Wexner Medical Center, Columbus, OH.
Background: Acute pancreatitis (AP) is the leading cause of gastrointestinal hospitalizations in the United States. Characterization of AHRQ PSI’s in hospitalized patients represents an opportunity to improve patient outcome and decrease costs of care. Our aim was to measure the prevalence and association of PSI with outcomes in patients with AP.
Methods: All adult patients (≥18 years of age) in the Healthcare Utilization Project Nationwide Inpatient Sample (NIS) hospitalized from 2007-2012 for a primary diagnosis of AP (ICD-9 577.0) were analyzed. The prevalence of AHRQ PSIs and their associations with mortality, length of stay, and cost were evaluated using multivariate logistic or linear regression, as appropriate, adjusting for significant demographic and hospital factors.
Results: There were 1,569,432 admissions for AP during the study period. A total of 116,641 PSI’s occurred, with 91,627 (5.8%) patients experiencing ≥1 PSI. The most common PSIs (prevalence per 10,000) included: acute respiratory failure (223), sepsis (192), diabetes with complications (120), pressure ulcers (47), and central venous line infections (27). In patients with ≥1 PSI during hospitalization, there were significant increases in mortality rate (adjusted OR 26.66, 95% CI, 24.31, 29.25, p<0.001), length of stay (adjusted OR 9.22, 95% CI 8.91, 9.52, p<0.001), and cost (adjusted OR 26.017, 95% CI 24,981, 27,053, p<0.001).
Conclusions: The development of ≥1 AHRQ PSI during admission for AP is associated with worse inpatient survival and increased utilization of healthcare resources. Additional studies are needed to identify methods of decreasing the incidence of PSIs in acute pancreatitis, particularly for conditions such as respiratory failure and sepsis, which are likely related to the underlying severity of disease.
Does a Pancreatic Duct Above 3 mm Warrant Diagnostic Workup? Clues From a Population Based Cohort
G. Beyer,1 F. Kasprowicz,1 A. Aghdassi,1 H. Völzke,2 T. Kohlmann,2 M.M. Lerch,1 J. Kühn,3 J. Mayerle.11Department of Medicine A; 2Department of Community Medicine; 3Center of Radiology; University Medicine, Ernst-Moritz-Arndt-University, Greifswald, Germany.
Introduction: Changes in diameter of the pancreaticobiliary ducts are hall-marks of a variety of diseases including benign conditions such as chronic pancreatitis as well as pancreaticobiliary malignancies. Transabdominal ultrasound (US) and MRCP are the first line non-invasive imaging modalities to access duct configuration, with broad availability and ever increasing accuracy. However, the reference ranges in asymptomatic individuals and their change with age and after medical procedures have so far only been studied in small cohorts.
Aim: To define new reference ranges for the pancreatic duct on MRCP in the general population.
Methods: Study subjects were recruited from the general population based SHIP and underwent whole body MRI with optional MRCP +/− secretin (1,5T MRI system). PD and was measured in the head of pancreas on MRCP by expert investigators who were blinded to other study findings.
Results: 1005 patients were initially scanned but only 683 included in a first analysis. Median age was 57y (31-89y) and 52.7% were female. On both imaging modalities the diameters increased with age and MRCP revealed that up to 10% of participants had PD widths exceeding the upper limit of 3mm previously accepted upper reference value and thus warranting further examinations to exclude malignancy. Cholecystectomy did lead to increased CBD (p≤0.05) but not PD. No difference was seen between men and women.
Conclusions: The width of the pancreaticobiliary ducts increases in an age dependent manner in asymptomatic volunteers. To reduce healthcare expenditure and unnecessary workup age related adjustments of the reference values might be needed to avoid unnecessary diagnostics.
Deregulation Β-Catenin Gene Expression in Pancreatic Cancer
A. Botezatu,1 I.V. Iancu,1 A. Plesa,1 C.C. Diaconu,1 G. Anton,1 R. Florea,2 V. Ilie,2 A. Sorop,2 N. Bacalbasa,2 V. Tica,2 D.G. Duda,3 S.O. Dima,2 I. Popescu.21“St. S. Nicolau” Institute of Virology, Bucharest, Romania; 2Fundeni Clinical Institute, Gastroenterology and Hepatic Transplantation Center, Bucharest, Romania; 3Edwin L. Steele Laboratory for Tumor Biology, Charlestown, MA.
Pancreatic cancer remains a major challenge and early detection could decreases the mortality caused by this disease. Nuclear β-catenin protein accumulation has been correlated with late stages of tumor progression and metastasis. β-catenin regulation might be performed through epigenetic modification, especially DNA methylation at promoter level. A key factor for β-catenin gene regulation is considered ERBB2, which is over expressed in several cancer types.
The aim of this study was to identify the factors involved in β-catenin gene expression deregulation in pancreas oncogenesis. For this purpose we evaluated the β-catenin gene expression and promoter methylation status, and ERBB2 gene expression in 60 paired samples (normal/pancreatic adenocarcinoma-PDAC). Promoter methylation status was quantified in qMS-PCR using bisulphite treated DNA samples (EpiTect Bisulfite Kit – Qiagen) while β-catenin and ERBB2 genes expression was determined in qRT-PCR.
A decreased methylation pattern of β-catenin promoter was found in advanced disease stages. Significantly lower methylation frequencies (p<0.05) were noted in PDAC stage III and IV (0-20%, median=0.0128%) which correlated with increased β-catenin and ERBB2 gene expression. In PDAC III-IV stages, the fold change expression of β-catenin was between 0.1219-5.907 (median=1.8462) while for ERBB2 gene it displays levels between 0.07118-9.651 (median=1.7524).
These results suggest an epigenetic regulation of β-catenin gene expression in PDAC, through promoter demethylation, correlated with an increase of ERBB2 gene expression. These factors may represent a trigger for pancreatic oncogenesis process.
Analysis of Novel Ciliate Lipases as Candidates for Enzyme Substitution in Pancreatic Exocrine Insufficiency
A. Brock,1 I. Aldag,2 S. Edskes,2 M. Hartmann,2 J. Schnekenburger.11Biomedical Technology Center of the Medical Faculty, University of Muenster, Germany; 2Cilian AG, Muenster, Germany.
Pancreatic exocrine insufficiency (PEI) caused by inflammation or pancreatic tumors results in a lack of digestive enzymes and neutralization compounds. Despite satisfactory clinical results with current enzyme therapies, a normalization of fat absorption in patients is rare. An individualized therapy is required with high dosage of enzymatic units, usage of enteric coating, and addition of gastric proton pump inhibitors. The key goal to improve this therapy is to identify digestive enzymes with high activity and stability under physiological conditions in the gastro intestinal tract. Here we analyzed three novel ciliate lipases derived from Tetrahymena thermophila. Using highly precise pH-STAT-Titration and colorimetric methods, we determined stability and lipolytic activity under physiological conditions in comparison to commercially available porcine and fungal enzyme preparations. We measured from pH 2 to 9, with different bile salts concentrations, and substrates such as olive oil and fat derived from pig diet. The ciliate lipases CL-A, CL-B and CL-C revealed activities up to 220-fold higher than Creon©, pancreatin, and rizolipase Nortase© and an activity range reaching from pH 2 to 9. They are highly active under the presence of bile salts, complex pig diet substrate and more stable after incubation in human gastric juice compared to porcine pancreatic lipase and rizolipase. These novel enzymes are therefore promising candidates for an enzyme replacement therapy for pancreatic exocrine insufficiency.
A New Panel of Serum Markers for Diagnosis and Differentiation of Inflammatory Gastrointestinal Diseases and Tumor
A. Brock,1 T. Erben,1 T. Herzog,2 W. Uhl,2 E. Rijcken,3 R. Mennigen,3 R. Ossig,1 J. Schnekenburger.11Biomedical Technology Center of the Medical Faculty of the University Muenster, Germany; 2Clinic for General and Visceral Surgery, St. Josef Hospital, Bochum, Germany; 3Department of Surgery, University Hospital Muenster, Germany.
Pancreatic diseases such as inflammation and tumor result in heavy pain, digestive disorders, weight loss and not often death. Pancreatic cancer remains a leading cause of cancer death since it spreads fast and is rarely detected in early stages. An early diagnosis is crucial for a consequential and successful treatment of chronic pancreatitis and ductal adenocarcinoma. Currently, the gold standard is in vivo diagnostic imaging since available biomarkers are often insensitive or not specific enough for a reliable diagnosis. In order to improve diagnostic approaches for detection and differentiation of chronic pancreatitis and ductal adenocarcinoma, we selected a range of biomarkers to indicate inflammatory as well as tumorigenic processes in pancreatic tissue. Here we present the use of a panel of five different tumor and inflammation derived molecular serum proteins. We determined serum protein concentrations of biomarker panels with enzyme-linked immunosorbent assays (ELISA) in blood plasma from 400 patients either with gastrointestinal diseases such as chronic pancreatitis, ductal adenocarcinoma, Crohn´s disease and ulcerative colitis or from healthy donors. Statistical analysis of the data was performed with ANOVA using SPSS.
Our results reveal that the tested serum marker panels proofed significantly to distinguish chronic pancreatitis, inflammatory bowel diseases, as well as ductal adenocarcinoma and healthy patients from each other. The use of this analyzed marker panel may enable a high significance in diagnostics of tumors and inflammation and can be used to discriminate between chronic pancreatitis and ductal adenocarcinoma. The marker panel showed superior performance compared to established serum markers.
The CILIP-Project – Ciliate Enzymes With Recombinant Lipase for Treating Pancreatic Exocrine Insufficiency
A. Brock,1 I. Aldag,3 S. Edskes,3 T. Herzog,2 W. Uhl,2 M. Hartmann,3 J. Schnekenburger.11Biomedical Technology Center of the Medical Faculty of the University Muenster, Germany; 2Clinic for General and Visceral Surgery, St. Josef Hospital Bochum, Germany; 3Cilian AG, Muenster, Germany.
Therapy of pancreatic exocrine insufficiency (PEI) with enzymes derived from pancreatin requires high dosages, risk of impureness, viral contamination as well as intolerance and refusing for ethical reasons. Due to enzyme inactivation and suboptimal reaction conditions, identifying enzymes with high stability and activity in the gastrointestinal tract maintains a key challenge in PEI therapy. The specific aim of the CILIP-Project – “Ciliate enzymes with recombinant lipase for treating pancreatic exocrine insufficiency” is to create new therapy approaches for patients with pancreatic exocrine insufficiency. Endogenic digestion enzymes with recombinant lipases are derived from Tetrahymena thermophila and characterized in vitro and in vivo. Tetrahymena thermophila lipases were cloned, overexpressed and screened for high lipolytic activity and stability. The project team did an extensive search for lipase homologs in Tetrahymena thermophila genome to identify 37 possible candidates. 19 were cloned and six showed lipolytic activity under our test conditions. After characterization of the cloned lipases for pH depending enzyme activity and stability, we selected a combination of three ciliate lipases for a drug formulation in an animal study using Minipigs as a model organism.
Tetrahymena thermophila digestive enzymes allow an ethical unproblematic and controlled production of enzyme preparations with high activity and stability, low costs, no infection risks, and therefore a high biological safety, while reducing drug volume. The successful testing of the CILIP enzymes will be an important progress towards a safe and efficient way to use pancreatic enzyme substitution.
Insulin Protects Pancreatic Acinar Cells During Experimental Acute Pancreatitis
J.I.E. Bruce,1,2 M.D. Sans,2 H. Durairaj,2 B. Holz,2 B. Nielson,2 S. Ernst,2 J.A. Williams.21Faculty of Life Sciences, The University of Manchester, Manchester, United Kingdom; 2Department of Molecular and Integrative Physiology, University of Michigan, Ann Arbor, MI.
Acute pancreatitis is an inflammatory disease whereby the pancreas digests itself. Our previous studies have shown that insulin directly protects isolated pancreatic acinar cells from pancreatitis-inducing agents. The aim of the current study was to translate these findings to experimental animal models of acute pancreatitis using the following strategy:
1-Determine the protective effect of endogenous insulin release by comparing pancreatitis in type-1 diabetic Akita mice vs wild-type mice.
2-Determine the protective effect of endogenous insulin effectiveness, specifically in pancreatic acinar cells, during pancreatitis in pancreatric acinar (PA) cell-specific conditional (C) insulin receptor (IR) knockout mice (KO) mice (PACIRKO). IRs were deleted in adult mice using a tamoxifen-inducable Cre-recombinase gene deletion system.
Pancreatitis was induced either using caerulein (8 hourly intraperitoneal (IP) injections of caerulein (50 ug/kg) over 2 days), or the novel alcohol/fatty acid model (2 hourly IP injections of 100 mg/kg palmitoleic acid (POA)/ 0.8 g/kg ethanol). Histological and biochemical features of pancreatic injury and inflammation were assessed by harvesting pancreatic tissue and plasma at 2 and 24 hours after the last injection.
Initial results show that pancreatitis was exacerbated in both Akita diabetic mice and PACIRKO mice compared to corresponding controls of the same genetic background. This was assessed using H&E staining, which allowed visualisation of edema, cell damage and recruitment of inflammatory cells. In addition, measurement of pancreatic tissue mRNA by qPCR was also used as a measure of early inflammatory markers, TNFa and IL-6.
These data provide initial indications that endogenous insulin directly protects pancreatic acinar cells during experimental pancreatitis and further corroborating.
Inhibition of Hedgehog Pathway in 2D and 3D Co-cultures of CAFs and PDAC Cell Lines
S. Brumskill,1,2,4 L. Barrera,1 F. Campbell,3 C. Halloran,1,2 W. Greenhalf,1,2 C. Ghirelli,4 R. Sutton,2 M.-A. Campbell,4 E. Costello.1,21Liverpool NWCR Centre, Molecular and Clinical Cancer Medicine; 2NIHR Pancreas Biomedical Research Unit; 3Department of Pathology, Royal Liverpool University Hospital, Liverpool, United Kingdom; 4Redx Oncology, Liverpool, United Kingdom.
Background and Aims: In Pancreatic Ductal Adenocarcinoma (PDAC) it is believed that developmental signalling pathways such as Hedgehog (Hh) and Wnt contribute to the activation of Cancer Associated Fibroblasts (CAFs) and maintenance of the tumour microenvironment. We sought to determine the effect of pharmacological inhibition of Smoothened (Smo) on downstream Hh and Wnt pathway targets to identify a molecular crosstalk mechanism between these pathways.
Methods: CAFs were isolated from patient samples using the outgrowth method. Basal expression of Hh pathway components in CAFs (Smo, Ptch, Gli1, Gli2) and Wnt (Axin2, CTNNB1, LEF1) was detected using RT-PCR. This was then compared with the expression levels in cells co-cultured with PDAC cell lines using a 0.3μm transwell. The secretion of Sonic Hedgehog (Shh) ligand by PDAC cell lines was determined by ELISA. PDAC cell lines and CAFs were cultured in 3D and the effect of this culture system on Hh and Wnt pathway was determined using RT-PCR.
Results: PDAC cell lines secrete Shh ligand. Culturing CAFs in the presence of Shh secreting cells causes an upregulation of Hh target genes (Smo, Gli1, Gli2, p<0.05) and a down regulation of Wnt target genes (Axin2 p<0.05). A 3D model has been developed in which to test the effect of chemotherapeutic agents to compare with the transwell co-culture system. Ongoing work will include determining the effect of Redx Smo inhibitors on Hh and Wnt pathway using both transwell and 3D models.
Conclusion: Unravelling crosstalk mechanisms between Hedgehog and Wnt signalling in PDAC using 3D models which take into account the presence of CAFs within the tumour microenvironment could help identify novel chemotherapeutic interventions.
Small Intestinal Bacterial Overgrowth in Chronic Pancreatitis: Systematic Review and Meta-Analysis
G. Capurso, M. Signoretti, L. Archibugi, S. Stigliano, M. Piciucchi, G. Delle Fave. Digestive & Liver Disease Unit, Sapienza University, Rome, Italy.
Background and Aim: Small intestinal bacterial overgrowth (SIBO) has been proposed as a factor causing symptoms and potentially impairing the nutritional status in chronic pancreatitis (CP) patients, but evidence is conflicting. A systematic review and meta-analysis were conducted on the prevalence of SIBO in CP.
Methods: Medline was searched up to November 2014. Case series/case-control studies investigating SIBO in adult CP patients were considered eligible. Prevalence of positive tests was pooled and the rate of positivity between CP cases and controls, relative OR and 95% confidence interval (CI) calculated.
Results: Eight studies were identified from a list of 1579, containing 300 CP patients. The pooled prevalence of positive tests (glucose or lactulose H2 Breath Test) was 34% (95% CI 13%-62%) with high heterogeneity (I2=92%). Sensitivity analyses, respectively excluding the 2 studies employing lactulose BT or the 3 studies including patients with previous surgery, gave a pooled prevalence of 21% (95% CI 13%-34%) and of 20% (95% CI 4%-60%) with reduced heterogeneity. The pooled odds ratio for positive tests in CP cases compared with controls was 5.7(95% CI 1.6-20.7; p=0.007; I2=67%), being reduced to 2.8 (95% CI 1.2-6.8; p=0.017; I2=32%) excluding the study employing lactulose. Data on differences between SIBO positive and negative CP patients were available for 6 studies, heterogeneous in terms of symptoms and nutritional status. Three studies evaluated the response to treatment in SIBO-positive CP patients and suggested improvement after therapy.
Conclusion: At least 20% of CP patients have SIBO with a significantly increased risk over controls. The rate of SIBO is higher in studies employing lactulose BT or including operated patients. The relation of SIBO with symptoms, nutritional status and effects of treatment in CP deserve further investigation. These results might suggest that SIBO should be excluded in all CP patients.
Development of the Spanish Pancreatitis Quality of Life Instrument (SPANQOLI): A Pilot Study
Y. Carrasquillo, S. Han, J. Kheder, L. Bocelli, W. Wassef, P. Hernandez. Department of Gastroenterology, University of Massachusetts Medical School, Worcester, MA.
Introduction: Chronic pancreatitis represents a global issue that remains difficult to treat as it affects many different aspects of a patient’s health. The Pancreatitis Quality of Life Instrument (PANQOLI) was developed and validated to assess the quality of life of patients with chronic pancreatitis. This study presents the development of the SPANQOLI, which is the Spanish version of this instrument.
Methods: The PANQOLI which has been previously validated, was translated using the back translation method (Translated to Spanish and then to English). The SPANQOLI was administered to 7 patients with chronic pancreatitis who are Spanish-speaking only. These scores were then compared with previous PANQOLI scores from the same patients who completed the instrument with the aid of an interpreter.
Results: In this study patients had an average age of 51 ± 11.3. In terms of the SPANQOLI, patients scored an average of 56.8 ± 14.7, with a higher score representing an improved quality of life. On the original PANQOLI, these patients scored an average of 56.2 ± 14.6. There was no statistically significant difference between the two scores.
Conclusion: The PANQOLI was the first instrument developed to evaluate quality of life in chronic pancreatitis patients. This study presents the first instrument designed to assess quality of life in Spanish speaking patients and demonstrates relative consistency in scoring with the PANQOLI. Further larger studies will be needed to validate this instrument in a Hispanic chronic pancreatitis population, which will prove useful in addressing the growing Hispanic population in the United States.
NEMO/ IKKγ Ablation Promotes Fibrosis and Impairs Regeneration During Pancreatitis
L.K. Chan, T. Wirth, H.J. Maier. Institute of Physiological Chemistry, University of Ulm, Ulm, Germany.
Introduction: The role of IKK/ NF-κB signaling during pancreatitis has remained controversial, with contradicting results from several studies showing both beneficial and detrimental effects when interfering this pathway.
Aims: To determine the contribution of IKK/ NF-κB signaling in the parenchymal compartment to the pathogenesis of both acute and chronic pancreatitis by targeting a component of the IKK complex essential for canonical NF-κB activation.
Methods: Pdx-1-Cre mice were crossed with mice bearing floxed NEMO/IKKγ transgenes, resulting a targeted deletion of NEMO in the epithelial compartment of the pancreas (NEMOΔPanc). These animals were subjected to both acute and chronic cerulein pancreatitis. The pancreata were analyzed at several time points after pancreatitis induction.
Results: During acute pancreatitis, NEMO deletion did not result in major changes in the early phase (24 hours), but led to sustained fibrosis in the recovery phase (7 days). In chronic pancreatitis, NEMO ablation led to an exacerbation of fibrosis and an increase in acinar to ductal dedifferentiation. While wild type animals showed a strong early compensatory acinar cell proliferation, such effect was impaired in NEMOΔPanc animals, resulting slower pancreas regeneration. Gene expression analysis revealed sustained activation of pro-fibrogenic genes and the CXCL12/CXCR4 axis in NEMOΔPanc animals treated with cerulein. Administration of the CXCR4 antagonist AMD3100 attenuated the deleterious effects of NEMO ablation.
Conclusions: Our results show that inhibition of canonical NF-κB signaling in parenchymal compartment through the ablation of NEMO results an exacerbation of fibrosis and a defect in regeneration during the course of pancreatitis. The CXCL12/CXCR4 axis contributes to some of these deleterious effects.
Pancreatic Cancer Development is Dramatically Accelerated by a High Fat, High Calorie Diet in Male and Female Mice
H. Chang, A. Moro, K. Hertzer, A. Stark, C.E. Chou, M. Xu, X. Jung, A.I. Schmidt, D. Dawson, O.J. Hines, G. Eibl. Department of Surgery, David Geffen School of Medicine at UCLA, Los Angeles, CA.
Epidemiologic data has related obesity to higher risk of pancreatic cancer (PaCa), but the underlying mechanisms are poorly understood. To allow for detailed mechanistic studies in a relevant model mimicking diet-induced PaCa, a high-fat, high-calorie diet (HFCD) was given to LSL-KrasG12D/+;p48-Cre (KC) mice carrying a pancreas-specific oncogenic Kras mutation. The mice were allocated to a HFCD or control diet (CD), and weighed weekly. Cohorts were sacrificed at 3, 6, 9, and 12 mos. and tissues harvested for histological analysis. HFCD-fed KC mice gained more weight (g) than CD-fed KC mice at 3- (9.3 vs 7.1), 6- (14.9 vs 8.5), 9- (18.5 vs 13.1) and 12-mos. (18.2 vs 14.7) (p<.05 for all ages). Interestingly, males gained more weight than females at each time point (p<.05). Histologically, KC mice fed the HFCD showed robust signs of inflammation and more advanced PanIN lesions, compared to CD-fed animals. Importantly, the occurrence of cancer was remarkably accelerated by the HFCD. The tumor incidence (%) for CD-fed KC mice at 3, 6, 9, and 12 mos. was 0, 0, 0, and 27, respectively, and 8, 20, and 40 for HFCD-fed mice at 3, 6, and 9 mos. Most KC mice on the HFCD had to be euthanized before 12 mos. of age. It is noteworthy that male mice had a higher rate and an earlier onset of malignancy than females. For HFCD-fed KC males, the tumor incidence (%) was 12, 42, and 50 at 3, 6, and 9 mos., respectively, and 0, 0, and 33 for HFCD-fed KC females. To sum up, the HFCD led to greater weight gain and markedly accelerated PanIN progression as well as PaCa development in the conditional KrasG12D mouse model, with noticeable gender difference. These findings will provide the basis for more robust studies investigating the mechanisms underlying the obesity-cancer link in males and females, as well as to evaluate interventions targeting obesity-associated PaCa development.
Pre-Diagnosis Cachexia Rather Than BMI is Associated With Worse Survival Outcomes in Patients With Pancreatic Cancer
J.I. Chang,1 B.Z. Huang,2 B.U. Wu.31Internal Medicine, Kaiser Permanente Los Angeles, CA; 2Research and Evaluation, Kaiser Permanente Southern California, Pasadena, CA; 3Center for Pancreatic Care, Gastroenterology, Kaiser Permanente Los Angeles, CA.
Objective: Both obesity and cachexia are established risk factors for worsened survival in patients with pancreatic ductal adenocarcinoma (PDAC). Our aim was to characterize the relationship between BMI class, cachexia at diagnosis and survival in patients with PDAC.
Methods: In this longitudinal retrospective cohort study (2006-2014), cachexia was defined by the international consensus for cancer cachexia and calculated by weight change 6 months prior to diagnosis. BMI class was determined 6 months prior to diagnosis. We assessed both the independent effects of cachexia and BMI class on survival, and possible effect modification across the two variables. Patients were censored at death, last follow-up, and end of study. Kaplan-Meier estimates and Cox regression with adjustment for age, race, stage, surgery, chemotherapy, and Charlson comorbidity index were used in survival analysis.
Results: Among 977 individuals, 611 (63%) had cachexia at diagnosis. Individuals with cachexia had lower survival (median 4.3 months, CI 3.8-4.9) compared to those without (median 5.2 months, CI 4.4-6.0), log-rank p=0.036. In multivariate analysis, cachexia was independently associated with increased mortality (HR 1.26, CI 1.08-1.48). This did not differ across BMI class, but varied with chemotherapy. Cachectic patients who did not receive chemotherapy had a 53% increase in risk of death compared to non-cachectic patients (HR 1.53, CI 1.24-1.91), while those receiving chemotherapy were unaffected by cachexia (HR 1.00, CI 0.80-1.26), pinteraction=0.009. Pre-diagnosis BMI class did not significantly affect survival outcomes.
Conclusions: Cachexia not obesity at PDAC diagnosis was associated with worse survival, an effect mitigated by chemotherapy. Pre-diagnosis cachexia is a significant negative prognostic factor that should be considered in newly diagnosed PDAC.
Correlation Between CEACAM6 Expression and Clinicopathological Characteristics, Progression of Pancreatic Cancer
J. Chen, N. Lv, K. Jiang, C. Dai, W. Gao, Q. Li, J. Wu, J. Wei, F. Guo, Z. Lu, Y. Miao. Pancreas Center, The First Affiliated Hospital of Nanjing Medical University, Nanjing, China.
Background: Carcinoembryonic antigen-related cell adhesion molecule 6 (CEACAM6) as a member of CEA family,has an oncogene function of molecular adhesion protein.
Aim: To analyze CEACAM6 expression in pancreatic cancer and its prognostic value.
Methods: Clinical data of 99 patients with surgically resected pancreatic ductal adenocarcinoma in our institution from Jul. 2008 to Dec. 2010 was retrospectively analyzed. The expressions of CEACAM6 and E-cadherin in tumor specimens were detected by immunohistochemical staining. Univariate and multivariate analyses were performed to analyze the relationship between pancreatic cancer prognosis and the clinical parameters.
Results: Follow-up study showed that overall survival (OS) time ranged from 1.0 to 46.0 months and median OS was 16.0 months. CEACAM6 expression was not directly related to patient's gender, age, tumor size, location, neural invasion or tumor T stage, but closely related to lymph node metastasis and histological grading. Patients with CEACAM6 positive expression had poorer prognosis. The median survival time of patients with CEACAM6 positive expression and negative expression was 15.0 months and 37.0 months (p <0.001), respectively. Multivariate analyses showed that lymph node metastasis, histological grade, neural invasion and CEACAM6 expression were independent prognostic factors for pancreatic cancer. CEACAM6 expression was negatively correlated with E-cadherin expression (P < 0.01).
Conclusion: CEACAM6 expression was in close relation with disease progression and patients’ prognosis. Tumor lymph node metastasis, neural invasion, histological grade, CEACAM6 expression are the independent prognostic risk factors for pancreatic cancer. CEACAM6 might have a potential relationship with epithelial-to-mesenchymal transition.
Using Affymetrix Gene Chip, Screening the Biomarkers to Evaluate the Severity of Acute Pancreatitis in the Early Stage
W.W. Chen,1,2 L.H. Deng,1 X.X. Zhang,1 N. Shi,1 Y. Ma,1 X.N. Yang,1 Q. Xia.11Department of Integrated Traditional Chinese and Western Medicine, West China Hospital, Sichuan University, Chengdu, China; 2 Department of Gastroenterology, Northern Jiangsu People's Hospital, Yangzhou, China.
Aim: Screening new biomarkers to evaluate the severity of AP in the early stage.
Methods: We conducted a prospective trial and researched gene expression profiles of peripheral blood leucocyte in AP patients. 87 participants (MAP 22, MSAP 43 and SAP 22) were recruited in West China Hospital from September 1st to November 30th, 2014. Ten healthy participants were also included as control group. Peripheral blood was obtained within 24 hours on admission. Leukocytic gene expression (n = 10 for each AP group and n = 6 for control group) was detected and analyzed. Meanwhile, demographic characteristics, laboratory and imaging examinations as well as treatment were investigated.
Results: There were no statistical differences in demographic characteristics. All the samples’ RNA were qualified. The differentially expressed (DE) genes were filtered to include those with fold-change (FC) ≥ 2 and p-value ≤ 0.05. Compared to control group, MAP group induced 3252 DE genes, MSAP group induced 4099 DE genes and SAP group induced 5361 DE genes. Compared to MAP group, MSAP group induced 11 DE genes and SAP induced 600 DE genes. Compared to MSAP group, SAP induced 207 DE genes. Average expression values were analyzed for MAP3K5. Compared to control group, the FC of MAP was 1.33, MSAP was 1.47, SAP was 1.50; Compared to MAP group, FC of MSAP was 1.11, SAP was 1.13; and FC between SAP and MAP was 1.02.
Conclusion: AP can effect on peripheral blood leucocyte gene expression profiles, and the expression quantity has a relation with the severity of AP. It was just the same to MAP3K5. Human mRNA of white blood cell could be the biomarkers to evaluate severity of AP in the early stage.
Hypocalcemic Tetany - A Simple Bedside Marker of Worst Outcome in Acute Pancreatitis: A Prospective Cohort Study
P. Chhabra, S.S. Rana, V. Sharma, R. Sharma, D.K. Bhasin. Dept. of Gastroenterology, PGIMER, Chandigarh, India.
Background: Hypocalcemia is considered a marker of poor prognosis in acute pancreatitis (AP). However the prognostic significance of hypocalcemic tetany, which is not commonly observed in patients with AP, has not been described. Hence we conducted this prospective study to evaluate the role of tetany in prognosis of patients with AP.
Methods: Consecutive patients of AP presenting within 7 days of onset of symptoms were included & followed up till recovery or death. The serum calcium levels were done at admission & patients were divided into two groups based on the presence (group 1) or absence of hypocalcemia (group 2). The clinical signs of hypocalcemia (Chvostek sign or Troussueau’s sign) were looked for in all patients with hypocalcemia & outcome measures were compared between patients with normocalcemia, asymptomatic & symptomatic hypocalcemia (tetany). The outcome parameters assessed were POF (persistent organ failure), the need for intervention & mortality.
Findings: Of the 105 patients (53 M; mean age 37.34 ± 12.62 years) included, 37 (35.2%) had hypocalcemia [group 1] & 68 (64.8%) had normal corrected serum calcium levels [group 2]. Patients with hypocalcemia had significantly higher frequency of POF, mortality & need for intervention (p<0.05). Twelve of 37 (32.4%) patients with hypocalcemia had tetany. Patients with tetany had significantly low serum corrected calcium & ionized calcium as compared to patients with asymptomatic hypocalcemia (p<0.05). Patients with tetany when compared to patients with asymptomatic hypocalcemia had significantly higher mortality (100% vs. 32%; p=0.000) as well as POF (100% vs. 32%; p=0.000). Tetany predicted mortality with a sensitivity of 75 % & specificity of 100%.
Interpretation: Patients of AP with hypocalcemic tetany have a very poor prognosis & its presence can be used as a simple bedside marker of increased mortality.
Dual Inhibitor of HDAC and GSK-3β Prevents Pancreatic Cancer Progression
C. Chheda, B. Soufi, C. Fall, R. Hu, M. Laayouni, R. Murali, S.J. Pandol, M. Edderkaoui. Cedars-Sinai Medical Center, UCLA, and West LA VAGLAHS, Los Angeles, CA.
Background: Pancreatic cancer cells are highly proliferative and metastatic, and extremely resistant to current treatments. We have developed a novel dual inhibitor that targets both the glycogen synthase kinase 3 beta (GSK-3β) and histone deacetylase (HDAC), important mediators of cancer progression.
Methods: KrasLSL-G12D/+;Trp53LSL-R172H/+;Pdxcre (KPC) mice were treated (ip) with the dual inhibitor ALB-185357 (5mg/Kg) 3 times/week from age 2 months until death or euthanasia at age 7 months. Pancreatic tumors, fibrosis, inflammation, and metastasis markers were measured by immunohistochemistry. In vitro, ALB-185357 was applied to different cancer and normal cells, and cell survival and apoptosis were measured by MTT and DNA fragmentation assays, respectively. Markers of EMT and cancer stemness were measured by Western. Invasion of the cells was measured by Matrigel Invasion Assay.
Results: Treatment of KPC mice with ALB-185357 significantly increased mice survival by 2-fold, decreased tumor size, fibrosis and inflammation in pancreatic tissue, and prevented metastasis. In vitro, ALB-185357 significantly (at 300nM) and dose dependently decreased cell survival and increased apoptosis in several pancreatic cancer cells. A similar effect was observed in lymphoma, liver and breast cancer cell lines. ALB-185357, however, did not affect survival of the normal pancreatic ductal cells and hepatocytes. ALB-185357 increased histone acetylation, inhibited GSK-3β activity, decreased expression of markers of EMT and cancer stemness, and prevented invasion and sensitized the cancer cells to gemcitabine.
Conclusion: We have designed and synthesized a novel drug that prevents growth, metastasis, and resistance to death of pancreatic cancer cells in vivo and in vitro with no significant toxicity to normal cells. Treatment with ALB-185357 increased the survival of KPC mice suggesting its usefulness for treatment of malignancies.
An international PCT patent application was filed for this drug.
Gasotransmitters in Acute Necrotizing Pancreatitis: Friend or Foe?
S. Chooklin, Y. Pidhirnyy, S. Chuklin, O. Usach. Department of Surgery, Regional Clinical Hospital, Lviv, Ukraine.
Introduction: Nitric oxide (NO) and hydrogen sulfide (H2S) have emerged as dominant redox regulators of numerous aspects of cellular and physiological functions within several organ systems included cardiovascular, immune and neurological tissues. Recent studies have begun to reveal that these two gaseous molecules may have redundant or overlapping pathophysiological functions often involving similar molecular targets. H2S and NO have been extensively studied for its various roles in cardiovascular and neurological disorders. However, the role of NO and H2S in pancreas inflammation is still controversial.
Materials and Methods: The study involved 25 acute necrotizing pancreatitis patients. The concentration of gas mediators were determined in the blood serum: H2S using N,N-dimethyl-p-phenylenediamine and NO, which was expressed as NO2−+NO3−, was measured in reaction with Griess reagent.
Results: A significant decrease in the H2S level was observed in patients with AP (the level was 9.5% less than in control group). Furthermore in these patients the level of NO (NO2−+ NO3−) was 31.5% less than that of the control group. Changes of H2S levels correlate with changes of NO levels. Concentration of gasotransmitters depends on the severity of the pathological process. Concentrations of H2S negative correlated with C-reactive protein, and positive with thrombin time.
Conclusion: These data can be an evidence of interaction of these two gaseous transmitters in inflammation of the pancreas. They may have anti-inflammatory and anticoagulant effect, but further research is required.
DPYSL3 Variant 1 Regulates Cell Survival in Pancreatic Cancer
S. Chowdhury,1 M. Ali,1 D. Maroni,1 N. Woods,2 S.P. Thayer.11Department of Surgery, Fred and Pamela Buffett Cancer Center at University of Nebraska Medical Center, Omaha, NE; 2Eppley Institute, University of Nebraska Medical Center, Omaha, NE.
Background & Aims: DPYSL3, a cell adhesion molecule has recently attracted attention as a metastatic modulator where it has been reported to function either as a tumor promoter or suppressor. DPYSL3 has 2 transcriptional variants, a 684 amino acid long form (DPYSL3 variant 1) and a 570 amino acid short form (DPYSL3 variant 2). The specific roles of the two DPYSL3 variants have not been characterized. In the current study, we investigated the variant specific functions of DPYSL3 in pancreatic cancer cell lines.
Methods: RNA and protein analysis was performed to determine the variant specific expression of DPYSL3 in pancreatic cancer cell lines. DPYSL3 variant 1 and 2 was overexpressed under in normal and serum starvation condition to determine the downstream survival, proliferation and invasion/migration signaling in pancreatic cancer cells.
Results: DPYSL3 variant 1 and 2 were differentially expressed in pancreatic cancer cell lines. DPYSL3 variant 1 (long form) was expressed in all pancreatic cancer cell lines tested. DPYSL3 long form has an alternative transcriptional start site. This additional sequence in the long form possess several short linear motifs (SLiMs) suggesting the potential recognition by cell cycle regulators, CDKs and cyclins. Cell cycle arrest by serum starvation of pancreatic cancer cells leads to a robust downregulation of the long form of DPYSL3 in a concentration dependent manner. However, no change was observed in the short form of DPYSL3. Transfection of the long form of DPYSL3 under serum starvation condition led to an increase in cell survival responses by the upregulation of survivin (inhibitor of apoptosis) and the downregulation of cell cycle arrest genes such as cyclin dependent kinase inhibitor p27kip1.
Conclusion: DPYSL3 variant 1 (long form) might be involved in regulating cell survival and proliferation of pancreatic cancer cells.
TFF2/SMAD4 Tumor Suppressor Signaling in Pancreatic Cancer is Regulated by Promoter Methylation
S. Chowdhury,1 D. Klinkebiel,2 J. Yamaguchi,3 A.S. Liss,3 S.P. Thayer.11Department of Surgery, Fred and Pamela Buffett Cancer Center at University of Nebraska Medical Center, Omaha, NE; 2Department of Biochemistry, Fred and Pamela Buffett Cancer Center at University of Nebraska Medical Center, Omaha, NE; 3Andrew L. Warshaw Institute for Pancreatic Cancer Research, MGH, Boston, MA.
Background & Aims: Loss of TFF2 is thought to play a role in progression of PDAC. Previous work revealed TFF2 functions as a tumor suppressor. Overexpression of TFF2 decreased PDAC cell line proliferation via a SMAD4 dependent mechanism. TFF2-KO mice in the background of mutant Kras resulted in the accelerated formation of both IPMN and PDAC. To date little is known about the mechanism of this loss. This study looks at promoter methylation as a possible mechanism of TFF2 inactivation.
Methods: In Aspc1 and Panc1 cell lines methylation of the native TFF2 promoter was assessed by pyrosequencing. The transcriptional significance of this promoter methylation was assessed by using a DNA methylation inhibitor, decitabine. siRNA knockdown were used to determine downstream pathway transcriptional dependency on TFF2.
Results: A total of 5 CpG islands within the TFF2 promoter, from −181 to −149 from the transcriptional start site were examined by pyrosequencing. TFF2 expressing (Aspc1) and nonexpressing (Panc1) cell lines revealed differential levels of TFF2 promoter methylation. In TFF2 nonexpressing cell lines, the 5 CpG sites were hypermethylated. In contrast, in TFF2 expressing cell line these CpG sites were hypomethylation. Decitabine treatment resulted in a reduction in methylation in all 5 CpG sites, which was accompanied by an increase in TFF2 expression. TFF2 demethylation in both cell lines also resulted in an increase in SMAD4 expression. SMAD4 was upregulated by >2 fold in Aspc1 and Panc1 cells. This upregulation was significantly reduced when TFF2 was knock-down. This data suggests the transcriptional upregulation of SMAD4 by decitabine is dependent on TFF2.
Conclusion: These data support the hypothesis that methylation of the TFF2 promoter may be an early mechanism in tumor suppressor inactivation in PDAC.
DPYSL3 Regulates TFF2/SMAD4 Tumor Suppressor Signaling in Pancreatic Cancer
S. Chowdhury,1 M. Ali,1 J. Yamaguchi,2 A.S. Liss,2 A. Sontheimer,2 S.P. Thayer.11Department of Surgery, Fred and Pamela Buffett Cancer Center at University of Nebraska Medical Center, Omaha, NE; 2Andrew L. Warshaw Institute for Pancreatic Cancer Research, MGH, Boston, MA.
Background & Aims: TFF2-expressing pancreatic duct glands (PDGs) have been identified to be a progenitor stem cell niche important in regeneration of the pancreatic duct epithelium. Dysregulation of PDG compartment by inflammatory injury is believed to result in metaplasia and cancer. Loss of TFF2 is thought to be important in carcinogenesis. Previous work suggested that TFF2 functions as a SMAD4-dependent tumor suppressor. Little is known about TFF2 cellular signaling mechanisms. In the present study, we interrogated the interaction of TFF2 and Dihydropyrimidinase-like 3 (DPYSL3) in mediating SMAD4-dependent tumor suppressor functions in PDAC.
Methods: LCM of the PDG compartment was analyzed by microarray analysis. TFF2/DPYSL3 interaction was determined using IP/WB. Knockdown (KD) of TFF2 and DPSYL3 was performed to test for downstream regulation of SMAD4 signaling. TFF2 methylation was manipulated in Panc1 and Aspc1 cell lines using a DNA methylation inhibitor (decitabine) and its effect on DPYSL3 was determined.
Results: Minimal sample microarray analysis revealed 4-fold upregulation of DPYSL3 in the PDG compartment compared to the main duct. IP of TFF2 followed by WB of DPYSL3, and vice versa showed that TFF2 and DPYSL3 interact with each other in Panc1 and Aspc1 cells. TFF2 KD down regulates DPYSL3 mRNA. Demethylation of the TFF2 promoter by decitabine resulted in upregulation of TFF2, DPYSL3 and SMAD4 mRNAs. Decitabine treatment in the presence of TFF2 KD significantly decreased the up-regulation seen in DPYSL3 and SMAD4 mRNA. Knockdown of DPYSL3 led to SMAD4 downregulation. However, demethylation of the TFF2 promoter in the presence of DPYSL3 KD significantly reduces the up-regulation of the SMAD4 mRNA.
Conclusion: This data suggests that DPYSL3 is a necessary component of the TFF2/SMAD4 tumor suppressor-signaling axis in pancreatic cancer cells.
Mechanistic Studies in the Inflammatory Response of Pancreatitis and Pancreatic Cancer: Role of Myeloid Derived Suppressor Cells
N. Cieza-Rubio,1 T. Jie,1 R.L. Heimark.1,21Department of Surgery, University of Arizona, Tucson, AZ; 2University of Arizona Cancer Center, Tucson, AZ.
Background: Tumor-infiltrating myeloid-derived suppressor cells (MDSCs), are important mediators of a tumor-permissive microenvironment that contributes to tumor growth and could account for the limited success of immunotherapeutic strategies. MDSCs suppress adaptive immunity by blocking T cell activation, inducing Treg accumulation, and inhibiting natural killer cell cytotoxicity against tumor cells.
Aim: We investigated the roles of MDSCs in the regeneration of the exocrine pancreas associated with acute pancreatitis and the progression of acinar to ductal metaplasia.
Results: Acute pancreatitis was induced in wild type and P48+/Cre;LSL-KRASG12D mice using caerulein; an early influx of MDSCs into the pancreas was observed with flow cytometry and IHC. Numbers of Gr1(+)CD11b(+) MDSCs increased over 20-fold in pancreata of mice with acute pancreatitis to account for nearly 15% of intrapancreatic leukocytes and have T cell suppressive properties. This marked accumulation of MDSCs returned to normal values within 24 hours of the insult in wild type mice; however, in the oncogenic KRAS mice, MDSCs levels remained elevated. When intrapancreatic MDSCs were depleted by administration of a CCR2 antagonist (SB265610) in wild type mice, the severity of acinar damage was increased. This was also accompanied by a delayed regeneration determined morphologically and with the mitotic immunomarker phospho-histone H3. Isolated intrapancreatic MDSCs from treated mice induce naïve acinar cells to undergo acinar ductal metaplasia when co-cultured in collagen 3D cultures. Purified splenic MDSCs failed to induce the phenotypic transdifferentiation.
Conclusion: MDSCs are required for adequate pancreatic regeneration in wild type mice with acute pancreatitis and their persistent elevation in oncogenic KRAS mice is not only associated with immune-evasion, but may also function as direct enhancer of malignant proliferation.
Amelioration of Experimental Acute Pancreatitis With FK-506 and Tamoxifen
D.L. Clemens,1,2 K.J. Schneider,1 C.K. Arkfeld,1 S. Singh.11Department of Internal Medicine, University of Nebraska Medical Center, Omaha, NE; 2VAMC Omaha, NE.
Introduction: Post-ERCP pancreatitis remains a common cause of significant morbidity and mortality despite prophylactic use of currently available strategies i.e. pancreatic duct stenting and rectal indomethacin. Two inflammatory pathways involved early in the course of acute pancreatitis are mediated by nuclear factor of activated T-cells (NFAT), which is activated by the calcium sensitive phosphatase, calcineurin, and the inducible transcriptional activator nuclear factor-κB (NF-κB). Both of these regulate the expression of cytokines and chemokines, important mediators of the damage associated with the inflammatory response characteristic of acute pancreatitis. Targeting these pathways individually to prevent acute pancreatitis has yielded mixed results. To test the effectiveness of targeting more than one inflammatory pathway, we treated mice with the calcineurin inhibitor tacrolimus (FK-506) and the NF-κB inhibitor tamoxifen.
Methods: Mice were treated with FK-506 (1 mg/kg) and tamoxifen (20 mg/kg) for 2 days before inducing pancreatitis. Pancreatitis was induced by 8 hourly injections of caerulein (50 μg/kg). Mice were sacrificed 1 hr after the final injection.
Results: Combination treatment with FK-506 and tamoxifen resulted in reduced serum amylase and lipase levels. Compared with control animals, treated animals also displayed decreased mRNA expression of the proinflammatory cytokines IL-1β, TNF-α and IL-6, as well as increased expression of the anti-inflammatory cytokine IL-10. Additionally, these biochemical changes were associated with significantly less inflammation in the pancreata of treated animals.
Conclusions: Attenuation of the inflammatory response reduces the severity of acute pancreatitis. Concurrently targeting multiple inflammatory pathways with FDA approved pharmaceutical agents may be a viable approach to prevent post-ERCP pancreatitis or reduce the severity of this disease.
NFATc1-Dependent Downregulation of GLI1 Underlies Polyunsaturated Fatty Acids Cytotoxic Properties in Pancreatic Cancer
A. Comba,1,2 M.E. Pasqualini,2 E. Iguchi,1 M.V. Messler,2 R. Silva,2 M.G. Fernandez-Barrena,1 E.J. Tolosa,1 E. Enriquez-Hesles,1 A.L. Vrabel,1 L.L. Almada,1 D.L. Marks,1 B. Botta,3 L. Di Marcotullio,3 V. Ellenrieder,4 A.R. Eynard,2 M.E. Fernandez-Zapico.11Schulze Center for Novel Therapeutics, Mayo Clinic, Rochester, MN; 2INICSA, CONICET and Universidad Nacional de Córdoba, Córdoba, Argentina; 3Department of Molecular Medicine, Sapienza University, Rome, Italy; Gastroenterology and Gastrointestinal Oncology, University Medical Center Goettingen, Goettingen, Germany.
Numerous reports have demonstrated a growth inhibitory effect of Polyunsaturated Fatty Acids (PUFAs) in multiple tumors including pancreatic cancer. However, the molecular mechanisms modulating this phenomenon remain elusive. Here, we provide evidence of a novel anti-tumoral mechanism of the PUFA arachidonic acid (AA). In vivo and in vitro experiments showed that AA treatment decreases tumor growth and metastasis, and increases apoptosis. Molecular analysis of this effect showed reduced expression of a subset of anti-apoptotic proteins, BCL2, BFL1/A1 and 4-1BB in AA-treated cells. We demonstrated that the downregulation of the transcription factor GLI1 is the underlying mechanism controlling BCL2, BFL1/A1 and 4-1BB expression. Using chromatin immunoprecipitation and expression studies, we demonstrated that GLI1 binds to the promoter of these antiapoptotic molecules, and regulates their expression. Further characterization showed that AA regulates GLI1 expression through the modulation of its promoter via NFATc1-mediated repression of GLI1 transcription. AA promotes NFATc1 activation through an oxidative stress-Calcineurin dependent axis. Finally, we provide evidence that AA-induced apoptosis and gene repression can be inhibited by overexpressing GLI1 in AA-sensitive cells. Collectively, these results define a novel mechanism underlying AA anti-tumoral functions that may serve as a foundation for the future PUFA-based therapeutic approaches.
Are the Current Physician Quality Reporting System (PQRS) Measures Adequate for Academic Pancreatic Centers of Excellence (APCOE)?
D.L. Conwell,1 P. Hart,1 M. DiMagno,2 M. Alsante,3 C.M. Wilcox,4 J. Barkin,5 B. Kisloff,6 D.C. Whitcomb,6 the PancreasFest 2015 Academic Pancreatic Centers of Excellence Ad Hoc Working Groups. 1The Ohio State University Wexner Medical Center, Columbus, OH; 2University of Michigan, Ann Arbor, MI; 3National Pancreas Foundation, Bethesda, MD; 4University of Alabama-Birmingham, AL; 5University of Miami, FL; 6University of Pittsburgh, PA.
Background: In 2017, as part of Medicare’s new Value-Based Payment Modifier Reimbursement Program, physicians who do not report PQRS measures for 2015 will be subject to a payment adjustment of −2.0%. Physicians in groups of 10 or more will be subject to a payment adjustment of −4.0%. Recently, IBD quality measures group was adopted by the AGA. There are currently no quality measures for pancreatic disease.
Aim: Determine if current quality measures can be “bundled” for PQRS reporting by APCOEs to avoid the negative payment penalty.
Methods: An ad hoc working group of academic physician-scientist from multiple academic societies was formed to review measures and develop standards for PCOEs. Over 400 PQRS quality measures were reviewed to identify those that might be reflective of quality care administered to both pediatric and adults.
Results: 17 quality measures (PQRS number) were identified that could be assessed during evaluation of pancreatic disease patients. The quality metrics were distributed across 4 domains as follows: Effective Clinical Care 7/17 (41%): diabetes control adult/pediatrics (001,365), osteoporosis screen, therapy, fracture follow-up (039,040,041), alcohol/drug treatment (305), and depression screen (371). Communication and Care Coordination 3/17 (18%): medication reconciliation (024), osteoporosis communication (024), biopsy follow-up (265). Community and Population Health 6/17 (35%): tobacco use, screening, cessation (226), unhealthy alcohol use (173), depression screening (134), pain assessment (131), BMI, weight assessment children/nutrition (120, 239). Patient Safety 1/17 (6%): medication reconciliation (130).
Conclusion: Currently, no pancreas-specific PQRS measures are available. Only 17 of 400 measures are relevant to the care of pancreatic disease patients. Systematic review of the elements of quality practice and recommendations for pancreas-specific measures (i.e. exocrine insufficiency, pain medication use/abuse, cancer screening, genetic/family counseling) are needed to align future reimbursement with quality pancreatic disease care.
The Role of Prolactin and its Cognate Receptor in Pancreatic Cancer Progression
A. Criscimanna,1 M. Socorro,1 M. Tandon,1 A. Singhi,2 F. Esni.1,3Departments of 1Surgery and 2Pathology; 3University of Pittsburgh Cancer Institute, University of Pittsburgh, Pittsburgh, PA.
Prolactin is perhaps best known as the hormone that stimulates lactation in mammals, but it has many functions beyond reproduction and lactation. Prolactin binds to a specific membrane receptor, the prolactin-receptor (PRLR), which induces several intracellular signaling cascades that are mediated via Jak/STAT, and Jak/Ras/MAPK components. Macrophages are monocyte-derived myeloid cells that produce an array of cytokines, chemokines, growth factors, and hormones, many of which possess tumor-promoting activities. Numerous studies in recent years have highlighted the involvement of stroma in general, and macrophages in particular in Kras-induced pancreatic neoplasia. However, the mechanism(s) by which macrophages influence pancreatic tumorigenesis is still under intense investigation.
Here, we provide evidence that in Ptf1aCre;KrasG12D mice PRLR is expressed in a subset of early mPanINs, however its expression becomes more homogeneous as these lesions progress and transform to PDA. Interestingly, PRLR is also detected in human PDA. Furthermore, we show that PanINs respond to prolactin, as increased systemic prolactin levels promote mPanIN progression and transformation to PDA in a dose dependent manner. Additionally, we demonstrate that during the course of PanIN development in Ptf1aCre;KrasG12D mice, prolactin is produced locally by macrophages surrounding the neoplastic lesions. Finally, an FDA-approved antagonist of PRLR has been shown to induce autophagy-related cell death in ovarian cancer cells. We are currently studying the effect of this drug on pancreatic cancer cells in vitro and in vivo.
In conclusion, we have identified macrophage-derived prolactin as a key stromal factor necessary for PanIN to PDA transformation. Moreover, targeting prolactin/PRLR may have clinical implications in pancreatic cancer.
Dectin-1 Signaling Promotes Pancreatic Oncogenesis by the Induction of Peritumoral Immune Suppression
D. Daley, L. Tomkötter, A. Ochi, D. Tippens, S. Jonnadula, S. Chang, N. Akkad, R. Barilla, A. Torres-Hernandez, G. Miller. Department of Surgery, New York University School of Medicine, New York, NY.
Aim: To determine the significance of Dectin-1 signaling in pancreatic oncogenesis.
Background: Toll-like receptors (TLRs) are the most well studied family of pattern recognition receptors (PPRs) and specific TLR signaling has been shown to play a critical role in promoting chronic pancreatitis and pancreatic oncogenesis. C-type Lectin receptors (CLRs), including Dectin-1, are a family of trans-membrane PRRs that are required for recognition of fungal or mycobacterial pathogens. However, while TLR ligation has been implicated in pancreatic adenocarcinoma (PDA), the role of Dectin-1 in sterile inflammation, and specifically in the development of PDA, has not been studied.
Methods: We evaluated the effects of deletion or blockade of dectin-1 signaling in pancreatic cancer on tumor size and survival, peritumoral fibro-inflammation, and epithelial transformation. We utilized p48Cre;LSL-KrasG12D (KC) mice as our murine pancreatic oncogenesis model and we crossed KC mice with Dectin-1-/- mice to create a Dectin-1-/- pancreatic cancer mouse model. Components of the dectin-1 signaling pathway and the immune infiltrate within the pancreatic TME were assessed and characterized using immunohistochemistry, flow cytometry and western blotting.
Results: The Dectin-1 receptor and endogenous ligands for the receptor are highly expressed in inflammatory and epithelial compartments in the pancreatic TME in humans and mice. Dectin-1 deletion or signaling blockade significantly delayed the progression of PDA in mice, while dectin-1 ligation accelerated oncogenesis. Dectin-1 signaling promoted the infiltration of pro-tumorigenic tumor associated macrophages (TAMs) in the pancreatic TME. Furthermore, Dectin-1+/+ TAMs directly inhibited anti-tumor T lymphocytes and induced differentiation towards immune suppressive T-cell phenotypes compared to Dectin-1-/- TAMs.
Conclusion: Dectin-1 receptor ligation drives inflammatory cell activation, inducing immune suppressive inflammatory changes in the pancreatic tumor microenvironment thereby contributing to the progression of pancreatic oncogenesis. This implicates the Dectin-1 pathway as a target for the treatment of pancreatic oncogenesis.
Molecular Evidence for Monoclonal Skip Progression in Main Duct Intraductal Papillary Mucinous Neoplasms of the Pancreas
K. Date,1 T. Ohtsuka,1 T. Fujimoto,1 Y. Gotoh,1 Y. Nakashima,1 H. Kimura,1 T. Matsunaga,1 N. Mochidome,2 T. Miyazaki,2 Y. Oda,2 M. Tanaka,1 M. Nakamura.1Departments of 1Surgery and Oncology, 2Anatomic Pathology, Graduate School of Medical Sciences, Kyushu University, Fukuoka, Japan.
Background: It has been recently suggested that main duct (MD) intraductal papillary mucinous neoplasm (IPMN) might have a feature of monoclonal skip progression, and the aim of this study was to clarify clonality of distinct MD-IPMNs using microarray, mutational, and immunohistochemical analyses.
Methods: Total RNA was extracted from fresh-frozen tissue samples of 2 metachronous MD-IPMNs and matched non-neoplastic pancreas tissues of the same pancreas in 2 individuals, and whole human genome microarray analysis was carried out. Formalin-fixed paraffin-embedded tissue specimens from synchronous and/or metachronous distinct 28 IPMNs were collected from 12 patients who had 2 or more distinct IPMNs, and genomic DNA was extracted and GNAS/KRAS mutational status was investigated. Then, immunohistochemical analysis was also performed to validate the expression pattern of the indicated proteins.
Results: According to the gene expression profiles by microarray data, 2 metachronous MD-IPMNs of the same individual had a pairwise correlation coefficient of 0.9523 and 0.9512, whereas the same histological grade MD-IPMN of the different individual had 0.8092 and 0.8211. Heatmap and hierarchical cluster analysis revealed that 2 metachronous MD-IPMNs of the same individual were divided into the same cluster and the gene expression patterns under the same branch were very similar. Eight of 12 patients had 2 or more distinct MD-IPMNs and the GNAS/KRAS mutational status of distinct MD-IPMNs were consistent with each other. Immunohistochemical assessment using 6 specific proteins showed the same expression pattern was observed in 40 of 48 (83%) between 2 distinct MD-IPMNs in the same individual.
Conclusions: These findings indicated that MD-IPMNs might have a feature of monoclonal skip progression.
The Unfolded Protein Response and Overcoming Chemoresistance
P. Dauer, O. McGinn, X. Zhao, N. Arora, M. Singh, V. Dudeja, S. Banerjee, A.K. Saluja. Division of Basic and Translational Research, Department of Surgery, University of Minnesota, Minneapolis, MN.
Pancreatic cancer is a devastating disease with poor survival statistics that have remained relatively unchanged for the past 30 years. There are multiple factors contributing to these statistics, including late detection, no effective therapeutics, and a highly chemoresistant phenotype. It has been reported that a condition known as endoplasmic reticulum (ER) stress and the unfolded protein response (UPR) correlate with advanced stage, shorter survival and chemoresistance in multiple cancers, but this is unclear in pancreatic cancer.
Glucose regulatory protein (GRP78) regulates UPR in non-stressed conditions. Our studies show that the average GRP78 serum levels from 4 patients with pancreatic cancer was 15,023 pg/mL/μg protein (SEM = 5419). These values were significantly greater than the serum levels of healthy volunteers (4.32 pg/mL/μg protein, SEM = 1.77). Murine in vivo analyses indicate a three-fold increase in GRP78 mRNA expression of KPC tumor samples versus KPC pancreas without tumors. Additionally, the KPC tumors have increased GRP78 expression in the ducts as tumors progress from pre-cancerous lesions to pancreatic adenocarcinoma, detected by immunofluorescence. One possible route of chemoresistance in pancreatic cancer is through efflux of xenobiotics with ABC transporters. Our studies show that mRNA expression of NRF2, a transcription factor downstream of UPR activation, is increased in KPC tumors versus KPC pancreas without tumors. Further, mRNA of ABC-C transporters (transcriptionally regulated by NRF2) are also increased in KPC tumors versus that KPC pancreas without tumors. Based on our studies, UPR could provide multiple selective targets for future therapeutics, which may also decrease the chemoresistance in pancreatic cancer.
Targeting the Tumor-Associated Microenvironment: The Clinical Impact of Integrin Alpha 5 (ITGA5) in Patients With Pancreatic Ductal Adenocarcinoma
S.W.L. de Geus,1 P.J.K. Kuppen,1 H.A.J.M. Prevoo,1 R.L. Vlierberghe,1 R-J. Swijnenburg,1 J.S. Mieog,1 B.A. Bonsing,1 H. Morreau,2 C.J.H. van de Velde,1 A.L. Vahrmeijer,1 C.F.M. Sier.11Department of Surgery, University of Leiden, Leiden, The Netherlands; 2Department of Pathology, University of Leiden, Leiden, The Netherlands.
Background: There is cumulative evidence that the tumor-associated microenvironment is crucial to tumor development and progression. Integrin αv (ITGA5) and α-smooth muscle actin (α-SMA) are important players in the tumor-associated microenvironment. Therefore, the aim of this study is to examine the expression of ITGA5 and α-SMA in relation to their potential for tumor targeted therapy and imaging of pancreatic cancer patients.
Methods: Expression of α-smooth muscle actin (α-SMA) and integrin αv (ITGA5) was evaluated by immunohistological analyses in 137 patients with pancreatic ductal adenocarcinoma. In addition, the tumor-stroma ratio was assessed based on hematoxylin and eosin (H&E) stained tumor sections retrieved from the same patient population. Chi-square, fisher-exact and student t-tests were used to evaluate the association between clinical and pathological characteristics and biomarkers expression and survival analysis was performed using a Cox proportional hazard model.
Results: A high tumor-stroma ratio was found in 89.2% of the patients, α-SMA expression was over-expressed in 84.8%, membranous and stromal ITGA5 expression was abundant in 39.3% and 65.5% of the patients. High α-SMA expression was positively associated with up-regulated stromal ITGA5 expression (p < 0.001), but not with membranous ITGA5 expression (p = 0.161). ITGA5 expression of the tumor-associated microenvironment was also correlated with vascular invasion (p = 0.023). On multivariate analyses high α-SMA expression (p = 0.041) and combined stromal and membranous ITGA5 expression were of independent predictive value for OS (p = 0.025) and DFS (p = 0.051) in pancreatic ductal adenocarcinoma patients.
Conclusion: The results of this study suggest that the tumor-associated microenvironment and in particular ITGA5 play a critical role in the progression of pancreatic cancer. Therefore, ITGA5 holds great promise as target for targeted-therapy and personalized medicine in pancreatic cancer patients.
In Vivo Imaging of Non-Radioactive Near Infra-Red 2-Deoxyglucose During Mild and Severe Acute Pancreatitis (AP) in Rats
C. de Oliveira, K. Patel, P. Noel, R.N. Trivedi, A. Singh, V.P. Singh. Department of Medicine, Mayo Clinic - Arizona, Scottsdale, AZ.
Background: While 2-Deoxy-2-[18F] fluoroglucose (FDG) uptake is increased in human AP, its relevance to AP severity is unknown. We thus studied the uptake of the non-radioactive, near infrared fluorescence labelled 2-deoxyglucose analog, IRDye® 800CW 2-DG probe (2-DG; Li-Cor) during mild (cerulein; CER) and severe (intraductal glyceryl trilinoleate; ID-GTL) AP.
Methods: Wistar rats (300g; 3-6/group) were administered 2-DG (44mcg/kg; I.V.). CER AP (50μg/kg, I.V) and severe AP (ID-GTL 50μL/0.1kg) were induced within 10 minutes of giving 2-DG. DL group had only duct ligation and controls (CON) only received 2-DG. Imaging was done every 10-15 minutes over 2 hrs. Average Radiant Efficiency [p/s/cm2/sr]/[μW/cm2] was measured over the pancreas using the IVIS 200 in-vivo imaging system (PerkinElmer) using the Living Image® software and verified in ex vivo pancreata. Serum amylase, lipase and pancreatic edema measured after 6-8 hours of AP. Values are mean ± SEM and p<0.05 was considered significant.
Results: 2-DG uptake significantly increased over controls between 40-100 minutes in both AP models. The peak was 2-fold CON in the CER and 6-fold CON in ID-GTL groups (p<0.05), with a 1.2-fold increase in DL group. Ex vivo pancreas imaging confirmed the increased signal of ID-GTL (∼4-fold) and CER (∼1.7-fold) vs DL and control. ID-GTL but not CER rats were moribund and had hemorrhagic pancreatic necrosis when sacrificed. Both CER and ID-GTL AP increased serum amylase (4901±793 U/L; 9841±1530 U/L vs 2642±294 U/L in DL and 408±22 U/L in CON; p<0.01), lipase (3734±695 U/L; 6455±1684 U/L vs 486±71 U/L in DL and 23±3.4 U/L in CON; p<0.05) and pancreatic edema (87±2.1%; 88%±1.6% vs 79±1.4% in DL and 67±2.3% in CON; p<0.05).
Conclusion:In-vivo fluorescent imaging system using a non-radioactive 2-DG optical probe may be useful alternate to FGD and predict the AP severity early in the disease.
Role of Preoperative CA19-9 in Predicting Long and Short Term Progression Free Survival in Patients Undergoing Pancreatic Cancer Resection
S. Desai,1 C. Langmead,2 H. Zeh,3 A. Zureikat,3 N. Bahary,1 R. Brand.11Department of Medicine, University of Pittsburgh, Pittsburgh, PA; 2School of Computer Science, Carnegie Mellon University, Pittsburgh, PA; 3Department of Surgery, University of Pittsburgh, Pittsburgh, PA.
Background: A major advancement in the management of pancreatic adenocarcinoma (PC) patients would be identifying those patients who will not achieve significant benefit from attempted curative resection due to the presence of unrecognized metastatic disease at surgery or a short-time (<1yr) to PC recurrence. A pre-operative serum biomarker that could predict these patients would be of great benefit by avoiding unnecessary surgeries. Our aim was to determine if serum CA19-9 could predict long or short-term progression free survival (PFS) for those patients undergoing attempted curative resection.
Methods: We retrospectively reviewed 287 patients from our PC registry undergoing attempted curative resection from January 1, 2006 through February 28, 2014. Pre-surgical CA 19-9 levels, total bilirubin, neo-adjuvant or adjuvant chemotherapy, PFS and overall survival were collected. Analysis was limited to those patients who did not receive neoadjuvant therapy (50 patients). Long-term PFS was defined either as ≥3 yrs or ≥ 2 yrs without radiologic evidence of disease and short-term PFS was defined as <1 yr without radiologic evidence of disease. Statistical p-values were calculated with a Mann Whitney U test.
Results: Twenty-five of 50 patients had short-term PFS, 7 patients had PFS ≥ 3 yrs and 14 patients had PFS ≥ 2 yrs. There was no statistically significant difference in the serum CA19-9 between those with < 1 yr and either ≥ 2 (p=0.77) or 3 yrs PFS (p=0.82).
Conclusions: Pre-operative CA19-9 did not distinguish between long and short-term PFS. These results support the need for future studies aimed at evaluating different serum biomarkers alone, or in combination with CA 19-9 and/or pre-operative clinical factors, to predict those patients with a short (<1 yr) PFS.
Impact of the Anatomical Location of Necrosis on Outcome in Patients of Acute Pancreatitis
N. Dhaka,1 A. Munit,4 J. Samanta,1 R. Prasada,1 P. Gupta,2 V. Gupta,3 T.D. Yadav,3 S.K. Sinha,1 R. Kochhar.1Departments of 1Gastroenterology, 2Radiodiagnosis, 3Surgery, Postgraduate Institute of Medical Education And Research, Chandigarh, India; 4John H Stroger Hospital of Cook, Chicago, IL.
Aim: To evaluate the impact of anatomical location of necrosis on outcome in patients of acute nercrotizing pancreatitis (ANP).
Methods: 161 ANP patients were classified according to the site of necrosis [pancreatic (I), peripancreatic (II) or both (III)]. Patients with pancreatic necrosis were further divided in three groups based on anatomical location of necrosis [necrosis involving only head region (A), involving body with or without tail region (B), involving the whole pancreas (C)]. Site of necrosis was correlated with outcome (hospital stay, need for ICU care, need for ventilator, pigtail catheter drainage (PCD), surgery and mortality).
Results: Pancreatic necrosis was seen in 115 (71.5%) and peripancreatic necrosis alone in 46 (28.5%) patients. Out of 115 patients with pancreatic necrosis, necrosis at location A was seen in 35 (30.5%), at location B in 48 (41.7%) and at location C in 32 (27.8%) patients. Patients with location A and C necrosis were significantly associated with more hospital stay (38.82±25.8, 27.99±22.1 vs. 13.04±7.6 days, p<0.001), need for ICU care [n=23(65.71%), n=18(56%) vs. n= 6(12.50%), p<0.001], requirement of ventilator [n=3(8.57%), n=16(50%) vs. n=0(0%),p<0.001], surgery [n=3(8.57%), n=4(12%) vs. n=0(0%),p=0.06] and PCD requirement [n=32(91.42%), n=22(68%) vs. n=0(0%),p<0.001] as compared to location B necrosis . Outcome comparison between patients of location A and C necrosis showed that necrosis at location A was associated with more hospital stay (38.82±25.8 vs. 27.99±22 days), need for ICU care [n=23(65.71%) vs. n=18(56%)] and PCD [n=32(91.42%) vs. n=22(68%)], but it was statistically non-significant. However need for surgery [n=3(8.57%) vs. n=4(12%), p=0.067) and ventilator [n=3(8.57%), n=16(50%),p<0.001) were seen more in location C necrosis. Mortality was exclusively seen in patients with necrosis at location C [(n=14(43%)].
Type III was the commonest type of necrosis (n=69, 42.8%), followed by type II (n=46, 28.57%) and type I (n=46, 28.57%). Patients with type III necrosis had significantly increased hospital stay [34.28±24.8 vs. 13.26±7.4, 18.19±10.5 days, p<0.001] and need for ICU care [n=41(59.4%) vs. n=6(10.86%), n=16(34.7), p<0.001). Need for ventilator (n=19, 27.5%), PCD (n=54, 78.2%), surgery (n=7, 10.14%, and mortality (n=14, 20.28%) were exclusively seen in type III patients. Comparison between patients with type I and II necrosis showed that type II necrosis had significantly more hospital stay (18.19±10.5 vs. 13.26±7.4 days, p=0.011) and need for ICU care [n=16(34.78%) vs. n=6(13.04%), p<0.001) and PCD [n=15(32.06%) vs. n=0(0%), p<0.001].
Conclusion: Necrosis involving pancreatic head region had worse outcome as compared to body and tail regions. Patients with necrosis involving both pancreatic and peripancreatic region had poorer outcome than patients with only peripancreatic necrosis or pancreatic necrosis.
Roles of Extracellular ATP in Promoting Systemic Inflammation During Acute Pancreatitis
A.K. Dixit, J. George, Y. Ryu, Z. Yuan, A. Sareen, V. Dudeja, R. Dawra, A.K. Saluja. Division of Basic and Translational Research, Department of Surgery, University of Minnesota, Minneapolis, MN.
Introduction: Pancreatitis is an inflammatory disorder of the pancreas. Severe acute pancreatitis (SAP) is associated with multi organ dysfunction, which is main cause of death in SAP. How local injury leads to systemic inflammation and multi organ failure in SAP not understood till now.
Aim: In the current study we explored the role of extracellular ATP (eATP) in causing multi organ failure.
Method: For in vitro pathological insult, acinar cells were stimulated with 100 nM cerulein or various concentrations of taurolithocholic acid-3-sulfate (TLC-S) or 1 mM Carbacol for 3 hr at 37 °C. For In vivo studies mild acute pancreatitis was induced by 7 or 10 hourly injection of 50 ug/kg cerulein. Severe model of acute pancreatitis was induced by 8 hourly injection of 50 ug/kg cerulein for 2 days. ATP concentrations was measured in serum or media supernatant using bioluminescence assay.
Results: In acute pancreatitis micro molar concentrations of eATP was detected in serum (40 uM in 2 day cerulein model, 8 uM in 10 hr cerulein model and 170 nM in control model) and the amount of eATP correlates with severity of disease. Acinar cells treated in vitro with cerulein or different concentration of bile acid releases 2-3 folds ATP in the medium. More over acinar cell treated with micro molar concentrations of ATP release significant amount TNF-α and IL-1β. Injection of ATP (40 mg/kg i.p.) alone causes increase in serum cytokines and causes migration of neutrophils in mice peritoneum. Apyrase; an ecto enzyme which degrades eATP, when injected in mice significantly reduced pancreatitis associated serum IL-6, TNF-α and lung MPO during acute pancreatitis.
Conclusions: Extracellular ATP is released in serum during acute pancreatitis and it causes activation of immune system. The levels of eATP correlate with disease severity and degradation of eATP using apyrase reduces pancreatitis associated serum cytokines and lung injury.
Role of miR-21-3p in Promoting Inflammation During Acute Pancreatitis
A.K. Dixit, J. George, U. Barlass, Z. Yuan, A. Sareen, V. Dudeja, R. Dawra, S. Subramanian, A.K. Saluja. Division of Basic and Translational Research, Department of Surgery, University of Minnesota, Minneapolis, MN.
In pancreatitis, initiating events of the disease originate in acinar cells. Understanding these events is important for deciphering pathophysiological mechanism of the disease. miRNA are small noncoding single stranded RNA which play a significant role in post-transcriptional regulation of protein expression. Although, ubiquitous each cell type has a distinct expression signature of miRNA. Dysregulation of miRNA has been reported in various inflammatory conditions. However, not much information is available on the role of miRNAs in pathophysiology of pancreatitis.
Aim: To identify the dysregulated miRNA involved in acute pancreatitis and its role in regulating inflammation.
Method: Human and murine pancreatic acinar cells were prepared by standard collagenase digestion method. For in vitro pathological insult, acinar cells were stimulated with 100 nM cerulein or various concentrations of taurolithocholic acid-3-sulfate (TLC-S) or 1 mM Carbacol for 1 hr at 37 °C. For In vivo studies acute pancreatitis was induced by 7 hourly injection of 50 ug/kg cerulein, for L-arginine model pancreatitis was induce by two 4.5 mg/kg L-arginine one hour apart and mice was sacrificed after 72 hrs. For cerulein and LPS model, pancreatitis was induced by 6 hourly injection of 50 ug/kg of cerulein followed by 10 mg/kg of LPS. Total small RNAs were isolated using mirVanaTM kit. Targets for miR-21-3p was screen-using miRDB.
Results: miR-21-3p was up regulated in human and mouse acinar cell treated with TLCS or carbacol or cerulein invitro. In all murine models acute pancreatitis (cerulein, cerulein and LPS, L-arginine) miR-21-3p was up regulated. miR-21-3p levels negatively correlates with cylindromatosis (CYLD) levels, one of its predicated target. CYLD is a deubiquitinase, which negative regulator of NF-kB and AP-1 pathway.
Conclusions: miR-21-3p is upregulated in all models of acute pancreatitis and miR-21-3p potentially regulates CYLD, which is responsible for activation of inflammatory pathways mediated via NF-kB and AP-1 during acute pancreatitis.
Pancreatic Surgery Trends in the United States Between 1998–2011
A. Dudekula,1 S. Munigala,2 A.H. Zureikat,3 D. Yadav.1Division of 1Gastroenterology and 3Surgical Oncology, University of Pittsburgh Medical Center, Pittsburgh, PA; 2Department of Internal Medicine, St. Louis University, MO.
Aim: To study the trends in number and type of surgeries performed for pancreatic indications in the United States.
Background: The incidence and burden of pancreatic diseases is changing. We hypothesized that pancreatic surgery trends will parallel the population distribution of pancreatic diseases, and new advances in knowledge, technology and treatments.
Methods: Using the Nationwide Inpatient Sample, we identified patients ≥18 years who underwent pancreatic surgery between 1998-2011. We calculated age- and sex-adjusted rates per million adult population using the 2010 US population as a reference. Changes in patient characteristics and outcomes were analyzed.
Results: Of 191,012 surgeries, 151,454 (79%) were performed for a primary pancreatic indication. Of these, 82% were resections and 64% performed for tumors (malignant 52%, benign 12%). Surgery rates per million population increased non-significantly by 1.5 folds, but significant trends for individual operations and indications were noted. Population rates increased significantly for distal pancreatectomy, were borderline significant for pancreaticoduodenectomy, and decreased significantly for drainage procedures. Surgery rates increased significantly for tumors and cysts/pseudocysts, but decreased significantly for acute pancreatitis. During the study period, the use of the laparoscopic approach increased (3.8 to 9.1%, p-trend <0.001). The mean age and comorbidity burden for patients increased significantly, coupled with a significant decrease in the length of stay and in-hospital mortality.
Conclusions: The number of pancreatic surgeries performed in the US is increasing. Although surgeries are being offered to older patients with more concomitant medical problems, they are being performed with increasing safety, better outcomes and use of laparoscopic techniques.
A Personal History of Pancreatic Cancer and Another Lynch-Related Cancer: A New Indication for Germline Genetic Panel Testing
B. Dudley,1 F. Monzon,2 A. Singhi,3 S. Lincoln,2 N. Bahary,1 R. Brand.11Department of Medicine, University of Pittsburgh, Pittsburgh, PA, 2Invitae Corporation, San Francisco, CA; 3Department of Pathology, University of Pittsburgh, Pittsburgh, PA.
Introduction: About 10% of pancreatic adenocarcinomas (PC) are attributed to hereditary causes. Multi-gene panel tests for cancer susceptibility genes can help identify a genetic basis for an individual’s PC diagnosis, direct chemotherapy, and provide information for family members. Guidelines for genetic testing for an individual with PC are limited and primarily focus on family history (FH), although the Bethesda guidelines for microsatellite instability testing include individuals with a personal history of two Lynch syndrome (LS)-related cancers. The primary aims of our study were to determine the prevalence of mismatch repair (MMR) and other germline genetic mutations in individuals with PC and another LS-related cancer.
Methods: Histories from all PC patients enrolled in our registry (n = 1296) were reviewed for a personal history of a Bethesda-designated LS-related cancer. A 29 gene panel-test was performed in a research setting for patients without clinical testing.
Results: Forty-six patients were identified. Genetic test results were available for 31 of these individuals: 7 had syndrome-directed testing, 2 had clinical panel testing, and 22 had the research panel. Eleven of 31(35%) patients carried a deleterious mutation in a gene associated with PC risk: 1 BRCA1; 1 BRCA2; 1 PALB2; 2 ATM; 1 CDKN2A; 1 TP53; 4 MMR genes. Five of the 11 patients had no FH of cancer in a first-degree relative. If the 15 untested patients were considered to have negative genetic tests, 11 of 46 patients (24%) had a deleterious mutation. MMR mutations accounted for only one-third of identified mutations (4/11).
Conclusion: At least 24% of individuals with PC and another LS-related cancer have mutations in genes associated with PC risk. If validated, future genetic testing guidelines for PC should include those patients with PC and LS-related cancers.
Oncolytic Adenovirus for Radioiodine Therapy of Pancreatic Cancer
B. Eidenschink,1 J. Sell,1 C. LaRocca,1 K. Jacobsen,1 M. Fernandez-Zapico,2 J. C. Morris,2 M. Yamamoto,1 J. Davydova.11Department of Surgery, University of Minnesota, Minneapolis, MN; 2Mayo Clinic, Rochester, MN.
Radioiodine therapy has successfully treated and diagnosed thyroid cancers over the last 50 years. However the clinical scope of radioiodine in other cancers is limited due to restricted iodine uptake. Our lab proposes to expand the clinical use of radioiodine into pancreatic cancer. We hypothesized that the oncolytic adenovirus-based expression of the sodium iodine symporter (NIS) will allow iodine to concentrate into targeted cancer cells providing diagnostic imaging with I-123 and therapy with I-131.
The adenovirus (Ad) vector design is the infectivity-enhanced Oncolytic Adenovirus which expresses the NIS and adenovirus death protein (ADP) from the E3 region (NIS-OAd). The Ad5/Ad3 fiber modification enables the infection of CAR-negative cells. Vector replication is controlled through a tumor specific promoter COX2.
We have shown the specificity of our viruses in vitro with multiple pancreatic cancer cell lines. The expression of ADP resulted in an increase in oncolytic activity and viral spread. The functionality of NIS-OAds was tested with an in vitro radioiodine uptake assay and showed up to a 10-fold increase in radioiodine uptake in pancreatic cancer cell lines.
NIS-OAds specificity and functionality have also been evaluated in human pancreatic cancer and normal pancreas tissues ex vivo. The viral copy numbers have shown an increase of COX2 controlled NIS-OAds in cancerous tissue with no increase in normal tissue. This observation suggests that the COX2 controlled virus activation is specific to human pancreatic cancer. Furthermore, radioiodine uptake evaluation showed specific uptake of radioiodine in ex vivo slices as well. Overall, this study supplies data to support the specificity and functionality of NIS-OAds in human pancreatic cancer tissues.
Current projects include therapeutic and imaging studies with radioiodine in immuno-competent Syrian hamsters bearing syngeneic pancreatic cancer, human cell line-derived xenografts in nude mice and in the patient tumor-derived xenografts (PDX) in SCID mice. Together the virus specificity, imaging, and therapeutic data support the potential of NIS-OAds as cancer theranostics. Ultimately, the goal of our research is to form a multimodal therapy with radiation and oncolytic virus for diagnosis and therapy of pancreatic cancer.
Histone Deacetylases Act as Molecular Switches to Promote Pancreatic Regeneration Following Pancreatic Injury
J.F. Eisses,1 A. Criscimana,2 Z.R. Dionise,1 T.A. Javed,1 J.A. Ozolek,3 A. Davis,3 A.I. Orabi,1 S. Sarwar,1 S. Jin,1 S.P.S. Monga,4 F. Esni,2 S.Z. Husain.1Department of Pediatric 1Gastroenterology, 2Surgery, 3Pathology, Children’s Hospital of Pittsburgh of UPMC, Pittsburgh, PA; 4Department of Pathology, University of Pittsburgh School of Medicine, Pittsburgh, PA.
Identifying the molecular switches that permit proper regeneration of the pancreas after major injury, such as with pancreatitis, could yield vital insight for future therapies to mitigate pancreatic injury. We exploited a clinical clue that valproic acid (VPA), an anti-epileptic drug that is strongly associated with pancreatitis, is known to inhibit the histone deacetylases (HDACs), which are epigenetic regulators of transcription. Because HDACs are known to modulate embryonic development of the pancreas, and there is re-emergence of embryonic signaling following pancreatic injury, we hypothesized that HDACs function as a crucial switch to drive proper regeneration and recovery after injury. Using a novel machine learning tool to quantify acinar dropout, we found that VPA delayed recovery of the pancreas following CCK hyperstimulation, and it reduced acinar cell proliferation. Importantly, expression of class I HDACs (which are the primary VPA targets) rose in the mid-phase of pancreatic recovery. VPA administration led to the persistence of acinar-to-ductal metaplastic complexes (ADMs) and prolonged the expression of the embryonic transcription factors Sox9 and Pdx1 within recovering acini. In addition, HDACi with VPA prolonged β-catenin association with the nuclear TCF4 complex. These effects were not observed with valpromide, a non-HDACi analog of VPA. The data suggest that HDACs serve as an epigenetic switch to permit the redifferentiation of ADMs to mature acinar cells by silencing β-catenin signaling and thereby orchestrating proper regeneration following pancreatic injury.
Cytidine Deaminase (CDA) Transcript Analysis Complements hENT1 Protein Staining in Predicting Gemcitabine Response in the ESPAC-3 Pancreatic Cancer Cohort
N. Elander,1,2 K. Aughton,1 P. Ghaneh,1 J.P. Neoptolemos,1 E. Costello,1 D. Palmer,1 T. Cox,1 F. Campbell,1 E. Garner,1 A.S. Evans,1 N. Rimmer,1 C. Halloran,1 M.W. Büchler,3 W. Greenhalf.11NIHR Liverpool Pancreas Biomedical Research Unit, Department of Molecular & Clinical Cancer Medicine, University of Liverpool, United Kingdom; 2Depepatrment of Oncology & Department of Clinical & Experimental Medicine, Linköping University, Linköping, Sweden; 3Department of Surgery, University of Heidelberg, Heidelberg, Germany.
Background: Pyrimidine based chemotherapy offers benefit in both resectable and advanced pancreatic cancer, but which type of treatment is best is patient dependent. Treatment predicting biomarkers are needed in order to personalize chemotherapy.
Materials and Methods: Tissue microarray sections representing 380 patients from the adjuvant ESPAC-3 study cohort underwent in situ hybridization and quantification with the RNAscope® SpotStudioTM technology. Associations with survival data and transcript levels of CDA, DCK, hENT1, RRM1, and RRM2 were assessed. Previously known hENT1 protein expression data were included in analyses.
Results: In gemcitabine treated patients with high hENT1 protein staining, low proportion of CDA-positive cells was associated with improved overall survival ([OS] CDA-low v high quartiles, Mantel-Cox p = 0.041). In 5-FU treated patients, regardless hENT1 status, a similar trend was revealed (OS CDA-low v high p = 0.053) and if average CDA level was measured (rather than proportion of positive cells) this reached statistical significance (HR = 1.672, p = 0.0143). No correlation between hENT1 transcript and protein levels was evident, suggesting post-transcriptional regulation of hENT1 in pancreatic tumor cells. No correlation with DCK, hENT1, or RRM1/2 transcript levels and survival was observed.
Conclusion: CDA transcript level analyzed with the RNAscope® technology, alone or co-analyzed with hENT1 protein expression, is a treatment predictive marker and a potential tool for tailored adjuvant chemotherapy in pancreatic cancer.
Fully Covered Lumen Apposing Metal Stent for the Management of Walled off Necrosis (WON): A Single Center Experience
S. Elwir, S. Amateau, S. Mallery, M. Freeman, M. Arain. Department of Gastroenterology, University of Minnesota, Minneapolis, MN.
Aim: To report results associated with the use of a short, dumbbell shaped fully covered lumen apposing metal stent (LAMS) in the management of necrotizing pancreatitis (NP) complicated by walled off necrosis (WON).
Methods: Data were extracted from a prospectively maintained database of patients (pts) with WON. Pts treated with a 15 mm inner diameter, 1 cm length EUS guided LAMS were analyzed.
Results: 22 pts (82.6% males, mean age 49 yrs) with symptomatic WON underwent drainage/ endoscopic transluminal necrosectomy (ETN) utilizing LAMS from Apr 2014 – Nov 2014. Median time from presentation of NP to intervention was 44 days (range 23 – 733). Drainage was achieved via EUS guided cystenterostomy in all: single cystgastrostomy (CG) in 15 pts, dual site CG in 3 pts, and CG plus cystduodenostomy (CD) in 4. 6 pts required percutaneous drainage in addition to LAMS placement, 2 pts eventually underwent surgery. LAMS placement was successful in 28/29 attempts. There were no immediate complications. Fever developed within 6 days of LAMS placement in 2/5 initial pts treated with LAMS; both pts had LAMS occlusion due to impacted necrotic tissue. When feasible, all subsequent pts were treated with one (12 pts) or more (1 pt) 10 F double pigtail plastic stents through the lumen of the LAMS to maintain patency, however fever occurred within first 2 weeks in 6/13 and stent occlusion in 7/13. To date, ETN has been done in 19/21 pts. ETN was done after LAMS removal in 7 pts and through the LAMS lumen in 9 pts. LAMSs were removed in 16 pts after a mean of 2.5 procedures (range 2-5) and remained in situ for a mean of 25 days (range 3 – 88 days).
Conclusions: Endoscopic drainage of WON using EUS-guided LAMS stents is a safe and effective for initial drainage; however, despite the large lumen, stent occlusion by solid necrosis occurs early and frequently, so that scheduled ETN is advisable. LAMS offer a convenient conduit for ETN.
The Effect of an Integrated Health System Algorithm Based on 2012 International Consensus Guideline on the Local Practice Pattern for the Management of Pancreatic Cystic Neoplasms
A. Eskandari,1 E. Alonso,1 A. Ko,1,3 B. Lim.1,21School of Medicine, University of California, Riverside, CA; 2Department of Gastroenterology, Kaiser Permanente Riverside Medical Center, Riverside, CA; 3Department of Surgery, Kaiser Permanente Riverside Medical Center, Riverside, CA.
Aim: To examine the local practice pattern, i.e. gastroenterology (GI)/surgery referrals and endoscopic ultrasound (EUS), for pancreatic cystic neoplasms (PCL) after a region-wide incorporation of an algorithm based on the 2012 international consensus guideline (ICG) in an integrated health system.
Background: PCLs have become an increasingly common challenging clinical entity for primary/acute care physicians, gastroenterologists, radiologists and surgeons. ICG has been developed to aid in the management of PCLs; revised version was published in 2012. Based on this, Kaiser Permanente Southern California (KPSC) published a regional algorithm in October 2013 with radiology reports including a brief summary of recommendations.
Method: Retrospective review was performed; patients with PCL diagnosis given between April 2012 and April 2015 (18 months before and after the publication of the algorithm) at a single medical center within KPSC were identified.
Results: 237 patients (142 pre and 95 post-algorithm) received a new diagnosis of PCLs in the study period. There was no difference in the mean cyst size for pre (19.6 mm) and post (17.3 mm), p = 0.26. The mean age of the post population was slightly greater (69 vs 66 years, p = 0.05). Compared to pre, post period had significantly less proportion of patients with EUS (40% vs 60%, p = 0.0026), GI consults (55.7% vs 85.4%, p < 0.0001), and surgery consults (23.7% vs 55.3%, p < 0.0001). Fewer patients underwent surgical resection for post (3.2%) vs pre (8.5%), p = 0.1010.
Discussion: In current healthcare climate, there is increased pressure to optimize resource utilization. Dissemination of an algorithm for PCL management an integrated health system resulted in fewer EUS and GI/surgery referrals, possibly due to increasing the confidence level of physicians ordering imaging studies, mostly primary and acute care physicians, in managing PCLs.
Conclusion: There was a drastic change in local practice pattern in the management of PCLs after a region-wide incorporation of an algorithm based on ICG-2012 in an integrated health system, namely significantly fewer EUS and GI/surgery referrals.
Open and Minimally Invasive Pancreaticoduodenectomy for Pancreatic Cancer: Perioperative, Oncologic, and Survival Outcomes
M.F. Eskander, S.W.L. de Geus, L.A. Bliss, S.C. Ng, A.J. Moser, J.F. Tseng. Surgical Outcomes Analysis & Research, Beth Israel Deaconess Medical Center, Boston, MA.
Background: Although minimally invasive pancreaticoduodenectomy (PD) has been described favorably in single-institution studies, national data is lacking.
Methods: 2010-2011 National Cancer Data Base was queried for patients with pancreatic adenocarcinoma who had a PD, excluding those with metastatic disease. Open surgery was compared to robotic or laparoscopic minimally invasive surgery (MIS) based on intention to treat. Outcomes were overall survival from diagnosis, margin status, post-operative length of stay (LOS), and 30-day unplanned readmission rate. Patient characteristics compared by chi-square. Kaplan-Meier analysis and Cox proportional hazards models used to evaluate overall survival.
Results: Of 5302 PDs, 725 (13.7%) were begun via MIS and of those, 232 (32.0%) were completed open. MIS group vs. open more likely to have comorbidities (38.8% vs. 34.4%, p = 0.0226), surgery at an academic center (72.8% vs. 64.1%, p < 0.0001), and poorly differentiated tumors (40.7% vs. 35.1%, p = 0.0119). There was a higher rate of R0 resection margins for MIS (79% vs. 75.3%, p = 0.0317) and a shorter median LOS (8 vs. 9 days, p < 0.0001); 30-day readmission rates did not significantly differ (p = 0.5757). Overall median survival was 20.2 months for MIS vs. 20.4 months for open (log-rank p value 0.6911). In patients who received neoadjuvant therapy, open surgery was associated with a significant survival benefit (25.8 vs. 21.7 months; HR 1.50, 95% CI 1.13-1.99) which lost significance when cohort was limited to high volume MIS centers.
Conclusions: In this national study, MIS demonstrated a superior R0 resection rate and shorter LOS but no difference in 30-day readmission rate or overall survival compared to open surgery. Particularly in the neoadjuvant setting, MIS PD should be limited to centers of excellence. Further investigation into oncologic outcomes after pancreatic surgery is warranted.
New Therapeutic Approach in the Successful Surgical Therapy of Pancreatic Cancer
G. Farkas, Jr, L. Leindler, G. Lazar. Department of Surgery, University of Szeged, Szeged, Hungary.
Introduction: Even nowadays the only successful therapy for pancreatic cancer treatment is surgery, but postoperative mortality still can be occur decreasing the number of survivals.
Aim: Postoperative grade Bassi C pancreatic fistula is still the main reason for postoperative death. Our aim was to decrease or stop pancreatic leakage with new conservative therapy.
Materials and methods: After operation catabolism is the main metabolic change in human therefore proper postoperative therapy has absolute necessity. We used 20% soil oil and 80% olive based 3 CBS (three chambers bag) central vein parenteral infusion in 2000 ml/day dosage. Between 01.2014-05.2015 16 patients received this therapy. The mean age was 62.3 and 65.2 years, male and female respectively. Operations were: 10 Whipple, 3 distal resections and 3 atypical pancreas resections. For pancreatic fistula detection we measured the amylase content of the drains. We checked the drain amount and content from the onset until healing.
Results: The amount of juice was 200-500 ml/day and the amylase content was higher than 50000U in all cases from the second or third postoperative days. Total amount of 2000 ml/day 3 CBS parenteral infusion was used from second postoperative day, with Saline and RL infusion in extra. Sandostatin was performed 3 times per day. The drain amount started to decrease 6-8 days after this treatment. Enteral feeding started 10-12 days after with overlap. No reoperation was necessary. All patients left the hospital 21-28 days after surgical intervention.
Conclusion: With this new therapy we could treat problematic cases successfully with whom in the past reoperations should have been done and death could have been detected. We hope due to this therapy the successful surgical therapy can be widened moreover new patients can be involved for surgical intervention, which in the past were excluded just because old age week physical status and soft pancreatic tissue to prevent fatal complications.
Total Pancreatectomy and Islet Autotransplantation: A Decade Long Nationwide Analysis
R. Fazl Alizadeh, Z. Moghadamyeghaneh, A.N. Demirjian, D.K. Imagawa, C.E. Foster, M.J. Stamos, H. Ichii. Department of Surgery, University of California, Irvine, CA.
Background: Total pancreatectomy (TP) is the last resort to control the severe pain in patients with chronic pancreatitis due to the morbidity of the operation and the frequent severe resultant diabetes. Islet autotransplantation (IAT) following TP is reported, by well experienced groups, to be an effective therapy to prevent post-surgical diabetes. However, there is limited nationwide data analysis during the last few decades. The aim of our study was to investigate outcomes, predictors of in-hospital morbidity, and mortality after TP+IAT.
Methods: The nationwide inpatient sample (NIS) database was used to identify patients who underwent TP+IAT during 2002-2012 in the US. Multivariate regression analysis was used to identify predictors of in-hospital morbidity.
Results: A total of 923 patients underwent IAT after pancreatectomy during 2002-2012. There were 754 patients who had TP+IAT. The most common indication of surgery was chronic pancreatitis (86%) followed by acute pancreatitis (12%). The number of patients undergoing TP+IAT annually significantly increased during the 11 years of study from 53 cases in 2002 to 155 cases in 2012. Overall mortality and morbidity of patients were 0% and 57.8 %, respectively. Post-surgical hypoinsulinemia was reported in 42.3% of patients, indicating 57.7% of patients were insulin independent during hospitalization. Predictors of in-hospital morbidity were obesity (AOR: 3.024, P<0.05), fluid and electrolyte disorders (AOR: 2.71, P<0.01), alcohol abuse (AOR: 2.63, P<0.01), and weight loss (AOR: 2.43: P<0.01).
Conclusion: During 2002-2012, the overall number of patients who underwent TP+IAT has been increasing. Our study showed TP+IAT is a safe and feasible procedure with no mortality, acceptable morbidity rates and achieved high rate of early insulin independence. Obesity, fluid and electrolyte disorders, and alcohol abuse are the most significant predictors of in-hospital morbidity.
Pancreas-Specific Secretory Pathway Ca2+-ATPase 2 Affects GPCR-mediated Ca2+ Signalling
M. Fenech,1,2 C. Pin1,2,3Departments of 1Pediatrics, 2Physiology & Pharmacology, 3Oncology, Western University, Children's Health Research Institute, London, Ontario, Canada.
Background: Enzymes are released from acinar cells through regulated exocytosis, which involves a signalling network that regulates spatial and temporal accumulation of Ca2+. Cytosolic Ca2+ levels are lowered, in part, by Ca2+ATPase pumps. Our laboratory has previously identified a novel isoform of secretory pathway Ca2+-ATPase 2 (SPCA2) that appears to be pancreas specific. However, this isoform, designated SPCA2c (c for carboxy) contains only the last 136 amino acids of SPCA2, making it unlikely that SPCA2c functions as a Ca2+-ATPase. Other studies report that SPCA2 can increase cytosolic Ca2+ concentrations via interaction with the plasma membrane Ca2+ channel, Orai1. The goals of this study were to determine if SPCA2c could affect cytosolic Ca2+ levels and Ca2+ movement in response to GPCR-mediated signaling.
Methods and Results: SPCA2c tagged with FLAG (SPCA2c-FLAG) was transiently transfected into HEK293 with and without the stable expression of Orai1 or AR42J cells in combination with GFP. Co-immunofluorescence analysis (IF) for FLAG and cell organelle markers was performed to localize SPCA2c within the cell. Also, transfected cells were identified by GFP expression, and then assessed for cytosolic Ca2+ levels using Fura2 ratiometric analysis before and after stimulation with carbachol. IF analysis revealed SPCA2c localization to both the endoplasmic reticulum and golgi apparatus in AR42J and HEK 293 cells. Transient expression of SPCA2c in HEK293 cells, both with and without the stable expression of Orai1, resulted in elevations in resting cytosolic Ca2+ levels, and altered responses to carbachol stimulation, indicating a functional role for SPCA2c in Ca2+ homeostasis.
Conclusions: Our findings suggest SPCA2c plays a unique role in maintaining cytosolic Ca2+ that involves interacting with Orai1. Further work is aimed at determining SPCA2c’s role in affecting the acinar cell response to injury.
Evaluation of Proposed Biomarkers in Early Stage Pancreatic Ductal Adenocarcinoma
M.A. Firpo,1 A. Rosati,2 G.D. Khanderao,1 D.G. Adler,1 C.S. Scaife,1 K.M. Boucher,1 M.C. Turco,2 S.J. Mulvihill.11Huntsman Cancer Institute, University of Utah, Salt Lake City, UT; University of Salerno, Fisciano, Italy.
Development and deployment of an accurate, blood-based assay that could be used to screen asymptomatic patients for pancreatic ductal adenocarcinoma (PDAC) risk would likely improve outcomes by identifying treatable disease. We evaluated serum levels of 30 analytes previously identified as potential circulating biomarkers in at least two prior studies involving pre-malignant rodent models, gene expression studies of PanIN lesions in clinical samples, or specifically evaluated in serum from early stage PDAC cases. Analytes were measured in each of 180 serum samples from healthy control subjects, chronic pancreatitis cases, and stage IA, IB, or IIA PDAC cases. Nineteen analytes (ALCAM, AXL, CA 19-9, CEACAM1, COL18A1, EPCAM, HA, HP, ICAM1, IGFBP2, IGFBP4, MMP2, MMP7, PRG4, SPP1, TGFBI, THBS1, TIMP1, TNFRSF1A) had significant differences between the three classes, while and additional four analytes (BAG3, BSG, LCN2, PARK7) trended towards significance. We evaluated the 30-analyte panel using a technique that maximizes area under the receiver operating characteristic curve and identifies analytes that most contribute to class discrimination using the lasso procedure. For the comparison of healthy controls from early stage PDAC, an optimal panel included analytes BAG3, CA 19-9, CEACAM1, HA, IGFBP2, PARK7, and SPP1 yielding an area under the ROC curve (AUC) of 0.92. For the comparison of healthy controls and chronic pancreatitis from early stage PDAC, an optimal panel included analytes BAG3, CA 19-9, CEACAM1, EPCAM, LCN2, MSLN, PARK7, PRG4, SPP1, and TNFRSF1A, yielding an AUC of 0.88. These data suggest that suitably accurate algorithms (>90% accuracy) can be devised using subsets of the 30 analyte panel. It is expected that increased accuracy can be realized by incorporating additional analytes into the resulting algorithms.
The Effect of Cystathionine-Gamma-Lyase Gene Deletion on the Renin-Angiotensin System in Cerulein-Induced Acute Pancreatitis in Mice
R.R. Gaddam,1 A. Badiei,1 S.T. Chambers,1 I. Ishii,2 M. Bhatia.11Department of Pathology, University of Otago, Christchurch, New Zealand; 2Department of Biochemistry, Graduate School of Pharmaceutical Sciences, Keio University, Tokyo, Japan.
Background: Cystathionine-γ-lyase (CSE) is an endogenous hydrogen sulfide (H2S)-producing enzyme involved in inflammation. We previously demonstrated that, H2S synthesized by CSE has a pro-inflammatory role in cerulein-induced acute pancreatitis and associated lung injury. The renin-angiotensin system (RAS) components (ACE, ACE2, Ang II, and Ang-(1-7)) play an important role in the regulation of inflammation. The present study investigated the effect of CSE gene deletion on RAS metalloproteases and their bioactive peptides in caerulein-induced acute pancreatitis and associated lung injury in mice.
Methods: Wild-type and CSE-deficient (CSE-KO) C57BL6 mice (male, 25-30 g) were each assigned into control (saline) or interventional (cerulein) groups. Acute pancreatitis was induced by hourly cerulein injections (50 μg/kg) for 10 hours. Mice were sacrificed 1 h after the last saline/cerulein injection. Pancreas and lung tissues were collected and processed to measure ACE, ACE2, Ang II and Ang-(1-7) activity/levels.
Results: The RAS components in pancreas (ACE2 activity and levels, Ang-(1-7)) and lung (ACE and ACE2 activity and levels, Ang II and Ang-(1-7)) were significantly lower in CSE-KO mice. The cerulein treatment resulted in significant decreases in ACE2 activity (pancreas and lung) and Ang II levels (lung) in wild-type mice but not in CSE-KO mice.
Conclusion: These findings suggest that acute inflammation inhibits activation of the RAS system. However, in the absence of baseline H2S production (CSE deletion) both ACE and ACE2 and their bioactive peptides were reduced suggesting H2S has a role in the maintenance of normal homeostasis of the RAS system. This requires further study.
Laparoscopic Pancreaticoduodenectomy (LPD) With Uncinate Process First Approach
W. Gao, K. Jiang, J. Wu, Q. Li, F. Guo, J. Chen, J. Wei, Z. Lu, M. Tu, X. Dai, C. Dai, Y. Miao. Pancreas Center, The First Affiliated Hospital of Nanjing Medical University, Nanjing, China.
Background: Complete resection for pancreatic head cancer is of prognostic relevance; however, most patients will develop recurrence around superior mesenteric artery (SMA) and celiac trunk (CT). PD with uncinate process first approach dissect the pancreatic head in a caudal-cranial direction, aiming to improve exposure and complete dissection of SMA. LPD has flexible and unique view advantage from caudal, posterior and lateral side, which is ideal for uncinate process first approach.
Technique: After initial exploration, pancreas is tunneled above the portal vein; laparoscope was transferred to left lateral trocar, which provides a left lateral and posterior view. Then SMA trunk below pancreas was reached by incise Treitz ligament, the dissection followed SMA cranially, it is possible to follow SMA at this stage all the way downward towards its origin. The proximal jejunum is then transected and pulled to right. Laparoscope was transferred to right lateral trocar, which provides a right lateral and posterior view. Kocher’s maneuver is well facilitated by this view, pancreas head was complete mobilized, SMA root were dissected and looped above left renal vein. The uncinate was then completely dissected in a caudocranial direction along SMA axis, tissue between SMA and CT was resected. Finally laparoscope was transferred to infra-umbilical trocar, pancreas neck is transected, resection is complete after bile duct is dissected all the way up and transected.
Results: LPD with uncinate process first approach were completed in 2 cases. Operative times were 550 and 610 minutes, 1 patient discharged uneventfully at POD9, the other discharged POD35 after multiple CT guided drainage for grade C pancreas fistula.
Conclusion: LPD has flexible and unique view advantage from caudal, posterior and lateral side, giving excellent exposure and dissection of SMA. Uncinate process first approach are ideal for LPD.
Increased Nuclear Transport of β Catenin and LRP 6 Phosphorylation as Novel Prognostic Biomarkers for the Progression of Pancreatic Tumor
B. Garg, S. Modi, K. Majumder, S. Banerjee, V. Dudeja, A.K. Saluja. Department of Surgery, University of Minnesota, Minnieapolis, MN.
Background: WNT/β-catenin cascade regulates cell fate, proliferation, survival and migration, however its role in carcinogenesis is still unclear. Therefore, the present study explores molecular signatures of this pathway to unravel the onset, progression and therapeutic regression of pancreatic cancer.
Methods: WNT/ β catenin pathway genes were studied in human pancreatic cell lines, KPC mice, Syngenic Tumor Implantation (STIM) and Xenograft models by q-RT-PCR, immunoblotting and confocal microscopy, in presence and absence of Minnelide, a novel chemotherapeutic drug developed by our group, and its combination with Gemcitabine and Paclitaxel. Immunoprecipitation-MS analysis with p-LRP 6 was done to elucidate the downstream molecules leading to nuclear transport of β catenin.
Results: Our study elucidated the upregulation of WNT1, FZD1, LRP6, GSK3β, Axin2 and βcatenin and nuclear translocation of beta catenin in S2VP10 and Mia-PaCa compared to normal human ductal cells. Reduced TCF/LEF1 reporter activity upon FZD1 & LRP6 silencing advocated their key roles in WNT/β catenin signaling. LRP6 phosphorylation was important to trigger WNT pathway. Nuclear transport of β catenin, pLRP6, and upregulation of c-Myc, cyclin-D were linked with tumor progression in KPC mice model. Immunoprecipitation-MS analysis revealed an increase in novel binding partners of pLRP6, presumed to be involved in tumor progression. Interestingly, 30 days Minnelide treatment in KPC models down-regulated of WNT pathways genes, and reduced β catenin nuclear transport and decrease in the pLRP6 binding partners, Similar effects were also observed with other chemotherapeutics drugs in combination with Minnelide.
Conclusion: (1) β catenin nuclear transport and LRP6 phosphorylation are keys to trigger WNT/β catenin pathway. It can be used as biomarkers for pancreatic cancer progression. (2) Minnelide and its combination with Gemcitabine and Paclitaxel synergistically regress tumor regression via inhibiting WNT/β catenin pathway.
The MET Receptor Tyrosine Kinase is Required for the Repair of Pancreatic Acinar Cells Following Acute and Chronic Injury
I. Gaziova,1 D. Jackson,2 P. Boor,3 D. Carter,2 Z. Cruz-Montserrate,4 C. Elferink,2 A. Joshi,2 B. Kaphalia,3 C.D. Logsdon,4 L. Soong,3 X. Tao,1 S. Qiu,3 L. Elferink.1Departments of 1Neuroscience and Cell Biology, 2Pharmacology, 3Pathology, University of Texas Medical Branch, Galveston, TX; 4Department of Cancer Biology, University of Texas MD Anderson Cancer Center, Houston, TX.
Introduction: Pancreatitis results from inflammatory injury to acinar cells. Our understanding of the signaling pathways guiding acinar cell repair following injury is incomplete. In this study, we examined the importance of MET signaling as an intrinsic repair mechanism for acinar cells following acute damage and chronic alcohol-associated injury.
Methods: We conditionally deleted MET in adult mouse acinar cells (MET−/−). Using MET−/− and METfl/fl mice, acute pancreatic injury was induced with cerulean and chronic injury by feeding mice Lieber-DeCarli diets containing alcohol. We examined the exocrine pancreas of these mice histologically for acinar death, edema, inflammation and fibrosis and changes in the transcriptional program using next generation sequencing.
Results: We report that MET expression was routinely low in normal adult pancreas, yet elevated in ductal and acinar cells in human pancreatitis specimens, consistent with a role for MET as an adaptive repair mechanism. We showed that genetic deletion of MET in adult murine acinar cells was linked to increased acinar cell death and chronic inflammation. Of note, increased pancreatic fibrosis was detected in MET−/− mice following alcohol-associated injury. Finally, we identified specific alterations of the pancreatic transcriptome mediated by MET signaling during acute and chronic injury, key for tissue repair and inflammation.
Conclusion: MET signaling is important for acinar cell survival and regeneration, and attenuates the symptomology of pancreatic injury. Strategies that transiently activate MET signaling may promote repair of the exocrine pancreas in patients with acute or chronic pancreatitis.
Morphine Analgesia Worsens Chronic Pancreatitis in Mice
J. George, U. Barlass, A. Dixit, Z. Yuan, A. Sareen, S.K. Garg, V. Dudeja, R. Dawra, S. Roy, A.K. Saluja. Division of Basic and Translational Research, Department of Surgery, University of Minnesota, Minneapolis, MN.
Background: Chronic pancreatitis (CP) is a progressive inflammatory disease of the pancreas, characterized by irreversible morphologic changes, gradual fibrotic replacement with associated severe abdominal pain, exocrine insufficiency and diabetes mellitus. Narcotics such as morphine and its derivatives are widely used for pain management in CP. We have earlier shown that morphine increases severity of acute pancreatitis (AP) in murine model of AP.
Aims: To study the effect of morphine in a cerulein induced mouse model of chronic pancreatitis.
Methods: Chronic Pancreatitis was induced by administration of cerulein (50ug/kg, i.p., hourly x6x 2 days/week x 6) to mice (C57BL/6). Morphine (25 mg/kg i.p., bid) was started in the treatment group and was given for 4 weeks. All mice were sacrificed after 11 weeks from start.
The effect of morphine treatment on CP was evaluated by measuring changes in pancreas to mouse weight ratio, pancreatic histology and sirius red staining for collagen.
Results: Morphine treated mice showed increased collagen staining compared to cerulein alone CP group, suggestive of increased fibrosis in chronic pancreatitis. Morphine treated mice also had a significant reduction in pancreas to mouse weight ratio compared to control group (4.35 ± 0.09 vs 5.72 ± 0.08; p < .01). Pancreatic histology of morphine treated mice had significantly increased chronic inflammatory infiltrate and fibrotic changes compared to cerulein group.
Conclusions: Our study shows that morphine worsens chronic pancreatitis in mice. This is the first study to evaluate the effects of opioids analgesics in chronic pancreatitis disease progression. Our results suggest, opioids should be used with caution in the management of CP.
Pancreatic Adenocarcinoma of the Young: Impaired Survival Caused by Genetic Changes?
A. Gluth,1 F. Bergmann,2 U. Hinz,1 N. Giese,1 J. Werner,1 M.W. Büchler,1 W. Hartwig.11Department of Surgery, 2Institute of Pathology, University of Heidelberg, Germany.
Background: Patients below the age of 40 yrs. who present with pancreatic cancer are rare and their long-term outcome is unclear. The present analysis aims to correlate age with survival and pre- and postoperative morbidity in a large cohort of patients with pancreatic adenocarcinoma. It furthermore aims to identify possible genetic variances between young and old patients.
Methods: Between October 2001 and June 2012 data from 2462 patients with adenocarcinoma of the pancreas and their characteristics were prospectively collected in an electronic database. Six different groups were defined by decades of age. Survival curves were estimated using Kaplan-Meier analysis and were compared using the log-rank test. Differences in patient and tumor characteristics were analyzed using the chi-square test. A matched pair analysis of the <40 yrs. old and 60 to 70 yrs. old patients was performed. K-Ras mutation analysis was performed in the <40 yrs. old patients.
Results: 1658 patients underwent resection of pancreatic adenocarcinoma and 804 patients underwent exploration or bypass surgery. Of those, 27 and 14 were younger than 40 yrs., respectively. Survival after resection differed significantly between age groups (p = 0.01), whereas survival after exploration or bypass was comparable between groups (p = 0.25). Patients >80 yrs. had the shortest median survival after resection and after exploration (14.4 and 6.9 months, respectively). In the matched pairs analysis, no significant differences in survival were identified between <40 yrs. and 60-70 yrs. old patients, but the young had a longer duration of symptoms (p = 0.012) and a trend of higher weight loss preoperatively (p = 0.07). Importantly, the subgroup of patients <40 yrs. and AJCC stage III/IV disease had a worse outcome compared to the corresponding group of 60-70 yrs. old (9.1 vs. 15.5 months, respectively, p = 0.04). K-Ras mutations were identified in only 25% of <40 yrs. old patients.
Conclusions: Young patients who undergo resection for pancreatic cancer have a longer duration of symptoms, more weight loss, and in advanced disease a shorter survival as compared to older patients. Whether genetic variations, with K-Ras mutations in only 25% of the young, are responsible for this impaired survival requires further analysis.
Covering the Cystic Duct Orifice With Self Expanding Metal Stents for Malignant Biliary Obstruction: Is There an Increased Risk of Cholecystitis?
V. Gohil, Y. Wu, S. Rashid, V. Chitnavis, P. Yeaton, A. Brijbassie. Department of Gastroenterology, Virginia Tech Carilion School of Medicine, Roanoke, VA.
Background: Fully covered self-expanding metal stents (FCSEMS) are utilized to restore biliary flow in malignant biliary obstruction. It remains unclear whether cholecystitis is as a result of Cystic Duct (CD) involvement by tumor or from cystic duct occlusion secondary to FCSEMS placement.
Methods: Using an IRB-approved database, we conducted a retrospective analysis of 49 patients with malignant biliary obstruction over a two-year period that subsequently underwent ERCP with FCSEMS placement. Development of cholecystitis was evaluated between patients with suspected cystic duct involvement by tumor and cystic duct occlusion secondary to FCSEMS placement.
Results: Between November 2012 to November 2014, 29 (59.2%) males, mean age 68.3 ± 12.4 years underwent ERCP with placement of FCSEMS for palliation of obstructive jaundice in the setting of pancreatic adenocarcinoma (43)(87.8%). Thirteen patients developed cholecystitis of which 9 (69.3%) (p < 0.01) had suspected tumor involvement of the cystic duct. Four patients developed cholecystitis where the stent covered the cystic duct orifice (30.8%) (p = 0.42). Logistic regression analysis was performed to determine predictors for cholecystitis. The odds of having subsequent cholecystitis for a patient with cystic duct involvement by tumor were 8.6 times than a patient without CD tumor involvement (OR 8.6 [95% CI 2.1 ± 35.5], p = 0.003). Additionally, the odds of having subsequent cholecystitis for a patient who had a FCSEMS covering the cystic duct orifice was 0.61 than that of a patient without a stent covering (OR 0.61 [95% CI 0.14 ± 2.69], p = 0.51).
Conclusion: The risk of cholecystitis appears to be increased from tumor involvement of the cystic duct rather than from coverage of the ductal orifice by fully covered self-expanding metal stents. Further long-term prospective data are required to confirm this observation.
Covering the Cystic Duct Orifice With Self Expanding Metal Stents for Biliary Obstruction Secondary to Pancreatic Adenocarcinoma: Is There an Increased Risk of Cholecystitis?
V. Gohil, Y. Wu, S. Rashid, V. Chitnavis, P. Yeaton, A. Brijbassie. Department of Gastroenterology, Virginia Tech Carilion School of Medicine, Roanoke, VA.
Background: Fully covered self-expanding metal stents (FCSEMS) are utilized to restore biliary flow in malignant biliary obstruction. It remains unclear whether cholecystitis is as a result of Cystic Duct (CD) involvement by tumor or from cystic duct occlusion secondary to FCSEMS placement.
Methods: Using an IRB-approved database, we conducted a retrospective analysis of 43 patients with malignant biliary obstruction secondary to pancreatic adenocarcinoma over a two-year period that subsequently underwent ERCP with FCSEMS placement. Development of cholecystitis was evaluated between patients with suspected cystic duct involvement by tumor and cystic duct occlusion secondary to FCSEMS placement.
Results: Between November 2012 to November 2014, 29 (59.2%) males, mean age 68.3 ± 12.4 years underwent ERCP with placement of FCSEMS for palliation of obstructive jaundice in the setting of pancreatic adenocarcinoma (43) (87.8%). Thirteen patients developed cholecystitis of which 9 (69.3%) (p < 0.01) had suspected tumor involvement of the cystic duct. Four patients developed cholecystitis where the stent covered the cystic duct orifice (30.8%) (p = 0.42).
Logistic regression analysis was performed to determine predictors for cholecystitis. The odds of having subsequent cholecystitis for a patient with cystic duct involvement by tumor were 8.6 times than a patient without CD tumor involvement (OR 8.6 [95% CI 2.1 ± 35.5], p = 0.003). Additionally, the odds of having subsequent cholecystitis for a patient who had a FCSEMS covering the cystic duct orifice was 0.61 than that of a patient without a stent covering (OR 0.61 [95% CI 0.14 ± 2.69], p = 0.51).
Conclusion: The risk of cholecystitis appears to be increased from tumor involvement of the cystic duct rather than from coverage of the ductal orifice by fully covered self-expanding metal stents. Further long-term prospective data are required to confirm this observation.
IPMN and Cyst Fluid CEA: The Journey Beyond Diagnosis
V. Gómez, S. Majumder, T.C. Smyrk, W.S. Harmsen, F.T. Enders, F. Gleeson, B.K. Abu Dayyeh, P.G. Iyer, R.K. Pearson, B.T. Petersen, E. Rajan, S.S. Vege, K.K. Wang, S.T. Chari, M.D. Topazian, M.J. Levy. Mayo Clinic, Rochester, MN.
Introduction: Pancreatic cyst fluid carcinoembryonic antigen (CEA) is a useful marker for identifying mucinous pancreatic cystic lesions (PCLs). However, data indicate that the cyst fluid CEA level does not distinguish malignant from nonmalignant PCLs nor provide any prognostic information. The aims of this study were to investigate the potential associations of cyst fluid CEA with IPMN subtype and degree of histologic dysplasia.
Methods: Retrospective, single center study consisting of patients who underwent EUS guided cyst fluid aspiration with CEA concentration and surgical resection of IPMNs. The surgical pathology specimens were examined in a protocolized manner to verify the IPMN subtype and degree of dysplasia.
Results: PCL surgical specimens were reviewed from 85 patients [39 (45.9%) female, mean age 67 years] with IPMN; including branch duct IPMN (bIPMN, n = 68) and mixed main duct with side branch IPMN (mbIPMN, n = 17). bIPMN were associated with higher CEA concentration compared to mbIPMN (median CEA 634 ng/mL vs 112 ng/mL; P = 0.006). When the entire group was stratified according to IPMN subtype, intestinal IPMN was associated with significantly higher CEA concentration (median CEA 982.5 ng/mL; P = 0.02); however, gastric, pancreatobiliary and oncocytic subtype IPMN were not (all P > 0.05). When stratified based on the degree of dysplasia, no statistically significant linear correlation was seen with CEA concentration and increasing degree of dysplasia (P = 0.80).
Discussion: Our data suggest that the pancreas cyst fluid CEA concentration may be useful in predicting both the gross morphologic and epithelial subtype of IPMN, which may provide additional diagnostic and prognostic utility. However, the degree of dysplasia did not correlate with CEA concentration, thereby limiting the prognostic value of CEA. These findings merit additional evaluation.
Cysts in the (de)Nude: What is the Significance of Pancreatic Cyst Epithelial Denudation?
V. Gómez, S. Majumder, T.C. Smyrk, W. Harmsen, F. Enders, F. Gleeson, B. Abu Dayyeh, P. Iyer, R. Pearson, B. Petersen, E. Rajan, S.S. Vege, K. Wang, S.T. Chari, M. Topazian, M. Levy. Mayo Clinic, Rochester, MN.
Introduction: Pancreatic cystic lesions (PCLs) are common and may require treatment with intracystic alcohol and/or chemotherapy. Many consider the finding of denuded epithelium within the resected specimen as a key measure of successful therapy. However, little is known regarding the incidence and degree of epithelial denudation among untreated PCLs. The aims of this study were to determine the prevalence, extent and predictors of epithelial denudation in PCLs.
Methods: Retrospective, single center study of patients who underwent surgical resection of PCLs. All cysts were reviewed by a GI pathologist and the presence and extent (%) of cyst epithelial denudation determined by protocolized histopathology examination. The CT/MRI- measured cyst dimensions were used to determine the number of cyst wall biopsies required to accurately assess each lesion.
Results: PCL surgical specimens were reviewed from 164 patients; neuroendocrine tumors (n = 13) and pseudocysts (n = 11) were excluded due to their inherent lack of epithelium. Of the remaining 140 patients [84 (60%) female, mean age 62.7 years], 85 (60.7%) were IPMN, 33 (23.5%) were MCN, 11 (7.9%) were SCA, and 11 (7.9%) were other cyst types. Denuded epithelium was identified in 95 (68%) of all CPLs. A greater extent of epithelial denudation was seen in MCN compared to IPMN and SCA (mean % denuded epithelium 45.1%, 10.8%, and 22.4%, respectively (P < 0.0001). PCLs resected from the neck/body/tail of pancreas were associated with greater extent of denuded epithelium than PCLs resected from the head/uncinate (23.9% versus 13.4 %; P = 0.035).
Discussion: Our study supports the contention that the presence and extent of cyst epithelial denudation of treatment naïve CPLs vary with cyst histology and other factors. These data suggest that the finding of denudation following intracystic therapy may not provide an adequate measure of successful intervention. Further studies are needed to determine whether denudation correlates with meaningful clinical endpoints.
Wnt-Mediated Autophagy is Inhibited by Pigment Epithelium-Derived Factor
J. Gong,1,2 G.S. Belinsky,1,2 U. Sagheer,1,2 A. Rhim,3 C. Chung.1,21Sections of Digestive Diseases; Endocrinology, Department of Medicine, Yale University School of Medicine, New Haven, CT; 2Veterans Affairs Connecticut Healthcare System, West Haven, CT; 3University of Michigan Medical School, Ann Arbor, MI.
Purpose: Autophagy is a survival mechanism for cancer cells and deletion of autophagy genes can inhibit the development of pancreatic ductal adenocarcinoma (PDAC). Regulation of autophagy in pancreatic intraepithelial neoplasia (PanIN) cells is not well understood. We have a longstanding interest in the broad tumor suppressor, pigment epithelial derived factor (PEDF), a recently identified modulator of Wnt/β-catenin signaling. PEDF blocks experimental pancreatic cancer and its expression is associated with improved survival in human pancreatic cancer (Cancer Research 2004, Clin Cancer Research 2005, GUT 2011). In this study, we tested whether PEDF-mediated Wnt blockade modulates autophagy in PanIN cells.
Methods: Two murine PanIN cell lines, PI5505 (PanIN-2) and PI34 (PanIN-3) were derived from Pdx-Cre; LSL-KrasG12D; p16fl/fl; YFP mice at 6 weeks of age. Primary PanIN cells were treated with PEDF (300 ng/ml) protein for 24 h. Autophagic and Wnt signaling were determined by biochemical assays, qPCR, and immunoblotting. Levels of reactive oxygen species (ROS) were determined by biochemical assays. Human PanIN lesions were assessed for superoxide dismutase (SOD2) and PEDF expression.
Results: Small molecule activators of Wnt/β-catenin signaling and canonical Wnt ligands enhanced autophagy, while inhibitors of Wnt signaling decreased autophagy. Induction of autophagy in turn negatively regulates canonical Wnt signaling. Inhibiting autophagy led to activation of Wnt signaling through upregulation of the active Wnt receptor, pLRP6, and active β-catenin levels. PEDF inhibited autophagy induced by starvation, rapamycin, and wnt3a. PEDF-mediated autophagy inhibition led to increased H2O2 levels. Consistent with the elevated H202 levels, PEDF exposure increased SOD2 and reduced catalase levels in PanIN cells. SOD2 expression was diminished in PEDF KO mice. In human PanIN lesions, SOD2 expression was mostly absent in high-grade PanIN (PanIN-2/3), while PanIN-1 lesions retained SOD2 labeling.
Conclusion: Our study demonstrates that PEDF inhibits autophagy in PanIN cells by targeting Wnt/β-catenin-mediated autophagy and modulating levels of enzymes that regulate the intracellular balance of ROS.
Angiogenic Signature Points to JAK/STAT and TGF-β as Therapeutic Targets in Pancreatic Cancer
J. Gore,1,3 K.E. Craven,2 J.L. Wilson,1 M. Korc.1,2,3Departments of 1Medicine, 2Biochemistry and Molecular Biology, Indiana University School of Medicine, Indianapolis, IN; 3Pancreatic Cancer Signature Center at Indiana University Simon Cancer Center, Indianapolis, IN.
Pancreatic ductal adenocarcinomas (PDACs) have an abundance of driver mutations and are often viewed as hypovascular, yet they exhibit regions of microvasculature and overexpress several pro-angiogenic cytokines and growth factors. Using data from The Cancer Genome Atlas (TCGA), we have determined that ∼35% of PDAC cases exhibit a strong pro-angiogenic gene signature and that the same patients also exhibit a gene signature that reflects active TGF-β signaling. Similar gene signatures are present in PDACs arising in a genetically engineered mouse model (GEMM) that we established in which oncogenic Kras is combined with loss of RB (KRC), and these murine PDACs are rich in endothelial cells (ECs) and sinusoidal-like vessels that have blood flow, as determined by intravital confocal microscopy. By contrast, tumors in the KPC GEMM lack these signatures and have few ECs and vessels. Array, in silico and functional analyses of pancreatic cancer cells (PCCs) derived from KRC tumors revealed that multiple pro-angiogenic genes are up-regulated in the PCCs in a TGF- -dependent manner. Moreover, conditioned media from KRC PCCs activate endothelial STAT3 to enhance EC growth and migration. Co-culture of KRC PCCs with murine ECs, or human PCCs established from a patient-derived orthotopic model with human ECs revealed that PCCs and ECs cooperate to enhance each other’s growth. Importantly, blocking JAK/STAT or TGF-β signaling pathways suppresses this mitogenic cross-talk and attenuates angiogenesis and tumor growth in vivo. Therefore, targeting these pathways could be a beneficial therapeutic strategy that could be particularly useful in PDAC patients who have a pro-angiogenic gene signature.
Profibrogenic Effects of Endothelin Axis in Pancreatic Cancer
S. Gupta,1 S. Rachagani,1 X. Wang,2 C. Guda,2 S.K. Batra,1,3 M. Jain.1,31Departments of Biochemistry & Molecular Biology, 2Department of Genetics, Cell Biology & Anatomy, 3The Fred and Pamela Buffett Cancer Center, University of Nebraska Medical Center, Omaha, NE.
Introduction: Signaling along endothelin (ET) axis is involved in wound healing and promotes pathologic hepatic and pulmonary fibrosis. Pancreatic cancer (PC) epitomizes the complexity of tumor microenvironment (TME) characterized by intense desmoplasia that contributes to pathobiology and therapy resistance. We observed significant overexpression of the ET-axis components PC cells and TME and hypothesized that ET axis promotes fibrosis in PC.
Methods: TCGA database was analyzed to identify gene signatures associated with the upregulation of ET axis components (ET-1, ETAR and ETBR) in PC. Human and murine [KPC (KrasG12D, P53, Pdx1-Cre)] PC tissues were analyzed by confocal microscopy to determine the expression of ET axis in pancreatic stellate cells (PSCs). The cellular cross-talk between PC and PSCs along ET axis was studied in vitro using selective ETAR (BQ123), ETBR (BQ788) and dual (Bosentan) ET receptor antagonists. KPC mice were treated with Bosentan and the impact on desmoplasia in autochthonous tumors was studied by IHC.
Results: Bioinformatics analysis of TCGA database revealed high positive correlation of fibrosis associated genes (Col 1A2, 3A1, 5A2, 6A3; PDGFRB, FAP, LUM, and FBN1). Pathways regulating hepatic fibrosis, SHh signaling, and tumor growth and metastasis, were upregulated in tumors exhibiting overexpression of ET axis. In tumor tissues, cancer cells expressed both ET receptors, while PSCs predominantly expressed ETBR. ET axis antagonism resulted in dose dependent inhibition of PSCs proliferation and downregulation of genes associated with PSC activation (α-SMA) and fibrosis (Fbn1, CTGF) via inhibition of ETBR signaling. Bosentan treatment of KPC animals resulted in significant decrease in desmoplasia and ECM proteins in tumors.
Conclusions: ET-1 axis promotes fibrosis in PC and can be pharmacologically targeted to improve the delivery of therapeutic agents.
Evaluation of Minnelide on Granulocyte Differentiation and Apoptosis
V.K. Gupta, A. Sareen, S. Modi, B. Giri, S. Banerjee, A.K. Saluja. Division of Basic and Translational Research, Department of Surgery, University of Minnesota, Minneapolis MN.
Background: Neutropenia, an abnormal low count of neutrophils, is an important dose-limiting toxicity of various cancer chemotherapy that often results in dose reduction from the initial dose. Minnelide, a water soluble pro-drug of triptolide, has shown a great promise as an effective drug against pancreatic cancer. This compound is currently being evaluated in a Phase I clinical trial against GI cancers at the University of Minnesota.
Aim: To study the effect of Minnelide, a potent chemotherapeutic drug against pancreatic cancer, on neutrophil population both in vitro and in vivo.
Methods: Neutrophils were isolated from bone marrow cells of C57BL/6 J mice using MACS and treated with triptolide and after 24 hr and 48 hr cell viability and cell death were measured by using CCK-8 and Annexin assay respectively. For studying the effect of Minnelide on granulocyte differentiation, maturation and release, C57BL/6 J mice or KPC mice were treated for 21 days with Minnelide 0.21 mg/kg QD and Saline intraperitoneally. Animals were sacrificed after respective treatment. Bone marrow cells were collected, fixed and stained for stem cell marker (CD34), neutrophil marker (Ly6G) and apoptosis markers. Cells were analyzed using flow cytometry using Trucount beads (BD Biosciences) to determine the absolute counts.
Results:In vitro studies using neutrophils isolated from bone marrow of C57BL/6 J mice showed that triptolide reduces neutrophil viability at high doses of 100nM and 200nM without causing any significant apoptosis. Our data show that Minnelide caused slight decrease in the neutrophil population in the peripheral blood after 13 day of treatment; however, neutrophil counts went up after 21 days. Similarly Minnelide treatment on KPC tumor mice did not show any significant neutropenia. Our analysis on the bone marrow cells showed that Minnelide treatment result in significant increase in myeloid progenitor cell population (CD34 + Ly6G+) without affecting stem cell population.
Conclusion: This study shows that Minnelide did not cause any neutropenia in normal and tumor mice at a dose of 0.21 mg/kg QD. Our results show accumulation of myeloid progenitor cell in bone marrow, which indicates that Minnelide probably interfered with their maturation.
Left-Sided Portal Hypertension following Pancreatoduodenectomy With Resection of the Superior Mesenteric-Portal Vein Confluence for Locally Advanced Pancreatic Ductal Adenocarcinoma: Benefit of Concomitant Resection of the Splenic Artery
K. Gyoten, S. Mizuno, H. Kato, A. Tanemura, Y. Murata, N. Kuriyama, Y. Azumi, M. Kishiwada, M. Usui, H. Sakurai, S. Isaji. Department of Hepatobiliary Pancreatic and Transplant Surgery, Mie University School of Medicine, Mie, Japan.
Background: For pancreatic ductal adenocarcinoma (PDAC) patients, we had undergone pancreatoduodenectomy (PD) with resection of the superior mesenteric-portal vein (SMV/PV) confluence, in which PV was reconstructed but the splenic vein (SV) and splenic artery (SA) was not. The aim of this study is to evaluate the influence of division of SV as well as SA on left-sided portal hypertension (LSPH) after PD.
Patients and Method: From April 2005 to June 2014, 65 PDAC patients who underwent PD with resection of SMV/PV confluence (SV was sacrificed) were classified into three groups: Group A (n = 18) in which SA and inferior mesenteric rein (IMV) were preserved, Group B (n = 36) in which SA was preserved and IMV was sacrificed, Group C (n = 11) in which SA and IMV were sacrificed. We analyzed the frequency of the development of collateral vessels and gastrointestinal (GI) hemorrhage, spleen volume ratio and platelet counts after PD.
Result: The development of collateral vessels was found in 10 patients (55%) in Group A, in 18 (50%) in Group B and in 3 (27%) in Group C. GI hemorrhage did not develop in Group C, but developed in 2 (11%) in Group A and in 3 (8.3%) in Group B. GI hemorrhage occurred in 4 patients within 6 months and in 1 at 24 months after operation. Spleen volume ratios at 6 and 24 months were 1.42 and 2.06 in Group A, 1.19 and 1.55 in Group B and 1.17 and 0.93 in Group C. Platelet counts (x103 /ul) at 6 and 24 months were 143 and 141 in Group A, 173 and 130 in Group B, and 155 and 157 in Group C.
Conclusion: PD with resection of SMV/PV confluence followed by no reconstruction of SV causes LSPH, which increases the risk of GI hemorrhage. In this situation, resection of the splenic artery might be a helpful treatment.
Postoperative Pancreatic Fistula: We Need to Redefine Grades B and C
T. Hackert, U. Hinz, T. Pausch, I. Fesenbeck, O. Strobel, L. Schneider, S. Fritz, M.W. Büchler. Department of Surgery, University of Heidelberg, Heidelberg, Germany.
Background: Postoperative pancreatic fistula (POPF) is the most important complication following pancreatic surgery. In 2005, the International Study Group for Pancreatic Surgery (ISGPS) introduced a standardized POPF definition with severity grading from A-C. In recent years, interventional drainage (ID) has become the standard of care for symptomatic postoperative fluid collections or undrained POPF. From the original definition, it is unclear whether ID is categorized as POPF B or C. Therefore, international authors shift ID between grades B and C. The aim of the study was to analyze patients with ID (proposed new grade B) vs. patients with re-operation (grade C) for POPF.
Methods: Between 2005 and 2013, all patients undergoing pancreatic resection were analyzed regarding POPF A-C. Demographic data, type of operation, postoperative complications, therapies and outcome were examined with focus on ID vs. re-operation.
Results: 2955 patients were included and 403 patients developed POPF (13.6%). 11% of all POPF were grade A, 17% grade B (clinically symptomatic without ID) and 72% grade C. These patients underwent either ID (n = 165) or re-operation (n = 123). Patients with ID had an average hospital stay of 33d and POPF-associated mortality of 0%. This was strikingly different to patients undergoing re-operation with a hospital stay of 47d and POPF-associated mortality of 37% (p < 0.0001).
Conclusion: After 10 years of the ISGPS classification, there is a clear cut outcome difference between patients undergoing POPF-associated ID or re-operation. We propose assigning all patients undergoing ID as POPF grade B. Patients undergoing re-operation should definitely remain within category C.
Overexpressed SMG-1 Inhibited the Proliferation, Invasion, and Chemoresistance of Human Pancreatic Carcinoma Cells by Regulating mTOR
L.L. Han,1 T.H. Hu,2 J. Wang,1 L. Jing,1 M.C. Wang,1 M.J. Liu,3 K.J. Nan.11Department of Oncology, 2Department of Respiratory, First Affiliated Hospital, College of Medicine of Xi'an Jiaotong University, Xi'an, China; 3Department of Ultrasound, Second Affiliated Hospital, College of Medicine of Xi’an Jiaotong University, Xi’an, China.
Background: Pancreatic carcinoma (PC) is one of the leading causes of cancer death, with less than 5% of patients surviving 5 years after diagnosis. Suppressor with morphogenetic effect on genitalia (SMG-1) have been demonstrated involved in tumorigenesis. But the role of SMG-1 in the proliferation, invasion and chemoresistance of PC cells remains unclear.
Results: In this study, the mRNA and protein expression of SMG-1 in PC patients has been investigated using real-time PCR assay and immunohistochemistry. The relationship between expression of SMG-1 and clinicopathological parameters, and PC patient survival was analyzed. We showed that SMG-1 level in tumor tissues of PC patients is a novel prognostic marker of overall survival. The clinical characteristics demonstrated that low SMG-1 correlated with larger tumor size, lymphatic invasion and poor prognosis. Moreover, functional study showed that overexpressed SMG-1 could significantly inhibit proliferation of PC cells by inducing apoptosis and G1-phase arrest. Meanwhile, overexpression of SMG-1 remarkably inhibited invasion of PC cells. Furthermore, overexpression of SMG-1 in pancreatic cancer cell lines suppressed the expression of mTOR, which plays a key role in the development of pancreatic cancer.
Conclusion: In this study, we revealed that SMG-1 might serve as a tumor suppressor via regulating mTOR expression to inhibit pancreatic tumor growth.
Extended Pancreatectomies in Locally Advanced Pancreatic Cancer: Are There Any Limits?
W. Hartwig,1* A. Gluth,1* D. Koliogiannis,1 U. Hinz,1 T. Hackert,1 J. Werner,1 M.W. Büchler.11Department of Surgery, University of Heidelberg, Germany. *Equal contribution.
Background: Margin-free resection is still the only potential chance for cure in patients with advanced pancreatic cancer. The aim of the study was to evaluate perioperative outcome and long-term survival after extended pancreatectomy (EP) in a large prospective cohort of patients.
Methods: Between October 2001 and July 2013, a total of 613 consecutive EPs, as defined by the new ISGPS consensus, were performed, prospectively documented, and analyzed, and were compared with 1218 standard pancreatectomies (SP). Uni- and multivariate analysis was performed to identify risk factors for perioperative morbidity and mortality. Likewise, patient, tumor, and resection characteristics were correlated with survival. Long-term outcome was assessed using Kaplan Meier analysis.
Results: The 613 EPs consisted of 286 extended pancreatoduodenectomies, 123 extended pancreatic left resections, and 204 extended total pancreatectomies. In these, 506 portal vein, 117 colon, 89 gastric, 65 arterial, 47 adrenal, 20 small bowel, and 14 kidney resections were performed. EPs had a significantly higher non-surgical and surgical morbidity and 30-day mortality compared to standard resections (42.6% vs. 34.2%; 37.0% vs. 28.2%; and 4.2% vs. 1.8%, respectively). Operating time ≥300 min, total pancreatectomy, and ASA-Score ≥3 were independently associated with increased hospital mortality in multivariate analysis. Colon or portal vein resection had no effect on hospital mortality. Median survival and 5-year survival rates were significantly decreased in EPs vs. SPs (16.5 vs. 24.3 months and 10.7 vs. 20.3% respectively). Ca-19-9 levels ≥250 U/ml, a high ASA Score (ASA 3/4), G3 tumors, more than 8 affected lymph nodes, R2 resections, operating time ≥420 min, and type 4 portal vein resections were independently associated with decreased overall survival in multivariate analysis.
Conclusions: Extended pancreatectomy, if needed, is associated with good long-term outcome in pancreatic cancer, but has increased perioperative morbidity and mortality, in particular when extended total pancreatectomy has to be performed. Tumor and to a lesser extent also resection characteristics impair survival which is markedly better than with best palliative therapy available.
Sex-Specific Responses of the Corticotropin-Releasing Factor (CRF) System in Exocrine Pancreas
B. Hasdemir,1 J.A. Oses-Prieto,2 A.L. Burlingame,2 A. Bhargava.11Department of Ob-Gyn and The Osher Center, 2Pharmaceutical Chemistry, University of California, San Francisco, CA.
Defective acinar cell secretion is a hallmark of pancreatitis and Ca2+ signals regulate acinar cell secretion. The CRF family includes CRF and urocortins (Ucn1-3) and mediates stress and immune responses via activation of two GPCRs CRF1 and CRF2. We have shown sex-specific actions of Ucn1 on cellular stress responses during acute pancreatitis. The aims here were to ascertain 1) differential actions of CRF and Ucn1 on acinar cell Ca2+ signaling, and 2) sexspecific mechanisms of Ucn1 action.
Ucn1, but not CRF stimulation resulted in amylase secretion from acinar cells, a novel finding.
Primary acinar cells were loaded with Fura-2 AM and intracellular Ca2+ [Ca2+]i in response to agonist(s) stimulations was measured. Acinar cells isolated from wildtype (WT) mice of both sexes showed similar Ca2+ responses to CRF or Ucn1 stimulation. Haploinsufficiency of CRF2
(Ht) revealed several sex-specific differences: 1) only acinar cells from female mice showed increased [Ca2+]i responses to CRF. 2) Astressin2b, a CRF2-specific antagonist, decreased Ucn1-induced Ca2+ responses in male cells alone. 3) Pre-treatment with caerulein exacerbated the CRF response in male, but not in female acinar cells. Importantly, western blot analysis showed equal CRF2 expression in both sexes. Mass spectrometry identified several CRF2- interacting proteins in co-immunoprecipitated lysates after CRF or Ucn1 stimulation including F-actin and heat shock proteins. Redistribution of F-actin is key for Ca2 + −mediated secretion from acinar cells. Ucn1, but not CRF stimulation triggered F-actin redistribution similar to cerulein stimulation. Finally, F-actin association with CRF2 was seen in male mice only after cerulein treatment and co-IP. Thus, our results identify a novel mechanism by which CRF2 interacts with cellular proteins in a context-and sex-specific manner to mediate stress coping cellular responses.
Intratumoral Expression of Tenascin C is a Potential Surrogate Marker for Histological Effect of Chemoradiotherapy in Locally Advanced Pancreatic Cancer Patients
A. Hayasaki,1 Y. Murata,1 M. Usui,1 M. Kishiwada,1 S. Mizuno,1 H. Sakurai,1 T. Yoshida,2 S. Isaji.11Division of Hepato-biliary Pancreatic and Transplant Surgery, 2Division of Matrix Biology, Mie University, Tsu, Mie, Japan.
The indication criteria for curative-intent resection after chemoradiotherapy (CRT), referred as adjuvant surgery, has not been established in locally advanced unresectable (UR-LA) pancreatic adenocarcinoma (PDAC) because of the lack of useful surrogate marker. Tenascin C (TNC) is an extracellular matrix that is upregulated in PDAC and associated with cell-matrix interaction facilitating tumor cell invasion. The aim is to determine outcome of adjuvant surgery and to assess usefulness of TNC as a surrogate for effect of CRT.
Methods: From February 2005 to September 2014, among 220 patients with cytologically/histologically proven PDAC, 96 with UR-LA PDAC who underwent CRT (gemcitabine/S-1 based) were enrolled. They were divided into high (tumor destruction > 50%) and low responder (<=50%) according to the histological effect of CRT. The intratumoral expression of TNC was evaluated by immunostaining of the stroma around the tumor cells using resected specimens for 32 with resection, and staining was scored as positive (>20%) or negative (<=20%) according to the percentage of the positively stained area.
Result: Among the 96 patients, 43 (44.8%) underwent resection, and R0 resection was achieved in 25 (58.1%). The patients with R0 had significantly longer overall survival (OS) than those with no resection (R0 vs. no: 21.2 vs. 10.1 months). The R0 rate was higher in high responder (n = 10) than in low responder (n = 33) (high vs. low: 80.0 vs. 51.5%). The positive expression rate of TNC was significantly lower in high responder than in low responder (high vs. low: 16.7 vs. 69.2%), and the patients with negative TNC had significantly longer OS than those with positive (positive vs. negative: 17.1 vs. 29.3 months).
Conclusion: Effective histological effect of CRT contributes to improving survival for UR-LA PDAC patients with resection. The intratumoral expression of TNC may serve as a surrogate for indicating candidate for adjuvant surgery.
Rates of Malignancy in Side Branch IPMN are Underestimated: A Systematic Review of Recent Publications
M. Heckler, C.W. Michalski, S. Schaefle, S. Fritz, M.W. Büchler, T. Hackert. Department of Surgery, University of Heidelberg, Heidelberg, Germany.
Background: The risk of malignancy in branch duct IPMN (BD-IPMN) is controversially debated. While the number of diagnosed and treated cases increases year by year, diagnostic accuracy is relatively low. Despite the consensus guidelines of Sendai (2006) and Fukuoka (2012), individual treatment decisions are often difficult. We thus systematically reviewed studies reporting on outcomes after conservative and/or surgical management of cysts that were diagnosed as BD-IPMN prior to treatment.
Methods: Studies reporting on more than 40 patients and published between 2006 (following the Sendai consensus) and 2014 were included if at least one Sendai / Fukuoka criterion was used for decision making. Definition of malignancy was assumed for patients revealing high-grade dysplasia or invasive cancer.
Results: 12 studies including 1727 patients (650 primarily resected, 1077 primarily conservative treatment) were included. The median follow up time ranged from 25 to 77 months. In the primary resection group, 23 % of the patients without any feature predicting malignancy according to the guidelines had a malignant lesion in the histopathological workup (compared to 34% of patients that expressed high risk (hrf) or worrisome features (wf) according to the guidelines). In 607 patients with absence of hrf or wf, BD-IPMN that were initially followed conservatively, resection was finally performed in 122 patients after a median follow up period ranging from 12.7 – 41 month. 17% of these patients had a malignant lesion in histopathological analysis.
Conclusion: This retrospective analysis demonstrates that the current consensus guidelines do not safely predict the risk of (future) malignancy in BD-IPMN. Until more accurate predictive diagnostic parameters are available, a more aggressive surgical approach should be discussed for such lesions.
A Common Deletion Variant of Chymotrypsin B2 (CTRB2) Causes Misfolding
E. Hegyi M. Sahin-Tóth. Department of Molecular and Cell Biology, Boston University Medical Center, Boston, MA.
Background: Naturally occurring structural variants of human chymotrypsins B1 (CTRB1) and B2 (CTRB2) have not been studied so far. A deletion of 585 nucleotides in the CTRB2 gene in >10% of the population has been described recently (Pang et al., Human Mutation 34, 345–354, 2013). The deletion affects the entire exon 6 of CTRB2 and portions of the flanking introns. Upon splicing of the remaining exons 5 and 7, the frame shift in the coding DNA results in a premature stop codon, thereby creating a C-terminally truncated CTRB2 protein.
Aims: Our aim was to investigate the impact of the deletion on mRNA expression, cellular secretion and proteolytic stability of CTRB2.
Methods: Expression of CTRB2 mRNA was measured in human pancreatic cDNA samples by RT-PCR. Secretion of CTRB2 was measured from the conditioned medium of transiently transfected HEK 293 T cells by SDS-PAGE and western blot.
Results: The mRNA for the deletion variant was expressed at normal levels in two unrelated cDNA samples from human pancreas. In contrast to wild-type CTRB2 which was readily secreted to the growth medium of transfected cells, the deletion variant was not secreted to a measurable extent. Accumulation of the deleted protein was detected intracellularly suggesting misfolding and ER retention. Consistent with this interpretation, the intracellularly accumulated deletion variant exhibited increased sensitivity to proteolytic degradation by trypsin.
Conclusions: A common deletion variant of human CTRB2 causes misfolding which results in intracellular retention with a consequent loss in secretion.
p120 Catenin Suppresses Basal Epithelial Cell Extrusion in Invasive Pancreatic Neoplasia
A.M. Hendley,1,4 Y.J. Wang,1 J. Alsina,1 K.J. Lafaro,2 A. Maitra,3 C.A. Iacobuzio-Donahue,2 S.D. Leach,2 J.M. Bailey.41The McKusick-Nathans Institute of Genetic Medicine, Johns Hopkins University School of Medicine, Baltimore, MD; 2The David Rubenstein Pancreatic Cancer Research Center, Memorial Sloan Kettering Cancer Center, New York, NY; 3Departments of Pathology and Translational Molecular Pathology, The University of Texas MD Anderson Cancer Center, Houston, TX; 4Division of Gastroenterology, Hepatology, and Nutrition, Department of Internal Medicine, The University of Texas Health Science Center at Houston, Houston, TX.
Aberrant regulation of epithelial cell extrusion has been shown to promote metastasis. We identify molecular requirements for early extrusion using a lineage-traced preinvasive mouse model of pancreatic cancer, KCiMist1G, with conditional knockout of p120 catenin (Ctnnd1). Mice with biallelic loss of p120 catenin progressively develop high grade PanIN lesions and invasive neoplasia accompanied by prominent acute and chronic inflammatory processes. Loss of p120 catenin in the context of oncogenic Kras also promotes remarkable apical and basal epithelial cell extrusion. Quantification of basally extruded isolated epithelial cells revealed a striking 832/7000 CK19+ cells in KCiMist1; p120f/f pancreata vs. 15/7000 CK19+ cells in KCiMist1; p120wt/wt pancreata. An analysis of the DNA content of isolated, basally extruded CK19+ cells in KCiMist1; p120f/f pancreata showed populations of cells that displayed aneuploidy and abnormal DNA content. In addition, quantification of apical and basal epithelial cell extrusion observed in metaplastic duct lesions in cerulean-mediated acute pancreatitis showed significant increases in CiMist1; p120f/f pancreata when compared to CiMist1; p120wt/wt pancreata. These results provide insight into mechanisms controlling early events in the metastatic process and suggest that p120 catenin enhances pancreatic cancer progression by regulating neoplastic epithelial cell invasion.
Suppression of CD51 in Pancreatic Stellate Cells Inhibits Tumor Growth by Reducing Stroma and Altering Tumor-Stromal Interaction in Pancreatic Cancer
K. Horioka, K. Ohuchida, M. Sada, B. Zheng, T. Ohtsuka, T. Ueki, E Nagai, K. Mizumoto, Y. Oda, M. Nakamura. Department of Surgery and Oncology, Graduate School of Medical Sciences, Kyushu University, Fukuoka, Japan.
Background: Pancreatic stellate cells (PSCs) are major sources of extracellular matrices (ECM) in pancreatic cancer stroma, which works a barrier to delivering chemotherapeutics to cancer cells. PSCs also enhance malignancy by interacting with cancer cells. CD51, also known as integrin αv, has been shown to play a crucial role in fibrosis of several organs, and some integrins on cancer-associated fibroblasts augment malignant progression. Here we investigated CD51 expression on PSCs and its functional roles in pancreatic cancer.
Methods: CD51 expression in human pancreatic cancer was evaluated by immunohistochemistry and immunocytochemistry. Knockdown of CD51 in PSCs was performed using lentivirus-mediated shRNA. Cell proliferation and motility were analyzed with CD51-silenced PSCs. To assess tumor growth in vivo, pancreatic cancer cells were implanted subcutaneously into nude mice with PSCs. The amount of tumor stroma was evaluated by the positivity of α-SMA and picrosirius red staining and cell proliferation was evaluated by PCNA index.
Results: CD51 was expressed in both the stroma and cancer cells in pancreatic cancer tissues. Individual PSCs showed diffuse expression of CD51 on the cell membrane. Knockdown of CD51 in PSCs decreased the expression of ECM genes such as collagen I as well as several growth factors, such as CTGF and PDGF-A. Cell proliferation and motility of PSCs were suppressed by CD51 knockdown. In the co-implantation models of pancreatic cancer cells and PSCs, tumor growth in vivo was inhibited by CD51 knockdown in PSCs (P < 0.05). We also found the reduced tumor stroma and decreased proliferation of cancer cells in implanted cancer tissue with CD51-silenced PSCs (P < 0.05).
Conclusion: The present data suggest that CD51 in PSCs could be a potential therapeutic target for pancreatic cancer.
Acute Pancreatitis Associated With Pancreatectomy: Clinical Analysis of 10 Cases
D. Huang, Q. Li, C. Dai, K. Jiang, J. Wu, W. Gao, J. Wei, J. Chen, F. Guo, Z. Lu, Y. Miao. Pancreas Center, the First Affiliated Hospital of Nanjing Medical University, Nanjing, China.
Objective: Pancreatectomy induced acute pancreatitis (AP) is less common compared to other causes of AP. We aimed to study the characteristics and treatment outcome of postpancreatectomy patients who developed AP in the following time.
Methods: In this retrospective study, records of postpancreatectomy patients admitted in our pancreas center with AP between January 2013 and May 2015 were studied. Clinical information and auxiliary examination was obtained by reviewing the records.
Results: Of 742 postpancreatectomy patients during the study period, ten of them complicated AP in the next days. The ten patients were all diagnosed of mild acute pancreatitis. Four patients received pylorus-preserving pancreaticoduodenectomy (PPPD), three patients received standard Whipple's procedure, and the rest three received middle segment pancreatectomy. Nine of the 10 cases were classified as Acute recurrent pancreatitis (ARP), and average number of recurrences was 2.8 ± 1.8. Average length of hospital stay was 8.9 ± 1.6 days. Average time from the first onset to postpancreatectomy operation was 225 (range 26-935) days. All the ten patients were managed conservatively, except one, who was reoperated in the end because of pancreatico-jejunal anastomotic stenosis. No deaths occurred during the study period, and all the cases were complete remission.
Conclusions: Knowledge regarding acute pancreatitis related to postpancreatectomy is of paramount importance in order to diagnose cases early and institute effective treatment in patients who have received pancreatectomy before.
Clinical and Biological Features of Patients of Acute Pancreatitis With Transient Organ Failure or Acute Peripancreatic Fluid Collection
W. Huang,1,2 D. Iglesia-Garcia,1 P. Szatmary,1 L. Fleming-Bird,1 K. Altaf,1 Q. Nunes,1 R. Mukherjee,1 W. Greenhalf,1 M. Raraty,1 R. Sutton.11NIHR Liverpool Pancreas Biomedical Research Unit, Royal Liverpool University Hospital, University of Liverpool, Liverpool, United Kingdom; 2Sichuan Provincial Pancreatitis Centre, Department of Integrated Traditional and Western Medicine, West China Hospital, Sichuan University, Chengdu, China.
Background: The moderate severity category of acute pancreatitis (AP) defined by Revised Atlanta Classification (RAC) is controversial. We sought to elucidate both clinical and biological features of transient organ failure (TOF) and acute peripancreatic fluid collections (APFC) in this group.
Methods: Patients with a first episode of AP admitted within 72 h after disease onset and aged 18-85 without advanced diseases were selected from a prospective E-database. Data of patients with mild and moderate severity defined by RAC were analyzed. Biochemical values were plotted from admission and days 2, 4, 7, 10, 14, 21 and 28 and cumulative values summated from each time point were compared among designated groups.
Results: There were 303 patients identified. Patients in the mild (n = 232) and TOF only (moderate; n = 16, all had TOF < 24 h) groups had significantly reduced overall infection, length of hospitalization, cumulative CRP and Δ-albumin (where Δ = sum of decrease in values at each time point) and neutrophils (all P < 0.05) compared to patients with local complications (LC, moderate; n = 55). Patients in the former two groups were not significantly different in the aforementioned parameters. The time-course pattern of CRP, albumin and neutrophils were similar between mild and TOF only groups, both were significantly different from the LC group. Further analyses revealed that there were no significant differences of outcome measures between APFC (n = 28) and acute necrotic collections (ANC; n = 27) groups, but these measures were dramatically reduced in the TOF only group (all P < 0.05). The time-course pattern of CRP, albumin and neutrophils were similar between APFC and ANC groups, both were significantly different from the TOF only group.
Conclusion: Our preliminary data suggest that in non-severe patients defined by RAC, patients in TOF only group behave clinically and biologically similar to the mild group, while patients with APFC have identical disease features to ANC. Patients in the APFC and ANC groups had significantly worse clinical outcomes compared with those in the mild and TOF only groups.
Early Prediction of Persistent Organ Failure in Patients With Acute Pancreatitis
W. Huang,1,2 P. Szatmary,1 D. Iglesia-Garcia,1 L. Fleming-Bird,1 S. Parente,1 G. Primo,1 E. Agnew,1 K. Altaf,1 Q. Nunes,1 R. Mukherjee,1 W. Greenhalf,1 M. Raraty,1 R. Sutton.11NIHR Liverpool Pancreas Biomedical Research Unit, Royal Liverpool University Hospital, University of Liverpool, Liverpool, United Kingdom; 2Sichuan Provincial Pancreatitis Centre, Department of Integrated Traditional and Western Medicine, West China Hospital, Sichuan University, Chengdu, China.
Background: Currently there are no satisfactory early predictors on or within 48 h of admission for persistent organ failure (POF). We sought to evaluate the predictive values of routine clinical measures for POF both on and within 48 h of admission.
Methods: Patients with a first episode of AP admitted within 72 h after disease onset and aged 18-85 without advanced diseases were selected from a prospective E-database. Severity was defined by Revised Atlanta Classification, while POF was defined as at least one of the systems (lung, cardiovascular and renal) with a SOFA score of 2 or more ≥48 h. The predictive values of routine clinical measures were calculated.
Results: Data were analyzed from 333 patients with 232 mild, 71 moderate and 30 severe cases, respectively. On admission, SIRS (≥2), BISAP (≥2), APACHE II (≥8) and SOFA (≥2) scores, urea, creatinine and hematocrit had low to fair AUC values ranging from 66.3% to 80.6%, while adjusted calcium, magnesium, bicarbonate, prothrombin time, CRP, albumin, total protein and hemoglobin barely had any predictive values for POF. Within 48 h of admission, the sensitivity, specificity and AUC for CRP (≥250 mg/L) were 88.9%, 82.7% and 90.4%, respectively; for Ä-albumin (≥9.5 g/L) were 82.8%, 77.4% and 85.2%, respectively; for Ä-total protein (≥13.5 g/L) were 90%, 82.2% and 91.3%, respectively; for urea (≥5.4 mmol/L) were 80%, 78.9% and 85.5%, respectively.
Conclusion: Our data indicate that there are no good predictors on admission for POF, consistent with published literature. However, CRP, Ä-albumin and Ä-total protein and urea have good predictive values within 48 h of admission. CRP and Ä-total protein are recommended for routine clinical use for predicting POF within 48 h of admission.
Cumulative Acute Pancreatitis Outcome Score for Clinical Trials in Acute Pancreatitis
W. Huang,1,2 P. Szatmary,1 D. Iglesia-Garcia,1 A. Sud,1 B. Lane,1 Q. Nunes,1 R. Mukherjee,1 W. Greenhalf,1 M. Raraty,1 R. Sutton.11NIHR Liverpool Pancreas Biomedical Research Unit, Royal Liverpool University Hospital, University of Liverpool, Liverpool, United Kingdom; 2Sichuan Provincial Pancreatitis Centre, Department of Integrated Traditional and Western Medicine, West China Hospital, Sichuan University, Chengdu, China.
Background: Despite several hundred randomized clinical trials in the treatment of acute pancreatitis (AP) no specific therapy has emerged. Regulatory agencies have advised development of outcome indices for AP as used in other diseases, to better demonstrate beneficial treatment effects in place of traditional end points. We sought to develop the Cumulative Acute Pancreatitis Outcome Score (CAPOS) to address this need.
Methods: Patients with a first episode of AP aged 18-85 without advanced pulmonary, cardiac, renal or malignant diseases were selected from a prospective E-database. Revised Atlanta and Determinant-based Classifications (RAC and DBC) of patients were made, other clinical features identified, and hematological and biochemical values plotted from admission and days 2, 4, 7, 10, 14, 21 and 28. Features and values that were abnormal in all disease classes and showed the most effective proportional differentiation between classes were incorporated into CAPOS.
Results: Data were analyzed from 333 patients; as features and values differentiated RAC classes more consistently than DBC classes, CAPOS was developed using RAC (232 mild patients; 71 moderate; 30 severe). Cumulative values summated from each time point for C-reactive protein, neutrophils, Ä-hematocrit (where Ä = sum of decrease in values at each time point), Ä-albumin and Sequential Organ Failure Assessment scores correlated closely with RAC classes and major clinical outcomes. These parameters together with opiate requirement, nutritional deficit, pancreatic imaging and length of stay were employed to form CAPOS using optimal cut-off values (0-4 for each parameter).
Conclusion: RAC was found to be more effective for severity stratification than DBC, providing the basis for development of CAPOS, an accurate measure of outcome in our patient cohort. Validation of CAPOS in further cohorts may provide a useful tool for outcome assessment in randomized clinical trials of treatment for AP.
Therapeutic Potential of Pyruvate in Acute Pancreatitis: In Vitro Findings and a Systematic Review
W. Huang,1,2,3 J.J. Xiong,4 C.R. Cheng,5 P. Szatmary,1 M. Chvanov,3 D.N. Criddle,1,3 Q. Xia,3 R. Sutton.11NIHR Liverpool Pancreas Biomedical Research Unit, Royal Liverpool University Hospital, Liverpool, United Kingdom; 2Department of Cellular and Molecular Physiology, University of Liverpool, Liverpool, United Kingdom; 3Sichuan Provincial Pancreatitis Center, Departments of Integrated Traditional and Western Medicine, West China Hospital, Sichuan University, Chengdu, China; 4Department of Pancreatic Surgery, West China Hospital, Sichuan University, Chengdu, China; 5School of Chemistry and Pharmaceutical Engineering, Sichuan University of Science and Engineering, Zigong, China.
Background: In acute pancreatitis (AP) there is an increase in glycolysis in pancreatic acinar cells (PACs), providing ATP during mitochondrial impairment. While there is evidence of protective effects from pyruvate and ethyl pyruvate (EP) on experimental AP, their effects on PACs remain unknown. We sought to address this question on PACs hyperstimulated with cholecystokinin (CCK) and also systematically appraise the evidence for protective effects in AP.
Methods: Freshly isolated PACs were stimulated with CCK (10nM) and effects of pyruvate and EP on cell fate were assessed using rhodamine 110/aspartic acid amide (20 μM, apoptosis) and propidium iodide (1ìM, necrosis) in a fluorescent plate reader. For the literature search the medical subject headings “acute pancreatitis”, “pancreatitis”, “pancreatic acinar cells”, “AR42J cells” and “pyruvate” were used.
Results: Both apoptotic and necrotic cell death pathways induced by CCK were significantly inhibited by pyruvate and EP (from 0.1-1 mM, all P < 0.001); 1 mM EP almost abolished any effects caused by CCK (n = 3 mice and >1 × 106 cell per experiment). In the review one study showed pyruvate pre-treatment significantly reduced serum amylase, pancreatic carbonyls and histological scores in rat cerulein AP. Nine studies demonstrated that EP (normally at a regimen of 40 mg/kg every 6 h) given after disease induction consistently reduced biochemical, immunological and histopathological parameters of injury in the pancreas, lung, intestine, liver and kidney in multiple murine AP models. Mortality was dramatically reduced even when EP was given 12 h-post disease induction, compared to untreated groups or those treated with lactate.
Conclusion: These data indicate that pyruvate and EP are protective in isolated PACs hyperstimulated with CCK, consistent with the marked protection effects in experimental AP.
The Impact of Early Initiation of High-Dose Pancreatic Enzyme Supplementation for Prevention of Nonalcoholic Fatty Liver Disease After Pancreaticoduodenectomy
Y. Iizawa, H. Kato, A. Tanemura, Y. Murata, A. Yoshinori, N. Kuriyama, M. Kishiwada, S. Mizuno, M. Usui, H. Sakurai, S. Isaji. Division of Hepatobiliary Pancreatic and Transplant Surgery, Mie University, Tsu, Mie, Japan.
We previously reported the risk factors of development of nonalcoholic fatty liver disease (NAFLD) after pancreaticoduodenectomy (PD), which didn’t focus on pancreatic enzyme supplementation (PES). From 2007, we standardized the intra- and postoperative management including pancreaticojejunostomy, routine PES and nutritional support, which allow us to obtain the true risk factor for development of NAFLD.
Patients and Methods: Among 222 consecutive patients receiving our protocol from 2007 to 2014, the 145 patients whose remnant pancreatic volumes (RPV) by CT volumetry could be followed more than 6 months were eligible in this study. According to RPV at one month after PD, the patients were divided into small RPV (SRPV: <10 ml in 51) and large RPV (LRPV: 10 ml or more in 94). The median CT attenuation value of the liver was measured pre and operatively (1 to 36 months after PD).
Results: Among 145 patients, 56 (39%) developed NAFLD (<40HU), and 88% (49/56) developed it within 6 months. Univariate analysis revealed age, gender, disease, preoperative chemoradiotherapy and SRPV as the significant factors. Multivariate analysis, however, identified younger age (OR: 1.01, P < 0.01), female (OR: 6.56, P < 0.01), SRPV (OR: 3.22, P < 0.01) and low-dose PES at 1 month (OR: 3.30, P < 0.05) as the significant independent risk factors. Combination analysis of independent risk factors was performed: the incidence of NAFLD was higher in female with low-dose (<pancreatin 6 g/day) than in female with high-dose (pancreatin 6 g/day or more) (71.4% vs. 59.6%), and also higher in SRPV with low-dose than in SRPV with high-dose (80.0% vs 52.2%).
Conclusion: Female, younger patient, SRPV and low-dose PES at 1 month are high risk groups of NAFLD, and high-dose PES might prevent NAFLD after PD.
Chronic Pancreatitis: An International Multidisciplinary Survey and Case Vignette Study
Y. Issa,1 H.C. van Santvoort,1 P. Fockens,2 M.G. Besselink,1 T.L. Bollen,3 M.J. Bruno,4 M.A. Boermeester.11Department of Surgery, Academic Medical Center, Amsterdam, The Netherlands; 2Department of Gastroenterology, Academic Medical Center, Amsterdam, The Netherlands; 3Department of Radiology, St Antonius Hospital, Nieuwegein, The Netherlands; 4Department of Gastroenterology, Erasmus Medical Center, Rotterdam, The Netherlands.
Background: The aim of the study was to gain more insight into the current opinion and clinical decision making of international pancreatologists, regarding the diagnosis and treatment of chronic pancreatitis (CP).
Methods: An online survey containing question regarding the diagnosis and treatment of CP and several controversial clinical case vignettes was send by e-mail to members of different international pancreatic associations: IHPBA, APA, EPC, ESGE and DPSG.
Results: A total of 288 pancreatologists participated with the survey. The majority (54%) was a registered specialist for over 10 years and 77% worked at an academic center. 48% of the specialists indicated to use a classification tool for the diagnosis of CP, especially the Mayo Clinic (28%), Mannheim (25%) or Büchler (25%). The CT was the most preferred imaging modality to assess an enlarged pancreatic head (59%), pseudocyst (55%), calcifications (75%) and peripancreatic fat infiltration (68%) and MRI for evaluating main pancreatic duct (MPD) abnormalities (60%). 59% indicated to use ESWL in the treatment of CP. TP-IAT was most preferred treatment in patients with parenchymal calcifications, without dilatation of the MPD (27%) and in patients with refractory pain despite maximal medical, endoscopic and surgical treatment (21%). In patients with an enlarged pancreatic head, 58% preferred surgery (PPPD) versus 42% endoscopy. In patients with a dilated MPD and intraductal stones 56% preferred endoscopic +/− ESWL treatment and 29% preferred surgical treatment.
Conclusion: There is little consensus regarding diagnosis and treatment of CP. Future studies and evidence-based guidelines should determine the role of diagnostic modalities and endoscopy and surgery (e.g. TP-IAT) in the treatment algorithm of CP.
Comparison of Different Classification Tools for the Diagnosis of Chronic Pancreatitis
Y. Issa,1 U. Ahmed Ali,1,2 S. van Dieren,1 H.C. van Santvoort,1 M.A. Boermeester.11Department of Surgery, Academic Medical Center, Amsterdam; 2Department of Surgery, University Medical Center Utrecht, Utrecht.
Introduction: Several classification tools for the diagnosis of chronic pancreatitis (CP) are used in daily practice and in clinical research. The aim of this study was to compare the Mannheim, Büchler and Lüneburg classification tools for CP.
Methods: A cross-sectional analysis of a prospectively collected multi-centre patient cohort including 669 patients 5 years after a first episode of acute pancreatitis was performed. We evaluated and compared the Mannheim, Büchler and Lüneburg classification tools for CP.
Results: The median follow-up period of the patients was 57 months (IQR 42-70). CP was diagnosed in 50 (7%), 60 (9%), 59 (9%), 61 (9%) and 46 (7%) of 669 patients by the Mannheim definite and propable, Lüneburg, Büchler and the treating physician, respectively. Differences between the classifications regarding the following criteria led to significant changes in the total number of diagnosis of CP: abdominal pain, recurrent pancreatitis, moderate to marked ductal lesions, endocrine and exocrine insufficiency, pancreatic calcifications and pancreatic pseudocysts. The overall agreement between the Mannheim, Büchler and Lüneburg was substantial (Kappa 0.75, range 0.69 – 0.79). The Büchler score had the highest sensitivity (94%), followed by the Mannheim (87%) and finally the Lüneburg classification (81%). The specificity ranged from 97% to 99%.
Conclusion: Differences in the diagnosis of CP by the different classification tools can be mainly attributed to whether abdominal pain or recurrent pancreatitis, exocrine and endocrine insufficiency, calcifications, ductal lesions and pseudocysts are included in the classification tools. A combination of these criteria in a modified classification tool may lead to a higher diagnostic accuracy for the diagnosis of CP.
Computer Tomography Versus Magnetic Resonance Cholangiopancreatography in Patients With Chronic Pancreatitis
Y. Issa,1 U. Ahmed Ali,1,2 T. Bollen,3 M. van der Hoek,1 Y. Keulemans,4 H.C. van Santvoort,1 C.Y. Nio,1 M.A. Boermeester.11Department of Surgery, Academic Medical Center, Amsterdam, The Netherlands; 2Department of Surgery, University Medical Center Utrecht, Utrecht, The Netherlands; 3Department of Radiology, St Antonius Hospital, Nieuwegein, The Netherlands; 4Department of Gastroenterology, University Hospital Maastricht, Maastricht, The Netherlands.
Introduction: Imaging of the pancreatic morphology is an indispensable and crucial part of the diagnostic and therapeutic evaluation of patients with chronic pancreatitis (CP).
Methods: A head-to-head comparison was made by 2 experienced radiologists of CT and MRI in patients with CP. All patients had undergone both CT and MRI within 6 weeks of each other, of the pancreas without any upflares of acute pancreatitis in the period between the CT and MRI.
Results: A total number of 75 patients were included in the study, with a mean age of 51 (±11) years. The majority had alcoholic (52%) or idiopathic (32%) CP. There were significant differences between the CT and MRI in the detection of: parenchymal calcifications (63% vs 7%, P < 0.001), intraductal stones (47% vs 29%, P = 0.01), peripancreatic infiltration (76% vs 55%, P = 0.001), atrophy (41% vs 59%, P = 0.001), pancreatic duct (PD) strictures (39% vs 71%, P < 0.001), and visibility of side branches (16% vs 81%, P < 0.001), respectively. Marked difference in the interobserver agreement between CT versus MRI were (prevalence adjusted bias adjusted kappa (PABAK): parenchymal calcifications (0.92 vs 0.70), intraductal stones (0.82 vs 0.62), compression of common bile duct (0.82 vs 0.68), peripancreatic infiltration ( 0.62 vs 0.36), presence of pseudocysts (0.76 vs 0.86) and the diameter of the largest pseudocyst (intraclass correlation coefficient 0.70 vs 0.97), respectively.
Conclusion: There are marked differences in the detection of morphological abnormalities between CT and MRI in patients with CP and interobserver agreement between the expert radiologists.
Imaging Modalities in Chronic Pancreatitis: A Systematic Review and Meta-Analysis
Y. Issa,1 M.A. Kempeneers,1 H.C. van Santvoort,1 S. Bipat,2 M.A. Boermeester.11Department of Surgery, Academic Medical Centre Amsterdam, The Netherlands; 2Department of Radiology, Academic Medical Centre Amsterdam, The Netherlands.
Background: The most frequent used imaging modalities for chronic pancreatitis (CP) are endoscopic retrograde cholangiopancreatography (ERCP), abdominal ultrasonography (US), endoscopic ultrasonography (EUS), magnetic resonance imaging (MRI) and computed tomography (CT). Aim of this study was to obtain summary estimates of sensitivity and specificity for imaging modalities for CP by means of a meta-analysis.
Methods: A systematic search was performed in Cochrane Library, MEDLINE, Embase and CINAHL databases for studies evaluating imaging modalities for CP up to January 2015. The search included terms for chronic pancreatitis, MRI, CT, EUS, ERCP and US. The inclusion criteria were: one or more imaging modality was evaluated in patients with CP (≥5 patients), the imaging technique was compared with a reference standard, and enough data was reported to construct a 2 X 2 table. All search hits were evaluated by two reviewers independently. The QUADAS-2 tool was used to assess the methodological quality of the included studies and a bivariate random-effects modeling to obtain summary estimates of sensitivity and specificity.
Results: A total of 7641 titles and abstracts were screened for eligibility. For 264 studies, the full text was retrieved, of which 45 studies fulfilled the inclusion criteria, with evaluation of 3629 patients. Sensitivity of ERCP 82% (95% CI: 76%-87%) was significant higher than that of US 67% (95% CI: 53%-78%) (p = 0.018). The sensitivity estimates of EUS, MRI and CT were respectively 82% (95% CI: 71%-90%), 78% (95% CI: 69%-85%) and 75% (95% CI: 66%-83%): did not differ significantly. There were also no significant differences between estimates of specificity for CT 91% (95% CI: 81%-96%), MRI 96% (95% CI: 90%-98%), EUS 91% (95% CI: 83%-95%), ERCP 94% (95% CI: 87%-98%) and US 98% (95% CI: 89%-100%).
Conclusion: ERCP and EUS have the highest diagnostic accuracy of all the imaging modalities in detection of CP, followed by the CT and MRI. US had the lowest diagnostic accuracy.
Treatment Strategy for Locally Invasive Pancreatic Cancer
J. Itakura, M. Watanabe, N. Hosomura, H. Amemiya, H. Kawaida, H. Okamoto, H. Kohno. Department of Surgery, University of Yamanashi, Cyuo-shi, Yamanashi, Japan.
Background/Aim: The treatment strategy for locally invasive pancreatic cancer is still controversial. We examined the adaptation of NAC for locally invasive cases by the classification as Borderline resectable (BR), Possible resectable (PR) and Regional lymph node metastasis (RL).
Methods: Fifty-five stage IIB and III pancreatic adenocarcinoma cases operated in our department from 1998 to 2014 were studied. These 55 cases were re-classified in two groups; BR group: Tumor abutment of the SMA or CA not to exceed >180 degrees of the circumference of the vessel wall, PR group: Venous involvement of the SMV/portal vein demonstrating tumor abutment with or without impingement and narrowing of the lumen, or metastases to lymph nodes beyond the field of resection. The tumor marker in every month and CT examination in every three months were evaluated as postoperative recurrence form and time.
Results: BR group was 12 cases, PR group was 43 cases. The operation methods PD/DP/TP were 2/10/0 in BR, 30/12/1 in PR. The degree of radical cure R0/R1/R2 was 5/4/3 in BR, 37/6/0 in PR. The recurrence was 10 cases (83%) of BR, 32 cases (74%) of PR. In recurrence form, local recurrence/distant metastases were 6/4 in BR, 8/24 in PR. The average CA19-9 peak value before operation was 924 mg/dl in the early distant-metastases case which sowed metastasis within 8 months after operation and 129 mg/dl in the other distant metastases cases. According to the ROC curve the cut-off value of CA19-9 to distinguish the early recurrence group and delayed recurrence group was 520 mg/dl.
Conclusion: As it is difficult to control the local recurrence for BR cases and the cases which showed high CA19-9 value have the possibility of potential distant metastasis, these cases should be considered as adaptation of NAC-RT or NAC.
Clinical Analysis of Agalactosyl Immunoglobulin-G (IgG) in Chronic Pancreatitis (CP) Patients
T. Ito,1 M. Hamano,1 T. Kawai,1 A. Ishimi,1 Y. Tokuda,1 C. Hibino,1 M. Kato,1 H. Saiki,1 K. Yamamoto,1 M. Naito,1 E. Miyoshi.21Japan Community Healthcare Organization, Osaka Hospital, Osaka, Japan; 2Department of Molecular Biochemistry and Clinical Investigation, Osaka University Graduate School of Medicine, Suita, Osaka, Japan.
Background: We previously reported fucosylated haptglobin was increased in both CP and pancreatic cancer. Agalactosyl IgG was reported to be increased in inflammatory bowel disease (IBD) similarly to rheumatoid arthritis(RA). The lectin complement pathway is shown to be activated through association of agalactosyl IgG with mannan-binding lectin in RA and IBD. However, changes of IgG agalactosylation in CP have not yet been clarified.
Methods: The ratios of the agalactosyl/non-agalactosyl fraction in fucosylated IgG oligosaccharides (G0F/G2F) were analyzed in 35 patients with CP, and 46 healthy volunteers (HV). The MBL levels associated with serum IgG were analyzed by enzyme-linked immunosorbent assay. Twenty-four CP patients consisted of 12 clinically possible or early CP and 12 probably or defined CP according to CP definition of Japan Pancreas Society. Correlations among G0F/G2F and blood chemistry markers were assessed.
Results: G0F/G2F levels were significantly increased in CP Patients compared with HV (1.84 ± 1.17 vs. 1.25 ± 0.50, p < 0.05). G0F/G2F levels in patients with probably or defined CP were relatively higher than those in patients with possible or early CP. G0F/G2F levels were weak significant linear correlations with serum amylase, trypsin, and IgG levels (P = 0.03, r = 0.363; P = 0.012, r = 0.421; P = 0.016, r = 0.404; restrictively). There were no differences of G0F/G2F levels in alcohol consumption and serum lipids levels.
Conclusions: G0F/G2F levels could be useful to diagnose CP in early stage, and might be a new biomarker for detection of CP. Agalactosyl IgG was supposed to be involved in chronic inflammation in CP progression like RA and IBD. Though additional studies on G0F/G2F levels in CP is supposed to be necessary, glycoprotein analyses could lead to clarify CP pathogenesis and early diagnosis.
Surgical Resection for Adenosquamous Carcinoma of the Pancreas
T. Ito, T. Sugiura, Y. Okamura, Y. Yamamoto, R. Ashida, K. Uesaka. Division of Hepato-Biliary-Pancreatic Surgery, Shizuoka Cancer Center, Shizuoka, Japan.
Background: Adenosquamous carcinoma of the pancreas is a rare malignancy of the exocrine pancreas representing less than 4% of pancreatic tumors. Previous series have demonstrated a poor prognosis for this disease, with a median survival in the range of 5 to 6 months. However, it is difficult to draw firm conclusions about the effectiveness of surgical treatment, because of the rarity of the disease.
Methods: Between 2002 and 2014, a total of 430 patients underwent surgical resection for invasive ductal carcinoma of the pancreas at our institute. Of them, twelve (2.8%) patients were identified as having adenosquamous carcinoma of the pancreas. Operative and pathologic evaluations were conducted. Survival was calculated by Kaplan-Meier method.
Results: Operative procedures included pancreaticoduodenectomy in three and distal pancreatectomy in nine. Combined resection due to macroscopic cancer invasion included stomach in four, colon in five, kidney in one, and celiac axis in three, respectively. R0 resection could be achieved in 11 (92%) patients. Six patients received postoperative adjuvant chemotherapy. The median size of tumor was 46 mm (19-96 mm). Lymph node metastasis was detected in seven patients. Microscopic cancer invasion to the other organs were detected in five patients. Three and five year disease-free survival rate and median survival time were 38.9%, 38.9% and 9 months. Three and five year overall survival rate and median survival time were 55.6%, 55.6% and not reached. At the time of analysis, five patients survived without any sign of recurrence. Among them, three patients survived for more than 5 years.
Conclusion: Compared to the previous reports, the present study of our single-center experience with resection for adenosquamous cancer of the pancreas has demonstrated a longer survival for patients that can undergo an R0 resection. Although this is a small series, we recommend surgical resection for these patients.
Determining the Role of RND2 in Pancreatic Cancer Progression
B. Jakubison, Y. Sun, S.F. Konieczny. Department of Biological Sciences & the Purdue Center for Cancer Research, Purdue University, West Lafayette, IN.
Introduction: Pancreatic ductal adenocarcinoma arises from activating K-RAS mutations in acinar cells where the cells dedifferentiate into duct-like cells and progress toward cancer. Upon K-RAS activation, acinar cells silence expression of the transcription factor Mist1. Previous studies have demonstrated that expression of MIST1 is sufficient to prevent precancerous lesion formation while a loss of MIST1 leads to cell transformation. In order to determine how MIST1 prevents tumorigenesis, we carried out RNA-Seq and ChIP-Seq studies to identify MIST1 targets. One gene target repressed by MIST1 is the Rho GTPase RND2, the only Rho GTPase transcriptionally controlled by MIST1. Given that regulation of the actin network is a critical component of cellular transformation, we investigated RND2 biological activity.
Results:Rnd2 transcripts are highly elevated in murine tumor cell lines derived from KrasG12D mice. Additionally, Rnd2 expression is elevated in pancreatitis and in K-RAS driven pancreatic cancer. To determine the functional consequence of RND2 expression, we generated tetracycline-inducible MIST1 and RND2 pancreatic cancer cells. Dox-induction of MIST1 directly led to repression of RND2 expression. Furthermore, MIST1-mediated repression of RND2 was dependent upon specific HDAC activity. When RND2 is induced, preliminary evidence suggests RhoA activity increases, which is in agreement with previous studies showing that increased RhoA activity leads to cellular transformation and increased metastatic properties. We are currently performing studies to examine the importance of RND2 expression in PDAC development.
Conclusions: MIST1 normally represses Rnd2 gene expression in acinar cells. However, when MIST1 is silenced during K-RAS-induced transformation, RND2 levels are elevated. Rho GTPases are known to drive both cell transformation and metastasis, providing a potential mechanistic link by which MIST1 prevents K-RAS mediated tumorigenesis.
Impact of EUS-FNA in the Assessment of Peri-Arterial Soft Tissue in Locally Advanced Pancreatic Cancer
N. Jamaluddin1 R. Watson.21Department of Internal Medicine, UCLA Medical Center, Los Angeles, CA; 2Division of Digestive Diseases, UCLA Medical Center, Los Angeles, CA.
Introduction: Pancreatic cancer most commonly presents as ‘locally-advanced’ (LAPC) signifying tumor association with local vasculature. As a result, ‘downstaging’ chemotherapy is increasingly being used. Imaging studies have limited use in reassessing resectability after chemotherapy. In contrast endoscopic ultrasound (EUS) allows for sampling of these areas via fine-needle aspiration (FNA). Our aim was to assess the diagnostic yield of EUS-FNA of persistent peri-vessel soft tissue identified on cross-sectional imaging after chemotherapy.
Methods: A retrospective analysis was performed of all patients at a tertiary care center from 2012 to 2015 who received EUS-FNA of persistent peri-vessel soft tissue following downstaging chemotherapy in patients with LAPC. The impact of EUS-FNA was determined by: 1) the diagnostic yield of the biopsy specimens and 2) the effect the results had on surgical decision making. We also compared the findings on imaging to the images and pathology results on EUS-FNA.
Results: 5/24 (20.8%) patients had biopsy proven peri-vessel involvement on EUS-FNA after chemotherapy. All five of these patients showed peri-vessel involvement on imaging as well. 9/24 (37.5%) patients had imaging that showed peri-vessel involvement that was not seen on EUS-FNA cytology. EUS accurately identified a lack of peri-vessel involvement in 6/7 (85.7%) of patients who went on to surgical resection. Overall, EUS correctly identified those patients as having no peri-vessel involvement in 8/10 (80%) of cases with 1 case of local vascular involvement missed respectively.
Discussion: This pilot series demonstrates the impact of EUS-FNA in the management of patients with LAPC undergoing downstaging chemotherapy. Specifically, EUS provided novel complementary information to cross-sectional imaging, while directly resulting in 6 additional successful surgical resections. Further prospective study of this novel use of EUS is warranted.
Fellows Symposium: Promoting Future Pancreatologists from Top Trainees
K. Jeffers M. Lewis. Mayo Clinic Jacksonville, Jacksonville, FL.
Background: The National Pancreas Foundation has sponsored an annual Fellows Symposium since 2006, with the primary goal of introducing the field of Pancreatology to young physicians and scientists at an early stage in their career to spark interest in research and clinical practice in the field. The curriculum has evolved over the last 10 years based on participant surveys and input from mentors.
Methods: Growth in participation, broadened diversity of specialties of trainees, improved knowledge of pancreatic diseases, and trainee retention was examined. Participation data and curriculum were reviewed retrospectively over a 10 year period, and pre- and post-test scores were recorded for the most recent year.
Results: Attendance by trainees and mentors grew by 189% over 10 years, with 20 fellows and 18 mentors in 2006 and 50 fellows and 22 mentors in 2015. Initially, the audience was primarily adult gastroenterologists. Starting in 2008 internal medicine residents were included. This was followed by inclusion of pediatric gastroenterology fellows in 2009 and general surgery residents and oncology fellows in 2010. The geographical background of these trainees broadened from primarily the Northeast and Midwest, to include the entire United States. 22% of trainees have attended more than one year. Pre- and post-test scores show a marked improvement after the completion of the course, with average score correct 36% prior to starting the symposium, to average 67% correct after the symposium.
Conclusion: A structured symposium aimed at reaching physicians and scientists early in their training is effective in improving the knowledge base in pancreas diseases, influencing retention of these trainees in careers in Pancreatology in a broad range of specialties, and increasing awareness of future directions of diagnosis and treatments of pancreatic diseases. The added benefit of one on one time with leaders in the field of Pancreatology provides mentoring and networking outside the trainee’s own institution for future collaboration.
Decreased Serum Thrombospondin-1 Levels in Pancreatic Ductal Adenocarcinoma Patients: An Early Indicator of Cancer or Diabetes Development?
C. Jenkinson,1,2,* V.L. Elliott,1,2,* A. Evans,1,2,* L. Oldfield,1,2 J.F. Timms,3 A. Gentry-Maharaj,3 U. Menon,3 T. Cox,1 F. Campbell,1 R. Sutton,1,2 S.P. Pereira,3 D.A. Tuveson,4 W. Greenhalf,1,2 J.P. Neoptolemos,1,2 E. Costello.1,21Department of Molecular and Clinical Cancer Medicine, University of Liverpool, United Kingdom; 2National Institute for Health Research Liverpool Pancreas Biomedical Research Unit, Royal Liverpool University Hospital, United Kingdom; 3Department of Women’s Cancer, Institute for Women’s Health, University College London, United Kingdom; 4Cold Spring Harbor Laboratory, Cold Spring Harbor, NY.
*Denotes equal contribution.
Aim: Identification of serum biomarkers enabling earlier diagnosis of pancreatic ductal adenocarcinoma (PDAC) could improve outcome. Serum protein profiles in patients with pre-clinical disease and at diagnosis were investigated.
Methods: Serum from cases up to 4 years prior to diagnosis of PDAC and controls enrolled on the UKCTOCS (n = 174) were studied, alongside samples from patients diagnosed with PDAC, chronic pancreatitis, benign biliary disease and healthy subjects (n = 199). iTRAQ enabled comparisons of pooled serum from a test set (n = 150). Validation was undertaken using MRM and/or western blotting in all 373 human samples and in a KPC mouse model.
Results: iTRAQ identified thrombospondin-1 (TSP-1) as down-regulated pre-clinically and in diagnosed patients. MRM confirmed significant down-regulation of TSP-1 up to 24 months prior to diagnosis. A combination of TSP-1 and CA19-9 (AUC = 0.86), significantly outperformed both markers alone (0.69 & 0.77 respectively). TSP-1 was also decreased in PDAC patients compared to healthy controls (P < 0.05), patients with benign biliary obstruction (P < 0.01) and in serum of KPC mice with PDAC. Low levels of TSP-1 correlated with poorer survival pre-clinically (P < 0.05) and at diagnosis (P < 0.02). Reduced TSP-1 levels were observed in PDAC patients with confirmed diabetes (P < 0.04). The decrease in TSP-1 compared to controls was only observed in PDAC patients with diabetes (P < 0.009).
Conclusion: Circulating TSP-1 levels decrease during the development of PDAC. The influence of diabetes on biomarker behavior should be considered in future studies.
Artery-First Approach Pancreaticoduodenectomy With En-Bloc Retroperitoneal Clearance (AFPD-ERC) for Pancreatic Head Cancer
K. Jiang, J. Wu, W. Gao, J. Chen, J. Wei, F. Guo, Z. Lu, P. Wu, Y. Miao. Pancreas Center, The First Affiliated Hospital of Nanjing Medical University, Nanjing, China.
Background: Curative resection is the only hope for long-term survival in pancreatic cancer, however, the oncological advantge of extended pancreaticoduodenectomy with intention of R0 resection is still unclear.
Aims: To investigate the feasibility and safety of AFPD-ERC.
Method: Extended retroperitoneal clearance began with an extensive Kocher’s maneuver, with left renal vein and origin of superior mesenteric artery (SMA) well exposed, No. 16th station of lymph nodes (LNs) were cleared at this step. After resectability assessed, transverse colon was lifted and peritoneum at the duodenojejunal junction was opened. SMA was identified and soft tissue around SMA was dissected with 1st jejunal branch and inferior pancreaticoduodenal artery divided. Proximal jejunum was transected according to the blood supply watershed line. After stomach transected and gall bladder mobilized, hepatoduodenal ligament was skeletonized with common hepatic duct divided. Pancreas was transected at 2-3cm left to the origin of splenic artery. No. 7/8/9 station of LNs were cleared with celiac trunk and common hepatic artery skeletonized. Tributaries to superior mesenteric-portal vein were ligated and divided. Specimen together with retroperitoneal soft tissue containing different stations of LNs and nerve plexus were removed en-bloc.
Results: From Dec. 2013 to Jun. 2014, a total of 8 patients underwent this procedure in our center. Median operative time was 390 (330-525) min with median blood loss 400 (300-600) mL. Postoperative morbidity included 2 cases of grade B pancreatic fistula with null mortality. Median postoperative hospital stay was 15 (10-30) d. Median number of harvested LNs was 15 (8-19). All surgical margins were free of tumor cells.
Conclusions: AFPD-ERC is a safe and feasible procedure, which may increase the possibility of R0 resection and improve survival in patients with pancreatic head cancer.
Continuous Glucose Monitoring Following Total Pancreatectomy and Islet Autotransplantation
J.M. Jimenez-Vega,1 D.A. Elder,1 R.S. Chima,2 M. Abu- El-Haija,3 J. Palermo,3 L.N. Hornung,4 J.D. Nathan.51Division of Endocrinology, 2Division of Critical Care Medicine, 3Division of Gastroenterology, Hepatology & Nutrition, 4Division of Biostatistics and Epidemiology, 5Division of Pediatric, General and Thoracic Surgery, Cincinnati Children’s Hospital Medical Center/University of Cincinnati, Cincinnati, OH.
Background: Total Pancreatectomy Islet Autotransplantation (TPIAT) is offered to patients with chronic pancreatitis with incapacitating pain not amenable to medical or endoscopic therapies. Successful islet engraftment can prevent the development of brittle diabetes. In the early post-surgical period, a hyperglycemic environment is detrimental to the transplanted islet.
Objective: To determine if a Continuous Glucose Monitoring (CGM) device can accurately monitor the concentration of blood glucose (BG) in the post-surgical period of TPIAT.
Methods: Two patients underwent TPIAT at our institution. As part of their intense glucose monitoring protocol, a CGM device (Dexcom G4 Platinum) was placed for the monitoring of BG trends. After IRB approval, a retrospective chart review was performed. BG data (serum glucose, Point of care (POC), and CGM data) from the first 7 days post-surgery was obtained.
Results: 25 time-matched measures on all 3 monitoring methods were found. 88% of CGM readings were within 15mg/dL and 76% were within 10mg/dL when compared to serum glucose (9.5% false positive readings). The mean absolute difference (MAD) was 9.04 mg/dL and the mean absolute relative difference (MARD) was 8.44%. 92% of POC readings were within 15mg/dL and 84% were within 10mg/dL of serum values (14.3% false positive readings). The MAD was 5.20 mg/dL and the MARD was 4.96%.
The CGM device and POC did not significantly differ from serum glucose (p=0.39, p=0.26 respectively), nor did the CGM significantly differ from the POC (p=0.07).
Conclusion: The CGM device was found to be a reliable tool in monitoring glucose values in the post-surgical ICU setting. Future studies are needed to validate these results.
Targetting Pancreatic Calcineurin to Prevent Post-ERCP Pancreatitis
S. Jin, A.I. Orabi, T.A. Javed, S. Sah, J.F. Eisses, S.Z. Husain. Department of Pediatric Gastroenterology, Children’s Hospital of Pittsburgh of UPMC, Pittsburgh, PA.
Pancreatitis is the most common and burdensome iatrogenic complication after endoscopic retrograde cholangiopancreatography (ERCP). Despite advances in current preventative approaches, post-ERCP pancreatitis (PEP) is still a problem. In recent work, we unraveled for the first time, that a fundamental mechanism PEP is the activation of aberrant Ca2+ and calcineurin (Cn) pathways through a combination of radiocontrast (RC) exposure to the pancreas and increased intra-pancreatic ductal pressure. However, Cn is expressed in a variety of cells and tissues, and it is unclear which source of Cn is necessary to transduce PEP. We hypothesized that Cn within the pancreatic acinar cell is a central node for PEP. In this study, we first demonstrate on a cellular level that RC-induced NF-κB is highly selective to pancreatic cells compared to an array of seven other tissue cell lines. Next, on an in vivo level, we generated acinar cell conditional knockout (CKO) mice of Cn by crossing an Elastase-Cre-ERT line with floxed alleles of the critical Cn subunit CnB1. PEP was induced by briefly infusing RC at high pressure within the pancreatic duct. Using appropriate littermate and sham-operated controls, the acinar Cn CKOs were largely protected against PEP. Surprisingly, the CKOs had a greater level of protection than global Cn KOs (CnAβ−/−). A single, brief intra-ductal application of the Cn inhibitors FK506 or cyclosporine (1-10 μM) given along with the RC infusion, protected against PEP more potently than multiple systemic doses for 24 hours, and there were no adverse effects with the former. These data demonstrate that acinar cell Cn is necessary for PEP, and they support therapies for PEP that target Cn within the pancreas.
Refine the Basis for Classification Systems for Severity of Acute Pancreatitis: A Retrospectively Analysis of 1094 Patients
T. Jin,1 W. Huang,1,2 L.H. Deng,1 X.X. Zhang,1 N. Shi,1 Z.Q. Lin,1 J. Guo,1 X.N. Yang,1 Q. Xia.11Sichuan Provincial Pancreatitis Centre, Department of Integrated Traditional and Western Medicine, West China Hospital, Sichuan University, Chengdu, China; 2NIHR Liverpool Pancreas Biomedical Research Unit, Royal Liverpool University Hospital, University of Liverpool, Liverpool, United Kingdom.
Background: Several key questions in Revised Atlanta Classification (RAC) and Determinant-based Classification (DBC) for severity stratification of acute pancreatitis (AP) remain unanswered: Should mixed subgroups of transient organ failure (TOF) and local complications (LC) be separated? What is the optimal cut-off time to define persistent organ failure (POF)? We sought to address these issues in this study.
Methods: Data of AP patients were retrospectively analyzed. Severe cases defined by RAC were paired with mild cases (approximate 1:1 ratio). Severity was stratified and internal group comparisons were conducted for both RAC and DBC. Pre-defined subgroups were also compared.
Results: There were 1094 patients with 294 mild, 476 moderate and 324 severe cases as per RAC, respectively. RAC performed better than DBC for severity stratification.
In the moderate group of RAC: patients with TOF only (n=50) had no intensive care need and surgical interventions, and were associated with significantly shortened length of hospitalization (LOS), compared with those in the LC only (n=250) or LC+TOF (n=176) groups (both P<0.001; LC only vs LC+TOF, NS); patients with acute peripancreatic fluid collections and other LC (APFC, n=311) had similar clinical outcomes to those with acute pancreatic necrotic collections (ANC, n=115), both had significantly longer LOS than TOF only group (P<0.001).
For all the patients: the rate of APFC, ANC, overall infections, surgical interventions and LOS were similar between patients with organ failure <24h-48h (n=96) and those <48h-3d (n=33), despite the latter had more intensive care need. Patients with organ failure <24h had better clinical outcomes than either of the aforementioned two groups and was with significantly reduced LOS (both P<0.001).
For patients in the mild and TOF only groups: patients with TOF <24h (n=30) and significantly shorter LOS (median: 8 vs 12d, P<0.001) compared with those had TOF <24h-48h (n=20), but was not significantly differ from those in the mild group (7d).
Conclusion: RAC stratifies severity better than DBC in our cohort. In the RAC, our data suggest to remove TOF only group from the moderate to the mild category and to define POF as organ failure duration >24h.
p120 Catenin is Required for Regeneration of the Exocrine Pancreas in Mice
N.C. Jones, A.M. Hendley, M.A. Pruski, J.M. Bailey. Division of Gastroenterology, Hematology, and Nutrition, Department of Internal Medicine, The University of Texas Health Science Center at Houston, Houston, TX.
Pancreatic cancer is amongst the most deadly and least treatable forms of cancers. Chronic pancreatitis is a known risk factor for pancreatic cancer. In pancreatic cancer patients, p120 catenin, an adherens junction protein and inflammatory suppressor, is loss or mislocalization and this pattern of expression has been correlated with poorer prognosis. Using an acinar cell-specific transgenic mouse model of injury and regeneration, we deleted p120 catenin and quantified injury sustained and the timeline of regeneration after induction of cerulean mediated acute pancreatitis. Significantly increased injury was observed CiMist1; p120f/f pancreata when compared to CiMist1; p120wt/wt pancreata from Day 3 to Day 11 post cerulean treatment, suggesting that p120 catenin null acinar cells display increased susceptibility to injury. Since p120 catenin is a known regulator of inflammation, we next examined the immune response in CiMist1; p120f/f pancreata. Using IHC, we observed that loss of p120 catenin lead to an increase in the number of F4/80+ macrophages present in the injured pancreas and promoted the retention of pSTAT3. This is consistent with previously published works showing that macrophages can serve as a source for IL-6, an activator of pSTAT3, and that loss of p120 catenin in the developing pancreas leads to upregulation of NF-êB, also an activator of pSTAT3. We further characterized the regeneration model by staining for CD3+ and CD16+ cells and observed no change at the site of injury. p120 catenin regulates pancreatic regeneration by modulating cell-autonomous and paracrine inflammatory signaling networks. We have identified a large shift in major immune cell markers, including an increase recruitment of macrophages. Continued experimental models to study pancreatic injury will provide insight on new therapeutic treatment strategies for pancreatitis and pancreatic cancer.
Intrabiliary Metastasis From Pancreatic Cancer: A Presentation Similar to Hilar Cholangicarcinoma
T. Kachaamy,1 J. Weber,1 V. Khemka,1 M. Kundranda.21WRMC-CTCA, Goodyear, AZ; 2Banner-MD Anderson, Gilbert, AZ.
A 61 year old male patient with a history of pancreatic ductal adenocarcinoma (PDAC), treated with irreversible electroporation for locally advanced disease six weeks prior, presented with abdominal pain and jaundice. Computer Tomography (CT) and Magnetic Resonance Imaging (MRI) showed a non-dilated common bile duct (CBD) with mildly dilated intrahepatic ducts (IHD) and no clear masses or stricture. An Endoscopic retrograde cholangiography (ERC) showed a less than 1 cm focal stricture at the bifurcation. Brushings were consistent with metastasis (mets) from PDAC. A 36 year old female patient with history of PDAC, treated with pancreaticoduodenectomy 18 months earlier followed by chemotherapy, presented with recurrent abdominal pain and abnormal hepatic panel. CT and MRI showed a normal CBD with a mildly dilated IHD and no clear masses or strictures. ERC showed a focal stricture at the bifurcation identical in appearance to the first patient. Cholangioscopy with biopsy was consistent with mets from PDAC. Mets from PDAC to the liver are not uncommon. Intraductal mets in the absence of liver mets are rare and can have a clinical picture similar to cholangiocarcinoma. These have been reported in colorectal, gastric, breast and hepatocellular carcinoma. We are reporting 2 cases of pancreatic cancer with isolated intrabiliary metastasis. The clinical presentations in both patients were very similar with signs and symptoms of biliary obstruction in the setting of prior treatment for PADC. Cross sectional imaging showed mild intrahepatic dilation and normal CBD and no masses. Endoscopic ultrasound showed nodular thickening at the area of the bifurcation. On ERC a very focal hilar stricture was seen. Tissue confirmed the diagnosis. Stenting resolved the signs and symptoms of biliary obstruction.
Isolated intrabiliary metastasis from pancreatic cancer should be considered in the differential of biliary tract obstruction in patient with history of pancreatic cancer.
The Role of nab-Paclitaxel Plus Gemcitabine Therapy for Recurrent Pancreatic Cancer Refractory to Gemcitabine and S-1
S. Kagawa, H. Yoshitomi, H. Shimizu, M. Ohtsuka, A. Kato, K. Furukawa, T. Takayashiki, S. Takano, S. Kuboki, D. Suzuki, N. Sakai, M. Miyazaki. Department of General Surgery, Graduate School of Medicine, Chiba University, Chiba, Japan.
Introduction: Gemcitabine (GEM) has been the standard first-line adjuvant therapy after pancreatic cancer resection based on CONKO-001 and ESPAC-1, 3 trial. Then, JASPAC-01 trial demonstrated a significant superiority of S-1, oral 5-fluorouracil prodrug, in the adjuvant setting. As a result, many of recurrent pancreatic cancer patients have already been refractory to GEM and S-1 in Japan. The aim of this study is to elucidate the feasibility and efficacy of nab-Paclitaxel (PTX) plus GEM for these multi drug resistant pancreatic cancer patients.
Methods: Ten recurrent pancreatic cancer patients treated with nab-PTX and GEM who had undergone pancreatic resection and adjuvant chemotherapy were retrospectively analyzed. The nab-PTX (125 mg per square meter of body-surface area) followed by GEM (1000mg per square meter) on day 1, 8, and 15 every 4 weeks was applied for standard protocol.
Results: nab-PTX plus GEM were administrated as second-line for 2, third-line for 5, and fourth-line for 3 patients. Previous chemotherapy regimens were GEM plus S-1 for 9, single-agent GEM for 4, single-agent S-1 for 4, S-1 plus CDDP for 2, GEM plus CDDP for 1, and docetaxel for 1 patient. The median duration of treatment and relative dose intensity were 5.9 months and 56.1%, respectively. The estimated median overall survival time after nab-PTX plus GEM was 12.7 months. The disease control rate was 60%. Grade3-4 leukopenia, neutropenia, and febrile neutropenia occurred in 20%, 20%, and 10% of patients, respectively.
Conclusion: nab-PTX plus GEM in reductive dose after prior treatments seems to be well tolerated and could be effective. nab-PTX plus GEM treatment may be considered as salvage chemotherapy after GEM and S-1 failure in good performance status patients.
Enucleation: A Safe Treatment Alternative for BD-IPMN
J. Kaiser,1 S. Fritz,1 M. Klauss,2 F. Bergmann,3 U. Hinz,1 O. Strobel,1 L. Schneider,1 M.W. Büchler,1 T. Hackert.11Department of General, Visceral and Transplant Surgery, 2Department of Radiology, 3Department of Pathology, University of Heidelberg, Heidelberg, Germany.
Objectives: Small branch-duct intraductal papillary mucinous neoplasms of the pancreas (BD-IPMN) are diagnosed with increasing frequency. With the intention to spare parenchyma and decrease postoperative complications, pancreatic enucleations are increasingly performed. The present study aimed to evaluate enucleation as an alternative surgical approach for BD-IPMN.
Methods: All consecutive BD-IPMN patients scheduled for pancreatic enucleation between January 2004 and September 2014 were analyzed regarding clinicopathological characteristics and surgical outcome. Enucleations were matched (case control) with formal oncologic resections.
Results: 115 patients with BD-IPMN (intention to treat: enucleation) were included. Among these, a total of 87 enucleations were performed in 74 patients. In 41/115 patients (36%), the planned enucleation was converted to a formal oncologic resection, due to location or size (46%), presence of a multicystic lesions (39%), or main-duct involvement (15%). No postoperative mortality was observed. 75/87 BD-IPMNs revealed low-grade dysplasia on histology (86%), 9% had moderate, and 5% high-grade dysplasia. The overall pancreatic fistula rate was 46%, mainly of grade A (41%). Enucleations had a significantly shorter operation time (146 vs. 270 min), blood loss (100 vs. 400 ml), and less postoperative endocrine and exocrine dysfunction compared to formal resections.
Conclusion: Given the high morbidity of pancreatic resections, enucleation is a safe procedure. It can be performed with low morbidity and no mortality in a high proportion of BD-IPMNs. Limitations may occur due to size, localization, multi-locularity, or main-duct involvement requiring conversion to more extended surgical procedures. Up to moderate and high-grade dysplasia, it is an important function-preserving alternative to standard resections.
A Surgical Approach to Extrahepatic Bile Duct Carcinoma With Intestinal Non-Rotation Presenting With Pre-Duodenal Portal Vein
H. Karasaki,1,2 Y. Mizukami,2 T. Kono,1,2 T. Maejima,1 S. Fukahori,1 N. Mukai,1 S. Kasai,1 Y. Matsubara,3 H. Amizuka,3 K. Nagashima.41Department of Surgery, 2Center for Clinical and Biomedical Research, 3Department of Gastroenterology, 4Department of Pathology, Sapporo Higashi Tokushukai Hospital, Sapporo, Japan.
Anatomical variation often annoys surgeons, and safe and curative intervention cannot be achieved without precise radiographic examination. Here we present a rare case with non-rotation type intestinal malformation suffering from biliary cancer. A 79 year-old female was admitted to our hospital with complaint of jaundice. Computed tomography demonstrated well-vascularized tumor, 20-mm in diameter, around the confluence between the cystic duct and distal bile duct. Anatomical variations associated with non-rotation type of midgut malrotation such as pre-duodenal portal vein, poly-splenia, left-sided inferior vena cava and persistent left superior vena cava were also identified. Pancreatic body was seen between portal vein and superior mesenteric artery, whereas tail of the pancreas was not visualized, suggestive of a short pancreas. Abnormal pancreatic biliary duct was not evident on MR cholangiography.
Pylorus-preserving pancreaticoduodenectomy was then performed by following steps. First, the pre-duodenal portal vein was detached from the hepatic hilum toward splenic vein. Second, the pancreas was split at right side of gastroduodenal artery branched from dorsal pancreatic artery via superior mesenteric artery. The second portion of the duodenum was also divided at the left of PV, and then the jejunum was cleaved at the right of the PV. Finally, the bile duct tumor was removed by retrieving the duodenum and pancreatic head from the behind of the PV.
Atypical procedures are unavoidable during the surgical intervention in patient with midgut malrotation due to complexed anatomical variations. For optimal preoperative planning, precise imaging navigation using 3D-reconstruction software is highly recommended. The “unusual” preoperative imaging and intraoperative photos will be presented.
Pancreatectomy With Artery-First Approach for Borderline Resectable Pancreatic Cancer: Surgical Technique to Secure Negative Tangential Margins and its Outcome
Y. Kawabata, Y. Tajima. Department of Digestive and General Surgery, Shimane University Faculty of Medicine, Shimane, Japan.
Background: The prognostic significance of complete tumor clearance at the resection margins, i.e., no residual tumor (R0) resection, has been demonstrated in pancreatic cancer surgery. Here we report an artery-first approach to secure complete negative tangential margins in borderline resectable cancers (BRPC) of the pancreas.
Methods: A total of 57 consecutive patients with BRPC underwent pancreatectomy between May 2006 and May 2015 at our institute. Seventeen patients received a conventional pancreatectomy (cPx), while 40 cases underwent an artery-first pancreatectomy (aPx). We defined patients as having BRPC based on the National Comprehensive Cancer Network guidelines on 2015. Margins were designated “R0” if no tumor cells were identified at any of the resection margins.
Results: The cPx included 15 pancreaticoduodenectomies (PD) and 2 distal pancreatectomies (DP), while the aPx consisted of 3 total pancreatectomies (TP), 26 PD and 11 DP. The aPx procedure was performed safely without any intraoperative complications. Intraoperative blood loss in the aPx was 640 ml (median, range: 40-2880) and it was significantly less than that (1370 ml; median, rang: 60-4359) in the cPx (p < 0.004). The total number of lymph nodes dissected was 21 (median, range: 5-58) in the cPx and 31 (median, range: 12-80) in the aPx (p < 0.022). R0 resection was accomplished in 65% and 94% of patients with the cPx and aPx, respectively, resulting in a significant decrease in the R1 rate in the aPx (6%) compared to that in the sPD (35%) (p < 0.019). Two years survival rates were 26.9% in cPx and 42.2% in aPx with median survival time of 10.5 months and 18.9 months, respectively.
Conclusion: Artery-first approach for BRPC achieved a high rate of negative tangential margins with favorable surgical outcome.
Long Term Surveillance and Risk of Progression of Low-Intermediate Risk Intraductal Papillary Mucinous Neoplasms
M. Kayal, L. Luk, F. Gress, A. Sethi, J. Poneros, E. Hecht, T. Gonda. Division of Digestive and Liver Diseases, Columbia University Medical Center, New York, NY.
Introduction: Intraductal papillary mucinous neoplasms (IPMNs) of the pancreas are potentially malignant epithelial neoplasms that involve the main duct, branch ducts, or both. Our goal was to understand the natural history and risk of progression of low-intermediate risk IPMNs that have been surveyed for > 4 years.
Methods: We conducted a retrospective review of radiology and endoscopy databases between 2001-2013. We identified 2400 patients with cystic pancreatic lesions, of whom 268 were followed > 4 years and 134 met clinical IPMN criteria. Data points such as demographics and cyst characteristics were collected. Progression was defined as an increase in cyst diameter > 2 mm or > 20% in the single largest dimension.
Results: 68 patients (50%) had interval cyst progression with an average increase of 64% in the single largest dimension. Of these patients, 15 developed worrisome features and one proceeded to surgery (pathology: high grade IPMN). Samples were analyzed via Kaplan Meier methodology; baseline cyst size and demographics were not predictive of risk of progression within the first five years of surveillance. In addition, there was no significant difference in rate of progression among cysts of different baseline size.
Conclusion: In a large retrospective cohort of patients with suspected low to intermediate risk IPMNs, nearly half progressed during long term follow-up as defined by >20% or >2 mm growth in the single largest dimension, however only a minority developed worrisome features. Baseline cyst characteristics and demographics were not predictive of progression or growth rate; cysts <10 mm and >30 mm grew at comparable rates in the five years after diagnosis.
Reprogramming Pancreatic Cancer to Induce Quiescence and Acinar Cell Fate
S.W. Kim,1,2,* J. van Niekerk,1,2,* T. Almaleh,1,2 R. Lahmy,1,2 P. Itkin-Ansari.1,21Department of Pediatrics, University of California, San Diego, La Jolla, CA; 2Development and Aging Program, Sanford-Burnham Medical Research Institute, La Jolla, CA. *These authors contributed equally to the work.
Pancreatic ductal adenocarcinoma (PDA) PDA is manifested by loss of exocrine acinar cell identity as well as uncontrolled proliferation. During progression to PDA the expression of basic helix-loop-helix (bHLH) transcription factors which regulate the acinar transcriptional program is lost, while expression of the bHLH inhibitor Id3 dramatically increases. We recently showed that restoring balance to the bHLH/Id3 axis by over expression of the bHLH protein E47 simultaneously induces acinar cell differentiation and cell cycle exit. Moreover, E47 causes feed forward induction of the acinar bHLH protein MIST1 and reduces tumorigenicity. Having found a genetic basis for reprogramming pancreatic cancer cell fate, we sought to translate our findings for clinical utility through drug discovery. Therefore, we developed a novel high-throughput screening assay to identify drugs which induce bHLH activity. Human PDA cells were transduced with a vector consisting of multimerized bHLH DNA recognition sites driving luciferase. As a positive control for the assay, a subset of PDA cells was engineered to overexpress E47. Ectopic E47 induced >70,000 fold increase in bHLH reporter activity within 48 hours. The dynamic range and reproducibility of the assay were demonstrated by achieving a z-factor = 0.585. Moreover, in a pilot screen, we identified a series of hits between 6 and 29 standard deviations from the mean. To conclude, we have discovered that restoring bHLH signaling in human PDA cells reprograms them toward a quiescent acinar state. From an initial screen, we have identified inducers of bHLH activity. A candidate drug shown to reprogram PDA in vitro and in animal studies would hold great promise as a novel therapeutic for PDA.
NecroX-7 Ameliorate the Severity of Cerulein-Induced Acute Pancreatitis (CIP) Through Inhibition of Necroptosis/Necrosis via Reciprocal Regulation of Mitochondrial ROS and Proinflammatory Cytokines
Y.T. Kim,1 M.Y. Kim,1 D.W. Jun,1 O.Y. Lee,1 B.C. Yoon,1 K.S. Yoo,1 H.S. Choi,1 K.G. Lee,2 D.H. Lee,3 S.H. Kim.4Departments of 1Internal Medicine, 2General Surgery, Hanyang University College of Medicine, Seoul, Korea; 3Department of Internal Medicine, Inha University College of Medicine, Incheon, Korea; 4LG Life Sciences Ltd., R&D Park, Daejeon, Korea.
Background/Aims: Recently, NecroX series has been reported to be necroptosis inhibitor that shows scavenger activity against mitochondrial reactive oxygen species (ROS). The aim of our study was to evaluate the protective effect of the strong antioxidant and anti necroptosis agent, NecroX-7 (tetrahydropyran-4-yl)-[2-phenyl-5-(1,1-dioxo-thiomorpholin-4-yl)methyl-1H-indole-7-yl]amine), on CIP in mice.
Methods: C57BL/6 mice were randomly divided into 3 groups, control group (n = 15), cerulein treated (CIP) group (n = 15), and NecroX-7 with cerulein treated group (n = 15). NecroX-7 was administered intraperitoneal at a dose of 20 μg/kg per mice 30 min before the first cerulein injection. Each groups were compared with biochemistry, real time PCR, western blotting, and histological parameters.
Results: The increase in amylase and lipase (8914.25 ± 741.83, 828.5 ± 252.75) were reduced after NecroX-7 administration (5632 ± 1231.33, 569.75 ± 215.04). The expression of TNF-α, IL-1β, IL-6, and MCP-1 were found to be substantially decreased in the NecroX-7 treated group, and NFκB was also suppressed by NecroX-7. As well as, the expression of necroptosis related genes were significantly enhanced and substantially reduced in NecroX-7 treated group. But, the expression of RIP3 was not changed. NecroX-7 also reduced level of HMGB1. There were also subtle decrease in procaspase 3 and caspase 8 and increase in Bcl-Xl and Bcl-2. Pancreas damage and lung injury substantially decreased in the NecroX-7 treated group.
Conclusions: NecroX-7 ameliorates the severity of CIP, suggesting that NecroX-7 may be attenuated CIP by inhibition of inflammation and necroptosis.
Evaluation of the Prognostic Significance of ‘High-risk Stigmata’ in the International Consensus Guidelines 2012 for Intraductal Papillary Mucinous Neoplasm
K. Kimura, R. Amano, S. Yamazoe, G. Ohira, K. Miura, K. Nishio, K. Sakurai, T. Toyokawa, M. Ohira, K. Hirakawa. Department of Surgical Oncology, Osaka City University Graduate School of Medicine, Osaka, Japan.
Background: In the International Consensus Guidelines 2012 for intraductal papillary mucinous neoplasm (IPMN), ‘high-risk stigmata’ (HRS) were described as indications for resection. The purpose of this study was to evaluate the prognostic meaning of HRS in the 2012 guidelines.
Method: Clinical and pathological data from 98 patients who underwent pancreatic resection for IPMN at our institution between 1994 and 2014 were retrospectively analyzed.
Results: The 98 resected IPMNs were categorized as demonstrating no criteria (NC) (n = 18), worrisome features (WF) (n = 39), or HRS (n = 46) according to the guidelines. By multivariate analysis, positive lymph node metastasis and HRS were significantly identified as independent prognostic factors. Five-year disease-specific survival rates for NC and WF were both 100%, whereas that for HRS was significantly poorer at 49.5% (p < 0.001 each).
Conclusions: In this retrospective analysis, HRS were an independent prognostic factor after surgical resection for IPMN. Moreover, HRS offered high diagnostic ability for detecting invasive IPMNs. These results strongly indicated that HRS had high possibility of invasive IPMN and worse prognosis.
Adequate and Timely Reduction of Pancreatic Ductal Hypertension as the Main Criterion for Successful Surgical Treatment of Chronic Pancreatitis
V. Klymenko, A. Klymenko, A. Steshenko. Zaporizhzhya State Medical University, Zaporizhzhya, Ukraine.
Introduction: Progressive course of chronic pancreatitis (CP) is characterized by severe fibrotization pancreatic parenchyma, causes pancreatic ductal hypertension, which is a powerful "catalyst" irreversible changes in the pancreas.
Aim: To increase the effectiveness of surgical treatment of CP on the basis of timely and adequate reduction of pancreatic ductal hypertension (before progression of irreversible changes in the pancreas).
Methods: Analyzed the long-term results (up to 10 years) of surgical treatment of 82 patients CP with pancreatic ductal hypertension who underwent conserving (not resection) operation (longitudinal total pancreatowirsungoduodenopapillotomy with longitudinal pancreaticojejunoduodenostomy). Male - 73 (73/82, 89.02%), women - 9 (9/82; 10.97%), the average age - 48 years. Wirsung’s duct diameter was 7-15 mm. Alcoholic origin of CP was in 57 (57/82, 69.51%), pain was observed in all patients. In 48 (48/82, 58.53%) patients (I group) was determined by mild exocrine insufficiency, in 34 (34/82; 41.46%) (II group) - was severe exocrine insufficiency (pancreatic fecal elastase - 1 < 100 mg / g). Quality of life was assessed by EORTC QLQ C-30, PAN28.
Results: in 73 (73/82, 89.02%) patients the size of enlarged head of pancreas turn to normal. All patients reduced abdominal pain. All patients in I group, as well as before surgery, remained mild exocrine insufficiency. In 31 (31/34, 91.17%) patient of II group still remained severe degree exocrine insufficiency, while 3 patients (3/34, 8.82%) - pancreatic elastase-1 in feces elevated and was varied from 129 ± 10 mg / g. All patients in Group II were forced to take enzyme preparations. Quality of life of patients in I group was equal to the category of healthy people.
Conclusion: Timely and adequate elimination of pancreatic ductal hypertension allows the long-term to maintain the original functional reserve of the pancreas (exogenous, endocrine function) as it was before the operation. Presented organ-preserving (not resection) operation solves the problem of surgical treatment of CP.
Pancreatic Stellate Cells Lead and Promote the Local Invasion of Cancer Cells, by Physically Remodeling the Extracellular Matrix With a Collagen Fiber Alignment in Pancreatic Cancer
K. Koikawa,1 K. Ohuchida,1 M. Sada,1 T. Abe,1 S. Endo,1 K. Horioka,1 T. Moriyama,1 Y. Miyasaka,1 T. Ohtuka,1 R. Ohuchida,2 T. Ueki,1 E. Nagai,1 K. Mizumoto,1 M. Nakamura.11Department of Surgery and Oncology, Graduate School of Medical Sciences Kyushu University, Fukuoka, Japan; 2Section of Fixed Prosthodontics, Division of Oral Rehabilitation, Faculty of Dental Science, Kyushu University, Fukuoka, Japan.
Background and Aim: A specific cell population that lead the local invasion in cancer, are called as “leading cells”. Regulating the leading cells may lead to the control of cancer cell invasion. However, the underlying mechanisms of this phenomenon have remained unclear. In this study, we identified the leading cells in pancreatic cancer and analyzed how the leading cells lead and promote cancer cell invasion in the extracellular matrix (ECM).
Methods: We established a 3D collagen invasion assay model, in which pancreatic cancer cells and pancreatic stellate cells (PSCs) were co-cultured. We investigated the movement of PSCs and cancer cells in collagen matrix, using 3D co-cultured model.
Results: We found that many PSCs invaded the matrix to extent, with cancer cells invaded always behind the invaded PSCs and that the number of invaded cancer cells was increased in co-cultures with PSCs compared with that of mono-cultured cancer cells (P < 0.05). Reflectant confocal microscopy and immunohistochemistry analysis showed that invaded PSCs changed the alignment of collagen fibers and promoted remodeling of ECM, compared with non-invaded PSCs and cancer cells. The expression level of Endo180 (CD280, MRC2), a collagen uptake receptor, was higher in PSCs than in pancreatic cancer cells. Endo180 knockdown by RNA interference attenuated the invasive abilities of leading PSCs (P < 0.05).
Conclusions: The present data suggest that PSCs lead the local invasion of pancreatic cancer cells, by physically remodeling ECM, possibly via the function of Endo180 in leading cells.
Management of Pancreatic Anastomotic Leakage by Measuring Drainage Amylase and Volume After Pylorus Preserving Pancreatoduodenectomy
M. Koizumi, N. Sata, M. Taguchi, A. Miki, H. Endo, H. Sasanuma, Y. Sakuma, H. Horie, Y. Hosoya, A.K. Lefor. Department of Surgery, Jichi Medical University, Shimotsuke, Japan.
Background: Postoperative pancreatic fistula (POPF) following pylorus preserving pancreatoduodenectomy (PpPD) is associated with significant morbidity and mortality. Early drain removal after PpPD is recommended to prevent POPF. In 2005, the International Study Group of Pancreatic Fistula (ISGPF) defined the diagnostic criteria of POPF by drain amylase concentration (D-AMY). The optimal timing of drain removal is still controversial. We evaluated the relationship between POPF and D-AMY after PpPD.
Aim: To evaluate D-AMY on POD1, 3 and 5 for predicting POPF and determine the optimal timing of drain removal.
Methods: 67 patients underwent PpPD from December 2012 to April 2015. We performed pancreatico-jejunostomy using Blumgart’s anastomosis (MB-A) or duct and parenchyma-to-whole layer of jejunum anastomosis (WL-A). POPF is defined by ISGPF criteria; D-AMY was measured on POD 1, 3 and 5, and classified into two groups, Group A: ISGPF grade A or less, Group B: grades B and C, respectively.
Results: 57 MB-A and 10 WL-A were performed. In patients undergoing MB-A, 53 (79%) and 4 (6.0%) were classified into Groups A and B, respectively. For WL-A, all patients were classified into Group A. On POD 5, D-AMY was 3618 mU/ml vs 13804 mU/ml (Group A vs B) in MB-A (P = 0.194) and D-AMY volume (D-AMY x volume mL/day) was 1.84X105 mU vs 24.6X105 mU (Group A vs B) in MB-A (P < 0.001). In Group A, D-AMY decreased sharply from POD 1 to POD 5. On the contrary, D-AMY was high even on POD 1 in Group B. The drain was removed on POD 5.9 in Group A and POD 29.5 in Group B. After drain removal, no adverse events occurred. In a grade C patient, re-anastomosis was required because of a persistent POPF. Length of stay was 26.4 days in Group A and 49.8 days in Group B.
Conclusion: D-AMY volume on POD 5 is valuable for predicting prognosis, and drain removal on POD 5 is reasonable in Group A.
Jak2/STAT3 Inhibition Limits the Activation of Pancreatic Stellate Cells InVitro
H.M. Komar,1 G. Serpa,1 T. Mace,1 O. Elnaggar,1 M. Bloomston,2 D.L. Conwell,3 G.B. Lesinski.11Division of Medical Oncology, 2Division of Surgical Oncology, Department of Surgery, 3Division of Gastroenterology, Hepatology, & Nutrition, The Ohio State University Wexner Medical Center, Columbus, OH.
Chronic pancreatitis is a devastating disease characterized by persistent inflammation and fibrosis of the pancreas. Symptoms, including severe abdominal pain and nausea, often progress to complete organ calcification and loss of function. Unfortunately, as the cellular and molecular mediators of this disease are poorly understood, no therapeutic options exist. Recent findings implicate a class of resident fibroblasts, pancreatic stellate cells (PSC), as key pathogenic mediators. The objective of this study was to elucidate the role of PSC-derived Jak/STAT signaling - a classic pro-survival and inflammatory pathway - in disease progression. In vitro, a variety of PSC samples were analyzed, including primary and immortalized mouse and human PSC from settings of both chronic pancreatitis and pancreatic cancer. Immunomodulatory cytokines including VEGF, M-CSF, and IL-6 were prominent in PSC supernatants at levels of up to 56, 811 and 820 pg/mL, respectively. Analysis of cell lysates by immunoblot demonstrated the activation of several pro-survival and inflammatory pathways, including Jak/STAT and MAPK. In culture, treatment of PSC with Ruxolitinib, a Jak1/2 inhibitor, resulted in reduced growth (40% growth after 72 hours of 50 μM) by MTT assay, reduced pSTAT3 indicative of target inhibition, and reduced expression of alpha-SMA, a marker of an activated phenotype. Treatment with a MAPK pathway inhibitor (MEK162) had no effect on growth or activation and resulted in a concentration-dependent increase in STAT3 phosphorylation. These data suggest that the Jak/STAT pathway but not the MAPK pathway functions to limit the cell autonomous inflammation driven by pancreatic stellate cells, thereby representing a viable therapeutic target.
Mean Brightness of Ultrasound Image Strongly Correlates With Degree of Pancreatic Steatosis
V. Kommineni,1 K. Patel,1 M. Belohlavek,2 D. Lam-Himlin,3 N. Gades,4 D. Faigel,1 M. Alloush,5 M. Sturek,5 V. Singh,1 R. Pannala.1Divisions of 1Gastroenterology, 2Cardiology, 3Pathology, 4Comparative Medicine, Mayo Clinic Arizona, Scottsdale, AZ; 5Cellular and Integrative Physiology, Indiana University, Indianapolis, IN.
Background/Aims: Intrapancreatic fat (IPF) has a role in pancreatitis, metabolic syndrome, and postoperative pancreatic fistulae. Ultrasound (US) assessment of IPF is based on subjective criteria such as diffuse hyperechogenicity. We performed image and texture analysis on US images of pancreata from Ossabaw miniature swine (animal model of metabolic syndrome) and two age groups of crossbred Yorkshire swine controls and correlated US measurements with IPF.
Methods: Mayo Clinic IACUC approved the protocol. Ex vivo surface US (10 MHz, range 5, depth 1.5 cm) was performed on freshly explanted pancreata from Ossabaw pigs (n = 11) and controls (n = 16, 2.5 and 5 month old pigs, n = 8 each and mean weights of 25 ± 1.5 and 81 ± 1.6 kg, respectively). Using Image J software, image and grey level cooccurence matrices (GLCM) texture analyses were performed on regions of interest (ROI) encompassing the pancreatic parenchyma. Fat % was calculated in a blinded manner on H&E stained slides by measuring the pixel area of fat as a % of total area. Wilcoxon Sign Rank test and Pearson coefficient were performed to compare means and assess correlation.
Results: Histologic fat % was markedly higher in Ossabaw pigs vs. controls (42.4 ± 9.8 vs. 7.9 ± 3.5, p < .01). Mean pancreatic ROI brightness was significantly higher in Ossabaw pigs, compared to controls (125.2 ± 6.9 vs. 93.9 ± 7.1, p < .01) and was strongly correlated with % fat (r = 0.83, p < .01). Mean fat % did not differ between 2.5 month (7.3 ± 3.6) and 5-month pig groups (8.6 ± 3.5, p = NS). Other aspects of GLCM texture analyses did not significantly differ between the groups.
Conclusions: Mean pancreatic brightness on high frequency US differentiates varying degrees of IPF and strongly correlates with histologic fat % in ex vivo porcine pancreata. Further validation in humans and comparison with subjective assessment and other imaging modalities is needed.
Low Dose Gemcitabine Plus TH1 Dendritic Cell Vaccination Generates Durable Cure in a KrasG12D/p53−/− Orthotopic Model of Pancreatic Ductal Adenocarcinoma
V. Konduri,1 D. Li,2 M.M. Halpert,1 D. Liang,1 J.M. Levitt,1,3,4 Q.C. Yao,1,2,4 W.K. Decker.1,4,51Department of Pathology & Immunology, 2Michael E. Debakey Department of Surgery, 3Scott Department of Urology, 4Dan L. Duncan Cancer Center, 5Center for Cell and Gene Therapy, Baylor College of Medicine, Houston, TX.
Pancreatic ductal adenocarcinoma (PDAC) is the fourth leading cause of cancer related death in the United States and exhibits a five-year OS of only 4-6% despite aggressive surgical and chemotherapeutic interventions. The recent application of immune-based therapies for the successful treatment of several different tumor types suggests potential application of immune-based treatment approaches to PDAC. To explore this concept further, we treated orthotopically-established K-rasG12D/p53−/− PDAC tumors with 40 mg/kg gemcitabine and a cell-based therapeutic vaccine previously shown to generate robust and durable cell-mediated (TH1) immunity. The efficacy of this combination therapy was compared to gemcitabine monotherapy and untreated controls. Tumor burden was assessed weekly by luciferase-based imaging (IVIS). The results indicated that the combination of chemotherapy and a TH1 dendritic cell-based vaccine was effective in eliminating tumor, preventing recurrence, and enhancing overall survival. None of the animals that received the combination therapy relapsed, while 4 of 5 mice that received only gemcitabine relapsed and progressed. Flow cytometry analysis of circulating PBMC demonstrated that mice receiving the combination therapy exhibited significantly elevated levels of IFN-γ+ and central memory CD8+ T-cells with significantly reduced levels of exhausted GITR+CD8+ T-cells several months after the cessation of treatment. In a different model system utilizing rapidly growing and highly metastatic PanC02 cells, TH1 vaccination administered as monotherapy significantly reduced tumor growth and eliminated subsequent metastasis to the liver, highlighting the potential efficacy of the TH1 vaccination regimen. The data suggest that TH1 vaccine immunotherapy is feasible, safe, and can be highly effective at controlling disease in both the adjuvant and monotherapeutic settings.
MUC5AC Mediated Oncogenic Signaling in Pancreatic Cancer
S.R. Krishn,1 S. Kaur,1 S. Ayala,1 S.L. Johansson,3 S.K. Batra.1,21Department of Biochemistry & Molecular Biology, 2Eppley Institute for Research in Cancer and Allied Diseases, 3Department of Pathology and Microbiology, University of Nebraska Medical Center, Omaha, NE.
Introduction: The outcome of patients with pancreatic cancer (PC) have not changed significantly in last four decades, reflecting our limited understanding of the molecular basis of its pathogenesis. Secretory mucin MUC5AC is one of the top differentially expressed genes in both genetic and clinical studies of PC, and is associated with shorter survival of PC patients. We hypothesize that overexpression of MUC5AC plays a critical role in growth and metastasis of PC, and investigated the phenotypic and molecular consequences of MUC5AC silencing during pathogenesis of PC.
Methods: MUC5AC expression was examined in the normal pancreas, primary, and metastatic PC cases by immunohistochemistry. MUC5AC expression was stably silenced in PC cell lines (FG/COLO357 & SW1990) and its oncogenic potential was deciphered utilizing in vitro functional assays and biochemical techniques.
Results: No expression of MUC5AC was observed in the normal pancreas tissues while its aberrant expression was observed in early lesions (PanINs), primary and metastatic cases of PC. MUC5AC inhibition results in significant reduction of PC cells growth associated with a significantly increased accumulation of PC cells in G1-phase, reduced progression to S-phase of cell cycle and increased apoptotic response. Furthermore, MUC5AC expression was also associated with significantly increased invasiveness and adhesion to extracellular matrix. Mechanistically, MUC5AC interacted with cell membrane-associated proteins (E-Cadherin, β-Catenin), impacts oncogenic signaling and its inhibition alter growth and metastasis associated genes (Cyclin-D1, Cyclin-E, p27Kip1, BAX, survivin, MMPs).
Conclusions: Our findings, for the first time provide molecular mechanistic evidence supporting the oncogenic potential of secretory mucin MUC5AC during PC cell survival and metastasis.
Pancreatitis After Pancreas Resection Predicts Clinically Relevant Postoperative Pancreatic Fistula
C.M. Kühlbrey, O. Sick, N. Samiei, F. Makowiec, U.T. Hopt, U.A. Wittel. Department of Surgery, Clinic for General und Visceral Surgery, Freiburg, Germany.
Introduction: Postoperative pancreatic fistula (POPF) is one of the major complications after pancreatic resection. The reasons for its development are still not known in detail but risk factors seems to be soft pancreatic tissue and a small main duct while in patients with exocrine insufficiency and solid tissue POPF is rare. Furthermore in patients with low postoperative systemic amylase concentrations, the occurrence of POPF is infrequent. The aim of this study was to examine the predictive value of the systemic amylase concentration on the occurrence of POPFs after pancreas resection.
Material and Methods: Perioperative data from 864 consecutive pancreas resections were assessed in prospectively maintained SPSS database. Pancreaticoduodenectomies as well as open and laparoscopic distal pancreatectomies were evaluated. Patients’ characteristics, operation details and perioperative clinical chemistry were collected. Serum and drain amylase concentrations were determined by routine clinical chemistry. POPFs were graded into A-C according to ISGPF definition. In addition to descriptive statistics, chi-square and nonparametric Kruskal-Wallis-test were performed.
Results: Between all groups gender, blood loss, surgical technique was equally distributed. In patients with reduced serum amylase (n = 87) on day 1 after pancreaticoduodenectomy clinically relevant POPFs were not observed in any case. In patients with normal serum amylase concentrations, POPFs grade B and C occurred only in 5,4% while in 38,2% of the patients with more than 3x elevated amylase concentrations a clinically relevant postoperative fistula developed in the following postoperative course (p < 0.001). Similar results were obtained for day 2 serum amylase values. The predictive value of increased serum amylase on day 1 after pancreaticoduodenectomy in ROC analysis showed an area under the curve of 0.797 (95%CI 0.757-0.838, p < 0.001) indicating that the systemic hyperamylasia detected on day one after pancreas resections is a predictor of clinically relevant POPFs.
Conclusion: Patients with a high risk for developing clinically relevant POPFs can be identified on the first postoperative day by increased serum amylase concentrations.
The Effect of Opioid Administration on the Severity of Experimental Acute Pancreatitis
B. Kui,1 Z. Balla,1 E.S. Kormányos,1 A. Molnár,1 B. Iványi,2 T. Takács,1 P. Hegyi,1 Z. Rakonczay, Jr.11First Department of Medicine, 2Department of Pathology, University of Szeged, Hungary.
Introduction: Acute pancreatitis (AP) most commonly presents with severe abdominal pain requiring the use of major analgesics (opioids). These drugs efficiently diminish the pain associated with AP, but there is scarce evidence on the effects of analgesia on the severity of the disease.
Aim: To investigate the effects of major analgesics (morphine, buprenorphine) on the severity of experimental AP.
Methods: AP was induced in SPRD rats by intraperitoneal (i.p.) injection of 3 g/kg L-ornithine (n = 8), control animals (n = 6) received physiological saline (PS) instead of the basic amino acid. 0.1-1 mg/kg i.p. buprenorphine (n = 6-8) or 5-20 mg/kg i.p. morphine (or PS) (n = 6-8) was administered 1 hour before AP induction. Animals were sacrificed 24 hours thereafter. Laboratory (serum amylase activity, pancreatic dry/wet weight ratio) and histological (necrosis, edema, inflammatory cell infiltration) parameters were measured.
Results: Administration of morphine dose-dependently reduced the severity of AP. All histological parameters like edema, inflammatory cell infiltration and necrosis were significantly reduced in the AP group treated with 20 mg/kg morphine. The serum amylase activity and pancreatic dry/wet weight ratio were significantly reduced in morphine-treated (20 mg/kg) AP group, compared with the PS-treated AP group. In contrast, all measured laboratory- and histological parameters were significantly elevated in AP rats treated with 1 mg/kg buprenorphine compared to the AP group treated with PS.
Conclusions: Different types of opioids can have varying effects on the severity of AP. Administration of morphine seems to have beneficial effects, whereas buprenorphine worsens the severity of the disease.
Deficiency of Lipocalin-2 Protect Against Cerulein-Induced Severe Acute Pancreatitis
S. Kumar,1 S. Rachagani,1 S. Joshi,1 S. Gupta,1 M. Varney,2 A.C. Cannon,1 K. Mallya,1 S. Kaur,1 M. Jain,1 S. Akira,3 R.K. Singh,2 S.K. Batra.11Departments of Biochemistry and Molecular Biology, 2Pathology and Microbiology, University of Nebraska Medical Center, Omaha, NE; 3Immunology Frontier Research Center (IFReC), Osaka University, Suita, Japan.
Background: Acute Pancreatitis (AP) remains uncomplicated and self-limiting necro-inflammatory disease; however, in 15-20% of patients, AP progresses to significant systemic organ dysfunction resulting in severe AP (SAP). We demonstrated that lipocalin-2 (LCN2) is significantly elevated in AP patients and is a predictor of severity. Further, serum LCN2 levels were significantly high in an experimental mouse model of AP. We thus, hypothesize that upregulation of LCN2 functionally contributes to the severity of AP.
Method: AP was induced in LCN2 knockout (KO) and wild type (WT) animals by eight hourly intra-peritoneal injections of cerulein (75 ug/kg body weight) on alternating days. Control animals were injected with normal saline. Pancreas, blood, lung, kidney and liver were isolated at 6, 12, 24 and 72 h time points.
Results: Cerulein administration induced AP in both LCN2 KO and WT animals, as demonstrated by increased serum amylase levels and necrotic area score (p < 0.05). However, LCN2 KO animals exhibited significantly reduced damage to pancreatic parenchyma compared to WT, as evident by significantly reduced cleaved caspase- 3 staining and lower number of TUNEL positive acinar cells in LCN2 KO animals, indicating decreased apoptosis in absence of LCN2. Further, there was a marked reduction in the infiltration of immune cells and decreased (p < 0.05) serum levels of inflammatory chemokines (Cxcl1, Cxcl5, Cxcl7) in LCN2 KO animals, suggesting its critical pathobiological role in AP. Moreover, pancreas of LCN2 KO animals recover much faster compared to WT animals.
Conclusion: Altogether, our results indicate that higher levels of LCN2 is associated with increased acinar cell apoptosis during pancreatitis and its targeting can potentially improve clinical outcomes in SAP.
Impact of Comorbidity on the Natural History of Pancreatic Cystic Neoplasms: A Competing Risk Analysis
K. Kwok, B.U. Wu. Center for Pancreatic Care, Kaiser Permanente Los Angeles Medical Center, Los Angeles, CA.
Introduction: Optimal management of pancreatic cystic neoplasms (PCN) remains controversial. A key gap is the lack of long-term, population-based natural history data to support management strategy. We theorized that comorbidities, reflected in the Charlson Comorbidity Index (CCI), impact PCN patient survival.
Methods: Eligible patients (retrospective longitudinal cohort) were identified from a pancreatic cyst registry (verified PCN patients diagnosed on cross-sectional imaging from 2006-2010, and followed until 2015). We excluded patients with acute/chronic pancreatitis, & with pancreatic cancer diagnosed in the first 6 months of index imaging. We performed 3 analyses: 1) comorbidity’s impact in overall mortality (OM), 2) pancreatic cancer mortality (PCM) risk factors, 3) subgroup analysis of non pancreatic cancer mortality (NPCM) risk factors, among patients with high-risk cyst features.
Results: 1774 patients were included. Median age was 69.5 years [IQR 58.8, 78.3], with 1158 (63.8%) females. Median CCI was 1 [0,3]. Median follow-up was 5.8 years [IQR 4.6, 7.2], with 27 (1.52%) PCM, and 357 (20.1%) NPCM.
1) In univariate analysis, high CCI (≥3) was associated with increased risk of OM (low vs high CCI 5% vs 40%, p < 0.0001).
2) PCM was more likely in those with high-risk cyst features at baseline than those without (3.6% vs 0.2%, p < 0.0001). In Cox proportional hazards regression, cyst ≥ 3 cm, main PD dilation, & age were independent PCM risk factors.
3) Subgroup analysis: 665 patients (37.5%) had ≥1 high-risk cyst features. Of these, 188 (28.2%) died during the follow up period – 163 (24.5%) from NPCM, & 25 (13.3%) from PCM. NPCM risk was higher in those with high CCI at the 2- (14.8% vs 1.3%, p < 0.0001) & 5-year (26.5% vs 4.9%, p < 0.0001) mark.
Conclusion: PCM is predicted by the presence of high-risk cyst feature (cyst size > 3 cm, main PD dilation) at baseline. However, a high CCI (≥3) impacts the risk of OM & NPCM, and should be considered in cyst management.
Post-pancreaticoduodenectomy Complications in High-Risk Patients Can Be Reduced With Hydrocortisone Treatment: A Randomized Controlled Trial
M. Laaninen, J. Sand, K. Vasama, J. Laukkarinen. Department of Gastroenterology and Alimentary Tract Surgery, Tampere University Hospital, Tampere, Finland.
Introduction: Previously we have shown that patients with >40% acini in the remaining pancreas are the ones developing complications after pancreaticoduodenectomy (PD), and in these patients inflammation of the pancreatic remnant preceding complications begins immediately after PD. We aimed to study whether complications among these high-risk patients can be reduced with hydrocortisone.
Methods: 102 patients undergoing PD were randomized to receive i.v. treatment with hydrocortisone 100 mg or placebo. First dose was given at the induction of anesthesia. Percentage of acini was calculated from a frozen sample at pancreatic transection line by a pathologist peri-operatively. The high risk patients with >40% acini continued to receive total of 8 doses of randomization-based treatment every 8 hours. The number of patients needed to complete the RCT was determined by power analysis. All complications were registered and analyzed according to the clinical significance (Clavien-Dindo). Post-operative pancreatic fistulas (POPF), hemorrhage (POPH) and delayed gastric emptying (DGE) were graded.
Results: 62/102 (61%) of the patients were determined as high-risk patients and continued in the RCT. 90-day mortality was zero. Hydrocortisone treatment reduced complications among the high-risk patients significantly (18 vs 41%; p < 0.05; Clavien-Dindo III-IV). The amount of clinically significant POPF (11 vs 27%; p = 0.118), PPH (14 vs 24%; p = 0.359) and DGE (29 vs 44%; p = 0.207) was reduced in the treatment group compared to the placebo group. Patients who were considered not high-risk (<40% acini) had almost nil complications.
Conclusions: Hydrocortisone treatment reduces complications among high risk patients undergoing PD. Furthermore, this study validates that >40% acinar cells in the transection line of pancreas is an excellent cut-off value to detect the high-risk patients.
Quality of Life (QoL) After Pancreaticoduodenectomy (PD) in Patients With Pancreatic Ductal Adenocarcinoma (PDA)
I. Laitinen, J. Sand, P. Peromaa, I. Nordback, J. Laukkarinen. Department of Gastroenterology and Alimentary Tract Surgery, Tampere University Hospital, Tampere, Finland.
Introduction: Survival for PDA is relatively short despite of PD, which carries a high risk for postoperative morbidity. The effect of PD on QoL is unclear.
Aim: We aimed to study QoL in PDA patients undergoing PD.
Patients and Methods: 60 patients with suspected PDA and planned PD were asked to fill EORTC:s QoL questionnaires QLQ-C30 and QLQ-PAN26 preoperatively and at 3-6-12-18-24 months postoperatively.
Results: Final diagnosis was PDA in 48 patients (64 (21-84) years, 52% men) and these patients were included in the final study population. Follow-up after PD completed in 90% (6 months) and 48% (24 months) of the survived patients. Compared to preoperative level, overall QoL tended to improve or remained the same in 63% of the patients during the follow-up. At 3 months after surgery a significant decrease in hepatic symptoms was seen (100%; p < 0.001), emotional functioning improved slightly (p = 0.016), pancreatic pain and sexuality symptoms tended to decrease by 33% and global and functional QoL tended to slightly improve. These parameters remained at the achieved level during the longer follow-up. A temporary rising tendency was seen in the digestive symptoms at 3 months but this returned back to the preoperative level later. However, altered bowel movements and sexuality symptoms tended to deteriorate during the longer follow-up. Nausea and vomiting increased significantly at 3 months (p = 0.001) but returned to preoperative levels later. In multivariate analysis no correlation between QoL and length of survival was seen.
Conclusions: PD does not worsen the QoL in most of the patients with PDA. The potentially beneficial effect on QoL is seen already at 3 months after surgery. This information may be helpful for the clinician and patient, when discussing and deciding on the operative treatment for PDA.
Molecular Architecture of Mouse and Human Pancreatic Zymogen Granules: Proteins Components and Their Copy Numbers
J. Lee, J. Caruso, G. Hubbs, P. Schnepp, J. Woods, P. Stemmer, B. Jena, X. Chen. Wayne State University, Detroit, MI.
Introduction: A molecular model of pancreatic zymogen granule (ZG) is critical for understanding its functions. We have extensively characterized the rat ZGs. In this study, we report the determination of the copy numbers per ZG of identified mouse ZG proteins. Similar studies on isolated human ZGs are in progress.
Methods: ZGs were purified from mouse pancreas or isolated acini of healthy human donors, lyzed and separated by SDS-PAGE. Gel slices were digested and peptides were analyzed by LC-MS/MS. For multiple reaction monitoring (MRM) experiments, the tryptic digest from selected gel slices were analyzed with peptide standards on a triple-stage-quadrupole instrument.
Results: 286 mouse proteins were confidently identified. Intact ZG lysate was used to minimize the protein loss, which is critical to the precise determination of protein copy numbers. Using MRM technology and isotope-labeled synthetic peptides, absolute quantities of Rab3D and VAMP8 were determined to be 7400 and 6500 copies per ZG of an averaged size of ∼750 nm in diameter. Copy numbers of other proteins in ZGs will be deduced accordingly based on their relative abundance. The emphasis of the human ZG analysis is to identify human ZG specific content and membrane proteins. In our initial pilot analysis, ∼150 proteins were identified. Preliminary analysis revealed that several proteins, including ZA2G, REG3A & 3G, SYTL2 and O2AG1, had not been reported present on rodent ZGs. Thorough LC-MS/MS analysis is ongoing and the results will be compared with published rodent ZG proteins as well as human pancreatic juice proteins. Copy numbers of human Rab3D, VAMP8 and other ZG proteins will be determined shortly.
Conclusions: These studies represent first proteomic analyses to identify species-specific ZG proteins. A comprehensive molecular model of ZGs can be developed from these results including the protein components as well as their membrane topologies and copy numbers.
Increased Neutrophil to Lymphocyte Ratio is a Poor Prognostic Factor in Patients Undergoing Gemcitabine and Erlotinib Combination Chemotherapy for Pancreatic Adenocarcinoma
J.M. Lee, H.S. Lee, J.J. Hyun, C.D. Kim. Division of Gastroenterology and Hepatology, Department of Internal Medicine, Korea University College of Medicine, Seoul, Korea.
Background: Cancer associated inflammation is one of prognostic factors predicting the outcome of cancer. The aim of this study was to investigate the prognostic significance of systemic inflammation-based markers in patients with advanced pancreatic cancer.
Methods: We reviewed the medical records of pancreatic cancer patients receiving adjuvant/palliative chemotherapy between January 2011 and December 2014. A total of 84 consecutive patients undergoing gemcitabine/erlotinib combination chemotherapy for pancreatic adenocarcinoma were retrospectively recruited. The associations between overall survival and clinically available factors including systemic inflammatory response markers. It was analyzed using Kaplan-Meier curves, univariate and multivariate Cox regression models.
Results: Median overall survivals were significantly different between two groups based on the inflammation-based prognostic markers; neutrophil to lymphocyte ratio (NLR) <5 versus ≥5 = 11.0 months versus 3.4 months (P < 0.001). Multivariate analysis identified increased NLR (HR = 3.046; CI = 1.021-9.083; p = 0.046) is an independent prognostic factor for poor prognosis and successful resection is a good prognostic factor (HR = 0.245; CI = 0.100-0.600; p = 0.002). However, platelet to lymphocyte ratio (PLR), pain score, and CEA were not significant factors for predicting the prognosis of gemcitabine/erlotinib combination chemotherapy.
Conclusion: There was a significant relationship between increased NLR and poor prognosis of gemcitabine/erlotinib combination chemotherapy. Our findings suggest that the NLR derived from routine blood tests before chemotherapy can be used as feasible biomarkers for predicting response of chemotherapy in pancreatic adenocarcinoma.
Long-Term Outcomes and Prognostic Factors in 78 Japanese Patients With Advanced Pancreatic Neuroendocrine Tumors: A Single-Center Retrospective Study
L. Lee,1 T. Ito,1 H. Igarashi,1 K. Kawabe,1 Y. Oda,2 R.T. Jensen.31Department of Medicine and Bioregulatory Science, 2Department of Anatomic Pathology, Graduate School of Medical Sciences, Kyushu University, Fukuoka, Japan; 3Digestive Diseases Branch, National Institutes of Diabetes, Digestive and Kidney Diseases, National Institutes of Health, Bethesda, MD.
Background: Despite an increase in the number of Japanese patients with pancreatic neuroendocrine tumors (PNET), long-term outcomes and prognostic factors, especially for those with advanced disease, remain unclear.
Methods: We retrospectively reviewed the medical records of 78 patients with unresectable PNET treated at our hospital from January 1987 to March 2015. Survival analyses were performed using Kaplan-Meier methods. Prognostic significance of several clinicopathological factors were analyzed by univariate and multivariate analyses using a Cox regression model.
Results: Median overall survivals of PNET (n = 64) and pancreatic neuroendocrine carcinoma (n = 14) were 83.7 months and 9.1 months, respectively (hazard ratio [HR]: 0.02, P < 0.001). Although no significant differences were observed using a Ki-67 cut-off value of 2% (HR: 2.05, P = 0.099) and 5% (HR: 2.25, P = 0.096), 10% threshold had a significant prognostic value in patients with PNET (HR: 9.95, P < 0.001). Presence of bone metastasis (HR: 4.38, P = 0.013) and the treatment after the advent of targeted therapeutic agents such as everolimus and sunitinib (HR: 0.07, P < 0.001) were significant prognostic factors in patients with PNET evaluated by univariate analysis. Multivariate analysis also revealed that a Ki-67 index ≥ 10% (HR: 38.8, P < 0.001), approval of targeted therapy (HR: 0.02, P < 0.001) and bone metastasis (HR: 5.56, P = 0.039) were independent prognostic factors.
Conclusions: We elucidated the long-term outcomes and prognostic factors in Japanese patients with advanced PNET.
Comparison of Cytological and Histological Examinations in the Diagnosis of Pancreatic Malignancy Using Endoscopic Ultrasound-Guided Fine Needle Aspiration
S.H. Lee, D.K. Jang, J.H. Son, J.W. Lee, J.K. Ryu, Y.T. Kim. Department of Internal Medicine and Liver Research Institute, Seoul National University College of Medicine, Seoul, Korea.
Background/Aims: Endoscopic ultrasound-guided fine needle aspiration (EUS-FNA) has become a crucial diagnostic technique for pancreatic malignancies. The specimen obtained by EUS-FNA can be prepared for either cytological or histological examinations. The aim of this study was to compare diagnostic performance of cytological and histological preparations using EUS-FNA in the same lesions when pancreatic malignancies were suspected.
Methods: One hundred and eighteen patients who underwent EUS-FNA for suspected pancreatic malignancies consecutively enrolled. All procedures were conducted by a single echoendoscopist under the same conditions. Four adequate preparations were obtained by 22-gauge needles with 20 to-and-fro movements for each pass. The 4 preparations included 2 cytological and 2 histological specimens. Sensitivity, specificity, and accuracy of the two examinations were compared by the McNemar chi-square test.
Results: The enrolled patients consisted of 62 males (52.5%), with the mean age of 64.6 ± 10.5 years. Surgery was performed in 23 (19.5%) patients. 116 (98.3%) lesions were classified as malignant, while 2 (1.7%) were benign. Sensitivity of cytology and histology were 87.9% and 81.9%, respectively, with no significant difference (P = 0.190). Accuracy was also not significantly different. Combining these two examinations, sensitivity and accuracy increased up to 94.0% and 94.1%, respectively. Cytological examination was more sensitive when the size of lesion was less than 3 cm (86.7% vs 68.9%, P = 0.033).
Conclusions: Our results suggest that the diagnostic performances of cytological and histological preparations are not significantly different for the diagnosis of pancreatic malignancies. However, cytological examination might be more sensitive for the pancreatic lesions < 3 cm.
Fisetin, a Flavonol, has Limited Inhibitory Effect on Human Pancreatic Cancer Cells
S.H. Lee,1 N. Kim,2 J.H. Son,1 D.K. Jang,1 J.W. Lee,1 J.K. Ryu,1 Y. Kim.11Department of Internal Medicine and Liver Research Institute, Seoul National University College of Medicine, Seoul, Korea; 2Asan Institute for Life Sciences, Asan Medical Center, University of Ulsan College of Medicine, Seoul, Korea.
Backgrounds and Aims: Fisetin (3, 7, 3’, 4’-tetrahydroxyflavone) is a flavonol from many fruits and vegetables, such as strawberries and apples. Recent reports show that fisetin possesses antiproliferative, apoptocic, neuroprotective and antioxidative activities in many cancer cell lines such as prostate and lung cancers. However the effect of fisetin on human pancreatic cancer cells has not yet been reported well. Thus we set up this study to investigate therapeutic effect of fisetin alone and in combination with gemcitabine.
Methods: Three human pancreatic cancer cell lines (Panc-1, AsPC-1, and Mia-PaCa2) were cultured and treated with fisetin alone, gemcitabine alone, or in combination of fisetin and gemcitabine. The doses for combinatory treatment of fisetin and gemcitabine were determined at the concentrations of single treatment which caused 70-80% viability compared with vehicle control within 72 hrs in incubation with the reagents. The cell viability was assessed by MTT assay. The underlying working mechanism was studied using Western blotting and Q-PCR.
Results: Panc-1 and Mia-PaCa2 cell lines were resistant to fisetin at nearly 200 μM. However, combinatory effect was observed in AsPC cells. The effect of fisetin appeared to be the results of reduced NF-kB activation, which was assessed by Western blotting. Furthermore it was observed that the expressions of KLF4 and OCT4 were decreased with fisetin and the expression of NOTCH1 was decreased with the combination of fisetin and gemcitabine.
Conclusions: Fisetin was not very effective as anti-tumor reagent for pancreatic cancer in general, but it showed combinatory effect in AsPC cell lines. It could be beneficial for some cases and needs further investigation.
Caveolin-1-ROS Feedforward in PSC Induces Stroma-Tumor Metabolic Coupling in Pancreatic Cancer
J.J. Lei, Q.Y. Ma, Z. Wang, Z. Wu. Department of Hepatobiliary and Pancreas Surgery, First Affiliated Hospital of Xi’an Jiaotong University, Xi’an, Shaanxi, China.
Aim: To explore whether there is metabolic coupling between PSCs and pancreatic cancer cells. Background: A recent clinical study has found that in breast cancer, cancer cells did not express high level glycolytic key enzymes; On the contrary, interstitial cells displayed high expression of glycolysis enzymes. This is inconsistent of the Warburg effect in cancer cells. We speculate that in the co-evolution of tumor cells and interstitial cells in tumor microenvironment, there is not only cytokine interaction between them, but also an energy shuttle between tumor cells and stromal cells, which is termed as the metabolic coupling.
Methods: Stellate cells and tumor cells were co-cultured in a chamber-based six well plates. The glycolysis and oxidative phosphorylation enzyme levels were detected by Western blot and Real-time PCR. The generation of lactic acid was determined by a lactic acid detecting kit. Si-RNA against Cav-1 was used to knockdown Cav-1 in stellate cells. NAC and BSO were applied to inhibit or upregulate oxidative stress under co-culture conditions.
Results: Under co-culture conditions, glycolytic enzyme level increased in stellate cells and oxidative phosphorylation enzyme level elevated in tumor cells. The lactic acid generation and discharge capacity increased in stellate cells but decreased in cancer cells. And the ability of lactic acid intake was upregulated in cancer cells. Upregulation of ROS in PSCs could suppress its Cav-1 expression, however, decrease in Cav-1 expression could in turn increase the ROS expression. Knockdown of Cav-1 in PSC could upregulate PSC glycolysis and reduce tumor cell oxidative phosphorylation, thus promoting metabolic coupling formation. Inhibition of ROS just had the opposite effect.
Conclusion: Cav-1-ROS feedforward in PSCs mediates the metabolic coupling between stromal and cancer cells in pancreatic cancer microenvironment.
Hypermethylation of the Mir-506 Gene Facilitates Pancreatic Cancer Progression and Chemoresistance via SPHK1/Akt/NF-Κb Signaling
J. Li, H. Wang. Institute of Hepatopancreatobiliary Surgery, Southwest Hospital, Third Military Medical University, Chongqing, China.
Objective: The aberrant expression of microRNAs (miRNAs) has emerged as an important hallmark of cancer. miR-506 has been reported to involve in several cancer types through distinct mechanisms, however, the role of miR-506 in pancreatic cancer remains unclear. Herein, whether and how miR-506 is involved in the pathogenesis of pancreatic cancer is investigated in this study.
Methods: The expression of miR-506 in pancreatic cancer specimens was examined by quantitative reverse transcriptase PCR. Pyrosequencing analysis was applied to study the methylation status in the promoter region of miR-506. Functional effect of miR-506 was analyzed by cell viability, cell cycle and apoptosis assays. The putative targets of miR-506 were predicted by bioinformatics, and verified by luciferase reporter assay, rescue experiments, Western blotting and immunohistochemistry assays, respectively.
Results: miR-506 promoter is highly methylated in pancreatic cancer tissues compared with non-cancerous tissues. Reduced miR-506 expression was significantly associated with decreased survival of pancreatic cancer patients. miR-506 inhibited cell proliferation, induced cell cycle arrest at the G1/S transition, and enhanced apoptosis and chemosensitivity of pancreatic cancer cells. Furthermore, we identified sphingosine kinase 1 (SPHK1) as a novel target of miR-506 that inhibited the SPHK1/Akt/NF-κB signaling pathway, which is activated in pancreatic cancer. High SPHK1 expression was significantly associated with poor survival in a large cohort of pancreatic cancer specimens.
Conclusions: Our data suggest that miR-506 acts as a tumor suppressor miRNA and is epigenetically silenced in pancreatic cancer. The newly identified miR-506/SPHK1 axis represents a novel therapeutic strategy for future pancreatic cancer treatment.
Initial Attempts at Enhanced Recovery After Surgery (ERAS) Program for Pancreatic Surgery in a High-Volume Center in China
Q. Li, N. Lv, Z. Lu, C. Dai, K. Jiang, J. Wu, W. Gao, F. Guo, J. Wei, J. Chen, Y. Miao. The First Affiliated Hospital of Nanjing Medical University, Nanjing, China.
Background: ERAS can reduce morbidity and length-of-stay (LOS) in orthopedics, colorectal and pelvic surgery. But its application in pancreatic surgery is poorly supported by current evidence, thus remains controversial. To clarify its role after pancreatic surgery, we initiated an ERAS program in a high-volume pancreas center in China, evaluated its implementation.
Methods: Based on international guideline and updated evidence, an institutional ERAS program for pancreatic surgery was developed in combination with our current practice. Our ERAS program included the following key aspects: patient education, early removal of drainage tubes, strict pain-control, early mobilization, early postoperative oral nutrition, prophylaxis anti-coagulation, enhanced glycemic control and near-zero fluid balance. Meanwhile, several measures were taken to improve its implementation, monitor its running, and assess clinical results. Achievement of management goals and short-term postoperative complications were analyzed.
Results: From Jan. to Mar. 2015, 47 patients were managed with the program. The percentage of goal achievements varied from 38% to 100% for different ERAS items, while more than 70% of items can be accomplished with an acceptable percentage (>60%). The proportion of patients achieving key targets were: 60% for NGT removal; 83% urethral catheter removal; 100% water intake on POD1; 62% tolerating diet on POD4; 83-89% meeting mobility targets, and 50% drain removal. Postoperative complication didn’t increase after program implementation. Mean postoperative LOS was 15.6 days.
Conclusion: ERAS is safety and feasible in an unselected patient population undergoing pancreatic surgery with significantly enhanced postoperative recovery. Monitoring of program running and appropriate interventions offers indispensable assistance in goal achievement to ensure effectiveness for implementing.
IL-8 and HGF in Predicting Development of Severe Acute Pancreatitis (SAP)
O. Lindström,1 A. Penttilä,1 K. Kuuliala,2 H. Mustonen,1 P. Puolakkainen,1 A. Kuuliala,2 M. Salmi,3 M. Hämäläinen,4 E. Moilanen,4 H. Repo,2 L. Kylänpää.11Department of GI Surgery, 2Department of Bacteriology and Immunology, Helsinki University Hospital and University of Helsinki, Helsinki, Finland; 3Department of Medical Microbiology and Immunology, University of Turku, Turku, Finland; 4The Immunopharmacology Research Group, University of Tampere, Tampere, Finland.
Background: About half of the acute pancreatitis (AP) patients who develop organ failure (OF) have OF already on admission. We recently found that high circulating levels of interleukin-8 (IL-8) and hepatocyte growth factor (HGF) are associated with OF in patients who present without OF [Nieminen et al. Crit Care. 2014 May 21; 18(3)].
Aim: To study IL-8 and HGF as predictors of OF among AP patients presenting without OF.
Patients and Methods: A prospective study of 176 nonconsecutive AP patients admitted, within 96 hours after the onset of symptoms, to Helsinki University Hospital between 2011-4. On-admission plasma samples from 23 severe, 25 moderately severe and 128 mild AP patients, according to the revised Atlanta classification, were analyzed for IL-8 and HGF by ELISA. Of the SAP patients, 13 had OF [Modified Marshall Score (MMS)>/=2)] on admission while 10 did not (MMS < 2).
Results: The median HGF and IL-8 values were higher in 23 SAP patients than in 153 patients with mild or moderately severe AP (p < 0.001). Both HGF and IL-8 predicted SAP among all patients: area under the ROC curve values were 0.82 (95% CI, 0.73-0.90) for HGF, 0.83 (95% CI, 0.74-0.92) for IL-8 and 0.62 (95% CI, 0.49-0.75) for CRP. In the forward stepwise logistic regression analysis of the markers, HGF was independent predictor of OF (OR 1.18; 95% CI, 1.05-1.33; p = 0.007). The subgroup analysis of the patients presenting without OF is in process.
Conclusion: HGF is an independent predictor of SAP on admission among all patients. The predictive value of HGF and IL-8 among the patients presenting without OF is currently being analyzed.
Gremlin Mediates Profibrogenic Activity of TGF-β in Chronic Pancreatitis
K. Liu, Y. Cao, T.C. Ko. Department of Surgery, University of Texas, Houston, TX.
Introduction: Transforming growth factor (TGF)-β is a key mediator of pancreatic fibrosis in chronic pancreatitis (CP) through activation of pancreatic stellate cells (PSCs) and promotion of excess extracellular matrix (ECM) production. Gremlin is a pro-fibrogenic factor in diseases of the kidney, lung and skin. We have reported that Gremlin is elevated in human and mouse CP and is induced by TGF-β in mouse PSCs (mPSCs). However, it is unknown whether Gremlin mediates TGF-β’s pro-fibrogenic activity in CP. This study tests the hypothesis that Gremlin is a downstream mediator of TGF-β during CP development.
Methods: Primary mPSCs were isolated from C57BL/6 mice and primary human PSCs (hPSCs) were isolated from a resected human pancreas. mPSCs and hPSCs were treated with TGF-β1 (1 ng/ml) for 24 for mRNA analysis and 48 hours for protein expression. Gremlin1/2 and fibronectin (FN, as a measure of fibrosis) expression were determined by quantitative real-time (q)PCR and immunofluorescence analysis. To inhibit Gremlin1 expression, mPSCs were transfected with Gremlin1 siRNAs (20 nM) for 5 hours prior to TGF-β1 treatment. To block TGF-β1/Smad3 signaling, mPSCs were pretreated with SB431542 (TGF-β1 receptor II inhibitor, 3 μM), or SIS3 (Smad3 inhibitor, 10 μM) for 45 minutes prior to TGF-β1 or Gremlin1 (500 ng/ml) treatment.
Results: TGF-β1 induced Gremlin expression in hPSCs (2.0-fold vs. control, p < 0.001) and mPSCs (1.5 fold vs. control, p < 0.05). TGF-β1 induced FN expression in mPSCs (1.6 fold vs. control, p < 0.05). Gremlin1 siRNAs blocked TGF-β-induced Gremlin1/2 and FN expression. Pretreatment with TGF-β1 receptor II inhibitor (SB431542) or Smad3 inhibitor (SIS3) also inhibited TGF-β1-induced Gremlin and FN expression but had no effect on Gremlin1-induced FN expression.
Conclusions: These results suggest that Gremlin mediates TGF-β’s pro-fibrogenic activity in the pancreas and Gremlin acts downstream of TGF-β1 receptor and Smad3 signaling. Gremlin may be an attractive therapeutic target in the prevention of CP development.
Early Prediction of Major Clinical Outcomes of Patients With Acute Pancreatitis by Circulating Histones
T. Liu,1,2 W. Huang,2,5 P. Szatmary,2 S.T. Abrams,1 W. Greenhalf,2 I. Welters,3 R. Sutton,2 G. Wang,1 C.H. Toh.1,41Institute of Infection and Global Health, University of Liverpool, Liverpool, United Kingdom; 2NIHR Liverpool Pancreas Biomedical Research Unit, 3Intensive Treatment Unit, 4Roald Dahl Haemostasis & Thrombosis Centre, Royal Liverpool University Hospital, Liverpool, United Kingdom; 5Sichuan Provincial Pancreatitis Centre, West China Hospital, Sichuan University, Chengdu, China.
Background: Circulating histones are elevated in both experimental and human acute pancreatitis (AP), correlating with disease severity. We aimed to evaluate whether circulating histones on admission predict major clinical outcomes in patients with AP.
Methods: Plasma obtained within 24 h of admission from patients with a first episode of AP aged 18-85 without advanced diseases were selected from a prospectively maintained biobank. Severity was defined by Revised Atlanta Classification. Circulating histones were determined by Western blot.
Results: There were 42 mild, 26 moderate and 20 severe patients. The median concentration of circulating histones was 2.1ìg/ml (IQR 1-4.8) for mild, 1.8ìg/ml (1-3.6) for moderate and 26.5ìg/ml (7-58.8) for severe class (severe vs mild or moderate, all P < 0.001; mild vs moderate, NS). At a cut-off of 7.8ìg/ml, the sensitivity, specificity and area under the curve (AUC) for severe class were 75%, 89.7% and 93.5%, respectively, superior to clinicobiochemical scoring systems: SIRS (≥2), BISAP (≥2), APACHE II (≥8) and SOFA (≥2) on admission and C-reactive protein (≥238 mg/L) at 48 h (AUC for these parameters range from 66.9% to 85.7%). At a cut-off of 26.5ìg/ml, the sensitivity, specificity and AUC for major infections (infected pancreatic necrosis and sepsis) were 71.4%, 93.8% and 83.8%, respectively; at a cut-off of 24ìg/ml, the sensitivity, specificity and AUC for mortality were 75%, 92.5% and 89.5%, respectively. However, the AUC of circulating histones for any local complications or pancreatic necrosis was below 65%.
Conclusion: On admission circulating histones accurately predict major clinical outcomes of patients with AP in our cohort and validation studies are warranted.
Preclinical Testing of Anti-EZH2 Targeting Therapy for Pancreatic Cancer
G. Lomberk, A. Mathison, A. Salmonson, M. Missfeldt, T. Christensen, R. Urrutia. Epigenetics and Chromatin Dynamics Laboratory, Translational Epigenomics Program (CIM), GIH Division, Department of Medicine, Mayo Clinic, Rochester, MN.
Epigenetic regulators transmit, extend, and modify signals of oncogenes and tumor suppressors and thereby are the ultimate effectors of the pancreatic cancer phenotype. Epigenetic events can be reversible, in a promising manner, by new pharmacology currently being tested in clinical trials. Here, we test the feasibility of targeting the epigenetic regulator, EZH2, for controlling PDAC cell growth, using both genetic and pharmacological approaches. We find that two drugs against EZH2 (GSK343 and EPZ005687) which are being tested against lymphoma, inhibit PDAC cell growth in vitro. For in vivo studies, we developed the first Pdx1-driven, conditional knockout of EZH2. Clinically, these animals appear normal, being born at accurate Mendelian ratios and with characteristics of good health, such as weight, coat, and color. Macroscopic examination of the pancreas at 3 months of age, however, reveals signs of atrophy with an organ-to-body weight ratio approximately 50% of controls. Histological examination reveals focal areas of fat infiltrate and hypoplasia-associated acinar-to-ductular metaplasia, confirming atrophy. Genome-wide profiling of these pancreata using RNA-Seq reveals deregulation of gene expression networks that are indicative of an epigenetic effect of EZH2 deletion. Conversely, EZH2 overexpression stimulates the growth of pancreatic cancer xenografts via ability to write 3MeK27H3 epigenetic marks. Biochemically, pancreatic EZH2 works by forming a complex with other chromatin proteins, including SUZ12, EED, AEBP2, PHF1, PHF19, MTF2, and JARID2. Combined, these experiments demonstrate that EZH2 inactivation inhibits PDAC cell growth by epigenetic mechanisms. The fact that anti-EZH2 drugs, already in clinical trials against other cancers, antagonize PDAC cell growth raises optimism for their potential in the treatment of this malignancy.
Mechanism of Necroptosis in Mouse Experimental Acute Pancreatitis
J. Louhimo,1 M.L. Steer,2 G. Perides.21Department of Surgery, University of Helsinki, Helsinki, Finland, 2Department of Surgery, Tufts Medical Center and Tufts University School of Medicine, Boston, MA.
Background: We have previously shown that necroptosis is the primary mode of cell death in biliary and secretagogue pancreatitis in mice.
Aim: To investigate the pathway by which necroptosis occurs during acute pancreatitis.
Methods: Pancreatic acinar cells were freshly prepared from wild type and RIP3K−/− mice and incubated with 100nM cerulein or 250 μM taurolithocholic acid 3-sulfate disodium salt (TLCS) in the presence or absence of the RIP1K inhibitor necrostatin. Necroptosis was assessed by the formation of necrosome containing RIP1K and RIP3K, and mixed lineage kinase domain-like protein (MLKL) using immunoblot analysis.
Results: Pancreatic acinar cells formed necrosome after stimulation with supraphysiological concentrations of cerulein or TLCS. The necrosome contained RIP1K, RIP3K and MLKL. Pancreatic acinar cells from RIP3K−/− mice did not form necrosome. Treatment of pancreatic acinar cells with necrostatin inhibited the formation of the necrosome. Treatment of pancreatic acinar cells with necrostatin inhibited cerulein- or TLCS-induced cell death. Treatment of mice with necrostatin after biliary pancreatitis had been induced, ameliorated the severity of the disease.
Conclusions: Necroptosis in pancreatitis occurs through the formation of a necrosome. The necrosome in mouse pancreatitis is formed by the aggregation of RIP1K, RIP3K and MLKL. Inhibition of necrosome formation after pancreatitis induction significantly reduced disease severity, which may lead to the identification of a new therapeutic target.
FGF21 Delays Pancreatic Cancer Formation and Prevents Liver Metastasis in Oncogenic Kras Expressing Mice Fed on High-Fat Diet
W. Lu,1 Y. Yang,2 Y. Luo,3,4 Y. Liu,2 X. Wang,3 M. Liu,2 R.A. Wolff,1 J.L. Abbruzzese,5 C.D. Logsdon.1,21Department of GI Medical Oncology, 2Department of Cancer Biology, UT MD Anderson Cancer Center, Houston, TX; 3Wenzhou Medical School, Wenzhou, China; 4Texas A&M University Health Science Center IBT, Houston, TX; 5Duke Cancer Institute, Durham, NC.
Pancreatic cancer is a lethal disease. About 95% of pancreatic cancer patients harbor oncogenic KRAS mutations. Obesity is a metabolic disorder and is one of the few modifiable risk factors for pancreatic cancer. One of the main factors that contributes to the development of obesity is high fat consumption. Recent studies have shown that High-Fat Diet (HFD) can promote oncogenic Kras mediated development of pancreatic intraepithelial neoplasia and PDAC. However, it is not clear why mice with oncogenic KrasG12D expression are vulnerable to HFD mediated pancreatic cancer initiation and development.
Here, we report that fibroblast growth factor 21 (FGF21), a key regulator in fatty acid oxidation and lipid metabolism, is highly expressed in pancreatic acinar cells and is a potential autocrine factor. However, the pancreatic expression of FGF21 was dramatically reduced (∼6 fold) upon one week resident oncogenic KrasG12D expression in pancreatic acinar cells in genetically engineered mouse model. The expression of FGF21 was diminished in aged oncogenic KrasG12D expression mice. To dissect the role of FGF 21 on HFD mediated PDAC formation in oncogenic KrasG12D expression mice, we fed the adult mice with HFD or HFD + FGF21. We observed that oncogenic KrasG12D expressing mice fed with HFD + FGF21 for 9 weeks significantly lower body weight and pancreatic triglyceride, as well as dramatically decreased inflammation, fewer PanIN lesions and tumor, compared with oncogenic KrasG12D mice on a HFD without FGF21. FGF21 treatment extended survival and prevented liver metastasis of KrasG12D expressing mice on a HFD. Our results suggest that pancreas derived FGF21 is an important metabolic regulator which is silenced by oncogenic KrasG12D expression and plays an important role in preventing HFD induced PDAC and liver metastasis in oncogenic KrasG12D expressing mice. Pharmacological administration of FGF21 in human may prevent PDAC formation and liver metastasis in patients associated with obesity.
Activation of Pancreatic Stellate Cell Involves EMT-like Process: In Vitro Experiments
Z. Lu, L. Tian, B. Cai, L. Zhao, D. Qian, Q. Xu, P. Wu, Y. Zhu, J. Zhang, Q. Du, K. Jiang, Y. Miao. Pancreas Center, the First Affiliated Hospital of Nanjing Medical University, Nanjing, China.
Background: Pancreatic adenocarcinoma (PDAC) and chronic pancreatitis (CP) are all characterized by desmoplastic reaction with activated pancreatic stellate cells (PSCs), which play an important role in these two diseases. However, mechanisms of PSC activation are still poorly understood.
Methods: Normal PSCs were isolated from Sprague-Dawley rat pancreas with enzyme digestion-gradient centrifugation method. Isolated PSCs were characterized by immunofluorescence and immunocytochemistry. To evaluate cell motility change after activation, PSCs after fresh isolation and late culture were compared in transwell migration assay and live cell imaging system. Expression of panel of EMT-related genes during the activation process was assessed both at the mRNA and protein level.
Results: One gram of rat pancreas tissue yielded 4-7 millions of PSCs. Activation of PSCs occurred after around 48 hours of in vitro culture, as indicated by its morphological change to myofibroblastic shape and less in the number of lipid droplets. Enhanced in cell migration ability was observed in activated PSCs compared with the quiescent ones. After PSC activation, expression of epithelial markers, including E-cadherin, BMP7 and desmoplakin decreased, while expression of mesenchymal markers, including N-cadherin, vimentin, fibronectin1, collagen1α1 and S100A4 increased. In addition, EMT-related transcription factors Snail and Slug were also up-regulated during that process.
Conclusions: The concurrence of increased cell migration ability and alterations in EMT-related gene expression indicated that transition of quiescent PSCs to its activated form might involve EMT-like process. Therefore, this should be considered in identifying potential therapeutic targets to alleviate pancreatic fibrosis in diseases like CP and PDAC.
Smoking and Alcohol Induce Pancreatic Acinar Cell Pathology via Inhibition of IRE1/XBP1
A. Lugea, R.T. Waldron, H.Y. Su, A. Go, A. Gerloff, S.J. Pandol. Cedars-Sinai Medical Center and UCLA-VAGLAHS, Los Angeles, CA.
Background: Smoking, an independent risk factor for chronic pancreatitis, accelerates the development of alcoholic pancreatitis. We reported that alcohol feeding in mice induces upregulation of the transcription factor spliced X-box binding protein (XBP1s) via the RNase activity of the endoplasmic reticulum (ER) regulator IRE1. XBP1s is essential for the secretory phenotype of the pancreatic acinar cell and for cell survival upon ER stress. Here we studied the combined effects of ethanol and cigarette smoke on ER-stress and IRE1/XBP1 regulation in acinar cells.
Methods: We used human and mouse primary acini (short-term studies) and the acinar cell line AR42J (long-term studies). Cells were treated for up to 72 h with ethanol (E; 50 mM), cigarette smoke extracts (CS; 10-40 ug/ml) or both (E + CS).
Results: Ethanol or CS alone had no significant effects on cell viability, apoptosis or ER-stress. However, E + CS treatment significantly decreased cell viability and induced cell death (necrosis and apoptosis) in both primary acini and AR42J cells. E + CS treatment decreased cellular ATP levels only modestly, but induced sustained activation of deleterious ER-stress signals, PERK and the proapoptotic transcription factor CHOP. Notably, while E increased XBP1s levels, CS alone or E + CS markedly decreased XBP1s expression. An IRE1 inhibitor that blocks XBP1s induction recapitulated the pathology induced by E + CS. In control and E-treated cells, pharmacologic IRE1 inhibition led to reduced CCK-stimulated amylase secretion, upregulated CHOP and augmented cell death. However, IRE1 inhibition did not further increase E + CS-induced CHOP upregulation and cell death, supporting the concept that E + CS pathology is mediated in part by diminished XBP1s expression.
Conclusion: Our data indicate that smoking promotes cell death and pancreatitis responses in ethanol-sensitized acinar cells by preventing an adaptive XBP1s expression needed to support ER function and the secretory pathway.
Second Primary Malignancy Among Pancreatic Cancer Patients
R. Luketina, C.W. Michalski, C. Tjaden, M.W. Büchler, T. Hackert. Department of Surgery, University of Heidelberg, Heidelberg, Germany.
Background: Development of second primary pancreatic cancer (SPC) after an index pancreatic cancer (PC) is fairly rare, primarily due to short survival. SPC should not be mistaken for recurrent PC, e.g. after positive margin resection. However, with advances in early detection and therapy, the number of long-term survivors is increasing, as is the hypothesized incidence of SPCs in this population.
Methods: All patients who underwent two pancreatic surgeries for PC, that were not associated with a positive resection margins after the initial procedure, at the Department of Surgery at the University Hospital Heidelberg, Germany between 2001 and 2014 were evaluated from a prospective database.
Results: During study period, 25 cases of SPC occurred after an initial PC. 17 (68%) patients were men and 8 (32%) women, with an average age of 67 years (range, 50-84 years). Most primary tumors were located in the head of the pancreas (15/25); consecutively these patients developed a SPC in the body (3/15) or tail of the pancreas (12/15). In 5/25 patients, where the primary lesion was located in the tail of the pancreas SPC was found in the remaining head. When the primary PC was located in the body/neck of the pancreas (5/25 patients), the subsequent lesion was found in the head of the gland in all 5 patients. Pancreatic ductal adenocarcinoma (16/25) was the most frequent histological type of the primary tumor and all these patients showed the same histology in the SPC (16/25). The interval between first and second PC ranged between <12 months (12/25 patients) and six years (1/25 patients).
Conclusion: Despite low survival among patients with PC and the difficulty of accumulating a sufficient number of patients with SPC, we show that the incidence of SPC in surgical PC patients in a specialized center is relatively frequent and can often be treated by re-resection. Consequently, structured follow-up is required after pancreas surgery for malignant diseases to improve the prognosis of these patients.
Enhanced Recovery After Surgery (ERAS) Versus Conventional Recovery Strategies for Pancreatic Surgery
N. Lv, Z. Lu, Q. Li, K. C. Dai, Jiang, J. Wu, W. Gao, F. Guo, J. Wei, J. Chen, Y. Miao. The First Affiliated Hospital of Nanjing Medical University, Nanjing, China.
Background: ERAS pathways have reduced morbidity and length of hospital stay (LOS) in orthopedics, colorectal and pelvic surgery. But its application in pancreatic surgery is poorly supported by current evidence, thus remains controversial. To clarify its role in pancreatic surgery, we initiated an ERAS program in a high-volume pancreas center in China, evaluated its implementation.
Methods: Based on the international ERAS guideline and the updated evidence, a unique institutional ERAS program for pancreatic surgery was developed in combination with our current practice. The ERAS protocol was applied in 47 consecutive patients undergoing pancreatic surgery, including PD, PPPD, DP and so on. Patients (n = 88) who received conventional recovery strategies during pancreatic surgery were included as the control group, Outcome measures included postoperative mortality, morbidity, hospitalization and 90-day readmission rate.
Results: No adverse effect related to ERAS items occurred. Compliance with postoperative items ranged between 38.3 and 100%. 12 of these 17 items could reach 60% goal achievement. The ERAS group had an earlier recovery of oral feeding (p < 0.01), mobilization (p < 0.01), NGT removal (p < 0.05), and urethral catheter removal (p < 0.01). There is no difference between ERAS and control group in mortality, overall morbidity, major complication rates. The mean postoperative hospital stay was 20.0 days in the conventional group and 15.6 days in the ERAS group p < 0.05).Meanwhile, the ERAS pathway significantly reduced hospitalization charge.
Conclusion: ERAS program can be safely implemented in pancreatic surgery with an earlier postoperative recovery and a shorter LOS than conventional recovery strategies. Achieving certain targets was challenging. Monitoring of program running and appropriate interventions can improve executive ability of the ERAS system during its initiation phase.
Survivin is a Potential New Therapeutic Target in the Treatment of Pancreatic Cancer
Q.P. Ly,1,2 G. Howell,2 M. Mendick,2 C. Murari,2 S. Laschanzky,2 M. Brattain.21Department of Surgery, 2Fred & Pamela Buffett Cancer Center, University of Nebraska Medical Center, Omaha, NE.
Background: Survivin promotes cancer cell survival by stabilizing X-linked inhibitor of apoptosis (XIAP). While highly expressed in most human cancers, survivin is mostly absent in mature, differentiated cells. YM155 attenuates survivin transcription and induces apoptosis in many tumor cell lines and xenografts, but has not been widely studied in pancreatic cancer. Smad4 mutation occurs in 50% of pancreatic cancer and correlates with poor prognosis. There is a desperate need for effective therapy for both Smad4 wild-type (wt) and mutant (mut) pancreatic cancer. Survivin represents a potential target for both phenotypes. We examined the effect of YM155 in both Smad4 wt and mut pancreatic cell lines in vitro and in vivo.
Methods: MiaPaCa2, T3M4, CAPAN1, CFPAC and BxPC3 (ATCC) were used to study YM155 activity in vitro and in vivo. We examined protein and mRNA expression of Survivin and XIAP after YM155 treatment by Western blot and QPCR. Cell proliferation and apoptosis were studied by MTT and DNA fragmentation assays. With IACUC approval, cell lines were implanted in athymic nude mice. Mice received YM155 at 5 mg/Kg/day via an osmotic pump. The xenografts were harvested, weighed and prepared for Ki-67 and TUNEL staining.
Results: YM155 treatment decreases the protein expression of Survivin and XIAP in all 5 cell lines. We confirmed that YM155 decreased survivin mRNA expression. All 5 cell lines responded to YM155 with decreased proliferation and increased apoptosis in both in vitro and in vivo but Smad4 mut cell lines were generally less responsive.
Conclusions: YM155 is efficacious in decreasing survivin expression, leading to reduced cell proliferation and increased apoptosis in both Smad4 wt and mut pancreatic cell lines in vitro and in vivo. Our data provide strong evidence that survivin may be a potential therapeutic target in the treatment of pancreatic cancer.
Evaluation of the Role of NF-κB in Tumor Microenvironment Using a Novel Mouse Model of Pancreatic Cancer
K. Majumder, N. Arora, S. Modi, A. Nomura, B. Garg, M.K. Singh, S. Ramakrishnan, S. Banerjee, A.K. Saluja, V. Dudeja. Department of Surgery, University of Minnesota, Minneapolis, MN.
Background: NF-κB activation in cancer cells plays a critical role in tumor growth and metastases. However, the role of NF-κB activation in the tumor microenvironment on tumor growth has not been systematically studied. We have evaluated the effect of p50 NF-ĸB subunit deletion in the tumor microenvironment on pancreatic tumor growth, using our recently characterized Syngeneic Tumor Implantation Model (STIM).
Methods: In the STIM model, tumor pieces from KPC mice (Pdx-Cre KrasG12D/+p53−/−) are implanted in pancreas of wild type (WT) or gene knock-out mice. STIM in WT mice has predictable tumor progression, robust stroma and intra-tumoral immune infiltration that is similar to human PDAC. By implanting KPC tumors (p50 replete) in a p50−/− background, we specifically evaluated the effect of p50 deletion in the tumor microenvironment (stroma and immune cells), on tumor growth. The growth rate of KPC tumors implanted in WT mice (STIM-WT) and p50−/− KO mice (STIM-NFKO) was compared and, their stromal (αSMA and collagen) and immune components were studied by IHC and flow cytometry.
Results: Significantly lower tumor volumes and tumor weights were observed in the STIM-NFKO mice as compared to STIM-WT mice [4 wks: 60 ± 34 vs 1101 ± 347 mm3, 8 wks: 269 ± 74 vs 1958 ± 593 mm3; p < 0.05]. Tumors from the STIM-NFKO mice had significantly decreased stromal collagen [38.1 % of STIM-WT, p < 0.05] and αSMA content [normalized to DAPI: 0.07 ± 0.02 vs 0.55 ± 0.05, STIM-NFKO vs STIM-WT, p < 0.05]. On flow cytometry analysis, there were no significant differences among the two groups in terms of intra-tumoral immune cell infiltration.
Conclusions: Using a novel immunocompetent model of pancreatic cancer, we have demonstrated the NF-κB in tumor microenvironment is important for tumor growth. Further evaluation of mechanisms by which NF-κB in tumor microenvironment impacts tumor growth can elucidate novel chemotherapeutic targets.
Diabetes Mellitus is Associated With an Exocrine Pancreatopathy (EP) That is Distinct From Chronic Pancreatitis (CP)
S. Majumder, L. Zhang, T.C. Smyrk, S. Mohapatra, Y.C. Kudva, A. Matveyenko, S.T. Chari. Mayo Clinic, Rochester, MN.
Background: Diabetes Mellitus (DM) is associated with exocrine pancreatic dysfunction. Our goal was to study the histopathology of the exocrine pancreas in type 2 DM (T2DM) patients with no clinical pancreatic disease and compare it with CP.
Methods: From our autopsy database we identified consecutive subjects without clinical pancreatic disease, history of alcoholism or autolysis on pancreatic histology with a premorbid diagnosis of T2DM and age- and gender-matched controls without DM. Data on smoking was recorded. Subjects with clinical CP included in this study had undergone pancreatic resection or autopsy. Pancreatic histology was reviewed independently by two expert pancreas pathologists and inter-observer disagreement resolved by consensus. Exocrine pancreatic fibrosis was graded as none, mild, moderate and severe (0 to 3) and exocrine pancreatopathy (EP) was defined as ≥2+ fibrosis in asymptomatic subjects. The pattern of fibrosis, presence of inflammatory infiltrate and ductal and vascular changes were compared in EP vs CP.
Results: The study cohort had 20 patients with T2DM, 22 controls and 20 patients with CP (10 autopsy); 50% of T2DM and control subjects were ‘never smokers’. EP was noted in 50% patients with T2DM versus 9% of controls (p < 0.05); the frequency was similar in smokers vs never smokers. All CP subjects had ≥2+ fibrosis. EP and CP subjects had similar frequency (100%) of interacinar fibrosis. CP was more likely to have interlobular fibrosis (100% vs 40%, p < 0.05), and an inflammatory stroma (100% vs 0%).
Discussion: DM is frequently associated with an EP that is asymptomatic and histologically distinct from CP. These two entities differ not only in the pattern of pancreatic parenchymal fibrosis, but also in the extent of stromal inflammation. EP is a novel entity whose pathogenesis needs further elucidation.
Atg5 Deficiency Worsens Cerulein Pancreatitis
S.R. Malla,1 Y. Qin,1 O.A. Mareninova,1 J.M. Elperin,1 E.M. Lotshaw,1 M. Ohmuraya,2 S.W. French,3 A.S. Gukovskaya,1 I. Gukovsky.11VA Greater Los Angeles Healthcare System and University of California at Los Angeles, CA; 2Kumamoto University, Japan; 3Harbor-UCLA Medical Center, Torrance, CA.
Background and Aims: Genetic ablation of Atg5, a key autophagy mediator, was reported to cause spontaneous pancreatitis – but also, to ameliorate cerulein-induced acute pancreatitis (CER-AP). To resolve this controversy, we investigated the role of Atg5 in pancreas homeostasis and CER-AP using different Cre recombinase drivers to ablate Atg5.
Methods: We studied 3-4 months old male mice of 2 transgenic strains in which pancreatic Atg5 is ablated with Cre under different promoters: Atg5Fl-;Spink3-Cre (termed A5-Spink) and Atg5Fl-;Pdx1-Cre (termed A5-Pdx); as well as the corresponding controls. In the first strain, Atg5 is excised in acinar cells only; in the second, in both acinar and islet cells. We measured parameters of autophagy and pancreas damage.
Results: The extent of Atg5 ablation (by immunoblot) was several-times less in A5-Spink than in A5-Pdx pancreas. Accordingly, conversion of cytosolic LC3-I into the autophagic LC3-II form – a key step in autophagosome formation, which is markedly induced in wild type CER-AP – was inhibited partially in A5-Spink and completely, in A5-Pdx pancreas. A5-Spink and (more prominently) A5-Pdx mice spontaneously developed mild pancreas damage manifest by some necrosis and inflammatory infiltrate. Independent of the Cre driver, Atg5 ablation worsened CER-AP. A5-Spink deficiency potentiated AP responses: trypsinogen activation, hyperamylasemia, and chemokines (CXCL2/MIP-2); whereas A5-Pdx deficiency upregulated mediators of acute-to-chronic disease transition: α-SMA, TGFβ and IFNγ, associated with lymphocyte/macrophage infiltration.
Conclusions: Autophagy inhibition caused by pancreatic Atg5 ablation worsens CER-AP. The extent, pattern, and cell type of Atg5 excision determine qualitative differences in the effects of Atg5 deficiency in pancreas. The results help explain controversies in previous studies.
Actinomycin D Sensitizes Chemotherapy Resistant Suit-2 Pancreatic Cancer Cell Lines to Gemcitabine
K. Mann, B. Rogoyski, P. Ghaneh, C. Rubbi, W. Greenhalf. Department of Molecular and Clinical Cancer Medicine, University of Liverpool, Merseyside, United Kingdom.
Background: Gemcitabine has a cytotoxic and a cytostatic mode of action. Cytostasis is brought about by inhibition of Ribonucleotide Reductase (RRM), which pauses the cell cycle in G1. We have developed resistant pancreatic cancer cell lines by exposure to increasing concentrations of gemcitabine. RRM1 was found to be significantly decreased and a cleavage band discovered. These findings are contradictory to published literature and we hypothesize that resistance to the S-phase dependent cytotoxicity results from hypersensitivity to cytostasis due to low RRM. Previous work has shown that the nucleolus is a cytoxicity target in all phases of the cell cycle and Actinomycin D (ActD) targets this specifically. We aim to target the nucleolus with ActD to establish whether sensitivity to gemcitabine is restored in our resistant cells.
Methods: Sensitive and resistant Suit-2 pancreatic cells were plated in a 96-well plate and after 24 hours, varying dosage combinations of gemcitabine (0.1 nm - 1um) and ActD (0.01 nm-5 nm) were added. These were refreshed after a further day giving a total treatment regimen of 48 hours. An MTS assay was performed and IC50 calculated.
Results: The initial assays demonstrated a gemcitabine IC50 of 17 nm and 83 nm and ActD IC50 levels of 0.5 nm and 1.4 nm for sensitive and resistant cells respectively. The isobolar curves at 1, 0.5, 0.1, 0.05, 0.01 nm of ActD demonstrate a synergistic relationship in sensitive cells with IC50 results of 16, 9, 8, 7, 13 nm respectively, although synergy was lost at 5nM ActD (22nM gemcitabine). For resistant cells the synergy is not seen at low concentrations of ActD but IC50 at 5nM ActD is just 14 nm gemcitabine.
Discussion: The relationship between gemcitabine and ActD is clearly complex but there is synergism in resistant cells at high concentrations and the reverse in sensitive cells. Further work is required to validate our hypothesis.
The Transcription Factor TFEB Supports Pancreatic Cancer Cell Growth
B. Marchand, D. Arsenault, M.J. Boucher. Gastroenterology Unit, Department of Medicine, University of Sherbrooke, Sherbrooke, Canada.
We recently demonstrated in pancreatic cancer cells that prolonged GSK3 inhibition triggers an apoptotic response that is limited by concomitant autophagy induction. The aim of this study was to identify cellular mechanisms contributing to the protective autophagic response to single out new addictive signals in pancreatic cancer cells. Given the newly suggested role of TFEB in regulating autophagy and lysosomal biogenesis, the impact of GSK3 inhibition on TFEB was tested. GSK3 inhibition accelerated the electrophoretic mobility shift of TFEB that was interconnected with TFEB dephosphorylation. Quantitative mass spectrometry (MS) analyses, co-immunoprecipitation and immunofluorescence studies showed that, upon GSK3 inhibition, the chaperones 14-3-3 dissociate from TFEB followed by TFEB accumulation within the nucleus. Accordingly, MS analyses unveiled increased interaction of TFEB with nuclear proteins such as histones and other DNA binding proteins. Unexpectedly, and in contrast with the cytoplasmic localization in non-tumoral pancreatic epithelial cells HPDE, TFEB was enriched in the nucleus of pancreatic tumoral cells in fed conditions. To evaluate the contribution of TFEB in autophagy and pancreatic cancer cell growth, a stable cell population with reduced expression levels of TFEB was generated. As compared with the shControl population, shTFEB cells were not responsive to the GSK3 inhibition-induced autophagy flux and displayed an impaired capacity (by 50%) to grow in an anchorage-independent manner that was further compromised (90% inhibition) upon GSK3 inhibition. Moreover, TFEB depletion exacerbated GSK3 inhibition-induced apoptosis. For the first time, our results unveil an aberrant TFEB nuclear localization in pancreatic cancer cells and suggest an active role for this transcription factor in supporting their anchorage-independent growth. Also, promotion of TFEB function upon GSK3 inhibition interferes with the GSK3 inhibition-induced pancreatic cancer cell death.
Effects of LC3 Overexpression on Pancreatic Acinar Cell Homeostasis and Pancreatitis Responses
O.A. Mareninova,1 W. Jia,1,2 J.M. Elperin,1 E.M. Lotshaw,1 M. Pimienta,1 G.E. Groblewski,3 A.S. Gukovskaya,1 I. Gukovsky.11VAGLAHS & University of California Los Angeles, CA; 2Beijing Hospital, Beijing, China; 3University of Wisconsin, Madison, WI.
Background & Aims: Despite accumulating evidence for important role of autophagy in pancreatitis, the underlying mechanisms are poorly understood. Conversion of Atg8/LC3 from cytosolic LC3-I to the autophagic LC3-II form is a key step in autophagosome formation. We used mice (over)expressing GFP-LC3 to study the role of autophagy in acinar cell physiology and pancreatitis.
Methods: Wild type and GFP-LC3 mice were subjected to starvation or pancreatitis induced by cerulein, L-arginine, CDE diet, or ethanol diet plus low-dose cerulein. We measured parameters of autophagy, acinar cell functions (eg, amylase secretion), and pancreatitis responses.
Results: LC3 overexpression markedly increased the basal number (but not size) of autophagic vacuoles in pancreas; and stimulated starvation-induced autophagy. By contrast, in GFP-LC3 mice with pancreatitis autophagy was inefficient, as manifest by accumulation of large vacuoles and p62-positive protein aggregates. One underlying mechanism could be decreased LC3-I to LC3-II conversion due to feedback down-regulation of Atg5 and Atg7, which we found in pancreas of GFP-LC3 mice. In all pancreatitis models studied, LC3 overexpression dramatically increased serum amylase; however, pancreatic levels of amylase and trypsinogen increased. The latter could be due to inhibition of autophagic degradation as well as activation of mTOR-dependent protein synthesis. LC3 overexpression worsened pancreatitis severity (eg, necrosis); but it had no effect on CCK-induced amylase secretion from acinar cells.
Conclusions: Results indicate a critical role for LC3-mediated autophagy in regulating exocrine pancreas homeostasis and pancreatitis responses. Although GFP-LC3 mice are widely used to monitor autophagy, caution should be exercised as LC3 overexpression may perturb both physiologic and pathophysiologic pathways.
Identification of a Novel Monomeric Form of the Histone Code Reader HP1γ in PDAC Cells Generated by Alternative Splicing of the CBX3 Gene
A. Mathison, M. Williams, T. Berent, S. Buttar, M. Hege, K. Bharucha, R. Urrutia, G. Lomberk. Epigenetics and Chromatin Dynamics Laboratory, Translational Epigenomics Program (CIM), GIH Division, Department of Medicine, Biophysics, Biochemistry and Molecular Biology, Mayo Clinic, Rochester, MN.
Our laboratory has been focused on identification and characterization of novel epigenetic readers, writers, and erasers of the histone code. In the current study, we report the existence of a novel HP1γ isoform, named sHP1γ generated by alternative splicing of the CBX3 locus. By reading the 3meK9H3 mark, HP1 proteins play a significant role in cell proliferation, differentiation, chromosomal stability, DNA repair, and cancer. Identification of sHP1γ mRNA was performed by analyzing RNA-Seq profiles of human PDAC samples. The translation of this protein was confirmed in PDAC cells by western blot analyses, using a specific antibody developed in our laboratory. In contrast to the 183 amino acid-long HP1γ the new isoform is composed of 101 residues and lacks the C-terminal domain involved in dimerization. Q-PCR analyses demonstrated that the mRNA is widely distributed in human tissues. Immunofluorescence studies demonstrate that this protein is localized to the nucleus. Molecular modeling, docking, and molecular dynamic simulations reveal that, in contrast to the conventional HP1γ the sHP1γ is monomeric but still binds to the 3MeK9-H3 mark. Thus, we conclude that alternative splicing of the CBX3 locus gives rise to distinct histone code readers, which are present in PDAC cells. Combined, these results demonstrate that an expanded repertoire of HP1γ readers can modulate histone code instruction during K9H3-mediated gene silencing. These data support the notion that the regulation of HP1γ-mediated gene silencing in PDAC is more complex than previously anticipated and should guide the design and interpretation of future studies aimed at understanding the biochemical and biological roles of this important family of epigenomic regulators.
GLI1-GLI2 Interaction is Required for the Regulation of Collagen Gene Expression in Pancreatic Cancer-Derived Stellate Cells
A.L. McCleary-Wheeler,1,2,* M.G. Fernandez-Barrena,1,* E. Iguchi,1,* L.L. Almada,1 D.L. Marks,1 E.J. Tolosa, M. Erkan,3 R.F. Huang,4 M.E. Fernandez-Zapico.11Schulze Center for Novel Therapeutics, Division of Oncology Research, Mayo Clinic, Rochester, MN; 3Department of Surgery, Technical University of Munich, Munich, Germany; 4Department of Surgical Oncology, University of Texas MD Anderson Cancer Center, Houston, TX. *These authors contributed equally to this work.
Changes in the expression of components of the extracellular matrix and its remodelers play a central role during early stages of fibrogenesis in cancer. Although this molecular event has been clearly established, the mechanism underlying this phenomenon remains elusive. In the current study, we report a previously undefined role for the zinc finger transcription factors GLI1 and GLI2 in cancer fibrosis involved in the regulation of collagen expression in pancreatic cancer-derived stellate cells. Expression studies generated from isolated pancreatic cancer stellate cells specimens demonstrated that the levels of GLI1 and GLI2 are increased in tumors along with collagen type 1 α1 (COL1A1). When combined, expression, luciferase, and chromatin immunoprecipitation assays indicate that GLI1 and GLI2 interact directly with the COL1A1 promoter in to activate its transcription in these cells. Mechanistically, GLI1 and GLI2 complex to regulate collagen gene expression. Experiments performed using GLI1−/− and GLI2−/− mouse embryonic fibroblasts reveal that both factors are required to modulate the expression and promoter activity of COL1A1. Finally, we demonstrate that this newly identified transcription factor complex is downstream of TGFβ signaling, and this pathway requires both factors to induce the expression of COL1A1. Thus, our findings expand the repertoire of chromatin-associated proteins involved in the transcriptional regulation of extracellular matrix proteins and define a novel pathway regulating fibrosis in pancreatic cancer.
TFII-I-Mediated Polymerase Pausing Antagonizes TGFβ Induction of the Pancreatic Oncogene GLI2
A.L. McCleary-Wheeler,1,2 L.L. Almada,1 D.L. Marks,1 A.L. Vrabel,1 R. Olson,1 M.E. Fernandez-Zapico.11Schulze Center for Novel Therapeutics, Mayo Clinic, Rochester, MN; 2College of Veterinary Medicine, Cornell University, Ithaca, NY.
GLI2 is an oncogenic transcription factor commonly upregulated in multiple tumors, including pancreatic cancer. In most cases, this overexpression is not due to genomic defects in the gene itself, suggesting dysregulation through alternative pathways. However, the identity of the pathways and mechanisms underlying this phenomenon remain elusive. In this study, we have identified TFII-I, an INR-binding transcription factor, as a repressor of GLI2. The GLI2 gene has a TATA-less promoter characterized by the presence of an INR element. Overexpression of TFII-I results in decreased GLI2 expression and promoter activity. Conversely, TFII-I knockdown substantially increases GLI2 expression. Mapping experiments using GLI2 promoter deletions suggest that TFII-I represses GLI2 gene expression through this regulatory sequence. Mutation of the INR resulted in a partial relief of this repression. ChIP assays demonstrate TFII-I binding to the INR region of the GLI2 promoter. Further analysis shows enrichment of phosphorylated RNAPII serine 2 (RNAPII Ser2) and loss of components of the RNAPII promoter-proximal pausing complex, NELFA and Spt5 following TFII-I knockdown. Immunoprecipitation studies demonstrate TFII-I interaction with SPT5, a component of the RNAPII pausing complex. Finally, we found TFII-I can antagonize TGFβ, a known inducer of GLI2 expression. Interestingly, this effect is exclusive of GLI2, TFII-I does not antagonize other known TGFβ-inducible genes. TFII-I binding to the INR decreased while RNAPII Ser2 was elevated in the GLI2 gene following TGFβ treatment. Moreover, TFII-I overexpression is able to antagonize TGFβ induction of GLI2. Together these results identify TFII-I as a novel repressor of GLI2 and support a key role for RNAPII pausing in gene expression regulation in pancreatic cancer cells.
Triptolide Results in Accumulation of HIF-1α but Reduces its Activity in Pancreatic Cancer
O. McGinn, N. Arora, P. Dauer, V. Gupta, A.K. Saluja, S. Banerjee. Division of Basic and Translational Research, Department of Surgery, University of Minnesota, Minneapolis, MN.
Introduction: The role of tumor hypoxia has become a major focus in cancer research since it influences both local and systemic tumor growth. The major transcription factor upregulated in response to hypoxia is HIF-1α. During hypoxia, HIF-1α stabilizes and transcribes genes involved in metabolism and other cellular functions. Previous studies from our laboratory have shown that triptolide, a compound derived from a Chinese herb, decreases viability of pancreatic cancer cells in vitro. Minnelide, the pro-drug of triptolide reduces the size of pancreatic tumors in several mouse models. Pancreatic tumors contain numerous hypoxic regions. Thus far, little is known about the effect of triptolide on HIF-1α in pancreatic cancer. The aim of this study is to address the relationship between triptolide and HIF-1α in pancreatic cancer.
Results: HIF-1α protein increased in pancreatic cancer cell lines (MiaPaca-2 and S2VP10 cell lines) in response to triptolide. Triptolide did not decrease HIF-1α binding activity, but HIF-1α transcriptional activity decreased after 24 h of triptolide treatment (22.0% of control). Downstream targets of HIF-1α decreased at the protein and mRNA level in both cell lines (11.0-32.0% of control). Analysis of levels of the binding partners of HIF-1α, namely p300 and HIF- 1β was performed. The mRNA levels of p300 (24.4%; 12.7%) and HIF-1β (27.9%; 16.3%) decrease in response to triptolide in S2VP10 and MiaPaca-2 respectively. CD31 staining revealed a significantly greater number of open blood vessels and less hypoxia in the Minnelide treated KPC (KrasG12Dtp53-pdxCre) mice. The HIF-1α staining in the Minnelide treated KPC mice was not significantly different from control. Minnelide treated human pancreatic tumor xenografts had significantly greater HIF-1α than controls.
Conclusion: Triptolide increases HIF-1α protein levels in pancreatic tumors. This accumulation of HIF-1α is due to reduced degradation rather than increased transcription. Despite the accumulation of HIF-1α, triptolide is able to decrease its transcriptional activity by inhibiting formation of the required co-activator complexes. Minnelide treated KPC tumors have more open blood vessels and less hypoxia than controls, with comparable HIF-1α levels.
A Non Dilated Main Pancreatic Duct Predicts AIP: A Comparison Study of Resected Focal AIP, Chronic Pancreatitis, and Pancreatic Adenocarcinoma
C. Medina,1 M. Peláez-Luna,1,4 L. Uscanga,1 C. Chan,2 E. Negrete,1 A. Ángeles.31Department of Gastroenterology, 2Department of Surgery, 3Department of Pathology, INCMNSZ, Mexico City, Mexico; 4Research Division, School of Medicine, UNAM, Mexico City, Mexico.
Autoimmune pancreatitis (AIP) may present as a focal pancreatic mass. Differential diagnosis includes focal chronic pancreatitis (FCP) and pancreatic cancer (PC). AIP diagnosis is based on international consensus diagnostic criteria (ICDC) with limited diagnostic accuracy in atypical presentations.
Aim: To compare clinical and imaging characteristics of resected focal AIP, FCP and PC cases and identify factors that could improve differential diagnosis.
Methods: Charts from patients that underwent pancreatic resection under suspicion of PC between 2000 and 2014 were reviewed. Clinical and Imaging, data were recorded and compared among groups.
Results: We included 79 cases; 41 men, mean age of 57.32 y/o ± 15.66 SD. Final pathology report was AIP 16, FCP 8, and PC 55 AP. All AIP cases were probable type 2 by ICDC. When patients were grouped according to the presence of malignancy, obstructive jaundice (OR 28.5; 8.18-79.5, 95% CI) and dilated main pancreatic duct (MPD) (OR 5.21; 1.9-14.6) predicted malignancy. When subjects were grouped as AIP and no-AIP, a non-dilated MPD was significantly associated with AIP (OR 9.3; 3.05-28.7), p < 0.001.
Conclusions: In the setting of undetermined pancreatic focal mass, a non-dilated MPD suggests the diagnosis of AIP.
Is “180° Rule” in Artery Invasion Still Validate for Defining Unresectable Pancreatic Cancer?
Y. Miao, C. Dai, K. Jiang, B. Cai, Z. Lu, J. Wu, W. Gao, Q. Li, J. Wei, J. Chen, F. Guo. Pancreas Center, the First Affiliated Hospital of Nanjing Medical University, Nanjing, China.
Introduction: Pancreatic ductal adenocarcinoma (PDAC) is the most lethal human malignancy. Surgery remains the only chance of cure. Guidelines define major artery invasion more than 180° as unresectable. In our experience, there is a plane between the adventitia and the surrounding tumor, thus the opportunity of resection should be considered.
Methods: Between June and December, 2014, we identified patients with PDAC that involve superior mesenteric artery (SMA) more than 180° with contrast-enhanced CT scan. With patients’ consent, a pancreaticoduodenectomy or distal pancreatectomy with divesting the artery of tumor was implemented. Intraoperative data, short- and long-term complications were reviewed to estimate the safety of this procedure.
Results: Six patients (median age: 57.5 years) were included. Intraoperative evaluation revealed all cases with more than 270° SMA involvement, 6 of which with SMA encirclement. Pancreaticoduodenectomy and distal pancreatectomy were performed in 3 cases respectively, four of which artery-first technique were utilized in. There were three organ-combined resections. Space between the tumor and adventitia was identified and divestment of the artery was accomplished. Operation duration was 279 ± 64 min. Intraopreative blood transfusion was 783 ± 523 mL. Major perioperative complication was ISGPF grade B pancreatic fistula. Diarrhea occurred in 50% cases as the only identified long-term complication and mortality was null till submission.
Conclusions: For some PDAC patients with artery invasion, the “holy plane” between tumor and artery make it feasible to divest the vessel. Divestment of artery is safe in selected patients. Further research should be carried out to identify patients who can potentially benefit from resections, and new criterion of resectability evaluation is warranted.
Modified One-Layer Duct-to-Mucosa Pancreaticojejunostomy Reduces Pancreatic Fistula After Pancreaticoduodenectomy
Y. Miao, C. Dai, K. Jiang, J. Wu, W. Gao, Q. Li, J. Wei, J. Chen, F. Guo, Z. Lu, X. Liu, W. Xu. Pancreas Center, the First Affiliated Hospital of Nanjing Medical University, Nanjing, China.
Objectives: Postoperative pancreatic fistula (POPF) is a major source of morbidity after pancreaticoduodenectomy (PD). The purpose of this retrospective study comparing modified one-layer duct-to-mucosa Pancreaticojejunostomy (PJ) with two-layer PJ after PD was to evaluate whether the one-layer duct-to-mucosa PJ after PD can reduce the incidence of POPF.
Methods: A total of 194 consecutive patients who underwent PD by one single surgeon (Y. Miao) from January 2011 and February 2014 were included in this study. And among those patient, 104 underwent one-layer PJ (one-layer group) and 90 patients underwent two-layer PJ (two-layer group), respectively. Preoperative clinicopathologic features, intraoperative parameters, postoperative morbidity with focus on POPF, were compared between the two groups.
Results: The overall incidence of POPF was 19.6% (38/194), and clinically relevant grade B/C POPF rate were 8.6% (16/194) and 3.1% (6/194), respectively. Between the two groups, no differences were noted in patients’ demographics and operation related factors. However, the incidence of POPF in one-layer group was significantly lower than that in two-layer group (13.5% [14/104 patients] and 26.7% [24/90 patients], respectively; P = 0.021). The median postoperative hospital stay was also significantly lower in one-layer group than two-layer group (13 days vs. 15 days, p = 0.035). One patient in two-layer group died due to postoperative hemorrhage.
Conclusions: Modified one-layer duct-to-mucosa pancreaticojejunostomy is a simple and easy technique for pancreaticojejunal anastomosis after PD, and can reduce POPF rate when compared to two-layer technique.
The Impact of Curative Resection for Pancreatic Adenocarcinoma With Lymph Node Metastasis Status
A. Miki, Y. Sakuma, H. Sasanuma, M. Koizumi, K. Endo, N. Sata. Department of Surgery, Jichi Medical University, Tochigi, Japan.
To analyze the meaning of R0 resection in patients with pancreas cancer, we evaluated 74 pancreatic cancer patients operated in our institute from 2007 to 2013.
Pancreatoduodenectomy were performed for 43 patients, pancreas tail resection were performed for 31 patients. Fifty percent patient underwent adjuvant chemotherapy after operation. Five year overall survival (OS) of R0 group was 85.8 months (m) and R1 group was 15.3 m (P = 0.09, Log-rank test). Lymph node metastasis significantly influenced the prognosis. R0 patient with no lymph node metastasis was better prognosis than lymph node metastasis patient (P = 0.002). Disease free survival (DFS) of R0 group with no lymph node metastasis was also better than lymph node metastases patient (P = 0.01). The significant difference did not exist between R0 group and the R1 group in patient of lymph node metastasis (P = 0.56).
OS of patients with adjuvant chemotherapy for no lymph node metastasis was better than no adjuvant chemotherapy. OS of patients with adjuvant chemotherapy for lymph node metastasis was not significant between R0 and R1 groups (0.94).
R0 operation is important to get good prognosis. In addition, prognosis was still poor for patients with lymph node metastasis.
Is Anaplastic Pancreatic Cancer Originated from Invasive Ductal Carcinoma of Pancreas?
K. Miura, K. Kimura, R. Amano, S. Yamazoe, G. Ohira, K. Nishio, M. Shibutani, K. Sakurai, H. Nagahara, T. Toyokawa, N. Kubo, H. Tanaka, K. Muguruma, H. Otani, M. Yashiro, K. Maeda, M. Ohira, K. Hirakawa Department of Surgical Oncology, Osaka City University Graduate School of Medicine, Osaka, Japan.
Introduction: Few studies were investigated whether anaplastic pancreatic cancer (APC) was derived from the invasive ductal adenocarcinoma (IDA). Therefore, we researched the origin and progression of APC from the expression of the lineage, epithelial-mesenchymal transition (EMT) and stem cell markers.
Materials & Methods: Using the resected specimen from 6 patients of APC (Ana) and 53 patients of the well, moderately or poorly differentiated IDA (Well, Mod or Por), immunohistochemistry of SOX9, E-cadherin, vimentin, Zeb-1, Snail, N-cadherin, CD24 and CD44 was performed. APC was divided into ductal lesion (DL) and undifferentiated lesion (UL). The staining score (0-3) was calculated from the staining intensity and percentage of the stained cells per field.
Results: The proportion of DL to all cancerous lesion was 0.5% to 32%. The staining scores of each antibody were showed as follows; the SOX9: UL / DL / Well/ Mod /Por = 2.7 / 2.0 / 2.5/ 2.5/ 2.1. E-cadherin: UL / DL / Well/ Mod /Por = 0.27 / 2.7 / 2.2/ 2.3/ 1.7.vimentin: UL / DL / Well/ Mod /Por = 2.3 / 0.83 / 0/ 0.42/ 0.78. Zeb-1: UL / DL / Well/ Mod/ Por = 2.0/ 1.0 / 0/ 0/ 0.38. Snail: UL / DL / Well/ Mod/ Por = 2.1 / 1.4 / 0.26/ 0.89/ 0.93. N-cadherin: UL / DL / Well/ Mod/ Por = 2.2 / 1.0 / 0.76/ 0.69/ 1.2. CD24: UL / DL / Well/ Mod/ Por = 0.56 / 0.30 / 0.4/0.54/ 0.61. CD44: UL / DL / Well/ Mod/ Por = 1.7 / 0.30 / 0.21/ 0.47/ 1.3.
Discussion: In the current study, it was suggested that APC might be derived from PDA by showing the expression of SOX9, whose expression is only identified in PDA among pancreatic neoplasms. Also, it was suggested that UL might change from DL through EMT by confirming the difference of the expression pattern of markers in DL and UL. As the expression of CD44 was relatively higher in UL than the others, the acquisition of stem cell property might be related to the progression to APC.
Conclusion: It was suggested that conventional PDA might progress to APC through dedifferentiation caused by EMT and the acquisition of stem cell property.
Multicenter Comparative Study of Laparoscopic and Open Distal Pancreatectomy Using Propensity Score-Matching
Y. Miyasaka,1,9 M. Nakamura,1,9 M. Tanaka,1,9 T. Morikawa,2,9 M. Unno,2,9 G. Wakabayashi,3,9 T. Beppu,4,9 T. Takahara,5,9 H. Yamaue,6,9 M. Miyazaki,7,9 T. Takada.8,91Department of Surgery and Oncology, Graduate School of Medical Sciences, Kyushu University, Fukuoka, Japan; 2Department of Surgery, Tohoku University Graduate School of Medicine, Sendai, Japan; 3Department of Surgery, Ageo Central General Hospital, Ageo, Japan; 4Department of Gastroenterological Surgery, Graduate School of Life Sciences, Kumamoto University, Kumamoto, Japan; 5Department of Surgery, Iwate Medical University School of Medicine, Morioka, Japan; 6Second Department of Surgery, Wakayama Medical University, School of Medicine, Wakayama, Japan; 7Department of General Surgery, Graduate School of Medicine, Chiba University, Chiba, Japan; 8Department of Surgery, Teikyo University School of Medicine, Tokyo, Japan; 9Japanese Society of Hepato-Biliary-Pancreatic Surgery and Japanese Society for Endoscopic Pancreatic Surgery.
Background: Many studies showed that Laparoscopic distal pancreatectomy (LDP) was associated with better postoperative outcomes compared to open distal pancreatectomy (ODP). However, bias cannot be denied in these studies. We conducted a multicenter study using propensity score-matching to minimize bias.
Aim: To compare perioperative outcome of LDP and ODP.
Methods: We retrospectively collected perioperative data of 2,266 patients who underwent distal pancreatectomy in 69 institutes of Japan. Among them, 2,010 patients were enrolled in this study. Perioperative outcomes were compared between LDP and ODP using propensity score-matched analysis.
Results: LDP was associated with higher rate of preservation of spleen and splenic vessels (P < 0.001); lower rates of intraoperative transfusion (P = 0.020), pancreatic fistula (ISGPF grade B and C; P < 0.001), and morbidity (P < 0.001); and shorter hospital stay (P = 0.001), but a longer operative time (P < 0.001).
Conclusions: LDP was associated with more favorable perioperative outcomes than ODP even after propensity score-matching.
Pancreatic Ductal Adenocarcioma Without High-Grade Pancreatic Intraepithelial Neoplasia (PanIN) May Develop via a Pathway Other Than PanIN-Carcinoma Sequence
T. Miyazaki,1,2 Y. Ohishi,1 Y. Miyasaka,2 K. Ozono,1,2 A. Abe,1,2 N. Mochidome,1,2 K. Saeki,1,2 E. Nagai,2 Y. Oda,1 M. Nakamura.21Department of Anatomical Pathology, Kyushu University, Fukuoka, Japan; 2Department of Surgery and Oncology, Kyushu University, Fukuoka, Japan.
Background: We previously reported pancreatic ductal adenocarcinoma (PDAC) without high-grade PanIN (PanIN-2, 3) in the vicinity had worse prognosis than PDAC with high-grade PanIN. However, molecular characteristics of PDACs with and without high-grade PanIN have not been compared.
Method: We reviewed all the consecutive series of 100 patients of PDAC and divided them into 2 groups: PDAC with high-grade PanIN in the background (HG-PanIN group) and PDAC with no high-grade PanIN in the background (NHG-PanIN group). In the invasive carcinoma components, p53, p16 and SMAD4 expression were evaluated by immunohistochemical stain. In 37 patients, KRAS mutation was also analyzed.
Result: The HG-PanIN group consisted of 60 patients, while the NHG-group consisted of 40 patients. The NHG-group showed significantly higher rate of vascular invasion and lymphatic invasion (P = 0.007 and P = 0.027) and lower overall and disease specific survival (P < 0.0001 and P < 0.0001), compared with the HG group. The NHG-PanIN group showed significantly higher rate of p53 accumulation and loss of SMAD4, compared with HG-PanIN group (P = 0.048 and P = 0.019). Loss of p16 was not significantly different between them. The rate of KRAS wild type was more common in the NHG-PanIN group than the HG-PanIN group (P = 0.019).
Discussion: We demonstrated that molecular characteristics in PDAC without high-grade PanIN were different from those in PDAC with high-grade PanIN, suggesting they may have a different carcinogenesis pathway. PDAC without high-grade PanIN may develop via a pathway other than PanIN-carcinoma sequence.
Plasma DNA Genotyping Using Digital PCR for Early Detection of Pancreatic Neoplasm
Y. Mizukami, Y. Ono, H. Karasaki, M. Ogata, T. Kono, K. Nagashima. Center for Clinical and Biomedical Research, Sapporo Higashi Tokushukai Hospital, Sapporo, Japan.
The genetic alterations that are responsible for the initiation and progression of pancreatic ductal adenocarcinoma (PDA) could serve as a new generation of biomarkers for early diagnosis of the tumor. Circulating cell-free DNA (cfDNA) released from tumor cells into the blood has been intensively studied as a novel way to monitor such genetic changes. There are two main types of pathologically and genetically distinct precursors for PDA — pancreatic intraepithelial neoplasias (PanINs) and intraductal papillary mucinous cystic neoplasias (IPMNs). Non-invasive markers for these precursor lesions have the potential to predict subsequent invasive neoplasia is highly desirable. The role of cfDNA genotyping targeting the major driver mutations in these tumors, such as KRAS and GNAS, are currently under investigation in Japanese patients who have pancreatic tumors (UMIN000012810). The major technical challenge is to specifically detect the small fraction of tumor-derived DNA in patient plasma and urine. Since sequencing of target mutant alleles in cfDNA has a limitation to detect very low frequency variants, we sought to establish protocols for super-sensitive and absolute quantification of the “key drivers” for pancreatic tumorigenesis using a droplet digital PCR platform. The primary endpoint of this multi-center prospective analysis is to evaluate whether such an approach can appropriately monitor the risk of IPMN progression and detect localized early-stage PDA. The preliminary results of these studies will be discussed.
Minnelide Synergizes With Standard Chemotherapy in Pancreatic Cancer
S. Modi, B. Giri, K. Majumder, V. Dudeja, S. Banerjee, A.K. Saluja. Translational Research, Department of Surgery, University of Minnesota, Minneapolis, MN.
Introduction: Addition of abraxane to gemcitabine offers a survival benefit of only 6 weeks over gemcitabine alone and is associated with increased toxicity in PDAC. Thus meaningful combinations that help reduce the doses of these toxic drugs while having better efficacy are urgently required. Minnelide (currently in phase I trials) have been shown to be effective against pancreatic cancer in preclinical studies. We have evaluated whether lower doses of Minnelide in combination with lowered doses of standard therapy in PDAC can provide similar anti-tumor effects.
Methods:In vitro: Highly aggressive pancreatic cancer cells (S2-VP10, S2-013 and PANC-1) were treated with low doses of paclitaxel (0-25nM), low dose triptolide (25nM) and a combination of both for 24-72 h. Effects of the combination therapy on Cell viability, apoptosis and Cell cycle were assessed.
In vivo: Xenograft subcutaneous and orthotopic models were established using S2-VP10 and S2-013 cells. Immunocompetent subcutaneous model was established using KPC derived cells. Due to development of fatal immune reaction in mice treated with abraxane (due to human albumin), equivalent dose paclitaxel was used in immunocompetent mice while immunodeficient mice were treated with abraxane. Minnelide was used as either 0.21 or 0.15 mg/kg/day, standard chemotherapy as Gemcitabine 250 mg/kg/week + abraxane 25 mg/kg/week, Combination with Minnelide as Gemcitabine 100 mg/kg/week + abraxane 10 mg/kg/week.
Results:In vitro, Combination of low doses of triptolide (25nM) and paclitaxel significantly decreased cell viability, increased apoptosis and induced G2/M phase arrest in all cell lines as compared to either drug alone.
In vivo: Combination of low doses of Minnelide and Gemcitabine + paclitaxel/abraxane significantly inhibited tumor progression and was effective in decreasing the tumor burden leading to an increased survival of tumor bearing mice (p < 0.05). Combination therapy significantly reduced cancer related morbidity by decreasing ascites and metastasis.
Conclusion: Minnelide synergizes with standard of care chemotherapy and helps in significantly reducing the doses of these toxic drugs all the while achieving better efficacy in treatment of PDAC.
Duodenal Display of Extended Field of Pancreatic Cancer: Potential Role of Galectin-3
N. Momi,1 H. Subramanian,2 R. Wali,1 M. Delacruz,1 V. Backman,2 H. Roy.11Department of Medicine, Boston University Medical Center, Boston, MA; 2Northwestern University, Evanston, IL.
Background: Peri-ampullary duodenum, which converges with pancreatic juice, may serve as a minimally-intrusive surrogate site for pancreatic cancer (PC) diagnosis. It empirically displays bio-optical markers of “field carcinogenesis” as demonstrated by our group using a novel optics technique, partial wave spectroscopy (PWS). To elucidate the biological rationale we hypothesize that PC-secreted factors via pancreatic duct may impact upon duodenal cells as indicator of field effect.
Methods & Results: Co-culture studies and trans-well/motility assay was conducted to monitor the impact of pancreatic-milieu on intestinal cells (IEC6), using human pancreatic epithelial cells (HPNE) and PC cells, [Miapaca-2 (Mia-2), Capan-2 (Cap-2)] and Panc1, representing the high to low aggressive PC. Intriguingly, IEC6 cells, trans-migrated more towards Mia-2 and Cap-2 vs HPNE and Panc1 (p < 0.04), suggesting that PC aggressiveness mirrors duodenal field carcinogenesis. Also, when IEC6 cells segregated based on their migration towards Mia-2 cells were subjected to PWS analysis, inflections in disorder strength, which is shown to parallel chromatin condensation/transcriptional-activity, were detected in migratory compared to non-migratory cells (Ld = 0.0008 vs Ld = 0.001, respectively) (p < 0.05), thus supporting circulating paracrine factors as determinants of aggressiveness. Further, a panel of well-documented markers for PC aggressiveness was tested both at translational and transcriptional levels in IEC6 cells conditioned with PC cells-derived media. Notably, Galectin-3, member of lectin family, known to bind mucin glycoproteins to facilitate cell-endothelium interactions in PC metastasis, was significantly induced in Cap-2 and Mia-2-conditioned IEC6 cells.
Conclusions & Impact: We demonstrate the biological basis for duodenal alterations in PC field carcinogenesis via paracrine signaling, as a promising approach for screening.
Recent-Onset Diabetes in IPMN: An Independent Predictor of Invasive Carcinoma and Intestinal-Type Histology
V. Morales-Oyarvide,1 M. Mino-Kenudson,2 C.R. Ferrone,1 A.L. Warshaw,1 K.D. Lillemoe,1 C. Fernández-del Castillo.11Department of Surgery, Massachusetts General Hospital, Harvard Medical School, Boston, MA; 2Department of Pathology, Massachusetts General Hospital, Harvard Medical School, Boston, MA.
Background: Diabetes mellitus (DM), smoking, obesity, and recent weight loss have all been associated with pancreatic ductal adenocarcinoma. However, their role in the progression of IPMN is not well defined. Our aim was to determine their prognostic value for the presence of invasive cancer in IPMN.
Methods: We evaluated 460 IPMN resected at our institution. Demographic, clinical, radiological, and pathological characteristics were assessed. A multivariate analysis was performed adjusting for age, sex, DM, obesity, recent weight loss >5 lb (WL), smoking status, and radiological main duct involvement to identify independent predictors of invasive cancer.
Results: There were 222 (48%) males; the mean age was 67, and 101 patients (22%) had invasive cancer. Eighty-nine (19%) patients had DM, 88 (23%) were obese, 112 (41%) had WL, and 248 (54%) were smokers. On univariate analysis, DM (p < 0.001) and WL (p = 0.027) were significantly associated with invasive cancer. On multivariate analysis, DM remained statistically significant (OR 3.61, 95% CI 1.61, 8.1, p = 0.002), and its prognostic value was strongest when it was restricted to cases with recent onset (≤5 years before surgery) (OR 5.93, 95% CI 1.81, 19.4, p = 0.003). Longstanding DM (>5 and >10 years) was not associated with invasive cancer. DM was also an independent predictor of intestinal-type predominant IPMN (RRR 2.59, 95% CI 1.07, 6.26; p = 0.035), but there was no association between DM or other risk factors and the histological type of invasive IPMN (colloid vs tubular vs oncocytic).
Conclusion: Recent-onset DM in patients with IPMN is a strong predictor of invasive cancer. Smoking, obesity, and weight loss are not independently associated with invasive cancer. We suggest recent-onset DM be considered as a potential high-risk stigma of malignancy in IPMN.
Factors Associated With the Development of Infected Pancreatic Necrosis: A Cohort Study From a Tertiary Referral Center
R.A. Moran, N. Yahyapourjalaly, T. James, S. Rao, A. Kamal, V.K. Singh. Department of Medicine, Johns Hopkins University, Baltimore, MD.
Introduction: Infected pancreatic necrosis (IPN) is associated with significant morbidity and mortality. Seeding of the pancreatic bed via hematogenous spread has been postulated as a key step in its development.
Methods: A retrospective cohort study was undertaken from ’00-‘14. Patients with parenchymal necrotizing pancreatitis (NP) were identified and included for analysis. Demographic and clinical characteristics were obtained from medical records. A multiple logistical regression (MLR) model was constructed for the known and potential factors associated with the development of IPN, including: >30% necrosis on CT; use of total parenteral nutrition (TPN); bacteremia and ileus.
Results: 142 patients were identified with NP, 61 with IPN and 81 with sterile necrosis (SN). IPN patients were more likely to have been: transferred from another hospital (75% vs 54%, p = 0.01); white (85% vs 65%, p < 0.01) and of a higher mean age (55.2 ± 13.5 vs 49.9 ± 14.3, p = 0.03). There was no difference in Sex (male, 75% vs 78%, p = 0.74) and mean Charleston comorbidity index (2.5 ± 2.3 vs 1.9 ± 2.0, p = 0.11) between the two groups. Causes of NP included; gallstones (42%), alcohol (32%) and other (26%). Patients transferred after the diagnosis of IPN (n = 13) were excluded from the MLR analysis. A history of bacteremia (OR: 3.0, 95% CI: 1.1-8.1, p = 0.03) and ileus (OR: 3.6, 95% CI: 1.5-8.4, p < 0.01) 30 days prior to the diagnosis of IPN were associated with the development of IPN on MLR analysis. Although > 30% necrosis was associated with the development of IPN on univariate analysis, neither it nor the use of TPN were associated with IPN on MLR analysis.
Conclusion: These results support that bacterial seeding of the pancreatic bed is the result of hematogenous spread. Furthermore, ileus, a manifestation of the gut dysfunction in AP may be a clinical indicator for ongoing bacterial translocation that puts patients at risk for developing IPN.
Characteristics and Predictors of Large Duct Chronic Calcific Pancreatitis: A Cross Sectional Analysis From a Tertiary Referral Center
R.A. Moran, T. James, N. Yahyapourjalaly, A. Sinha, P.H. Chen, R. Hernaez, V.K. Singh. Department of Medicine, Johns Hopkins University, Baltimore, MD.
Introduction: Management paradigms for chronic pancreatitis are based on the presence of a dilated main pancreatic duct (MPD). However, the characteristics and clinical factors associated with large duct chronic calcific pancreatitis (LDCCP) are unknown.
Methods: A retrospective cross sectional analysis of only patients with chronic calcific pancreatitis (CCP) based on CT imaging was conducted from ‘00-‘12. SDCCP Small duct chronic calcific pancreatitis (SDCCP) and LDCCP were defined as a MPD diameter of <5 and ≥5 mm, respectively. Demographic and clinical characteristics were evaluated as predictors of LDCCP using univariate and multivariate logistical regression.
Results: 527 patients were identified with CCP, with 149 (28.3%) patients classified as LDCCP. LDCCP patients were more likely to have intraductal calcifications [23.1% vs 38.3%, p = <0.01], pseudocysts [13.8% vs 20.8%, p = 0.04] and MPD strictures [1.3% vs 4.7%, p = 0.02] compared to SDCCP. There was no difference in the prevalence of diabetes in either group [37.6% vs 34.2%, p = 0.47]. Steatorrhea was more likely to be associated with LDCCP [45.5% vs 70.5%, p = <0.01]. White race was the only factor found to be predictive of LDCCP (POR: 1.61, p = 0.02) in the adjusted analysis. Age as a continuous variable [POR 0.99, p = 0.05], gender [POR 0.76, p = 0.184], acute recurrent pancreatitis [POR 1.33, p = 0.25], alcohol [POR 0.69, p = 0.25], smoking [POR 0.98, p = 0.93], idiopathic etiology [OR 0.95, 95%CI: 0.44 - 2.06, p = 0.90] and genetic etiology [OR 0.77, 95% CI: 0.15 - 3.91, p = 0.75] were not predictive of LDCCP. An additional stratified analysis was performed based on race and identified no effect modifiers.
Conclusion: LDCCP occurs in a minority of patients with CCP. White race was the only predictor of LDCCP in this cohort. It is unclear if this is related to underlying genetic factors or other variables associated with race.
High-Intensity Focused Ultrasound (HIFU) Therapy Combined With Chemotherapy has an Effect of Survival Time Elongation for the Pancreatic Cancer
F. Moriyasu, A. Sofuni, T. Sano, M. Fujita, T. Itoi. Department of Gastroenterology & Hepatology, Tokyo Medical University, Shinjuku, Tokyo, Japan.
Introduction: Recently, high-intensity focused ultrasound (HIFU) has been focused on as an ablation therapy for pancreatic cancer (PC).
Aims & Methods: We have evaluated the therapeutic effects and applicability of HIFU therapy in locally advanced PC.
This study took approval by ethical committee of Tokyo Medical University.
HIFU device used was FEP-BY02 (Beijing Yuande Bio-Medical Engineering Co. LTD., China). Emission frequency of HIFU was 1 MHz and power are controlled from 500 to 2,000 W. The subjects were 54 PC patients, i.e. 35 cases in stage III, 19 cases in stage IV.
Results: All tumors were visualized by HIFU monitoring ultrasound system, which is used to navigate targeting HIFU energy on the tumor area.
Clinical data of stage III and stage IV are as follows; mean tumor size was 34.4 vs 30.8 mm, mean treatment sessions: 3.0 vs 2.5 times, mean total treatment time: 2.6 vs 2.1 hours, mean total number of irradiation: 3,024 vs 1,838 shots, respectively.
The effects of HIFU therapy in Stage III and IV are as follows; the rate of complete tumor ablation was 85.7 vs 63.2%, the rate of symptom relief effect, represented by pain relief, was 65 vs 58%, the effectiveness of primary lesion was CR:0, PR:4, SD:29, PD:2 vs CR:0, PR:3, SD:14, PD:2, primary disease control rate (DCR) more than SD was 94.3% vs 89.5%. Comparison of mean survival time (MST) after diagnosis in Stage III and IV was 35.0 vs 15.2 months, respectively (p < 0.05, p =0.021). MST after diagnosis in HIFU with chemotherapy and chemotherapy alone (38 patients in our hospital) was 29.3 vs 12.2 months, respectively (p < 0.001).
Conclusions: This study suggests that HIFU has a high potential for palliative therapy of advanced pancreatic cancer. Also, HIFU could be applied to neo-adjuvant therapy for border line unresectable pancreatic cancers.
Adverse Outcomes of Pancreatic Cysts With Fukuoka Positive Features
S. Mukewar, N. de Pretis, R. Sah, A. Aryal, N. Takahashi, S. Vege, S. Chari. Department of Gastroenterology, Mayo Clinic, Rochester, MN.
Background: Fukuoka International Consensus Guidelines for intraductal papillary mucinous neoplasm (IPMN) provide indications for surgical resection in pancreatic cystic lesions (PCL) suspected to be IPMN. However, data concerning the outcome of conservative follow-up of PCLs with high-risk or worrisome features (Fukuoka positive) are limited. In such lesions we estimated the 5-year risk of adverse cyst outcomes defined as having pancreatic resection (PR) and/or developing pancreatic cancer (PC).
Methods: From Mayo Clinic Rochester’s electronic databases we identified 2000 randomly selected patients who had PCLs diagnosed on CT, EUS or MRI. Patients with inflammatory PCL, strongly suspected non-IPMN PCL and PCL without any Fukuoka features were excluded. We re-reviewed all cross-sectional imaging and abstracted clinical and follow-up data on Fukuoka positive PCLs suspected to be IPMN (n = 313).
Results: Of the 313 patients with suspected Fukuoka positive IPMN 50.5% were males. Their mean age at baseline was 65.5 ± 14.5 years. Of these 245 (78.3%) had worrisome features and 68 (21.7%) had high-risk features. They had a median follow up of 2.9 (0 to 15.6) years after development of Fukuoka positivity. For PCLs with worrisome features the 5-year probability of undergoing PR and developing PC were 30.2% and 4.3%, and with high-risk features they were 42.5% and 33.5%, respectively. Majority of adverse events occurred within 6-months of presentation. Following 6 month follow up, the 5-year risk of PR and PC for patients with worrisome features was 13.3% and 1.3%; and high-risk features was 11% and 4.4%, respectively.
Conclusions: While one-third of subjects with presumed IPMN with high risk features will develop PC within 5 years, those with worrisome (Fukuoka) features have a low risk of developing PC in a 5-year FU. If confirmed, these data would suggest that longer follow-up intervals may be safe for those without high-risk features.
Pancreatic Cysts: Diagnostic Accuracy and Risk of Inappropriate Resections
S. Mukewar, N. de Pretis, A. Aryal-Khanal, N. Takahashi, S. Chari. Department of Gastroenterology and Hepatology, Mayo Clinic, Rochester, MN.
Background: Pancreatic cystic neoplasms (PCN) frequently undergo surgery, given malignant potential. Pancreatic cyst surgery is associated with significant rates of morbidity and mortality. It is crucial to accurately characterize these cystic lesions to avoid surgery in patients with benign pancreatic cysts.
Aim: We aimed to assess the correlation between preoperative diagnosis (imaging and clinical) and postoperative diagnosis (histopathological) in patients undergoing pancreatic cyst surgery.
Methods: From January 2000 to January 2012, we randomly selected 2000 patients with ICD-9 code 211.6 and 577.2. Amongst these we identified patients undergoing pancreas surgery (n = 281). Patients with no pre-op imaging available for review or non-cyst indication for surgery were excluded (n = 107). Imaging details, demographics, pre-operative physician diagnosis and histopathologic details of pancreatic cysts were recorded in 174 patients.
Results: In 54 (31%) there was a discrepancy between the pre and postoperative pancreatic cyst diagnosis. Those with incorrect diagnosis had similar number of pre-operative imaging studies (CT, EUS or MRI) as the group with correct diagnosis. Preoperative diagnosis was incorrect in 46% of mucinous cystic neoplasms (MCNs), 33.3% of main duct/mixed-Intraductal papillary mucinous neoplasm (IPMN), 23.7% of branched duct-IPMNs, 12.5% of serous cystic neoplasms (SCNs). Misdiagnosis of MCN was more likely compared to non-MCN cysts (48% vs 24%; p = 0.04). 14% of all the resections were performed for asymptomatic benign cysts, preoperatively suspected to be potentially pre-malignant cysts.
Conclusion: In nearly 1 out of 3 patients undergoing pancreas cyst surgery, there is a discrepancy between pre- and post- operative diagnosis. Pre-operative diagnosis of MCN is more likely to be incorrect, compared to the other cysts.
Impact of National Pancreas Foundation’s (NPF) Animated Pancreas Patient (APP) Metrics on Pancreas Education and Awareness Nation and World Wide
S. Munigala,1 J. Holt,2 T. Gardner, MD,2 M. Alsante,2 A. Gelrud.21St. Louis University, St. Louis, MO; 2National Pancreas Foundation, Bethesda, MD.
Aim: To evaluate APP as an e-learning resource, consisting of multiple formats of visual learning (animations, videos and YouTube channel), patient outreach based on number and location in the world where the videos were seen.
Background: Pancreatic diseases account for substantial morbidity, mortality and cost. There are several reports on increasing burden of pancreatic diseases; data on internet based patient education is limited.
Methods: We used NPF’s APP metrics data from Sept 2013 to May 2015. We evaluated the number of sessions, pages viewed per session and average session duration on the APP website in the United States (US) and other countries. Total number of views, min watched, avg duration of time spent on the YouTube channel. We also calculated the metrics for top views per day, media type (Animation, Expert Video, Patient Video, and Slide Show) and top retention videos on YouTube.
Results: Overall, 23627 sessions (US-14407 and other- 9220) were viewed by 153 countries on the APP website (81039 pages viewed; avg session duration 5.24 min) and 294726 views (US-123100 and other- 171626) in 209 countries on YouTube during the study period. 48.1% of the viewers were patients, 17.3% were family/caregivers, 16.7% were health care providers and the rest were others. 61.9% of the views were from the US. Role and Anatomy of the Pancreas, Chronic Pancreatitis (CP), Acute Pancreatitis, Understanding ERCP and CP: what foods and beverages should I avoid were the top 5 videos viewed per day with 97034, 73663, 52753, 11607 and 10117 views on YouTube and 3499, 2583, 5938, 1504 and 453 website views respectively. Videos under management and understanding of non-malignant pancreatic diseases had highest retention (82% to 79%).
Conclusion: APP metrics had a wide reach for pancreas education. Continued efforts should be made to disseminate accurate and up to date information in the era of the internet.
Histological Effect of Chemoradiotherapy Contributes to Securing a Surgical Margin and Improving Prognosis in Patients With Locally Advanced Pancreatic Cancer
Y. Murata, M. Kishiwada, N. Kuriyama, Y. Azumi, S. Mizuno, M. Usui, H. Sakurai, S. Isaji. Division of Hepato-biliary Pancreatic and Transplant Surgery, Mie University, Tsu, Mie, Japan.
Aim: The clinical significance of histological effect of chemoradiotherapy (CRT) remains uncertain in patients with pancreatic adenocarcinoma (PDAC). The aim is to determine outcome of CRT followed by surgery and significance of histological effect of CRT in patients with PDAC.
Method: From February 2005 to September 2014, 220 patients with cytologically/histologically proven PDAC were enrolled in the gemcitabine or gemcitabine/S-1 based CRT protocol. According to NCCN guideline, they were classified into three groups: resectable (R, n = 18), borderline resectable (BR, n = 106), and locally advanced unresectable (UR-LA, n = 96). The histological effect of CRT was evaluated using resected specimens for the patients with resection and divided into two groups: high responder (tumor destruction > 50%) and low responder (<=50%). The results were correlated with rate of R0 resection and prognosis.
Results: Among the 220 patients, 135 patients (R:11, BR:81, UR-LA:43) underwent curative-intent resection after CRT. The patients with resection had significantly higher 3-year OS rate than those with no resection (resection vs. no resection in R, BR, UR-LA: 68.6, 37.9, 19.1% vs. 0, 0, 8.3%). The overall R0 rate was 78.5% (R:100, BR:86.4, UR-LA: 58.1%), and the patients with R0 had a significantly higher 3-year OS rate than those with R 1-2 (R0 vs. R1-2: 47.4 vs. 15.9%).The overall rate of high responder was 36.3%, and high responder achieved higher R0 rate than low responder in BR and UR-LA (high vs. low in BR, UR-LA: 93.3, 80 vs. 81.6, 51.5%). In UR-LA, high responder had significantly higher 3-year OS rate than low responder (high vs. low: 46.3 vs. 6.0%).
Conclusion: Effective histological effect of CRT contributes to increasing rate of R0 resection and improving survival for patients with BR and UR-LA PDAC.
Investigating Gemcitabine-Loaded Superparamagnetic Iron Oxide Nanoparticles Against Pancreatic Cancer Cells in Artificial Circulation
S. Nandi,1 P.D. Sykes,1 E. Hasan,2 M. Barrow,2 E. Costello,1 M.J. Rosseinsky,2 J.A. Hunt,3 C.M. Halloran.11NIHR Liverpool Pancreas Biomedical Research Unit; 2Department of Chemistry, University of Liverpool, United Kingdom; 3Department of Musculoskeletal Biology, University of Liverpool, United Kingdom.
Background: Nanoparticles can provide targeted therapy for pancreatic cancer. Manufactured micellar gemcitabine-loaded superparamagnetic iron oxide nanoparticles (G-SPIONs) have shown proven cytotoxic effect against static pancreatic cancer cells in vitro. Our model attempts to replicate how G-SPIONs could behave when administered in vivo.
Aim: Determining whether G-SPIONs can be drawn from a dynamic circulation by magnetic targeting; and if they have desired cytotoxic effect upon pancreatic cancer cell lines.
Materials/methods: Complete RPMI media was circulated through specifically manufactured 25 cm2 flasks containing 2x106 monolayers of MIA PaCa-2 cells at 37 °C/5%CO2. Cells were subjected to control media or 2% G-SPIONs, with/without a magnet (MAG) for 16/24/48/72 hours. Light and fluorescent microscopy was used to visualize the monolayer and G-SPION uptake. LDH assay was performed as a measure of cell lysis.
Results: Light microscopy confirmed destruction of monolayers and co-localization fluorescent microscopy, after up to 72 hours of flow, showed cellular uptake of rhodamine-tagged G-SPIONs, especially adjacent to MAG. LDH assay optical densities were higher, indicating increased cell lysis, in flow systems subjected to G-SPIONs with use of MAG: 16 h (0.751 v 0.139 nm; p<0.001); 24 h (0.798 v 0.226; p<0.05); 48 h (1.716 v 1.375; p=0.01) and 72 h (1.645 v 1.330; p=0.11). There was no significant difference in corresponding static experiments.
Discussion: G-SPIONs can be successfully drawn from circulation and destroy monolayers. Magnetic targeting provides greatest improvements over static conditions at early time points and is still superior at 72 h. Bio-magnetic targeting in custom-made nano-vehicles provides a potential novel theranostic solution in pancreatic cancer.
Islet Neogenesis Associated Protein (INGAP) Protects β-cells From Cytokine-Induced Toxicity
E. Nano L. Rosenberg. Department of Surgery, McGill University, Montréal, Quebec, Canada.
Diabetes is a metabolic disorder characterized by a deficiency in β-cell mas, which is believed to be mediated by pro-inflammatory cytokines. This cytotoxic assault is implicated in the pathogenesis of both Type 1 and Type 2 Diabetes. INGAP-peptide, a bio-active portion of the Islet Neogenesis-Associated Protein which is currently in clinical trials, is the first therapeutic neogenic agent for diabetes, capable of regenerating insulin-producing β-cells from endogenous pancreatic cells. Presently, very little is known about the mechanism of action of INGAP and any additional effects it could possess towards restoration of β-cell mass.
The objective of this study was to determine whether INGAP has a pro-survival effect on β-cells. We used an in vitro model of cytokine-induced β-cell death, Rinm5f rat insulinoma cell line treated with a cocktail of IL-1β and IFN-γ. Effects of INGAP pre-treatment were assessed by MTT viability assay, TUNEL, immunofluorescence, Griess assay and Western blot. Our results show that pretreatment with INGAP improved cell viability by 26% (p < 0.05), decreased caspase-3 activation and cytokine-mediated apoptosis after 48 hours. Additionally, we validated this model for the production of reactive oxygen species, known to cause detrimental effects on β-cells upon cytokine toxicity. Conversely, INGAP pre-treated cells expressed 59% less iNOS protein than cytokine control after 6 hours (p < 0.001) and reduced NO concentration detected in the medium by 53% after 24 hours (p < 0.001).
Together these findings are suggestive of anti-apoptotic effects of INGAP, offering novel insight into its full range of activity. Further studies characterizing the repertoire of anti-diabetic effects of INGAP and the signaling pathways implicated are critical for optimization of its therapeutic effects.
Robotic Cystogastrostomy With Pancreatic Necrosectomy
I. Nassour1 S. Kukreja.21Department of Surgery, University of Texas Southwestern, Dallas, TX; 2Department of Surgery, VA North Texas Health Care System, Dallas, TX.
Background: Pancreatic walled-off necrosis results from either acute or chronic pancreatitis. Endoscopic and open surgical internal drainage are well established treatments while minimally invasive techniques are still evolving. Laparoscopic cystogastrostomy has been described in many reports unlike robotic assisted cystogastrostomy.
Method: We report the case of robotic-assisted cystogastrostomy in a patient with symptomatic pancreatic walled-off necrosis secondary to biliary pancreatitis. Relevant clinical information, imaging, set-up, trocar placement, and surgical technique are described in detail in this video presentation
Results: 64 year old man with a history of biliary pancreatitis six months prior presented with 4 month duration of epigastric pain, poor PO tolerance, and 60 pound weight loss. CT showed an 18cmx7cm complex collection consistent with pancreatic walled off necrosis. EUS confirmed a heterogenous fluid collection with a large amount of debris. There was no associated mass or sepatations. Patient was taken to the OR and using three 8-mm robotic ports and one camera ports, cystgastrostomy was undertaken. The stomach was obviously compressed from the cyst in the lesser sac. After an anterior gastrotomy, the pseudocyst was identified using needle aspiration. A posterior gastrotomy exposed the pseudocyst wall which was entered using the hook cautery. Cyst fluid was aspirated and pancreatic necrosum was debrided. The pseudocyst was approximated to the posterior gastric wall to create a common channel using a running barbed suture. The channel was created large enough such that subsequent endoscopic necrosectomy could be undertaken without difficulty. The anterior gastrotomy was sthen utured closed. The patient tolerated the procedure well with near immediate resolution of his PO intolerance.
Conclusion: Robotic cystgastrostomy for pancreatic pseudocyst/pancreatic walled-off necrosis is safe and feasible in selected patients.
A Single p53 Mutation May Predict Survival in Intraductal Papillary Mucinous Neoplasms of the Pancreas
J. Nicholson,1 N. Howes,1 S. Harrison,1 T. Hanna,1 L. Yan,1 B. Lane,1 E. Garner,1 K. Bullock,1 C. Halloran,1 P. Ghaneh,1 R. Sutton,1 F. Campbell,1 M.W. Büchler,2 J. Neoptolemos,1 W. Greenhalf.11Liverpool Pancreas Biomedical Research Unit, Liverpool, United Kingdom, 2Department of General Surgery, University of Heidelberg, Heidelberg, Germany.
Background: IPMN of the pancreas are increasingly common entities being detected incidentally; their risk of malignancy can be as high as 85%. Consensus guidelines exist for the management of these lesions – the morbidity associated with pancreatic resection is as high as 50%. We set out to identify a marker which could be used to identify those IPMN which should be resected and those which may be safely observed.
Methods: Frozen section of pancreatic tissue from 73 patients who underwent resection for IPMN were retrospectively assessed (in a blinded fashion) for histological diagnosis and p53 status using whole genome sequencing. Once these analyses were complete the results were matched to survival.
Results: 27 cases were found to contain tissue consistent with a diagnosis of IPMN, of these p53 status was obtained in all cases. 23 individual mutations were identified, most cases had more than one mutation. 7 cases died of pancreatic cancer after resection. Kaplan-Meier survival analysis revealed that one mutation predicted survival regardless of histology (p = <0.0001). This mutation was present in 6 of the 7 cases who died and in none of those who survived to 5 years. The mutation was then validated against all 73 samples using mutation specific PCR.
Conclusions: The identified mutation could be used to differentiate those IPMN which result in poor survival at an earlier stage to facilitate potentially curative surgery. The mutation may be present in pancreatic juice which can be collected endoscopically as a screening tool.
Radical Surgery of Pancreatic Cancer With Concomitant Resesectable Metastasis
W. Niesen, U. Hinz, O. Strobel, A. Ulrich, M.W. Büchler, T. Hackert. Department of Surgery, University of Heidelberg, Heidelberg, Germany.
Background: Surgery remains the only chance for long-term survival in pancreatic cancer (PDAC). In metastatic disease (M1), chemotherapy (expected survival 6-9 months) is considered the only treatment option. Currently, the role of surgery in oligometastatic PDAC is discussed as many institutions combine PDAC resection with a small liver resection once in a while. Aim of the study was to evaluate the outcome of M1 PDAC resections.
Methods: Prospective data of all patients undergoing primary tumor and metastasis resection for stage IV PDAC during a 12-year period were analyzed regarding localization (liver or distant interaortocaval lymph nodes; ILN), morbidity and survival and compared to patients undergoing exploration and palliative treatment.
Results: 128 patients undergoing PDAC and metastases resection (liver n = 85; ILN n = 43) and 123 patients with exploration alone and palliative therapy were included. Surgical morbidity and mortality after synchronous resection of M1 tumors were 24% and 3.4%. The number of resected liver metastases was 1 or 2 in 76% and >3 in 24% of the patients. Overall median survival after M1 resection was significantly better than after exploration alone (liver 14.0, ILN 12.3 vs. exploration 8.6 months). In addition, also 5-year survival was significantly improved after M1 resection (liver 9.7%, ILN 10.1% vs. exploration 0%).
Conclusions: The present collective is the largest series of resected metastatic PDAC and shows that resection of liver or ILN metastases can be done safely and should be considered as it may be superior to palliative treatment in selected patients. Further studies to stratify patients for these procedures are warranted.
Prediction of the Severity of Acute Alcohol Pancreatitis by Systemic Levels of Soluble Urokinase-Type Plasminogen Activator Receptor (suPAR)
A. Nikkola,1,2 A. Janne,3 H. Reetta,4 R. Linnea,2 N. Isto,1 S. Juhani,1 J. Laukkarinen.11Department of Gastroenterology and Alimentary Tract Surgery, Tampere University Hospital, Tampere, Finland; 2University of Tampere, School of Medicine, Tampere, Finland; 3Fimlab Laboratories, Tampere, Finland; 4Department of Internal Medicine, Tampere University Hospital, Tampere, Finland.
Introduction: Systemic levels of soluble urokinase-type plasminogen activator receptor (suPAR) increase in various inflammatory and infectious diseases. Our aim was to study the activation and prognostic value of plasma suPAR in patients with first acute alcohol pancreatitis (AAP).
Methods: P-suPAR concentrations were measured from 104 patients with their first AAP during hospitalization and again 6–24 months after discharge. The severity of AAP was classified according to the revised Atlanta criteria.
Results: AAP was moderately severe in 29 (28%) and severe in 6 (6%) patients. For further analysis, the moderately severe and severe AAP were combined (non-mild AAP; n = 35, 34%). P-suPAR levels were significantly higher in patients with AAP during hospitalization than after discharge (median 4.8 ng/mL vs. 3.1 ng/mL; p < 0.001). Furthermore, P-suPAR levels were higher in non-mild compared to mild AAP (6.2 ng/mL vs. 4.2 ng/mL; p < 0.001). When the analysis was made within 1–4 days after admission (n = 68) AUC was 0.81 (95% CI, 0.70–0.92). At a cut-off level of 5.0 ng/mL, sensitivity and specificity to predict non-mild AAP were 79% and 78%, respectively. At a lower cut-off level (3.7 ng/mL), 35% of the patients received values below cut-off with sensitivity and specificity of 100% and 44%, respectively. P-suPAR was found to be a better prognostic marker for the severity of AAP than CRP, hematocrit or creatinine.
Conclusions: P-suPAR concentrations are elevated in AAP and correlate with the severity of the disease. These results suggest that P-suPAR may have potential to serve as a novel prognostic marker for AAP severity on admission.
The Current Management of Mucinous Cystic Neoplasms (MCN) of the Pancreas: A Systematic Review
L.N. Nilsson,1 A. Antila,2 J. Millastre Bocos,3 M. Marijinissen Van Zanten,4 A. Shamali,5 C. Verdejo Gil,6 M.G. Keane,7 J. Laukkarinen,2 M. Del Chiaro.11Department of Surgery, Karolinska University Hospital, Solna, Sweden; 2Tampere University Hospital, Tampere, Finland; 3Miguel Servet University Hospital, Zaragoza, Spain; 4Nijmegen University Hospital, Netherlands; 5Southampton University Hospital, Southampton, United Kingdom; 6Ciudad Real University Hospital, Real, Spain; 7Institute for Liver and Digestive Health, University College London, United Kingdom.
Background: The current management of MCN is defined by the consensus European and IAP guidelines which differ in criteria for surgical resection.
Aim: We performed a systematic literature review to define stronger evidence for the management of MCN.
Materials & Methods: A systematic review of existing literature from MEDLINE and EMBASE databases from 1970 to current.
Results: The systematic literature search resulted in 181 papers. After limiting to humans and English language, and excluding 87 papers following title and abstract review, 52 papers were analyzed. MCNs occur predominantly in middle-aged women (female: male 20:1), often found incidentally and usually located in the body or tail (93-95%). The current diagnostic tools (CT, MRI and EUS) are imperfect in differentiating MCN from branch duct IPMN and oligocystic serous adenomas. According to the literature the pre-operative accuracy in defining MCN is approximately 60% and generally not improved by cytology. In resected MCNs 12-23% were malignant, however in lesions <4.5 cm only 0.03% were malignant. If benign no follow-up is required after surgical resection (unifocal disease and recurrence not reported). After resection of invasive MCNs the 5 year survival rate was around 62.5% in large surgical series.
Conclusion: MCNs are solitary lesions in the body or tail, predominantly occurring in middle-aged women. They show low aggressive behavior when small, malignancy rate of almost nil when diameter <4.5 cm. Our systematic review supports the European Guidelines on the more conservative management.
Analysis of Clinicopathological Features of Early Recurrence After Curative Resection for Pancreatic Cancer
K. Nishio, K. Kimura, R. Amano, K. Miura, G. Ohira, S. Yamazoe, M. Ohira, K. Hirakawa. Department of Surgical Oncology, Osaka City University Graduate School of Medicine, Osaka, Japan.
Background: Early recurrence cases after resection of resectable pancreatic cancer were often found and prognosis of these cases were poor. Some reports said that the presence of minimally distant metastasis at the time of surgical resection caused early recurrence. If we would predict the factors for early recurrence of resected RPC, we should perform preoperative therapy rather than surgical resection for them.
Aim: We analyzed the clinicopathological factors and recurrence patterns within a year. Moreover, we consider the indication of preoperative therapy for RPC.
Method: From January 2000 to December 2014, we experienced 90 cases of RPC and carried out retrospective analysis. 32 cases recurred within a year (ER) and 58 cases survived with no recurrence over a year (NR).Of the 58 with NR, recurrences were observed in 28 cases (48.3%). Clinicopathological factors were assayed univariate analysis by Fisher exact test and multivariate analysis by multiple logistic analysis. Survival was calculated by Kaplan-Meier method and was compared for each group using log-rank test. Values of P < 0.05 were considered statistically significant.
Result: Univariate and multivariate analysis demonstrated G2-G4 in histological grade and preoperative CA19-9 ≥ 529 were significantly associated with early recurrence. 22 cases (68.8%) of ER had distant metastasis, whereas 6 cases (21.4%) of 28 cases with recurrence among NR had distant metastasis. Distant metastasis were more frequently observed in ER than NR (p < 0.001).
Conclusion: Even though curative resection was performed for RPC, ER was observed in cases of 35.6%. Recurrence pattern of ER was often more distant metastasis, which might imply that distant metastasis existed at the time of operation. G2-G4 in histological grade and preoperative CA19-9 ≥ 529 were predictors of early recurrence, and preoperative treatment might be suitable for such patients.
Circulating Inflammatory Cells (CICs) Undergo Lipotoxic Cell Death During Severe Acute Pancreatitis (SAP)
P. Noel, K. Patel, R.N. Trivedi, C. de Oliveira, A. Singh, V.P. Singh. Department of Medicine, Mayo Clinic, Scottsdale, AZ.
Background: We have previously reported multi system organ failure (MSOF) during SAP in obesity (PMIDs: 25500204, 25579844, 22049070) resulting from unsaturated fatty acid (UFA) mediated acute lipotoxicity. During SAP there is cell death and significant local and systemic injury. However the increase in inflammatory cell number does not mirror severity (PMIDs: 25500204) and leucopenia is a part of severe inflammatory response syndrome (SIRS) in SAP. We thus studied whether CIC cell death in SAP and the agents mediating this.
Methods: For AP induction rats received IP Cerulein (CER, 50 mcg/kg BID) ± glyceryl trioleate (GTO; 3gm/100gm) ± orlistat (50 mg/kg/day) and CICs were isolated. Rat peripheral blood mononuclear cells (PBMCs) were incubated with UFAs or saturated FAs (10μM), or cytokines IL-1β and KC/GRO (200ng/ml). CICs or PBMCs were immuno-phenotyped, stained for annexin V (AnxV+) and quantified using flow cytometry. Values depicted represent means±SEM and a p<0.05 indicates statistical significance.
Results: High levels of free fatty acids were noted in CER+GTO (539±116μM vs 252±91μM in controls p<0.003) with 97% mortality. In rats only the CER+GTO group had increased AnxV+CD11b+ (7.8±1.8% vs. 1.2±0.2% in controls) and AnxV+CD3+ (7.8±1.9% vs. 1.3±0.3% in controls, p<0.02) cells. At the time of mortality CER+GTO rats had a significant decrease in total white cell counts (3.5±0.5×103mm−3 vs. 12.2±1.2×103mm−3 in control, p<0.02). AnxV+ staining was not significantly increased in F4/80+ macrophages. Only UFAs increased AnxV+ staining (56.2±10.1% vs. 1.6±0.4% in controls, p<0.03) in PBMCs along with increased propidium iodide staining suggestive of necro-apoptotic cell death (7.2±0.2% vs. 0.4±0.1% in controls, p<0.03).
Conclusions: CICs may undergo necro-apoptotic cell death due to UFAs generated during SAP. This may result in the leucopenia noted in SIRS. Presence of injured CICs may be a valuable predictive marker of severity and MSOF in SAP.
Minnelide Decreases Neural Invasion and Pain Signaling in Pancreatic Cancer
A. Nomura, K. Majumder, S. Modi, P. Dauer, N. Aurora, S. Banerjee, S. Roy, A.K. Saluja. Department of Surgery, University of Minnesota, Minneapolis, MN.
Introduction: Cancer cell invasion into the neural spaces is especially common in pancreatic cancer, clinically correlating with poor prognosis and local recurrence. Pancreatic cancer neural invasion has also been associated with the severe neuropathic pain experienced by up to 85% of patients.
Aim: The aim of this study was to determine the efficacy of Minnelide against pancreatic cancer and neural cell signaling cross-talk, invasion, and pain signaling.
Materials & Methods:In vitro co-culture and Boyden chamber invasion assays of human (MIA PaCa-2 and S2-VP10) and mouse (KPC-tumor derived cell line) and mouse dorsal root ganglia (DRG) primary cultures were used to determine changes in gene expression and invasion of pancreatic cancer cells in the presence of neural cells. In vitro DRG Matrigel outgrowth and in vivo sciatic nerve assays were utilized for the effect of Minnelide on invasiveness. Control and Minnelide treated KPC mice were used
Results: Pancreatic cancer cell lines co-cultured with DRG, upregulated several genes associated with invasion (SNAI1, SNAI2, TWIST1, ZEB1, CDH2, VIM, MMP7) as well as in vitro invasiveness. However, with triptolide treatment these genes were drastically reduced and invasion inhibited. DRGs treated with triptolide decreased neural growth factors (NGF, Artemin, and BDNF) and pain signaling molecule (Substance P) and inhibited the neural outgrowth in Matrigel. Treatment in vivo of Minnelide in the KPC tumor model decreased the presence of mast cells, which has been shown to clinically correlate with pain.
Conclusions: Minnelide reduces neural signaling growth factors and pain signaling in DRG primary culture and in vivo within the KPC tumor model.
MMPs-7, −8, −9 and TIMP-1 in Acute Pancreatitis
E. Nukarinen,1 O. Lindström,2 K. Kuuliala,3 L. Kylänpää,2 V. Pettilä,1 P. Puolakkainen,2 A. Kuuliala,3 M. Hämäläinen,4 E. Moilanen,4 H. Repo,3 J. Hästbacka.11Department of Anesthesiology and Intensive Care Medicine, 2Department of GI Surgery, 3Department of Bacteriology and Immunology, Helsinki University Hospital and University of Helsinki, Helsinki, Finland; 4The Immunopharmacology Research Group, University of Tampere, Tampere, Finland.
Background: Systemic levels of matrix metalloproteinases (MMPs) and their tissue inhibitor-1 (TIMP-1) have found to be elevated in critically ill patients and to associate with outcome.
Aim: To investigate systemic levels of MMPs and TIMP-1 in AP patients, and to analyze their ability to detect severe AP.
Patients and Methods: This was a prospective study of 176 non-consecutive patients with AP admitted to Helsinki University Hospital between 2011 and 2014. Plasma samples from 23 severe, 25 moderately severe and 128 mild AP patients, according to the revised Atlanta classification, and 32 healthy volunteers were analyzed for MMP-7, −8, −9 and TIMP-1 by ELISA. The patient samples were taken within 96 hours after the onset of symptoms.
Results: MMPs and TIMP-1 levels were higher in AP patients than healthy volunteers (p<0.001). MMP-8 levels were significantly higher in severe than in mild (p<0.001) or moderately severe cases (p=0.013). The AUC-ROC values (with confidence intervals) for MMP-7, −8 and −9, and TIMP-1 to detect severe AP patients were as follows: 0.590 (0.465-0.714)(p=0.175), 0.939 (0.894-0.984)(p<0.001), 0.722 (0.606-0.838)(p=0.001) and 0.737 (0.614-0.860)(p<0.001). In predicting severe AP the MMP-8 cutoff value for optimal classification was 16.53 ng/ml and at this point the sensitivity was 86%, the specificity 91% and the positive likelihood ratio 10.05 (95% CI 5.3-15.39).
Conclusion: We found that MMP-7, −8, −9 and TIMP-1 levels were significantly higher in AP patients than healthy volunteers. High plasma levels of MMP-8 may predict development of severe AP.
Preoperative CT Assessments of the Pancreas Predict Fatty Liver After Pancreaticoduodenectomy
K. Ohgi, Y. Okamura, Y. Yamamoto, T. Sugiura, T. Ito, R. Ashida, K. Uesaka. Division of Hepato-Biliary-Pancreatic Surgery, Shizuoka Cancer Center, Shizuoka, Japan.
Background: Fatty liver (FL) after pancreaticoduodenectomy (PD) has been attracting attention. Although pancreatic exocrine insufficiency has been reported to be a main cause of FL after PD, a clinically practical examination to assess the pancreatic exocrine function has not been established. The aim of this study was to evaluate risk factors for FL after PD, with a focus on preoperative CT assessments of the pancreas.
Methods: A retrospective review was conducted using the data of 138 patients who underwent PD for primary pancreatic tumor. They were followed for more than six months after PD. FL was defined as a liver-to-spleen attenuation ratio less than 0.9 on postoperative unenhanced CT images around six months after PD. We evaluated several pancreatic CT parameters, including the pancreatic parenchymal thickness, pancreatic duct-to-parenchymal ratio and pancreatic attenuation on preoperative CT images.
Results: There were 83 (60%) males and 55 (40%) females, with a median age of 68 years (range, 38–88 years). Of these patients, 113 (82%) had invasive ductal carcinoma, 21 (16%) had intraductal papillary-mucinous neoplasm, two (1%) had neuroendocrine tumor and two (1%) had serous cystadenoma. The incidence of FL after PD was 30.4%. A multivariate analysis identified three independent risk factors for FL after PD: preoperative pancreatic attenuation of < 30 Hounsfield Units (Odds ratio [OR] 8.86, p < 0.001), a female gender (OR 6.41, p < 0.001) and a preoperative serum CA 19-9 level of ≥ 160 U/mL (OR 4.65, p = 0.003).
Conclusion: The degree of preoperative pancreatic attenuation significantly influenced the development of FL after PD. However, correlation between pancreatic attenuation and pathological changes has not been fully elucidated, which should be further investigated.
Therapeutic Management of Disconnected Pancreatic Duct Syndrome (DPDS)
H. Okamoto,1,2 J. Itakura,2 H. Fujii.21Department of Surgery, Tsuru Municipal Hosital, Yamanashi, Japan; 2Department Gastrointestinal Surgery, University of Yamanashi, Yamanashi, Japan.
Introduction: Disconnected pancreatic duct syndrome (DPDS) is a complication of necrotizing or traumatic pancreatitis.
Purpose: Therapeutic management of the DPDS, including endoscopic intervention and surgical exploration, is clarified on the basis of our clinical experience.
Patients and Methods: Three traumatic DPDS patients; Case 1; A 17-year-old man received an abdominal trauma in a rugby game, revealing the pancreatic neck laceration on CT. Case 2; A 34-year-old man received a blunt abdominal trauma caused by a car traffic accident, showing the laceration of the pancreas head with surrounding hematoma on CT. Case 3; A 32-year-old woman received an trauma by falling down the stairs, revealing the laceration of the pancreas body on CT. Each patient was treated and managed by endoscopical intervention or surgical exploration.
Results: Endoscopic pancreatic duct stent was chosen in pancreatic neck injury of Case 1. The stent treatment was succeeded and the patient was uneventfully healed.
Surgical exploration of external drainage and bilary drainage was initially conducted in Case 2. Subsequently, the longitudinal pancreaticojejunostomy was carried out 10 months after the initial operation. The pancreatic function was preserved.
Distal pancreatectomy was performed in Case 3. Although postoperative pancreatic fistula (Grade B) was occurred, the patient was healed.
Discussion: Proximal DPDS; incomplete disruption of the MPD or complete disruption of the MPD without the obstruction would be the best candidate for the endoscopic stent therapy. Complete disruption of the duct with the obstruction on the condition of the difficulty of stent placement, surgical exploration should be considered. Distal DPDS; on the condition of the difficulty of conservative therapy or stent placement, surgical operation such as distal pancreatectomy should be chosen.
Potential Biomarkers of Pancreatic Ductal Adenocarcinoma-Associated Diabetes
L. Oldfield,1 C. Jenkinson,1 T. Purewal,2 R. Sutton,1 J. P. Neoptolemos,1 W. Greenhalf,1 E. Costello.11National Institute for Health Research, Liverpool Pancreas Biomedical Research Unit, Royal Liverpool University Hospital, United Kingdom; 2Department of Diabetes and Endocrinology, Royal Liverpool University Hospital, United Kingdom.
Introduction: Cancer-related diabetes can precede PDAC diagnosis by up to 3 years. Only 1% of patients with new-onset diabetes have PDAC, it is therefore necessary to differentiate pancreatic cancer-associated diabetes (type IIIc) from the more common type II, for screening to be viable in this high risk group.
Aim: To investigate whether asymptomatic PDAC patients, newly diagnosed with diabetes, can be distinguished from individuals newly-diagnosed with type II diabetes.
Methods: Discovery work was performed using Luminex and isobaric tags for relative and absolute quantification (iTRAQ) on 161 pre-diagnosis and PDAC samples. Ingenuity pathway analysis (IPA) on proteins from discovery work identified those associated with metabolic disease pathways and diabetes. Candidate biomarkers were assessed using immunoassays on 135 serum samples categorized into various disease groups; PDAC (with/without diabetes), chronic pancreatitis (with/without diabetes), long-term type II diabetes, and healthy controls.
Results: A discovery program revealed 141 markers for early detection of PDAC. IPA found an association with metabolic disease pathways in 45 of the 141 possible candidate proteins identified. Twenty-five of these were significantly enriched for an association with diabetes. Serum levels of several candidate biomarkers significantly differed in pancreatic cancer-associated diabetes (PDAC-DM) compared with diabetes (DM), further comparison of markers revealed a difference between the protein profile of PDAC and PDAC-DM patients to that of type II diabetics.
Conclusions: Proteins associated with metabolic disease may behave differently in type II and type IIIc diabetes, a comprehensive analysis will be necessary to identify a type IIIc specific protein profile.
The Role of Receptor Interacting Protein Kinase 1 (RIPK1) in Fatty Acid Ethyl Ester (FAEE)-Acute Pancreatitis
Y. Ouyang,1,2 L. Wen,1,2 J. Bertin,3 P.J. Gough,3 R. Mukherjee,2 R. Sutton,2 D.N. Criddle.1,21Department of Cellular and Molecular Physiology, University of Liverpool, Liverpool, United Kingdom; 2NIHR Liverpool Pancreas Biomedical Research Unit, Royal Liverpool University Hospital, Liverpool, United Kingdom; 3Pattern Recognition Receptor Discovery Performance Unit, Immuno-Inflammation Therapeutic Area, GlaxoSmithKline, Collegeville, PA.
Background: Recent studies demonstrating the involvement of RIPK1 in programmed cell death have highlighted its importance as a potential therapeutic target in diverse pathologies. We have recently developed a new experimental model of alcoholic acute pancreatitis based on non-oxidative metabolism of ethanol (FAEE-AP), however, the potential involvement of RIPK1 is unknown.
Aim: To assess the effects of novel and established RIPK1 inhibitors in alcoholic acute pancreatitis using in vivo and in vitro approaches.
Methods: The established RIPK1 inhibitor necrostatin 1 (Nec-1; administered via minipump) was tested in in vivo FAEE-AP (induced by 1.35g/kg ethanol and 150 mg/kg palmitoleic acid (POA) injections) in C57BL/6J mice. The effects of Nec-1, Nec-1s and novel RIPK1 inhibitors (GSK’963A, GSK’728A and GSK’481A) were compared on palmitoleic acid ethyl ester (POAEE)-induced necrosis of isolated murine pancreatic acinar cells (PAC) using confocal microscopy (Propidium iodide).
Results: In vivo studies showed that histopathological scoring (edema, infiltration and necrosis) and biochemical parameters (MPO in lung and pancreas, serum amylase) at 24 hours after AP induction were significantly decreased in FAEE-AP in the Nec-1 treatment group compared with controls (≥6 mice/group). Nec-1 and Nec-1s (10μM) decreased POAEE-induced necrotic cell death in vitro from 24.17±1.48% to 13.68±2.34% and 12.29±1.25%, respectively (p<0.05; n=3). GSK’963A (10μM) reduced POAEE-induced necrotic cell death from 18.51±2.81% to 11.64±0.83%, whereas GSK’728A and GSK’481A (10μM) were without effect (n=3).
Conclusion: RIPK1 plays an important role in FAEE-AP, involving pancreatic acinar necrotic cell death pathway activation. Development of novel RIPK1 inhibitors may therefore provide potential for therapy in acute pancreatitis.
Acute Pancreatitis is Diminished by a Pre-treatment With the Hepatocyte Growth Factor in a Mouse Model
M. Palestino-Dominguez,1 M. Peláez-Luna,2 R. Lazzarini,3 L.E. Gomez-Quiroz,1 J. Marquardt,4 M.C. Gutierrez-Ruiz.11Department of Health Sciences, Universidad Autonoma Metropolitana-Iztapalapa, Mexico City, Mexico; 2Department of Gastroenterology, INCMNSZ, Mexico City, Mexico; 3Department of Biology of Reproduction, Universidad Autonoma Metropolitana-Iztapalapa, Mexico City, Mexico; 4Department of Internal Medicine, Johannes Gutenberg University I, Mainz, Germany.
Introduction: Oxidative stress plays an important role in the pathogenesis of acute pancreatitis (AP). Hepatocyte Growth Factor (HGF) induces protection by triggering an antioxidant response trough the transcription factors NF-κB and Nrf2. Evidence indicates that HGF is increased in AP and can protect against free fatty acids toxicity.
Aim: To address the protective effect induced by HGF in a mouse model of cerulein (Cn)-mediated AP.
Methods: Male CD1 mice, 12-14 weeks-old were randomized into groups to receive (I) 50 μg/kg, at 1 h intervals by 8 consecutive intraperitoneal (ip) injections and (II) HGF (20 μg/kg), iv, 12 h prior and just before the first injection of Cn. Mice were sacrificed 24 h after the first dose of Cn.
Results: Treatment with HGF decreased the ratio pancreas/body weight versus Cn treatment as well as serum amylase activity. Nrf2, NF-κB, AP1 were activated by HGF, judged by EMSA analysis, leading to the modulation of antioxidant and anti-apoptotic proteins such as GSTM, SOD1, Gpx1/2, HO-1, Bax, p38 Mcl1 respectively. In addition, ROS increment induced by Cn was abrogated by HGF treatment. Conclusions: Our data strongly support that HGF display a protective response against Cn-induced AP.
Intralobular Fat and Fibrosis Predict Development of Fistula After Pancreatic Resection
R. Pannala,1 V. Kommineni,1 K. Patel,1 D. Lam-Himlin,2 A. Mathur,3 N. Katariya,3 A. Moss,3 M. Crowell,1 V. Singh.1Divisions of 1Gastroenterology, 2Pathology, 3Surgery, Mayo Clinic, Scottsdale, AZ.
Background/Aims: Postoperative pancreatic fistula (POPF) occurs in 10-20% of patients after pancreatic surgery. Mechanical properties of pancreatic tissue, including fat and fibrosis influence POPF development. Lipolysis and consequent tissue damage from fatty acids may influence POPF development. We thus objectively quantified percentage fat and fibrosis at resection margins and assessed the relationship between these parameters and POPF development.
Methods: We included all patients who underwent pancreatic resection at our institution from January 2010 -December 2013 (n=152); demographic and clinical data, including POPF development were abstracted. Pathology slides from the resection margins were retrieved; serial sections stained with H&E and Masson’s trichrome were scanned into an electronic image format. In a blinded fashion, the pixel area of interlobular, intralobular, and total pancreatic fat and fibrosis were measured and calculated as % of total area. Multivariate logistic regression was performed to assess the relationship between fat, fibrosis, and POPF.
Results: Mean age and BMI were 65±13 years and 28±6 kg/m2; 53% were female. 48% and 43% underwent a pancreaticoduodenectomy and a distal pancreatectomy respectively. POPF was noted in 33/152 patients (22%). POPF patients, compared to those without POPF, were younger (59±13 vs. 67±12 yr, p<.01), had higher BMI (30±5 vs. 28±6 kg/m2, p<.01), higher % intralobular fat (4.7±2.3 vs. 3.1±1.8, p<.01) and lower % fibrosis (17±12 vs. 30±19, p<.01). In multivariate analyses, % intralobular fat was an independent positive predictor (p<.01) and % fibrosis was a negative predictor (p<.01) of POPF. Interlobular and total fat % were not significant predictors of POPF.
Conclusions: Accurate assessment of intralobular fat and fibrosis at pancreatic resection margins may help predict POPF. Evaluation of mechanical versus chemical role of these factors may allow for preventative and therapeutic strategies.
A Patient Specific PA Bio-Bank to Improve Personalized Treatment and Understand Chemotherapy Resistance Mechanism
E.M. Parasido,1 P. Sripadhan,2 J. Brody,3 R. Schlegel,2 J. Winter,3 C. Albanese.1,21Department of Oncology, Georgetown University, Washington, DC; 2Department of Pathology, Georgetown University, Washington, DC; 3Department of Surgery, Thomas Jefferson University, Philadelphia, PA.
Background: Pancreatic adenocarcinoma (PA) is the fourth leading cause of cancer related death in the USA and the 5 years mortality rate remains at approximately 94%. Therefore, a better understanding of PA tumor progression and resistance mechanisms will enhance treatment choice and clinical response. Much of the data available on PA drug targets and efficacy come from commercially available pancreatic cell lines and this represents the main limitations in PA research. In fact, these lines suffer from being unable to recapitulate a given patients’ tumor, underscoring the need for patient-specific primary epithelial PA cells.
Methods: Patients’ biopsies were collected after surgery and long-term cultures of PA cells were established using the conditional reprogramming of cells (CRCs) approach and have been continuous culture for over 6 months. The PA origin of the patient sample and matched CRC lines were verified by KRAS sequencing. CRC karyotyping was also performed. To assess patient-specific differences in treatment response, the IC50’s for Gemcitabine and Abraxane were determined. To better understand treatment resistance mechanisms, several drug-resistant clones were established and their resistance verified.
Results: We established KRAS-mutant primary cell lines derived from patients’ PA specimens. Their proven genetic stability, the differences between patients sensitivity to FDA approved drugs and the possibility of further investigation on resistance mechanism confirmed the power of this technology for on-demand in vitro use.
Conclusion: The establishment of a living PA biobank will enhance personalized medicine and will enable the high-throughput experiments necessary to better understanding drug sensitivity and resistance.
Risk Factors Associated With Early Readmission Following Sentinel Admission With Acute Pancreatitis
J. Park, J. Kuo, J. Skeans, A. Hinton, S. Krishna, D. Conwell, P. Hart. The Ohio State University Wexner Medical Center, Columbus, OH.
Background: Acute pancreatitis is the most common gastrointestinal cause of hospitalization in the United States and responsible for substantial healthcare costs. Readmissions are relatively common, but our ability to identify patients at high risk remains limited. We sought to determine clinical factors associated with early readmission following a sentinel admission with acute pancreatitis.
Methods: We analyzed consecutive admissions for a first episode of acute pancreatitis at our medical center from December 2012 through December 2013. Early readmissions were defined as an evaluation in the emergency department or inpatient admission within 30 days of hospital discharge. Univariate and multivariate analyses were performed to identify risk factors for early readmission.
Results: A total of 132 patients admitted with a sentinel episode of acute pancreatitis during the study period were selected for analysis. Early readmissions occurred in 22 (17%) patients. Factors associated with an increased risk of early readmission included: younger age at admission, antiemetic ordered within 24 hours of discharge, antibiotics administered during admission, and increase in body weight (>5kg) during admission. Younger age and excessive weight gain remained significant factors in a sensitivity analysis, excluding all hospital transfer patients. Likewise, using a multivariate model, age (adjusted OR 0.78, 95% CI 0.66-0.92, p<0.01; per 5 year increase) and weight gain (adjusted OR 4.08, 95% CI 1.32-12.58, p=0.01) were associated with the risk of early readmission.
Conclusion: Early readmissions are relatively common following a sentinel episode of acute pancreatitis. A younger age at diagnosis and excessive weight gain during the sentinel admission were associated with early readmissions. Additional investigation into the association of these and other novel factors with early readmission is warranted and could lead to decreased healthcare costs.
Vascular Enhancement Pattern of Mass in Computed Tomography May Predict Chemo-Responsiveness in Advanced Pancreatic Cancer
J. Park,1 S. Kim,1 S. Jeong,1 D.H. Lee,1 H.J. Choi,2 J.H. Moon,2 C. Kwon.31Department of Internal Medicine, Inha University School of Medicine, Incheon, Korea; 2Digestive Disease Center and Research Institute, Department of Internal Medicine, SoonChunHyang University School of Medicine, Bucheon, Korea; 3Digestive Disease Center, CHA Bundang Medical Center, CHA University, Seongnam, Korea.
Background/Aim: Chemo-responsiveness in pancreatic cancer with liver metastases may be different among the patients. The purpose of this study is to assess vascular enhancement of pancreatic ductal adenocarcinoma with liver metastasis and to analyze the correlation between enhancement and chemo-responsiveness.
Patients and Methods: From January 2007 to May 2013, 58 patients with advanced pancreatic adenocarcinoma were selected in three institutions. Chemo-responsiveness of all patients was assessed with CT before and after receiving at least 3 cycles of gemcitabine-based chemotherapy. Hounsefield unit (HU) was measured in pancreatic mass and the largest metastatic liver mass using region of interest (ROI). HU difference between arterial and pre-contrast phase (Delta value) could reflect vascular enhancement. According to chemo-responsiveness, the patients were divided into 2 group, Responder group (Partial response and stable disease) and Non-responder group (Progressive disease).
Results: Total 58 patients (28 men and 30 women; median age 64 years; range 41-84 years) were included in the study. 41 patients belonged to pancreatic responder group (70.7%) and 17 patients belonged to pancreas non-responder group (29.3%). Mean delta value (± SD) in pancreas responder group and non-responder group was 48.9 ( ±16.8) and 24.6 ( ± 14.2) (p < 0.001). 29 of 58 patients had liver metastasis. 19 of 29 patients belonged to liver responder group (65.5%) and 10 patients belonged to liver non-responder group (34.5%). Mean delta value in liver responder group and non-responder group was 36.68 ( ± 11.04) and 12.30 ( ± 4.00) (p=0.031). Four patients showed inconsistency between pancreas and liver chemo-responsiveness. They all belonged to pancreas responder group (Median delta value 35; range 30-44) and liver non-responder group (Median delta value 13; range 6-16).
Conclusion: Vascular enhancement pattern of the mass in CT may predict chemo-response in pancreatic cancer with liver metastasis. This prediction can play a role in planning individualized treatment.
Examining the Effect of Anacardic Acid Alone or in Combination With Chemotherapeutics on Pancreatic Cancer Cells
M. Park,1 M.M. Blackmon,1 S. Craver,1 V.S. Eversole,2 D. Upton.31Kentucky College of Osteopathic Medicine, University of Pikeville, Pikeville, KY; 2Department of Biology, University of Pikeville, Pikeville, KY; 3Department of Chemistry, University of Pikeville, Pikeville, KY.
Introduction: Pancreatic cancer is the 4th leading cause of cancer related death and its incidence has risen steadily. Although anti-cancer drugs have been developed based on the new molecular findings, the chemotherapy has produced unsatisfactory results due to chemotoxicity and chemoresistance. Thus, a new therapeutic regimen is in urgent need for pancreatic cancer patients.
Aim: The specific aims of the present study are to investigate the anti-tumor effects of Anacardic Acid (AA) on pancreatic cancer cell growth, to examine the synergistic effect of AA on chemotherapeutics Gemcitabine (GEM) or 5-Fluorouracil (5-FU), and to explore the underlying molecular mechanisms of AA.
Methods: After treatment with AA, GEM or 5-FU individually or in combination with AA and the chemotherapeutics, we determined cell viability with cell number count and MTT assays. In addition, we performed 3D spheroid formation assays to examine the effect of AA alone or in combination with the chemotherapeutics in a tumor environment. Western blot analysis and immunostaining were performed to investigate the underlying mechanisms of AA.
Results: Our preliminary data indicates that AA induced growth inhibition in pancreatic tumor cells in cell viability and 3D spheroid formation assays. In addition, AA potentiated the anti-tumor activity of Gemcitabine or 5-Fluorouracil in cell viability and spheroid formation assays. Mechanistically, AA appears to exert its anti-tumor activity via the activation of tumor suppressors, Chromatin Modifying Protein 1A (Chmp1A), Ataxia Telangiectasia Mutated (ATM), and p53.
Conclusion: Our data suggests that cashew nuts and mangos, which contain a number of Anacardic Acids, are promising complementary supplements to prolong the poor survival of pancreatic cancer patients.
Ethanol Worsens Acute Pancreatitis (AP) via Increasing Bioavailability of Fatty Acids (FA), and Not via Fatty Acid Ethyl Esters (FAEE)
K. Patel,1 P. Noel,1 A. Singh,1 C. Durgampudi,2 C. de Oliveira,1 R. Trivedi,1 V.P. Singh.11Department of Medicine, Mayo Clinic, Scottsdale, AZ; 2Department of Medicine, UPMC Passavant, Pittsburgh, PA.
Background: Alcohol consumption is a major risk factor for AP. Free fatty acids (FFA) are known to increase systemically in patients with severe acute pancreatitis (SAP). However, the role of ethanol and its ethyl esters in lipotoxic AP is ambiguous. In this study, we measured the impact of ethanol in FFA bioavailability and compared the relative toxicity of FFAs vs FAEEs in two mechanistically distinct AP models in rodents and in vitro
Methods: Mouse pancreatic acini were exposed to oleic acid (OA) or ethyl oleate (EO) in vitro. OA or EO were injected intraperitoneally (200 μl/30gram), with/out ethanol (100 μl/30gm) in CD1 mice and the parent triglyceride of OA-glyceryl trioleate (GTO) or EO were injected in the pancreatic duct in rats (50 μl/100gm). Outcomes included parameters for local and systemic injury. Values are reported as means±SEM and a p-value <0.05 was considered significant.
Results: OA but not EO caused LDH leakage, drop in ATP levels, cytochrome C leakage, mitochondrial depolarization and increased cytosolic calcium levels; resulting in acinar necrosis in vitro. Intraperitoneal OA caused 100% mortality (8/8) by 72 hours vs. EO (0/8, p<0.01). OA significantly elevated serum (Sr.) amylase (5795±129 vs. 515±35 U/L, p<0.01) and lipase (3032±1058 vs. 95±54U/L, p<0.01) vs. EO and increased pancreatic necrosis (14.2±4 vs. 0.6±0.5%, p<0.01) and the periphery of the pancreas. OA but not EO caused significant hypocalcemia (Sr.Calcium 7.4±0.3 vs. 8.9±0.3mg/dl, p=0.01) and lung, kidney injury (Sr.BUN 232±48 vs. 28±3.4mg/dl, p<0.01). Ethanol administration with OA increased sr. amylase, Lipase, sr. FFA’s more than OA alone (7.4±1.1 vs. 2.7±0.5mM, p=0.02), and caused earlier mortality (8 hours vs. 72 hours). Intraductal GTO caused worse local pancreatic necrosis, multisystem organ failure (MSOF), inflammation and mortality vs. EO.
Conclusions: FA’s are more potent than their ethyl esters in causing acinar necrosis, mortality, systemic inflammation and MSOF. Ethanol increases the bioavailability and related adverse outcomes of FFAs.
Increased Visceral Unsaturated Triglycerides (UTG) Result in Severe Acute Pancreatitis (SAP) in Diet Induced Obesity (DIO)
K. Patel, A. Singh, P. Noel, R. Trivedi, C. de Oliveria, V.P. Singh. Department of Medicine, Mayo Clinic, Scottsdale, AZ.
Background: Several studies from countries with high unsaturated fat consumption report SAP at lower BMI’s. Unsaturated fatty acids (UFA) are enriched in human pancreatic necrosis collections and worsen AP (PMIDs-25500204, 22049070). We thus, studied the impact of obesity induced by a UFA rich diet on outcomes of the classically mild cerulein AP.
Methods: C57B6 mice were fed normal laboratory chow (NLC; 5.6% fat) or a high UFA diet (HUD; 45% fat), ad libitum, for 24 weeks. AP was induced with cerulein (50ug/kg, by Alzet osmotic pumps). Outcomes measured were survival, pancreatic, systemic injury, lipolysis, serum (Sr.) calcium and cytokine levels. Values are reported as mean±SEM and p-values of <0.05 are considered significant.
Results: HUD mice gained more weight (49.4±0.8 vs. 26.4±0.2 gm, p<0.01), had higher body fat (28.2±0.5 vs. 10.6±0.3 %, p<0.01), higher visceral UTG (11.8±0.3 gm vs. 1.8±0.4 gm, p<0.01), increased intrapancreatic fat (26.2±5.6 vs. 0.57±0.2%, p=0.02) vs. NLC mice. AP caused 100% mortality by day 3(9/9) in HUD mice vs. the NLC mice (1/8, p<0.01), with gross visceral fat necrosis and lipolysis (Sr.Glycerol 5.6±1.5 vs. 0.5±0.07 mM, p=0.01). Pancreatic necrosis (19.7±3.2 vs. 1.2±0.9%, p<0.01) and peri-fat acinar necrosis (5.1±0.6 vs. 0.08±0.07%, p<0.01) were higher in HUD mice. However, Sr.amylase and lipase elevations were similar in both groups with AP. HUD mice had multi organ dysfunction syndrome (MODS) with persistent hypocalcemia (Sr.Calcium 6.1±1 vs. 9.9±0.3 mg/dl, p=0.01), lung injury and kidney failure (Sr.Blood urea nitrogen 57.5±9 vs. 23.9±2.3 mg/dl, p=0.03), and increased cytokines (Sr.IL6 52.6±12 vs. 3.1±8.4 ng/ml, p=0.01; Sr.Resistin 5049±1461 vs. 993±71 pg/ml, p=0.03).
Conclusions: High UFA diets increase unsaturated triglycerides in visceral fat. This converts mild →severe AP, via lipolysis, associated with worse markers, MODS and mortality. The adverse outcomes in countries with high UFA diets may thus be due to their accumulation in visceral fat.
Inflammatory Monocytes Regulate Acute Pancreatitis Resolution: A New Model of Chronic Pancreatitis
G. Perides,1 J. Louhimo,2 Z. Knotts,1 M.L. Steer.11Department of Surgery, Tufts Medical Center and Tufts University School of Medicine, Boston, MA, 2Department of Surgery, University of Helsinki, Helsinki, Finland.
Introduction: We previously showed that the Ly-6Chi inflammatory monocyte sub-set plays a critical pro-injurious role in regulating acute pancreatitis severity.
Aim: To determine whether inflammatory monocytes, activated at the time of pancreatitis induction, promote acute pancreatitis resolution and to develop a new model of chronic pancreatitis.
Methods: We induced secretagogue and biliary pancreatitis on mice. Twenty-four hours after pancreatitis was established we selectively depleted inflammatory monocytes by administering diphtheria toxin (DT) to transgenically modified mice that express the human DT receptor coupled to the CD11b promoter (CD11b-hDTR mice). Chronic pancreatitis was evaluated by quantitating edema, inflammation, cell injury/necrosis and fibrosis.
Results: Untreated mice completely resolved their acute pancreatitis a week after its induction. Mice in which the inflammatory monocytes were depleted could not resolve their pancreatitis. Their pancreas remained edematous and fibrotic, and it was reduced in size and infiltrated by inflammatory cells. The fibrosis and inflammation in the pancreas were maintained for at least 15 days after pancreatitis induction.
Conclusion: Inflammatory monocytes activated during the early phases of pancreatitis not only regulate pancreatitis severity but also pancreatitis resolution.
Serum Tumor Markers Are Not Useful to Select Patients With Mucinous Cystic Lesion Having Malignant Transformation
R. Pezzilli,1 L. Calculli,2 A. Barassi,3 G. Melzi d’Eril.31Pancreas Unit, Department of Digestive System, 2Department of Radiology, Sant’Orsola-Malpighi Hospital, Bologna, Italy; 3Department of Health Sciences, San Paolo Hospital, University of Milan, Italy.
Introduction: It has been reported that even if serum CA 19-9 is not a marker of cystic pancreatic neoplasm (CPN) malignancy, its determination provides additional information within their diagnostic work-up.
Aim: To assess both CA 19-9 and CEA serum concentrations in a consecutive series of patients affected by pancreatic diseases such as mucinous cystic pancreatic neoplasms (MPN), chronic pancreatitis CP) and pancreatic ductal adenocarcinoma (PDAC).
Methods: Ninety-one patients (54 males, 37 females, mean age 61.6 years, range 28-88 years) were studied. Thirty-two patients had MPN (2 patients had main duct IPMN, 2 mixed type IPMN, 2 mucinous cystadenoma and 26, branch duct IPMN), 35 had a firm diagnosis of chronic pancreatitis and 24 had a histologically confirmed pancreatic adenocarcinoma. Surgery was carried out in 10 of the 32 MPN patients (7 of them had severe dysplasia), in 5 patients with CP and 9 PDAC patients. Serum CEA and CA 19-9 were assayed in all patients at the time of the diagnosis using commercially kits. The upper reference limit (URL) of serum CEA is 3 ng/mL and the URL of serum Ca 19-9 is 37 U/mL.
Results: Abnormally high serum CEA concentrations were present in 6 patients (18.8%) with MPN, in 6 CP patients (17.6%) and in 12 (50.0%) PDAC patients (P=0.010). Serum CA 19-9 activities were abnormally high in 7 MPN patients, in 12 (34.3%) CP patients and in 12 (50.0%) PDAC patients (P=0.089). In the seven patients with MPN having histological confirmed severe dysplasia, abnormally high levels of CEA were present in 2 (28.6%) ad elevated serum activities of serum CA 19-9 were present in 3 patients (42.9%).
Conclusion: Serum determination of oncological markers does not add any useful information to select MPN patients having malignant transformation.
Leukotriene B4 Receptor Expression are Increased in Chronic Pancreatitis and Activated Primary Pancreatic Stellate Cells
H. Pham, R.T. Waldron, J. Yang, H. Su, S.J. Pandol, A. Lugea. Department of Medicine, Cedars-Sinai, Los Angeles, CA.
Background: Leukotriene B4 (LTB4) is a pro-inflammatory lipid mediator derived from arachidonic acid (AA) that binds to two LTB4 G protein-coupled receptors, BLT1 and BLT2. Over-expression of BLT2 confers growth and survival advantages in pancreatic intraepithelial neoplasia whereas antagonists to BLTs diminished proliferation and survival of pancreatic cancer cell lines. While inflammation is a pivotal process in carcinogenesis, it is unclear whether the LTB4 pathway is involved in the fibrogenesis of chronic pancreatitis.
Methods: Human pancreatic stellate cells were isolated from pancreatic specimen of chronic pancreatitis patients were designated as hCP and adjacent “normal” specimen were designated as hNP. Immortalized human pancreatic stellate cells were marked as ihPSCs. Primary pancreatic stellate cells isolated mouse pancreata are designated as mPSC. ALOX5, BLT1 and BLT2 were identified by qPCR using SYBR Green and corresponding antibodies by Western blotting.
Results: Screening of ALOX5 that catalyzes the biosynthesis of LTB4 by qPCR shows hCP exhibiting a 2.9 ± 0.5-fold and 16.6 ± 0.1-fold increase expression above, respectively, hNP and ihPSC. Nuclear transcription factor PDX1 involved in the development of chronic pancreatitis shows an increase mRNA expression in hCP by 2.0 ± 0.7-fold above hNP and 6.3 ± 0.2-fold above ihPSC. hCP exhibited an increase in BLT1 mRNA expression by 3.3 ± 0.3-fold above hNP and 3.8 ± 0.4-fold above ihPSC. Similarly, hCP exhibited increased BLT2 mRNA expression 3.5 ± 0.2-fold above hNP and 4.4 ± 0.2-fold above ihPSC. Control Panc-1 cells expressed BLT1 and BLT2 mRNA at 171.1 ± 0.2-fold and 5.5 ± 0.2-fold, respectively, above hCP. hCP exhibited increased ACTA2 mRNA expression 4.9 ± 0.3-fold above hNP and 5.9 ± 0.2-fold above ihPSC. hCP exhibit accelerated proliferative capacity at 72 hrs by 1.7 ± 0.1-fold above hNP and 1.4 ± 0.1-fold above ihPSC. Propagation of mPSC for 5 days, resulted in activation that correlated with an increase in BLT1 mRNA expression by 7.9 ± 0.5-fold and BLT2 mRNA expression by 10.0 ± 1.0-fold.
Conclusion: Stellate cells from chronic pancreatitis express elevated levels of ALOX5, BLT1 and BLT2 mRNA indicating the possible involvement of the LTB4 pathway in inflammation and fibrogenesis.
Pancreatic Cancer: Stromal Re-programming Offers a Novel Therapeutic Approach
S. Pothula, Z. Xu, D. Goldstein, R. Pirola, J. Wilson, M. Apte. Pancreatic Research Group, South Western Sydney Clinical School, Ingham Institute for Applied Medical Research and the Faculty of Medicine, The University of New South Wales, Sydney, Australia.
Pancreatic cancer (PC) stroma is now known to play a major role in cancer progression. Pancreatic stellate cells (PSCs, which produce PC stroma) interact with cancer cells to facilitate PC growth. A candidate pathway that may mediate this interaction is the hepatocyte growth factor (HGF)/c-Met pathway.
Aim: To determine the effects of inhibiting both the ligand (HGF) and receptor (c-MET) components of pathway with and without concomitant chemotherapy on PC progression.
Methods: A) Orthotopic PC produced by implantation of AsPC-1 cells +human PSCs (hPSC) into mouse pancreas. Mice treated for 6 weeks (n=7/group) with one of the following:
IgG (vehicle control IP Bi-weekly)
Anti-HGF antibody Rilotumumab/AMG102 (A@300ug IP Bi-weekly)
Gemcitabine (G@75mg/kgBW IP Bi-weekly)
Small molecule inhibitor for c-Met, Compound-A (C@60mg/kgBW, gavage daily)
Tumor volumes and metastasis assessed.
B) AsPC-1 cell proliferation and migration assessed in-vitro after exposure to hPSC secretions pre-treated with the above compounds.
Results: (Data expressed as % IgG-control)
A) C+A+G treated mice exhibited:
i) The greatest reduction in tumor volumes (A:42.3±7; G:55.7±8; C:39.6±8; C+A:39.5±7; C+G:52±9; C+A+G*21±4, p<0.0001)
ii) A virtual absence of metastasis, (Metastatic burden A:20±5; G:51±9; C:22±5; C+A:29±1; C+G:59±9; C+A+G*2.8±2.8, p<0.0001)
B) In-vitro studies: PSC secretions induce AsPC-1 proliferation and migration, which is significantly reduced (62.3±3% & 45.2±6% respectively) by C+A+G therapy (p<0.0001, n=5 hPSC preparations).
Conclusions: A 2-pronged approach targeting the HGF/c-Met pathway with relevant inhibitors and cancer cells with chemotherapy had significant anti-tumor effects and virtually eliminated metastasis, suggesting a novel therapeutic approach in PC.
Menstrual and Reproductive Factors and Risk of Pancreatic Cancer in Women
A. Pourshams, M. Bagheri, R. Shakeri, R. Malekzadeh. Digestive Oncology Research Center, Digestive Disease Research Institute, Tehran University of Medical Sciences, Tehran, Iran.
Introduction: Pancreatic cancer (PC) is a deadly disease with a 5-year survival of less than 5%. Worldwide PC incidence rates are lower among women than men. While this suggests a protective role for steroid hormones in PC risk, results from epidemiologic studies are not consistent.
Methods: Hazard ratios and 95% confidence intervals for menstrual and reproductive factors and PC were estimated using Cox regression methods in a prospective case –control study. A hundred twenty seven new incident PC cases and 178 controls were both recruited in the study from a referral center for endoscopic ultrasound between 2011 and 2015. A structured valid and reliable questionnaire was used for data collection by trained interviewers.
Results: Mean age (SD) of cases and controls were 65.2 (11.4) and 64.8 (11.8) years respectively. Age at menarche, age at menopause, number of parity, gravidity and abortion were not associated with PC risk.
Conclusions: This study does not support for the hypothesis that menstrual and reproductive factors are associated to PC risk.
PEDF Inhibits Pancreatic Tumorigenesis by Attenuating a Fibro-Inflammatory Reaction
D. Principe,1 B. DeCant,1 A. Diaz,1 R. Mangan,1 E. Wayne,1 B. Jung,1 B. Sreekumar,2 C. Chung,2 D. Bentrem,3 H. Munshi,3 F. Bishehsari,4 P. Grippo.11Department of Medicine, University of Illinois at Chicago, Chicago, IL; 2Department of Medicine, Yale University School of Medicine, New Haven, CT; 3Feinberg School of Medicine, Northwestern University, Chicago, IL; 4Department of Medicine, Rush University Medical Center, Chicago, IL.
Pancreatic cancer is characterized by a pronounced fibro-inflammatory stromal reaction that can contribute to cancer progression. Previous reports have demonstrated that pigment epithelium-derived factor (PEDF), a non-inhibitory serpin family member with potent neurotrophic and anti-angiogenic effects, can have tumor suppressor functions in pancreas. To identify a mechanism through which PEDF opposes pancreatic tumorigenesis, we employed human, mouse, and in vitro models, and explored the effects of PEDF on several cell types in the pancreatic tumor microenvironment. PEDF expression was found decreased in human pancreatic cancer samples, with loss of PEDF correlating with increased inflammation and fibrosis. Genetic ablation of PEDF in mice increased cerulein-induced inflammation and enhanced KRASG12D-induced fibrosis, inflammation, and metastasis in two separate models. Serum samples from PEDF-null mice show elevated levels of the pro-inflammatory and pro-fibrotic cytokines IL8 and TGFβ1, respectively, and reduced levels of the anti-inflammatory cytokine IL10. mtKRAS/PEDF null mice also display enriched populations of αSMA-expressing stellate cells and increased TGFβ1 expression. Recombinant PEDF neutralized macrophage migration and activity in vitro as well as inhibited macrophage-induced proliferation of tumor cells. Recombinant PEDF also suppressed both collagen I deposition and TGFβ1 synthesis by pancreatic stellate cells. Our results demonstrate that PEDF limits pancreatic cancer progression by attenuating the fibro-inflammatory reaction. Combined, these results suggest that restoring PEDF levels may be a potential therapeutic approach in pancreatic cancer.
p53 Gain-of-Function Mutations Promote Adenocarcinoma From Pancreatic Ductal Cells
M.A. Pruski,1 A.M. Hendley,1 N.C. Jones,1 M. Younes,2 A. Maitra,3 C. Iacobuzio-Donahue,4 S.D. Leach,4 J.M. Bailey.11Division of Gastroenterology, Hepatology and Nutrition, Department of Internal Medicine, The University of Texas Health Science Center at Houston, Houston, TX; 2Division of Pathology and Laboratory Medicine, The University of Texas Medical School at Houston, Houston, TX; 3Departments of Pathology and Translational Molecular Pathology, The University of Texas MD Anderson Cancer Center, Houston, TX; 4The David Rubenstein Pancreatic Cancer Research Center, Memorial Sloan Kettering Cancer Center, New York, NY.
Introduction: Selective activation of oncogenic Kras can help model the cell of origin for pancreatic ductal adenocarcinoma (PDAC). Previous data have shown that Kras activation in acinar cells can generate PanIN lesions, while similar activation in ductal cells has little to no effect.
Methods: We employed mouse models with one or two mutant p53 alleles (Tp53R172H), along with oncogenic Kras. In pancreatic acinar cells, oncogenic Kras along with one copy of Tp53R172H was sufficient for tumor formation; however, ductal cells required two copies of Tp53R172H. We used the Mist1:CreERT2 and Hnf1b:CreERT2 transgenic mice to examine the response of Kras activation along with Tp53R127H on acinar and ductal epithelial cells. Within the ductal and acinar cells, lineage labeling was engaged to study Ki67, cleaved Caspase 3 (CC3) and p21, to expose the differences of expression between these two cell types.
Results: When looking at Ki67 and CC3, we observed no significant difference in labeling between acinar and ductal cells. However, p21, known for its role in cell cycle arrest, was increased in ductal cells with Kras activation. Our extended analysis showed simultaneous activation of oncogenic Kras and two Tp53R172H alleles led to invasive PDAC formation from ductal cells in 75% of mice as early as 2.5 months following tamoxifen injection. These data demonstrate pancreatic ductal cells can serve as the cell of origin for pancreatic ductal adenocarcinoma.
Up-Regulation of CXCR2 and its Ligands in Severe Acute Pancreatitis
A. Purohit, S. Kumar, S. Kaur, S. Rachagani, M. Varney, S.K. Batra, R.K. Singh. Buffett Cancer Center, Department of Pathology and Microbiology, Department of Biochemistry and Molecular Biology, University of Nebraska Medical Center, Omaha, NE.
Introduction: Acute inflammation is a hallmark of acute pancreatitis and a variety of inflammatory cytokines and chemokines have been implicated in the pathogenesis of pancreatitis. CXCR2 ligands are the major regulator of neutrophil recruitment, activation and are critical in an inflammatory response. Limited studies have documented the role of CXC chemokines and their receptors in the severity of pancreatic pathophysiology. Our preliminary studies suggest that normal human pancreatic cells do not express CXCR2 ligands (CXCL 1, 2, 5, 8), but CXCR2 is constitutively expressed on pancreatic epithelial and acinar cells. Interestingly, higher serum levels of CXCR2 ligands have been reported in human and rodent pancreatitis that may be responsible for recruitment of inflammatory cells to the site of inflammation.
Our working hypothesis is that expression of CXCR2 ligands by acinar cells and recruitment of CXCR2+ neutrophils play a major role in the progression of acute pancreatitis.
Methods: We used C57BL/6 mice treated with cerulein intraperitoneally (75 μg/kg body weight, twice for 2 days) to investigate the levels of CXCR2 and its ligands at different time points. Cxcl1 (mouse homologue of human CXCL8) first showed 10 fold upregulation within 48 h and then decreased to 5 fold by day 7. Cxcl2 levels remain low at 48 h and later increased to 40 fold by day. We did not observe significant change in Cxcr2 levels following cerulein treatment.
Results: Immunohistochemistry and immune-fluorescence analysis of the pancreas from cerulein-induced pancreatitis demonstrated significant infiltration of neutrophils and macrophages at day 2 along with damage to pancreatic parenchyma. Majority of the immune cells express CXCR2 which is responsible for their recruitment after cerulean-induced pancreatic damage. Further analysis at day 7 showed clearance of infiltrating immune cells and recovery of damaged pancreatic tissue. Both acinar cells and infiltrating neutrophils stained positive for Cxcl1. initially, serum levels of CXCR2 ligands were evaluated in a small pilot study in acute pancreatitis patients stratified as severe acute pancreatitis (SAP) or mild acute pancreatitis (MAP). The serum levels of human CXCL8 (functional mouse homologue is Cxcl1) were higher in SAP patients compared to MAP patients, corroborating our analysis in the experimental mouse model of cerulein-induced acute pancreatitis.
Conclusions: Taken together, these data suggest that CXCR2 ligands are upregulated early (48h) during cerulein induced pancreatitis and CXCR2 ligands (CXCL8) are upregulated early during SAP compared to MAP in human patients.
Overexpression of DCLK1 in Pancreatic Cancer Cells Supports Tumorigenesis, Invasiveness, and Stemness
D. Qu, N. Weygant, W. Berry, R. May, P. Chandrakesan, S.M. Sureban, C.W. Houchen. Department of Medicine, University of Oklahoma Health Sciences Center, Oklahoma City, OK.
Aim: To determine whether overexpressing DCLK1 promotes pancreatic cancer cell tumorigenic activities.
Background: Pancreatic ductal adenocarcinoma (PDAC) has the worst prognosis of any major malignancy with less than a 6% 5-year survival rate. Doublecortin-like kinase 1 (DCLK1) is overexpressed and marks a population of tumor-initiating cells in PDAC. It regulates key oncogenes, pluripotency factors, angiogenic factors, and epithelial mesenchymal transition (EMT) related transcription factors. In this study, we evaluate the role of DCLK1 in pancreatic cancer cell tumorigenic activities.
Methods: Human pancreatic cancer cells (AsPC-1 and MiaPaCa-2) were infected with Lentivirus containing human DCLK1 cDNA to overexpress DCLK1 or green fluorescent protein (GFP) cDNA as control. Gene expressing levels in both cells were analyzed by real-time RT-PCR. The proliferative and invasive potential of these cells were compared using MTT assay for proliferation, wound healing assay for migration, soft agar assay for clonogenicity, and Matrigel coated transwell assay for invasion. The drug resistance of these cells was characterized by Gemcitabine induced toxicity assay.
Results: Overexpression of DCLK1 in MiaPaCa-2 and AsPC-1 cells resulted in dramatically increased cell proliferation in both lines (p<0.01) as well as a more than 2-fold increase in cell invasion (p<0.05). Moreover, DCLK1 overexpression results in 20% increase in migration and colony formation ability in soft agar (p<0.05). DCLK1 overexpression in MiaPaCa-2 cells confers significant resistance to gemcitabine in vitro (p<0.0001 at 200 nM).
Conclusion: These findings taken together demonstrate that DCLK1 expression modulates basic oncogenic processes in PDAC that fuel tumorigenesis, metastasis, and drug resistance. Targeting DCLK1 in PDAC with therapeutics may overcome these processes and increase the currently dismal PDAC patient survival.
Low Dose Ciprofloxacine Nanoparticles Decrease Bacterial Translocation During Severe Acute Pancreatitis
O. Rotar,1 I. Khomiak,2 V. Rotar,1 M. Fishbach,1 K. Taneja.11Bukovinian State Medical University, Chernivtsi, Ukraine; 2A.A.Shalimov National Institute of Surgery and Transplantology, Kiev, Ukraine.
Objective: Selective digestive decontamination is proven to prevent septic complication in patients with severe acute pancreatitis (SAP)  but may be followed with antibacterial resistance development. Diminishing a number of drugs could prevent it.
Aim: To investigate the ability of mucoadhesive chitosan nanoparticles (CNp) loaded with ciprofloxacine (CIPR) to decrease bacterial translocation (BT) during SAP.
Methods: In 200 Wistar rats SAP was induced by intraperitoneal injection of L-arginine. Enteral administration of 3 mg/kg of CIPR was applied in 1 group (n=60), 3 mg/kg of CIPR included in CNp in 2 group (n=60) and normal saline – in control (C) group (n=60). 5 ml of of 9 log CFU/g of K. pneumonia, P. mirabilis and E. coli HLY+ was introduced by gavage. Concentration of microorganisms in intestine and internal organs were investigated during 12-96 hours.
Results: In C group colonization of intestine by pathological bacteria occurred in all animals after 24 h in concentration 4.5-6.5 log CFU/g. BT started after 12 hours in 20% and appeared in 100% during 48-96 hours with E.coli, K. pneumonia, P.mirabilis, S. auereus, B. fragilis and C. albicans in concentration 4.2-7.5 log CFU/g. In 1 group IC was 28-36% less than C group (p>0.05) during all period and BT occurred in same frequency but in half concentration till 72 hour in portal blood and 96 hour in pancreas and mesenterial lymphatics. In 2 group IC was observed in 20%-40% during 12-48 h but amount of bacteria were less than 3.1 log CFU/g during all periods. BT to pancreas and other internal organs was found in 10-20% cases during 24-48 h by normal E. coli and S. epidermidis in amount 2.7-3.5 log CFU/g without development of serious dysbiotic changes in small intestine and colon.
Conclusions: Low level CIPR loaded CNp effectively decrease BT to internal organs during SAP in rats without dysbiotic changes.
The Prognostic Influence of Intrapancreatic Tumor Location on Survival After Resection of Pancreatic Cancer
D.A. Ruess,1 S. Chikhladze,1 O. Sick,1 H. Riediger,2 U.T. Hopt,1 F. Makowiec,1 U.A. Wittel.11Department of Surgery, University of Freiburg, Germany; 2Department of Surgery, Vivantes-Humboldt-Clinic, Berlin, Germany.
Introduction: Prognosis of pancreatic ductal adenocarcinoma (PDAC) is worse in case of primary tumor location in the pancreatic tail, compared to tumor location in the pancreatic head. However, for localized, resectable tumors survival seems to be at least similar. We analyzed the outcome after pancreaticoduodenectomy (PD) and distal pancreatectomy (DP) for PDAC at our institution.
Methods: Clinical, pathological and survival data from patients conveyed to pancreatic resection for PDAC 1994-2014 were explored retrospectively, accessing a prospective pancreatic database. Cases with primary total pancreatectomy were excluded.
Results: 413 patients were treated for PDAC: 347 (84%) underwent PD and 66 (16%) DP. Tumors located in the pancreatic tail were significantly larger than their counterparts located in the head (30.6 mm vs. 41.2 mm; p<0.001). However, distal tumors had significantly less nodal involvement (71% vs. 57%; p=0.03). Portal-vein resection (PVR) was performed more often in PD, multivisceral resection (MVR) was more frequent in DP (37% vs. 14% and 4% vs. 29%; p<0.001, χ2-test, 2df). Rates for negative resection margins and tumor grading were similar. Postoperative morbidity, rates of re-operation and mortality were comparable. Long-term outcome revealed no significant difference between PD and DP with 5-year survival rates of 17.8% and 22% respectively (p=0.284). Multivariate analysis confirmed positive resection margin, positive nodal status, blood transfusion and extended resection (PVR, MVR) as independent risk factors for poor survival after pancreatic resection.
Conclusion: Patients with resectable pancreatic ductal adenocarcinoma located in the body and tail of the pancreas display a similar postoperative oncological outcome despite larger tumors when compared to patients with resectable tumors located in the pancreatic head.
Is “Chronic” Long-term Intervention With Micronutrient Anti-Oxidant Therapy Required to Modulate the Disease Course of Chronic Pancreatitis?
S.N. Rupasinghe, A.K. Siriwardena. Hepatobiliary Surgical Unit, Manchester Royal Infirmary, United Kingdom.
Introduction: Micronutrient antioxidant therapy did not relieve pain in a large randomized trial of European patients with chronic pancreatitis (CP) without malnutrition. However, intervention was undertaken for 6 months only leaving unanswered the question of whether long term anti-oxidant therapy is required to modulate CP.
Methods: A single center clinical cohort analysis of patients with CP prescribed micronutrient antioxidant therapy and followed up for up to 10 years. International Classification of Disease (ICD) version 9 code 577.1 and ICD version 10 codes K86.0 (alcoholic CP) and K86.1 were sought. Charts were reviewed and data were collected on: gender, age at enrolment, disease duration in years, body mass index, cigarette smoking, alcohol use, insulin treatment and opiate analgesia. All patients received antox (Pharmanord, Morpeth, UK). The study was approved by regional ethics committee.
Results: 30 patients with a diagnosis of CP constitute final study population. Median (range) age at time of diagnosis was 40 (14 – 66) years; 19 (63%) were male and the median (range) duration of symptoms prior to referral was 2 (0-18) years. Alcohol was the dominant etiological agent in 22 (73%) and 16 (53%) were Cambridge stage 1. Twenty four (80%) had pain as a presenting symptom. Over a median duration of anti-oxidant treatment of 4 (1-10) years, pain decreased but the proportion with abdominal pain compared to pain-free remained constant (P=0.16; 2-way ANOVA with Bonferroni correction) with no treatment-related effect on pain. There was a significant increase in requirement for insulin (P=0.028) with time together with use of both endoscopic and surgical intervention
Conclusions: This is the first study to report long-term disease-specific outcome in patients with chronic pancreatitis prescribed micronutrient antioxidant therapy. There appears to be no effect of intervention on the natural history of chronic pancreatitis.
Effect of Chronic Ethanol Feeding on Expression and Enzymatic Activity of Pancreatic Digestive Enzymes
Y. Ryu, A. Bekolay, A.K. Saluja, R. Dawra. Division of Basic and Translational Research, Department of Surgery, University of Minnesota, Minneapolis, MN.
Background: Alcohol abuse is strongly associated with pancreatitis. It has been reported that in alcoholics, trypsinogens synthesis is perturbed. It is believed that altered synthesis and resulting change in intra-acinar cell trypsin activity might be sensitizing these cells to injury. Pancreatic digestive enzymes synthesis is also responsive to dietary changes, which can contribute towards observed changes in enzymes in patients. Therefore, there is a need to validate previous observations from human subjects using experimental animals fed alcohol under carefully controlled conditions and comparing it with control group kept under similar conditions and fed matched control diet.
Aims: The aim of this study was to evaluate changes in major pancreatic digestive enzymes expression and activity after chronic ethanol feeding.
Methods: Mice (C57BL/6) were fed ethanol (35% calories) containing Lieber-Decarli diet for 14 weeks, while control mice were pair-fed normal liquid diet. At the end of feeding pancreases were harvested and used for measuring mRNA expression levels and enzyme activity using respective biochemical assays.
Results: Expression levels of trypsinogen isoforms T7, T20 were comparable in control and ethanol diet groups, while T8 and T10 mRNA expression decreased in ethanol group. mRNA expression of chymotrypsinogens B and C; carboxypeptidases A and B, elastases, trypsin inhibitor SPINK 3 and amylase did not change as a result of chronic high ethanol consumption for 14 weeks. Total enzymatic activity of amylase, trypsin, chymotrypsin, carboxypeptidase A did not change in response to high ethanol consumption but elastase activity increased after 14 weeks of exposure.
Conclusions: Experimental feeding of ethanol at high levels altered mRNA expression of trypsinogen T8 and T10 but this change was not reflected in total trypsin activity. Trypsinogen T7 which contributes towards pathological responses was unaltered. Total elastase increased with ethanol exposure without any change in mRNA expression.
RCAD is a Key Post-Translational Modification for the Proper Sorting of Digestive Enzymes and the Secretory Function of the Exocrine Pancreas
M.E. Sabbatini,1 Y. Cai,2 H. Li.21Department of Biological Sciences, Georgia Regents University, Augusta, GA; 2Department of Biochemistry and Molecular Biology, Georgia Regents University, Augusta, GA.
Introduction: Chronic alcohol consumption is one of the main cause of acute pancreatitis. An excess of alcohol causes an increase in free radicals formation and endoplasmic reticulum (ER) stress in pancreatic acinar cells. There are a number of ER proteins that participate in the correct folding/sorting of proteins. One of these proteins is the Ufm1 (Ubiquitin-fold modifier 1) conjugation system, which is a novel ubiquitin-like modification system and part of the unfolded protein response. Regulator of C53 and DDRGK1 (RCAD) has recently been identified as an important regulator of several signaling pathways, including the Ufm1 conjugation system. Endogenous RCAD forms a complex with two proteins, C53 and DDRGK1, and promotes Ufmylation of DDRGK1.
Aim: To identify the role of RCAD for the proper sorting of pancreatic enzymes and the normal exocrine function of the pancreas.
Methods: The relative expression of RCAD and DDRGK1 in alcoholic-treated rats compared with non-treated rats was evaluated by qPCR analysis. Adult male SD rats were fed the Lieber-DeCarli liquid diet containing ethanol (36 % of total calories) or an isocaloric substitution with maltose-dextrin ad lib for 8 weeks. To determine the functional relevance of RCAD in the exocrine pancreas, isolated pancreatic acini from WT and RCAD inducible conditional KO (CKO) mice were prepared. CCK-induced amylase secretion was measured.
Results: Both RCAD and DDRGK1 transcript levels were down-regulated (50–60 %) in alcoholic-treated rats. To generate RCAD CKO mice, we crossed RCADTrap-F/+mice with FLPo deleter mice B6(C3)-Tg(Pgk1-FLPo)10Sykr/J to remove the gene trap cassette. The floxed RCAD mice were crossed with ROSA26-CreERT2 mice (B6.129-Gt(ROSA)26Sorotm1(cre/ERT2) Tyj4/J in which CreERT2 was inserted into ROSA26 locus. The constitutive and CCK-inducible amylase secretion was much higher (55–60 %) by isolated pancreatic acini prepared from RCAD CKO mice.
Conclusion: RCAD is likely to be involved in a post-translational modification system for the proper sorting of amylase and normal pancreatic secretion. The significance of RCAD will provide an important insight into the pathophysiology of acute pancreatitis.
Predictors of Malignancy in Main Duct and Mixed Type Intraductal Papillary Mucinous Neoplasms (IPMN)
R.P. Sah, N. Ahmed, S. Mukewar, N. Takahashi, S.T. Chari. Division of Gastroenterology and Hepatology, Mayo Clinic, Rochester, MN.
Background: Fukuoka guidelines for IPMNs recognize the following four predictors of malignancy- 1) presence of solid lesion, 2) biliary dilatation, 3) duct cut-off and 4) main duct dilatation. Here, we investigated the utility of these Fukuoka features in main duct and mixed type IPMNs.
Methods: In patients who underwent pancreatic resection at our institution with main or mixed type IPMN on pathology, a pancreas radiologist, blinded to histopathological diagnosis, re-reviewed the pre-operative CT scans to identify Fukuoka features; patients with pre-operative CT showing only branch-duct were excluded.
Results: The mean age of the 114 patients meeting study criteria was 69 ± 10 yr (57% M); 77% had main duct and 23% ad mixed type IPMN per CT review. Of these 40 (35%) had invasive adenocarcinoma (Ca) and 10 (9%) had high grade dysplasia (HGD). A solid lesion was seen in 48 (42%), biliary dilatation in 15 (13%) and duct cut-off in 45 (39%) patients, the prevalence of Ca or HGD in 3 groups being 79% (P < 0.0001), 87% (P = 0.0002) and 62% (P = 0.0014), respectively. The risk of Ca or HGD for main duct size (median 10 mm) was 47% for ≥ 10 mm and 41% for < 10 mm (AUC 0.54, P = 0.19). In multivariate analysis, only presence of solid lesion was an independent predictor of Ca or HGD (P < 0.0001). Among 66 patients without solid lesion, 6 (9%) had Ca and another 6 (9%) had HGD. In the group without solid lesion, 16 (24%) had duct cut-off with risk of Ca or HGD being only 6% (P = 0.27) while only 2 (3%) had biliary dilatation (1 Ca), attesting to the lack of predictive value of these Fukuoka features in the absence of solid lesion. Importantly, among patients without solid lesion, invasive cancer was only seen in those with main duct size >10 mm with a risk of Ca or HGD being 24% (6 Ca and 3 HGD of 38 patients); none of the 28 patients with main duct size <10 mm without solid lesion had Ca and 3 (11%) had HGD.
Conclusion: Patients with main duct or mixed type IPMN can be risk stratified based on presence or absence of solid lesion on imaging. Patients with solid lesion, regardless of main duct size, should undergo resection (risk of Ca or HGD ∼80%). Among those without solid lesion, those with main duct ≥ 10 mm should undergo resection (risk of Ca or HGD ∼25%) and those with main duct <10 mm (∼25% of all cases) can be conservatively managed as they have minimal risk of invasive cancer and low risk of HGD (∼10%).
Epigenetic Therapeutics of Pancreatic Cancer: Inhibition of PDAC Growth by Targeting the HP1-G9a/GLP Histone Methyltransferase Pathway
A. Salmonson, M. Missfeldt, A. Mathison, G. Lomberk. Epigenetics and Chromatin Dynamics Laboratory, GI Research Unit, Department of Medicine, Mayo Clinic, Rochester, MN.
By regulating gene expression networks that mediate neoplastic behavior, epigenetic protein complexes are the ultimate effectors of oncogenic pathways. This hierarchical position of epigenetic pathways within the mechanisms that regulate cancer makes them ideal candidates for therapeutic targets. The current work was designed to characterize the role of the K9H3 Histone Methyltransferase (HMT) pathway in mediating oncogenic signals downstream of Aurora Kinase A (AurkA), with the goal of designing efficient combinatorial therapies against PDAC. Affinity protein purification, combined with mass spectrometry, demonstrates that HP1γ isolated from mitotic cells interacts with AurkA and the HMTs, G9a and GLP. We also find that this complex is critical for mitotic progression and cell proliferation. Congruently, treatment of PDAC cells with individual drugs against AurkA or HP1-HMT inhibits cell growth by inducing senescence, a cytostatic response. However, the combination of these agents has a synergistic effect of reducing cell growth via a mechanism that bypasses senescence to result in a cytotoxic effect. Confocal and electron microscopy, along with cell cycle analysis, demonstrate that the cytotoxic effect of this combination is due to induction of mitotic catastrophe, a distinct form of apoptosis that occurs during mitosis. Molecularly, the combination of AurkA and HP1-HMT inhibition bypasses G2/M arrest with downregulation of the Chk1-Cdc25c pathway. In vivo treatment of PDAC orthotopic xenografts with the HP1-HMT inhibitor alone demonstrated reduced PDAC growth, and increased efficacy in PDAC growth reduction was observed in combination with the AurkA inhibitor over either individual treatment. Thus, our data demonstrate a novel AurkA-HP1-HMT mitotic pathway that holds promise for potential pharmacological targeting in combinatorial therapy for this malignancy.
Use of Oncolytic Adenovirus Expressing IFN-α to Treat Pancreatic Cancer
A. Salzwedel, C. LaRocca, J. Davydova, M. Yamamoto. Department of Surgery, University of Minnesota, Minneapolis, MN.
Unless found at early stage, there is no effective treatment against pancreatic cancer. Late diagnosis and high recurrence rate results in five-year survival of 6%. Notably, a Phase II trial showed 35% increase in five-year survival of patients treated with adjuvant therapy combined with systemic IFN-α (IFN) and chemoradiation. Nevertheless, the therapy showed high systemic toxicity and low IFN intratumoral levels. As IFN can act as chemoradio sensitizer, low intra-tumor levels of IFN hampered the efficacy of this promising therapy.
To improve tolerability and efficacy of IFN therapy we developed an oncolytic adenovirus expressing human IFN. To enhanced oncolysis and infectivity in pancreatic ductal adenocarcinoma (PDAC) cells we included Ad5/3 fiber modification and overexpression of Adenoviral Death Protein (ADP). We also created a vector with RGD fiber modification overexpressing ADP and hamster IFN to be used in hamster syngeneic immunocompetent model of pancreatic cancer. Both vectors (OAd-IFN) have replication controlled by Cox-2 promoter restricting replication and IFN production to PDAC cells.
MTS and crystal violet assays show Ad-IFN sensitizes PDAC cells to chemotherapy (5-FU, Cisplatin, and Gemcitabine), and radiation (RAD) 4 and 8Gy. Comparison between Ad-IFN with an identical counterpart not expressing IFN (Ad-LUC) showed that IFN sensitizes cells to chemoradion. Colony formation assays showed that combination of Ad-IFN + 5-FU + RAD resulted in reduced colony formation compared to Ad-LUC+ 5-FU + RAD and all other therapy groups.
In vivo studies shows that combination of OAd-IFN + RAD or OAd-IFN+ 5-FU + RAD resulted in higher tumor shrinkage compared to 5-FU, RAD, 5-FU + RAD, and Ad-LUC + 5-FU + RAD groups.
Our data shows OAd-IFN has potential to improve tolerability and efficacy of IFN therapy. Tumor specific expression of IFN by the virus can reduce systemic toxicity and increase tumor chemoradio sensitization, improving one of the few therapies effective against pancreatic cancer.
Elevated Levels of IL-6 and IL-8 Have Predictive Role in the Development of Respiratory Failure in Patients With Acute Pancreatitis
J. Samanta,1 S. Singh,2 R. Prasada,1 N. Dhaka,1 M. Ashat,5 S.K. Arora,2 S.K. Sinha,1 A.N. Aggarwal,3 V. Gupta,4 T.D. Yadav,4 R. Kochhar.11Department of Gastroenterology, 2Department of Immunopathology, 3Department of Pulmonary Medicine, 4Department of Surgery, Postgraduate Institute of Medical Education and Research (PGIMER), Chandigarh, India; 5John H Stroger, Jr. Hospital of Cook County, Chicago, IL.
Introduction: Acute lung injury (ALI) is the commonest organ failure in patients with acute pancreatitis (AP). It is a major cause of early mortality in such patients and cytokines play a major role in its pathophysiology.
Aim & Methods: To study the predictive role of inflammatory cytokines in development of ALI in patients with AP.
In this prospective study between July 2013 and December 2014 consecutive eligible patients of AP underwent complete demographic, clinical, biochemical and radiological evaluation. Severity classification was done using revised Atlanta classification and also systemic inflammatory response score (SIRS) and Bedside Index of Severity of Acute Pancreatitis (BISAP), CT Severity Index (CTSI) and APACHE II scores were used. Serial arterial blood gas (ABG) analyses were done and ALI severity defined as per Berlin Classification using PaO2/FiO2 ratio. Development of ALI was monitored in all the patients. Serum levels of interleukin (IL)-6, IL-8, IL-10, IL-1β and TNFα were measured at baseline (day 1) for all patients and on day 3 in those who had ALI. For comparative analysis patients were divided into 2 groups: with and without ALI. The ALI cohort was further subdivided into persistent ALI (P-ALI) and transient ALI (T-ALI). A subgroup of ALI patients who developed ALI later during hospital stay was defined as “late onset” ALI (LO-ALI) to devise a predictive model for ALI using cytokine levels. Statistical analysis was done using SPSSv22.0
Results: Of the 107 patients (mean age of 38.4 yrs, 64.5% males, etiology: alcohol 36.4% gallstone disease 26.2% and others 51.4%), ALI developed in 51 (47.7%) of whom 40 (78.4%) had ALI on admission while 11 (21.6%) had LO-ALI. T-ALI was seen in 16(31.4%) while 35 (68.6%) had P-ALI. Patients with ALI had significantly higher IL-1β (p < 0.0001), IL-6(p < 0.0001), IL-8 (P < 0.001) and TNFα(P < 0.0001) and lower IL-10 (p < 0.0001) levels on day 1, when compared to non-ALI group. In the ALI group, day 3 levels of IL-1β (p = 0.001), IL-6 (p = 0.02), IL-8 (p = 0.006) and TNFα (p = 0.006) were significantly higher than day 1 levels. Significant rise on day 3 of only IL-1β (p = 0.04) was observed in T-ALI group as compared to both IL-1β (p = 0.001) and TNFα (p = 0.02) in the P-ALI group. Day 1 levels of IL-6 and IL-8 had strong positive correlation with severity indices such as SIRS (p < 0.001), BISAP (p < 0.001) and CTSI (p < 0.0001) as also with outcome measures such as need for intervention (p < 0.0001), hospital stay (p < 0.0001) and intensive care stay (p < 0.0001).LO-ALI group had significantly higher levels of IL-6 (p < 0.0001), IL-8(p < 0.0001), TNFα (p < 0.0001) and IL-1β (p < 0.006).IL-6 at cutoff levels of 84.85 pg/mL (AUC = 0.94, sensitivity & specificity 91%) and IL-8 at cut off level of 112.5 pg/mL (AUC = 0.909, sensitivity 91% specificity 94.6%) predicted subsequent development of ALI.
Conclusion: Rising levels of IL-1β and TNFα suggest development of persistent ALI in patients with AP. IL-6 and IL-8 levels at admission can predict the future development of late onset ALI.
Identification of Met Inhibitor-Responding Tumors in Gastrointestinal Cancer
V. Sangwan,1,4 P. Peschard,1 M. Gigoux,1 N. Chughtai,1 A. Monast,1 M. Leimanis,2 V. Marcus,5 L. Ferri,1,2,4 M. Park.1,3,41Goodman Cancer Research Center, McGill University, Montreal, Canada; 2Department of Surgery, 3Department of Medicine, 4Department of Oncology, 5Department of Pathology, McGill University Health Center, Montreal, Canada.
The Met receptor tyrosine kinase (RTK) is known to be dysregulated following overexpression or amplification in many hard-to-treat cancers, including gastrointestinal tumors. However, clinical trials using Met inhibitors have failed, mainly due to flawed patient selection strategies. Criteria for patient selection has either been increased protein expression as assessed by immunohistochemistry (IHC) or gene amplification; however, the correct patient stratification strategy remains unclear.
In order to identify tumors that respond to Met inhibition, we have developed thirty gastrointestinal patient-derived xenografts. Pathological evaluation shows that the xenografts recapitulate the primary tumor. Using Met-expressing xenografts in animal models, we have identified tumors that either respond (show decreased tumor growth) or do not respond to the Met inhibitor, Crizotinib. In order to gain insight into responder characteristics we have identified two Crizotinib-resistant xenografts derived from a Crizotinib-responding xenograft tumor. Using IHC, immunoblotting and phospho-RTK arrays, we have shown that Crizotinib treatment leads to suppression of Met activation but increased levels of other RTKs in resistant tumors. In IHC studies, we observed that Met expression is heterogeneous, where Met is either present at the membrane or in the cytoplasm. Cytoplasmic localization of Met has previously been shown to correlate with poor prognosis. We are currently performing further studies to assess the importance of both Met expression and Met localization.
Our studies will help develop a method to identify patients that will benefit from treatment with Met inhibitors.
SBP-101 Induces Apoptosis in Mouse Pancreatic Acinar Cells and Inhibits Polyamines Induced Proliferation
A. Sareen,1 A.K. Saluja,1 A. Shah,2 R. Dawra.11Division of Basic and Translational Research, Department of Surgery, University of Minnesota, Minneapolis, MN; 2Sun BioPharma, Inc., Williston, FL.
Background: The polyamine (PA) uptake transport system is up-regulated in a number of neoplasms including pancreatic ductal adenocarcinoma (PDA) suggesting a promising therapeutic target. SBP-101, is a PA derived experimental drug developed to treat PDA. However, mechanism of effect of SBP-101 on pancreatic acinar cells is not known.
Aim: The aim of present study was to evaluate the effects of SBP-101 on pancreatic acinar cells.
Methods: Mouse primary pancreatic acinar or 266–6 cells were exposed to different doses of SBP-101. Cell viability was determined by CCK-8 assay. Mode of cell death was evaluated by determining the caspase 3/9 activation, annexin V positivity, PARP cleavage and lactate dehydrogenase (LDH) release. For mechanism of action, mouse 266–6 cells were treated with different polyamines with or without SBP-101.
Results: Treatment of mouse pancreatic acinar cells (266–6) with SBP-101 (1 μM to 100 μM) for 24 and 48 h did not cause significant decrease in cell viability. However, at 72 h, viability was significantly reduced. This response was saturated to 48 ± 3.3% at 3 μM. Annexin V positivity was detected at 48 h, which increased from 8.9 to 27.2 % at 72 h. Both caspase 3 activity and cleaved PARP were increased by 3-fold at 72 h. In primary acinar cells, there was a 1.5 ± 0.01-fold increase in caspase 3 activity with 3 μM dose of SBP-101 after 24 h. LDH release was comparable to untreated control. Treatment with putrescine or spermidine (10 μM) alone increased the viability of cells by 2.50 ± 1.5 and 2.00 ± 0.4-fold which in presence of SBP-101 was reduced to 1.50 ± 0.3 and 1.10 ± 0.34-fold, respectively indicating that the drug competes with these polyamines in its action.
Conclusions: SBP-101 induces acinar cell death by apoptosis and inhibits proliferation by competing with putrescine and spermidine. SBP-101 could be a promising candidate for treatment of PDA.
Prostaglandin E2 Reduces ERK Activation and Growth in Cultured Pancreatic Cancer Cells
A.I. Schmidt, J. Sinnett-Smith, S.H. Young, O.J. Hines, G. Eibl, H. Chang. Department of Surgery, David Geffen School of Medicine at UCLA, Los Angeles, CA.
A positive correlation of inflammation and cancer is widely accepted. Prostaglandin E2 (PGE2), a key mediator of inflammation, has been shown to induce pancreatic tumor growth in vivo. However, previous in vitro studies have demonstrated inconclusive and paradoxical effects of PGE2 on pancreatic cancer cell growth. The aim of this study was to investigate the effects of PGE2 on cell growth and signaling events in human pancreatic cancer cells in vitro.
Colony formation assays were performed to monitor cell growth in the absence and presence of PGE2 (0, 1, 10 μM) for 11 days. Increasing concentrations of PGE2 led to reduced cell growth in MIA PaCa-2 (MP2, reduction of average colony size by 11%, p < 0.05) as well as in PANC-1 cells. To investigate the signaling events underlying the growth-inhibiting effects of PGE2, we performed Western blot analysis for phosphorylated ERK (extracellular signal-regulated kinases). Interestingly, PGE2 (1 μM) inhibited diphospho-ERK (dpERK, T202/Y204) in MP2 and PANC-1 cells. PGE2 can bind to EP2 and EP4 receptors with subsequent activation of protein kinase A (PKA). The effect of PGE2 on ERK phosphorylation in PANC-1 and MP2 was inhibited by H89 (5 μM), a PKA inhibitor. Inhibition of phospho-CREB by H89 was used as a control for the efficacy of PKA inhibition.
Taken together, PGE2 led to inhibition of PANC-1 and MP2 human pancreatic cancer cell growth in vitro. In addition, PGE2 reduced ERK activation in those cell lines, which was mediated by PKA. Our results demonstrate direct anti-proliferative effects of PGE2 in human pancreatic cancer cells in vitro. Thus, the well-described tumor promoting effects of PGE2 in pancreatic cancer in vivo may result from effects on the tumor microenvironment instead.
Naïve T Helper Cell Subset Predominates in Severe Necrotizing Pancreatitis in Mice
A.I. Schmidt, R. Lauch, G. Wolff-Vorbeck, U.T. Hopt, U.A. Wittel. Department of General and Visceral Surgery, Universitätsklinik Freiburg, Freiburg, Germany.
The immune response of acute necrotizing pancreatitis (NAP) can be severe and the systemic immune activation correlates to the patient's outcome. We investigate changes of lymphocyte populations isolated from the blood, spleen and liver in mice with NAP induced by retrograde taurocholate injection.
Mice were treated in groups of 5 per treatment and time point with either taurocholate (NAP) or saline (SHAM) injection into the common bile duct. After 6, 12, 24 and 48 hours lymphocytes from the blood, the livers and spleens were stained for CD4, CD4CD45CD25, CD4CD45CD69, CD4CD45CD152, CD4CD45RB, CD8, CD8CD69. Gene expression of isolated CD4CD45RBhigh and -low cells was examined by quantitative RT-PCR array.
CD4+ cells were increased in severe pancreatitis in all of the examined compartments (Blood, 24 h: CD4+ cells in NAP 39%, SHAM 25%, p < 0.001). In addition, the ratio of naïve CD4CD45RB+ memory T cells to antigen experienced CD45RBlow cells was increased (Blood: CD45RBhigh 6-48 h NAP 50.7-69.7%; SHAM 31.8-57.4%; Liver: CD45RBhigh/low ratio at 24 h NAP 2.2, SHAM 0.6; p < 0.001). Quantitative RT-PCR revealed that these naïve memory T cells express reduced levels of inflammatory mediators; tumor necrosis factor or interferon-γ were downregulated in CD45RBhigh T helper cells (TNF 0.23fold; p < 0.01, IFN-γ 0.09 fold; p < 0.05). In the presence of pancreatitis however, the expression of CCR4, a receptor involved in the chemotaxis of regulatory T cells (0.04-fold, p < 0.05) was reduced in CD45RBhigh cells and the expression of toll-like receptor 6, a receptor involved in innate pathogen recognition (2.36 fold, p < 0.05) was increased.
These data indicate that necrotizing pancreatitis induces more complex immunological alterations in T cell populations than just cell activation and subsequent paralysis. A reduction from adaptive to innate immune mechanisms may be one of mechanisms explaining the anti-inflammatory response after necrotizing pancreatitis.
The First Series of Spiral Vein Graft Reconstruction Technique for Venous Resection in Borderline Operable Pancreatic Cancer
H. Seppänen, A. Kokkola, H. Mustonen, C. Haglund, I. Kantonen. Dept. of Surgery, Helsinki University Hospital, Helsinki University, Finland.
Background: We have used a novel reconstruction technique for portal or superior mesenteric vein resection in borderline operable pancreatic cancer. The postoperative outcomes from pancreatic surgery with different vascular reconstruction (VR) were analyzed.
Patients and Methods: There were 974 patients who had pancreatic surgery at the Helsinki University Hospital 2000–14. When cancer infiltration to the superior mesenteric (SMV) or portal vein (PV) was detected excision and reconstruction by either tangential, end-to-end anastomosis or a spiral graft from great saphenous vein (GSV) was performed. The GSV was excised and splitted open, side branches were ligated. It was sown around a 5–10 ml syringe. The spiral graft was anastomosed with SMV and PV. Splenic vein was anastomosed end-to-side to the graft.
Results: There were 123 (13 %) pancreatic cancer patients who had VR. Of these 108 (90 %) had pancreaticoduodenectomy, 11 (9 %) total pancreatectomy and 4 (3 %) distal resection. There were 45 (37 %) patients with spiral GSV graft reconstruction, 53 (43 %) with end-to-end anastomosis, 11 (9 %) with tangential suture or patch and 14 patients had other kind of reconstruction (including arterial). There was one in-hospital death (d 38) for surgical complications in end-to-end anastomosis group. This patient was the only one with a grade C fistula. None had grade B, and 3 (7 %) had grade A fistula postoperatively. There were no significant differences in re-operations with different kind of VR techniques.
Conclusions: This is a first series showing that patients with borderline resectable pancreatic cancer demanding a portal or superior mesenteric vein can be safely and feasibly reconstructed with a spiral graft from great saphenous vein.
Pancreatic Cancer Patients Express Elevated FVIII Levels and Metastasis-Associated Fibrin Turnover Prior to Their Diagnosis
H. Seppänen,1 N. Varkila,2 C. Haglund,1 R. Lassila.31Department of Surgery, Comprehensive Cancer Center, Helsinki University Hospital, University of Helsinki, Finland; 2University of Helsinki, Finland; 3Department of Hematology, Comprehensive Cancer Center, Helsinki University Hospital, University of Helsinki, Finland.
Background: Pancreatic ductal adenocarcinoma (PDAC) increases venous thromboembolism by 7-fold. The risk associates with Khorana score, which does not disclose the effects of cancer itself prior to oncological treatments (OT). Our aim was to evaluate associations between cancer stage, risk scores and preoperative biomarkers.
Patients and Methods: Between 2010–14 100 consecutively operated PDAC patients and 13 benign IPMN patients. Blood cell count and coagulation, inflammation and tumor markers were analyzed before surgery. Patients were divided into 5 groups: 1A: local PDAC no OT (n = 58), 1B: local PDAC with OT (n = 17), 2A: metastasized PDAC no OT (n = 12), 2B: metastasized PDAC with OT (n = 13) and 3: IPMN (n = 13).
Results: Mean FVIII:C was higher in PDAC vs IPMN, 222 % (SEM 9.75%) in 1A, 215% in 1B, 274% in 2A, 231% in 2B, but only 158% in 3 (p < 0.002 3 vs 1A, 2A and 2B). D-dimer was higher both in metastasized PDAC with OT and native local PDAC versus IPMN. Median D-dimer was 0.30 mg/L (0.09-4.5 mg/L) in 1A, 0.90 mg/L (0.19-4.5 mg/L) in 2B, but only in 0.19 mg/L (0.09-1.5 mg/L) group 3 (p < 0.002 3 vs 1A and 2B). CA 19-9 and D-dimer correlated in native PDAC (1A r = 0.38, p = 0.004) and especially metastasized PDAC (2A r = 0.98, p < 0.001). Khorana risk score did not correlate within the groups, and neither did other biomarkers differ.
Conclusions: Khorana risk score did not associate with cancer stage or OT. High preoperative FVIII:C predicted PDAC with and without metastasis. Fibrin turnover in form of elevated D-dimer associated with local and especially metastatic PDAC with OT, and with CA 19-9.
Prospective Study of Diabetes, Smoking, Diet and Risk of Pancreatitis: The Multiethnic Cohort
V.W. Setiawan,1,2 S.J. Pandol,3 J. Porcel,2 L.R. Wilkens,4 L. Le Marchand,4 M.C. Pike,1,5 K.R. Monroe.11Department of Preventive Medicine, Keck School of Medicine of USC, Los Angeles, CA; 2Norris Comprehensive Cancer Center, Keck School of Medicine of USC, Los Angeles, CA; 3Division of Gastroenterology, Departments of Medicine, Cedars-Sinai Medical Center and Department of Veterans Affairs, Los Angeles, CA; 4Epidemiology Program, University of Hawaii Cancer Center, Honolulu, HI; 5Memorial Sloan Kettering Cancer Center, New York, NY.
Background & Aims: In order to reveal modifiable risk factors that may contribute to increasing pancreatitis incidence in the United States, we conducted a prospective risk-factor analysis of 145,886 African Americans, Native Hawaiians, Japanese Americans, Latinos, and whites in the Multiethnic Cohort.
Methods: Pancreatitis cases were identified using hospitalization claim files (1993–2012) and categorized as gallstone-related acute pancreatitis (GS AP) (N = 1,065), non-GS AP (N = 1,222), and recurrent acute (RAP)/chronic pancreatitis (CP) (N = 523). We used exposure information on the baseline questionnaire to identify potential risk factors. Associations were estimated by hazard ratios (HRs) and 95% confidence intervals (CIs) using logistic regression stratified by risk set and adjusted for confounders.
Results: Diabetes was associated with all three types of pancreatitis, while obesity (BMI ≥30 kg/m2) was mainly associated with GS AP (HRmen = 1.99; 95% CI: 1.49, 2.65 and HRwomen = 2.02; 95% CI: 1.61, 2.53). Cigarette smoking was associated with non-GS AP and RAP/CP. Moderate alcohol intake was inversely associated with all types of pancreatitis in women (HRs = 0.66 to 0.81 for <1 drink/day), and only with RAP/CP in men (HR = 0.56; 95% CI: 0.40, 0.79 for <2 drinks/day). Dietary intakes of saturated fat and cholesterol, and their food sources (e.g. red meat, eggs, and shellfish), were positively associated with pancreatitis, whereas intakes of fiber, vitamin D, and coffee were inversely associated with variation by pancreatitis types.
Conclusions: Diabetes was associated with increased risk for all types of pancreatitis and smoking with non-GS AP and RAP/CP. Moderate alcohol intake lowers risk in women. Several dietary factors were associated with pancreatitis.
Neoadjuvant Chemoradiation Therapy in Borderline Resectable Pancreatic Adenocarcinoma
S. Shafi,1 A. Kaubisch.21Department of Medicine, Albert Einstein Montefiore New Rochelle Hospital, New Rochelle, NY, 2 Department of Oncology, Albert Einstein Montefiore Medical Centre, Bronx, NY.
Background: Pancreatic adenocarcinoma remains notorious for its poor prognosis. Annual deaths from pancreatic adenocarcinoma are rising. In 2014 there were 46,420 new cases and 39,590 pancreatic cancer deaths in United States. Surgical resection remains the only potentially curative treatment. Pancreatic tumors may be deemed ‘resectable’ or ‘borderline resectable’ based on anatomic characteristics. We present a single center experience of patients who received neoadjuvant therapy for borderline resectable pancreas cancer.
Methods: We identified 20 patients with borderline resectable pancreatic cancer treated from 08/2008 to 7/2014 with neoadjuvant chemoradiation therapy at Montefiore Medical Center. Inclusion criteria included patients with pancreatic cancer who were deemed borderline resectable based on NCCN 2014 criteria. Patients were grouped by type of treatment received: Group A received Folfirinox followed by chemoradiation therapy (CRT).Group B received gemcitabine based chemotherapy (gemcitabine alone or GTX- gemcitabine, docetaxel, and capecitabine combination) followed by CRT. We sought to determine rate of margin negative resection, pathological response and overall survival (OS), calculated from start of neoadjuvant therapy.
Results: Among a total of 20 patients with borderline resectable pancreas cancer 15 patients underwent surgical resection. Margin free resection (R0) was achieved in 100% and 66% patients in group A and B respectively. Patients with margin –ve resection (N = 13) had mean OS of 22 months, which was comparable to other studies in which it was 15–40 months. Grade 1 (marked) and Grade 2 (moderate) pathological responses were observed in 53% patients and was associated with a mean OS of 33 months. Those with + ve margins (N = 2) had OS of 16.5 months. Those who were not resected with POD after neo adjuvant therapy (N = 5) had mean OS of 19 months. Patients who did not undergo resection in other studies have shown median OS of 6–17 months.
Conclusion: Our institutional review of neoadjuvant multimodality treatment for this patient group shows outcomes which are comparable to those reported in the literature. Limitations of our review are the limited number of patients, a lack of uniformity with regard to pre- operative therapy, and the retrospective nature of data collection. Although the data set is small it is consistent with prior findings that pathologic response correlates with better outcomes. We are encouraged that in our group of patients 10 of 15 patients, who underwent surgical resection post neoadjuvant therapy, remain cancer free at the time of this report, and survival averages are likely to improve further over time. More prospective randomized controlled studies in a multi- institutional setting may be required to support the role of neoadjuvant therapy and identify the most promising treatment regimen.
Efficacy of SBP-101 With or Without Gemcitabine and/or Nab-paclitaxel in a Murine Xenograft Model of Human Pancreatic Ductal Adenocarcinoma
A. Shah,1 S. Gagnon,1 P. Milani de Marval,2 A. Kiorpes,1 M. Cullen.11Sun BioPharma, Inc., Gainesville, FL; 2Charles River Discovery Services, Morrisville, NC.
Background: SBP-101 is an analogue of the native polyamine (PA) spermine. PA analogues have been proposed as anti-cancer therapeutics. In this study the efficacy of SBP-101 administered alone and in combination with gemcitabine (GEM) and/or nab-paclitaxel (NAB) was evaluated in a murine xenograft model of pancreatic ductal adenocarcinoma (PDA).
Methods: BxPC-3 PDA tumor fragments were implanted into nude mice. When tumor volumes (TV) reached 100 mm3, mice were assigned to one of 7 groups (10/group): Vehicle, SBP-101 (5x/wk), SBP-101 (3x/wk), SBP-101 + GEM, SBP-101 + NAB, SBP-101 + GEM + NAB, or GEM + NAB for up to 4 wk. Body weights (BW, daily for 5 d then 2x/wk) and TV (2x/wk) were measured. The primary endpoint was % tumor growth inhibition (TGI) on Day 18. Groups were also assessed for partial regression (PR) and complete regression (CR) responses. The concentration of SBP-101 was evaluated in plasma (Cp) and in pancreas (P) in a satellite group (n = 3) treated with SBP-101 monotherapy.
Results: TGI occurred in all treatment groups ranging from 98.7% (SBP-101 + NAB) to 67.4% (SBP-101 alone, 3x/wk). Tumor regression occurred with SBP-101 + NAB (4 CR, 6 PR), GEM + NAB (5 PR), SBP-101 + GEM + NAB (1 CR, 3 PR) and SBP-101 + GEM (2 PR). Side effects included deaths (≤ Day 9, SBP-101 + GEM + NAB and GEM + NAB), and BW loss (all groups). Cp and P concentrations (Mean ± SD) were 35.3 ± 4.5 μg/mL and 177 ± 20.8 μg/g, respectively.
Conclusion: SBP-101 treatment combinations with GEM and/or NAB produced TGI of greater than 60% and tumor regression. P concentrations indicated good penetration of SBP-101 into tissue. SBP-101 has the potential to improve the standard of care for PDA. Further investigations into the best combination dosages and dosing regimens are warranted.
Curcumin Inhibits Angiogenesis Induced by Interaction of Pancreatic Cancer With Stellate Cells
S. Shao, X.H. Zhao, M.N. Luo. Department of Oncology, First Affiliated Hospital of Xi’an Jiaotong University, Xi’an, Shaanxi, China.
Aims: To evaluate antiangiogenic activity of curcumin and their relevant molecular mechanisms.
Methods: Pancreatic cancer cells (Panc-1 and Bxpc-3) and primary stellate cells (PSCs) were co-cultured with or without (20 μM) curcumin. Human umbilical vascular endothelial cells (HUVEC) were treated with the co-culture medium. Si-RNA against Cav-1 were used to knockdown Cav-1 in HUVEC. Tube formation assay was applied to evaluate angiogenesis ability. Caveolin-1 (CAV-1), HIF-1α, Notch1 and Hey1 mRNAs in HUVEC were measured by real-time PCR. VEGF-A, PDGF and COX-2 level in co-culture medium was determined by ELISA.
Results: Conditioned medium from pancreatic cancer cells (Panc-1 and Bxpc-3) and PSCs exposed to curcumin in vitro significantly inhibited HUVEC tube formation, compared with untreated cell medium. And obvious suppression of VEGF-A, PDGF, and COX-2 secretion was observed in co-culture cell lines treated with curcumin. Treatment with curcumin upregulated Cav-1 expression but inhibited HIF-1α, Notch1 and Hey1 expression in HUVEC. Knockdown of Cav-1 in HUVEC upregulated its HIF-1α expression and significantly reversed the effect of curcumin on inhibiting HUVEC tube formation. Moreover, silencing of Cav-1 up-regulated Notch1 and Hey1 expression in HUVEC.
Conclusions: Curcumin have antiangiogenic activity, determining the up-regulation of CAV-1 and the suppression of HIF-1α and Notch signaling.
Proteomic Characterization of Acinar Cells Isolated Using Laser Capture Microdissection From Pancreata of Mice With Cerulein-Induced Chronic Pancreatitis
J.P. Shapiro,1 H. Komar,2 P. Hart,1 D.L. Conwell,1 G.B. Lesinski.1,21Division of Gastroenterology, Hepatology & Nutrition, 2Division of Medical Oncology, Department of Internal Medicine, The Ohio State University Wexner Medical Center, Columbus, OH.
Diagnostic biomarkers for chronic pancreatitis at early stages are extremely limited. We hypothesized that analysis of pancreata from the cerulein model of pancreatitis may help identify novel factors with diagnostic utility. This model recapitulates inflammation, fibrosis and exocrine insufficiency observed in humans. C57BL/6 mice were injected intraperitoneally with 50 mg/kg cerulein (6x/day, 3x/week) for 1, 3 or 6 weeks. Mice treated with PBS (vehicle), or not injected were controls. We analyzed proteome profiles of acinar cells during the cerulein-induced progression of pancreatitis. At each time point, 10–20,000 acinar cells were collected using laser capture microdissection (LCM). Samples were processed for protein recovery, trypsin digested and peptides were analyzed by LC-MS/MS using liquid chromatography coupled to an OrbiTrap Fusion mass spectrometer. Peptide counts identified by LC-MS/MS were used to compare protein expression from individual cerulein treated vs. control mice. A total of 3,000 proteins were identified by LC-MS/MS. Consistent with acinar damage, secreted proteins found to have fewer detectable peptides included amylase and lipase, while increased peptides derived from trypsin were also detectable. More peptides from proteins associated with fibrosis including collagens, keratins and fibronectin were detected by LC-MS/MS. Together these data validate the biologic features of the system, showing hallmarks of damage and exocrine insufficiency. In addition, peptides from 180 other proteins were altered in acinar cells from cerulein treated mice by >3 -fold change. These data from a classic model of murine pancreatitis may provide insight into molecular processes of disease with diagnostic potential.
βIVb-Tubulin is a Novel Therapeutic Target for the Treatment of Pancreatic Cancer
G. Sharbeen,1,* J. Liu,1,* J. McCarroll,2,* L. Limbri, J. Youkhana,1 D. Goldstein,1 M. Kavallaris,2 P. Phillips.11Pancreatic Cancer Translational Research Group, 2Children’s Cancer Institute, Lowy Cancer Research Centre, UNSW Australia, Sydney, Australia; 3Australian Centre for NanoMedicine, UNSW Australia, Sydney, Australia.*Equal authors.
Introduction: Pancreatic cancer (PC) is a lethal disease, characterized by chemoresistance. Components of the cell cytoskeleton are therapeutic targets in cancer. βIV-tubulin is one such component that has two isotypes βIVa and βIVb; isotypes share high sequence homology. Studies have implicated high βIVb-tubulin expression with chemoresistance in breast and lung cancer. However, to date, no studies have examined the role of βIV-tubulin in PC or attempted to identify isotype specific roles in regulating cancer cell growth and chemosensitivity.
Aim: To determine the role of βIVa- or βIVb-tubulin on PC growth and chemosensitivity.
Methods: PC cells (MiaPaCa-2, HPAFII, AsPC1) were treated with siRNA (control, βIVa-tubulin or βIVb-tubulin). Suppression of βIVa- and βIVb-tubulin was >90% by qPCR. The ability of PC cells to form colonies ± chemotherapy (paclitaxel or vincristine) was measured by clonogenic assays.
Results: Silencing βIVb-tubulin significantly reduced the clonogenic capacity of HPAFII (36%, p < 0.001, n = 6) and AsPC1 cells (25%, p < 0.01, n = 5) versus control. In contrast, silencing βIVa-tubulin had no effect on PC cell growth. Notably, βIVb-tubulin silencing sensitized all 3 PC lines to vincristine (≥67% reduction vs. βIVb-tubulin silencing alone, p < 0.05, n = 5), and sensitized HPAFII and AsPC1 cells to paclitaxel (≥62% reduction vs. βIVb-tubulin silencing alone, p < 0.0001, n ≥ 3). βIVa-tubulin silencing had no effect on chemosensitivity.
Conclusions: We show for the first time that βIVb-tubulin, but not βIVa-tubulin, plays a role in regulating cell growth and chemosensitivity in PC cells. Therapeutic targeting of βIVb-tubulin in PC patients could reduce PC growth and increase chemotherapy drug efficacy, thus improving patient outcome.
βIII-Tubulin Regulates Sensitivity to Nab-Paclitaxel (Abraxane®) in Pancreatic Cancer
G. Sharbeen,1 J. McCarroll,2,3 J. Liu,1 J. Youkhana,1 D. Goldstein,1 M. Kavallaris,2,3 P. Phillips.1,31Pancreatic Cancer Translational Research Group, 2Children’s Cancer Institute, Lowy Cancer Research Centre, UNSW Australia, Sydney, Australia; 3Australian Centre for NanoMedicine, UNSW Australia, Sydney, Australia.
Introduction: Pancreatic cancer (PC) is lethal and is the 4th leading cause of cancer-related deaths. Albumin-bound paclitaxel (Abraxane®) combined with gemcitabine is the standard of care, but only increases median survival by 8 weeks. We have shown the microtubule protein βIII-tubulin is highly expressed in PC cells, and silencing its expression reduces PC tumor growth/metastases in vivo and sensitizes PC cells to chemotherapies in vitro including paclitaxel (Oncotarget. 2015;6:2235–49). However, the role of βIII-tubulin in regulating sensitivity to paclitaxel in vivo is unknown.
Aim: To assess the effect of silencing βIII-tubulin expression on PC sensitivity to Abraxane® in vivo.
Methods: MiaPaCa-2 PC cells expressing βIII-tubulin or control (Ctrl) shRNA were injected into the pancreas of mice (n = 7-9/group). 4 weeks later, mice were injected twice weekly with human albumin (control) or Abraxane® (10 mg/kg; Specialized Therapeutics Australia) for 4 weeks. βIII-tubulin levels assessed by qPCR, western blot and immunohistochemistry (IHC). Cell proliferation in tumors assessed by ki67 IHC.
Results: βIII-tubulin shRNA inhibited βIII-tubulin expression in PC tumors. Suppression of βIII-tubulin alone reduced PC tumor growth (128.3 ± 42.3 mm3; p < 0.05) compared to Ctrl (431.8 ± 170.3 mm3). Abraxane® alone also reduced PC tumor growth relative to albumin controls (93.8 ± 14.8 mm3, p < 0.05). Suppression of βIII-tubulin + Abraxane® significantly reduced tumor growth relative to single treatments (27.6 ± 12.1 mm3, p < 0.05) and was associated with a decrease in tumor cell proliferation.
Conclusions: Silencing βIII-tubulin sensitized PC tumors to Abraxane®. Therapeutic inhibition of βIII-tubulin combined with Abraxane® may improve the survival of PC patients.
Triptolide Induces Pancreatic Cancer Cell Death by Reactivating p53 via Aurora Kinase A
N. Sharma, M.K. Singh, V. Dudeja, S. Banerjee, A.K. Saluja. Division of Basic and Translational Research, Department of Surgery, University of Minnesota, Minneapolis, MN.
Background: Pancreatic ductal adenocarcinoma is the fourth leading cause of death in the United States. Triptolide and its water-soluble analog Minnelide, has shown promising results in phase I trial for pancreatic and other GI cancers. While Triptolide/Minnelide mediated killing of pancreatic cancer cells is mainly attributed to downregulation of Heat shock protein 70 (HSP 70) and transcription factor Specificity protein 1 (SP1), further mechanistic details are still unfolding.
Aim: The aim of the current study is to evaluate the effect of triptolide on Aurora kinase A (Aur-A), an important mitotic protein which is essential for cell proliferation, and is found to be up-regulated in many solid cancers including pancreatic cancer.
Method: Pancreatic cancer cell lines MiaPaCa-2, Panc-1, S2VP10 were treated with triptolide (25, 50, 100 nM) for 48 hours. Protein lysates were prepared from these cells and immuno-blotting experiments were performed using phospho-aurora A, total aurora A and other antibodies to detect other downstream proteins of aurora-A signaling. Mia-PaCa-2, PANC1 and S2VP10 cells were treated with triptolide for 24 hours and RNA was extracted using trizol method. Primers for β-Actin and TP53 were designed and obtained from Integrated DNA technologies (IDT)
Triptolide regulates kinase activity of Aur-A as evidenced by dose dependent decrease in phosphorylation at T288 of Aur-A protein. At higher concentration, triptolide also destabilizes Aur-A protein. Interestingly, Aur-A is known as a key regulatory component of the p53 pathway and overexpression of Aur-A leads to increased degradation of p53. In our experiments, we demonstrate that triptolide mediated Aur-A depleted cells do have increased expression of p53 as compared to untreated cells.
Conclusion: The tumor suppressor protein p53 plays a critical role in limiting malignant development and progression. Almost all cancers show loss of p53 function, through either mutation in the p53 gene itself or defects in the mechanisms that activate p53. In our study, we provide a novel mechanism to explain effectiveness of triptolide in limiting pancreatic tumor growth by possible reactivating p53 via Aurora kinase A.
Plasma MicroRNA-127’s Early Warning of Acute Pancreatitis With Respiratory Failure
N. Shi, L.H. Deng, W.W. Chen, X.X. Zhang, Y. Ma, R.J. Luo, K. Jiang, X.N. Yang, Q. Xia. Department of Integrated Traditional Chinese and Western Medicine, West China Hospital of Sichuan University, Chengdu, China.
Background and Aims: Prognostication in acute pancreatitis (AP) remains a challenging issue. Lung is the main organ of organ failure, early warning of respiratory failure might improve prognosis of severe acute pancreatitis (SAP). MicroRNA-127 (miR-127) plays a negative regulatory role in acute lung inflammation disease. We aim to determine the role of miR-127 in early predicting acute lung injury of SAP.
Methods: Rat AP model was induced with sodium taurocholate retrograde cholangiopancreatography injection (3.5% for SAP; 0.5% for mild AP). Sham-operated (SO) without injection was conducted as control. Sacrifice was made at 6 h, 12 h, 24 h to assess AP severity and collect lung tissue and plasma. A cohort of AP patients admitted to West China Hospital, China, within 48 h from the onset of disease was prospectively enrolled. Quantitative reverse transcription-polymerase chain reaction was used to detect miR-127 in lung and plasma and then measured in AP patients and in healthy volunteers.
Results: Histopathology scores of pancreas and lung, and biochemical markers in SAP rats were significantly higher than those in MAP and SO group (all P < 0.05). The expression of lung miR-127 of SAP rats at 24 h was significantly increased compared to that of SO and MAP group (P <0.05), while plasma miR-127 was decreased (P <0.05). Clinical study included 10 AP patients (5 with respiratory failure, 5 without respiratory failure), and 4 healthy volunteers. The expression of plasma miR-127 in AP patients with respiratory failure was significantly lower than that in healthy volunteers (P = 0.041), and those without respiratory failure (P = 0.030).
Conclusion: Plasma miR-127 might be a novel biomarker for early warning lung injury in SAP.
Pancreas Transection With Linear Stapler and Bipolar Cautery Forceps in Distal Pancreatectomy
Y. Shindo, T. Ueno, N. Suzuki, H. Matsui, M. Nakajima, S. Matsukuma, Y. Tokumitsu, Y. Tokuhisa, K. Sakamoto, T. Tamesa, S. Takeda, S. Yoshino, S. Hazama, H. Nagano. Department of Digestive Surgery and Surgical Oncology, Yamaguchi University Graduate School of Medicine, Ube, Yamaguchi, Japan.
Introduction: Pancreatic fistula (PF) is a major complication after distal pancreatectomy (DP). We usually use a stapler in combination with reinforcement. We recently prefer to use bipolar cautery forceps rather than using a surgical scalpel when transecting the pancreas, in which we could easily detect the main pancreatic duct and ligate it without fail. Furthermore, tiny branches of the stump would be sealed by the bipolar cautery forceps. Finally, the pancreatic remnant is cared using an additional absorbable reinforcement material and fibrin glue.
Patients and Surgical manipulations
Eight patients undergoing DP were enrolled in this pilot series.
Step 1: Reinforcement of staple line using 2-plys of commercial available polyglycolic acid (PGA) sheet (Neoveil ®).
Step 2: Guide of a Linear Stapler (Ethicon PROXIMATE® 60 mm) with a Penrose drainage tube.
Step 3: Compression of the pancreas parenchyma by the jaws of the stapler gently until the Gap force reaches to 1.5-2.0 mm.
Step 4: Ligation of the distal pancreas and wait for 5 minutes.
Step 5: Firing the stapler (as slowly as possible).
Step 6: Transection of the pancreas using SuperGliss® non-stick bipolar cautery forceps with VIO system.
Step 7: Detection and Ligation of the main pancreatic duct.
Step 8: Sealing of the pancreatic remnant incorporating the staples with PGA sheet and fibrin glue.
Results: The numbers of patients who developed grade A PF were 5 (62.5%). None of the patients developed grade B and C PF.
Conclusion: Our traditional procedure combined with this modification may become accepted as a unique approach in DP. With these early promising findings, clinical investigation of this technique is currently in progress.
Evaluation of Vitamin D, A, E Status in Patients With Pancreatic Disorders
S. Siminkovitch,1 M. Kovacheva-Slavova,1 B. Vladimirov,1 J. Genov,1 R. Mitova,1 P. Gecov,2 D. Svinarov,3 B. Golemanov.11Department of Gastroenterology, 2Department of Medical Imaging, University Hospital "Tsaritsa Ioanna-ISUL", Sofia, Bulgaria; 3Central Laboratory of Therapeutic Drug Management and Clinical Pharmacology, Alexandrovska University Hospital, Medical University of Sofia, Bulgaria.
Introduction: Deficiency of fat soluble vitamins D (VD), A (VA) and E (VE) is present in chronic pancreatitis (CP), recurrent pancreatitis (RP) and after pancreatic surgery (PS) but data about their extension are contradictory.
Aims & Methods: To determine the prevalence of fat soluble vitamin deficiency in patients with CP, RP and after PS and to assess its relationship to severity of morphological imaging data. 78 patients were enrolled (40 male, 38 female), mean age 52 yrs. 49 patients suffered from CP, 21– from RP, 8 underwent PS. Alcohol abuse was most common etiology. Imaging morphological data were assessed by Cambridge classification for CT/MRCP (grade I-IV). Pancreatic exocrine insufficiency (PEI) was found in 39 patients. Malnutrition was evaluated by serum prealbumin (preAlb) and retinol binding protein (RBP).VD was defined by LC-MS/MS method and VA and VE by HPLS. Statistical analysis was performed by SPSS v19.
Results: Vitamin A, D, E insufficiencies were present in 5; 93.6 and 70% of patients, respectively. Moreover 38.5% had VD deficiency (<25 nmol/L). Mean VD, VA and VE levels were 38.9 ± 25.7 nmol/L, 520 ± 306 μg/L and 4.36 ± 3.93 μg/L, respectively. VD status worsened with severity of morphological changes regardless of season or cause (p < 0.01). VD and VA levels were lower in patients with PEI, p < 0.05. VE insufficiency did not depend on PEI (p > 0.05). However, there is no significant difference for VA and VE within morphological groups. VA correlates with RBP (r = 0.69) and preAlb(r = 0.86).
Conclusion: Most of our patients regardless PEI were fat soluble vitamins (VE and VD) insufficient and there was a strong relationship only between VD levels and severity of morphological imaging changes.
Human Recombinant Activated Protein C (XIGRIS) as a Disease Modifier Early in Severe Acute Pancreatitis: Final Results of a Case-Controlled Study (XIGAP 1)
A.K. Siriwardena,1 C.J. Miranda,1 J.M. Mason,3 A.J. Sheen,1 J.M. Eddleston,2 M.J. Parker,2 B.I. Babu.11Hepatobiliary Surgical Unit, 2Critical Care Unit Manchester Royal Infirmary, 3School of Medicine, University of Durham, United Kingdom.
Introduction: Pancreatic microvascular thrombosis mediates necrosis in acute pancreatitis (AP). Xigris maintains microvascular permeability in small vessel beds and could modulate necrosis. Although it did not reduce overall mortality in a heterogeneous critical care population with sepsis, it was not evaluated as an early disease modifier in severe AP. This study is a preliminary evaluation of Xigris early in severe AP.
Methods: In a prospective case–control trial approved by ethics committee, patients with severe AP [fulfilling all of the following: APACHE II of ≥ 9 on admission and at least two of four SIRS criteria measured on two occasions separated by 24 hours] received Xigris for 24 hours by intravenous infusion (24 μg/kg/hr) within 72 h of disease onset. A matched control group treated within the prior 24 months in the same unit were comparators. Control data were matched by age, gender and severity. Of 166 consecutive patients, 43 met screening criteria and 19 were recruited. Mean admission APACHE II was 12.8 sd 5.0, 53% gallstone, 58% necrosis. Trial Registration: Eudract Number 2007-003635-23
Results: There was no treatment-related bleeding. Compared to controls there were fewer bleeding events in the Xigris group although not reaching significance (Xigris 0% vs. Control 21%, p = 0.13), similarly further intervention was less (11% vs. 47%, p = 0.07) in the treatment group. Length of stay was shorter for patients receiving Xigris (19 vs. 41 days, p = 0.03) as was inotrope use (5% vs. 32%, p = 0.02); mortality and incidence of infections were similar. Biomarker protein C increased while IL-6 decreased following infusion.
Conclusions: A 24-hr infusion of Xigris appears safe in severe AP. Further evaluation of this category of microvascular patency-modulating drugs in severe AP is indicated.
Immune Modulation of Pancreatic Cancer With CCK-receptor Blockade
J.P. Smith, S. Wang, S.A. Jablonski, L.M. Weiner. Departments of Oncology & Medicine, Georgetown Lombardi Comprehensive Cancer Center, Georgetown University, Washington, DC.
Introduction: CCK peptide stimulates growth of pancreatic cancer, accelerates PanIN progression, and activates stellate cells to release collagen. CCK has also been shown to have immunomodulatory effects that alter specific CD4+ T cell subsets and a positive regulatory effect on inducible T regulatory cells. The aim of this study was to investigate the role of CCK receptor & immune check point blockade on tumor infiltrating lymphocytes (TILs) in a syngeneic mouse model of pancreatic cancer.
Methods: Subcutaneous or orthotopic tumors were established using Panc02 murine pancreatic cancer cells in immune competent (WT) or immune deficient (SCID) mice. Mice were treated with PBS (control), L364,718 (CCK receptor antagonist, CCK-At), PD1- Ab or CTLA-4 Ab (immune checkpoint blockade, Im-Ab) or the combination of CCK-At and Im-Ab. Tumor growth and animal survival were evaluated. Tumors were subjected to immunohistochemical staining for TILs and flow cytometry.
Results: Orthotopic tumors of SCID mice were 5-fold larger and had more metastases than WT mice. In WT mice, TILs increased by 2-, 4-, and 6-fold in CCK-At, Im-Ab and combo therapy, respectively compared to controls (p < 0.03). Flow cytometry revealed a shift in the CD4:CD8 ratio from 1.6 to 0.4 in tumors treated with the combo therapy. On day 50 after tumor inoculation all control mice had died while 42%, 42% and 71% of CCK-At, Im-Ab mice, or combo-treated mice were alive (p = 0.02). By day 90 all mice died except one in the combo-treated group.
Conclusions: A competent immune system aids in suppression of pancreatic tumor growth and metastases. CCK receptor blockade promotes infiltration of TILs into pancreatic cancer. The combo of CCK-At and Im-Ab improve survival of pancreatic cancer in a mouse model. Strategies to empower the TILs through immune checkpoint blockade may be considered for future therapies in patients with advanced pancreatic cancer.
Dual PI3K/mTOR Inhibitors Induce Rapid Over-Activation of the MEK/ERK Pathway in Human Pancreatic Cancer Cells Through Suppression of mTORC2
H.P. Soares, Q. Xu, S.H. Young, J. Sinnet-Smith, E. Rozengurt. Division of Hematology-Oncology and Division of Digestive Diseases, Department of Medicine, David Geffen School of Medicine at UCLA; CURE: Digestive Diseases Research Center, University of California at Los Angeles, CA.
Background: The phosphatidylinositol 3-kinase (PI3K)/Akt/mTORC1/S6K pathway which is aberrantly stimulated in many cancer cells, has emerged as a target for therapy. However, mTORC1/S6K also mediates negative feedback loops that attenuate upstream signaling. Suppression of these feedback loops opposes the growth-suppressive effects of mTOR inhibitors and leads to drug resistance in a cell-type dependent manner. Here, we examine feedback loops in PANC-1 or MiaPaCa-2 pancreatic ductal adenocarcinoma (PDAC) cells.
Results: Exposure of PDAC cells (MiaPaca-2 and PANC-1) to the dual PI3K/mTOR inhibitor NPV-BEZ235 potently blocked mTORC1/S6K activation mTORC1/4E-BP1 and mTORC2-mediated Akt phosphorylation, in a concentration-dependent manner. Strikingly, NPV-BEZ235 markedly enhanced the MEK/ERK pathway in a dose-dependent manner. Maximal ERK over-activation coincided with complete inhibition of phosphorylation of Akt and 4E-BP1. ERK over-activation was induced by other dual PI3K/mTOR inhibitors, including PKI-587 and GDC-0980. The MEK inhibitors U126 or PD0325901 prevented ERK over-activation and the combination of NPV-BEZ235 and PD0325901 caused a more pronounced inhibition of cell growth than that produced by each inhibitor. Mechanistic studies assessing PI3K activity in single PDAC cells using a PIP3 fluorescent reporter indicated that NPV-BEZ235, PKI-587 and GDC-0980 act through a PI3K-independent pathway. Doses of PI3K/TOR-KIs that enhanced MEK/ERK activation coincided with those that inhibited mTORC2-mediated Akt phosphorylation on Ser473, suggesting a role of mTORC2. Knockdown of Rictor markedly increased baseline levels of ERK phosphorylation and treatment with NVP-BEZ235 did not produce further enhancement of ERK activation. These results imply that Rictor or mTORC2 exerts feedback inhibition of the MEK/ERK pathway in pancreatic cancer cells.
Conclusion: We propose that dual PI3K/mTOR inhibitors suppress a novel negative feedback loop mediated by mTORC2 thereby leading to enhanced MEK/ERK pathway activity in pancreatic cancer cells.
Multi-panel of Inflammatory and Nutritional Parameters in a Prospective Cohort of Pancreatic Cancer Patients
S. Stigliano, M. Piciucchi, G. Zerboni, L. Archibugi, V. Barucca, R. Valente, G. Delle Fave, G. Capurso. Digestive & Liver Disease Unit, Sant’Andrea Hospital, Sapienza University, Rome, Italy.
Background: Pancreatic Cancer microenvironment cells produce pro-inflammatory cytokines promoting cancer progression. Many biochemical markers, such as C-reactive protein (CRP) and Neutrophil/Lymphocyte Ratio (NLR) reflect this Immune System activation and predict poor prognosis. However, the prognostic role of NLR in patients with distant metastasis has not been extensively validated and the usefulness of NLR has not been compared to other inflammatory scores.
Aim: To assess the prognostic role of NLR and of other inflammatory markers and scores in different Pancreatic Cancer stage.
Methods: The association between inflammatory parameters (White Blood Cells, CRP, NLR, Odonera PNI score, Glasgow Prognostic Score, Platelets/Lymphocyte Ratio) and cachexia, nutritional status, tumor’s features was evaluated in a prospective PDAC cohort.
Results: 206 patients, 76 resectable, 130 not-resectable (81 metastatic) at diagnosis. Patients with NLR > 5 had lower albumin (3.1 g/dl vs 3.5 p < 0.0001) and hemoglobin (11.6 g/dl vs 12.3 p = 0.03) level than NLR < 5, but with similar rate of moderate (36.6% vs 40.4% p = 0.64) or severe cachexia (10% vs 13% p = 0.64). At multivariate Cox-analysis only NLR > 5 resulted associated with survival. Metastatic disease (HR 2.45; 95%CI 1.34-4.48 p = 0.0036), chemotherapy (HR 0.45; 95% CI 1.02-3.23 p = 0.042) and NLR > 5 (HR 1.82; 95% CI 1.02-3.03 p = 0.042) were associated with prognosis. NLR > 5 was the only significant biochemical marker in stage IV patients (HR 1.75; 95%CI 1.02-3.03).
Conclusion: Elevation of NLR at diagnosis (>5) is the only inflammatory marker able to predict poor prognosis in PC patients. Elevated NLR is associated to a more advanced tumor stage, elevated CRP and lower albumin level, but not to cachexia occurrence. In Stage IV patients, elevated NLR is associated with worse prognosis independently from chemotherapy.
Recurrent Acute Pancreatitis: Risk Factors of Re-admission
S. Stigliano, M. Piciucchi, M. Signoretti, G. Delle Fave, G. Capurso. Digestive & Liver Disease Unit, Sapienza University, Rome, Italy.
Background: There are limited data on the risk of hospital readmission after a first episode of acute pancreatitis (AP). The prevalence of Recurrent Acute Pancreatitis (RAP) in various retrospective studies on AP ranges from 10 to 30%, but the exact rate of recurrent acute pancreatitis is difficult to estimate. Risk of RAP seems higher in alcoholic and idiopathic AP, but most data were obtained in countries where alcohol is the most frequent etiology.
Objective: To evaluate the rate of RAP and possible risk factors associated with its occurrence.
Methods: Single-center prospective study of consecutive AP patients. Patients with previous biliary or pancreatic disorders were excluded. All patients had follow-up visits.
Results: 200 patients were included: 96 (48%) biliary, 30 (15%) alcoholic, 25 (12.5%) idiopathic, other causes 49 (24.5%). In a mean follow-up of 40 months, 34 patients (17%) had RAP. Most of RAP (62%) occurred during first year after hospital discharge and in particular 18% within first 30 days. Most frequent etiologies of RAP were: 17 (50%) biliary, 3 (8.8%) alcoholic and 5 idiopathic (14.7%). Age, gender and severity were not associated with RAP risk.
Of the 17 patients with biliary RAP, only 3 (17%) had received cholecystectomy after the first AP, compared to 36 (46%) of the 79 who did not have recurrence (p = 0.05). In this regard, failure to perform cholecystectomy was associated with the risk of biliary RAP (OR 4; 95% CI 1.05-15 p 0.02). In alcoholic AP, 33% of RAP had stopped drinking compared to 55.6% of not recurrent (p = 0.58). All alcoholic RAP had more than two attacks, compared to 12.5% of RAP due to other etiologies (p = 0.013).
Conclusion: RAP occurs in about 20% of cases, biliary etiology being the most prevalent. Failure to treat biliary etiology is associated with a 4-fold recurrence risk increase. Alcoholic AP patients seem to have a lower risk of recurrence if they quit drinking, but more often they experience multiple AP attacks.
Margin Clearance and Survival in Resected Pancreatic Adenocarcinoma in the Era of Adjuvant Chemotherapy
O. Strobel,1 T. Hank,1 U. Hinz,1 F. Bergmann,2 D. Jäger,3 T. Hackert,1 M.W. Büchler.11Department of General Surgery, 2Institute of Pathology, 3National Center for Tumour Diseases, Heidelberg University Hospital, Heidelberg, Germany.
Background: The definitions for R0 and R1 margin status after resection for pancreatic cancer are controversial and reported R0/R1 rates and associated survival are highly heterogeneous. A strict definition of R1 (margin clearance of ≤ 1 mm) is accepted in Europe, but not internationally. A prospective validation of this definition based on associated survival is missing. The impact of margin status on survival in the context of adjuvant therapy remains unclear.
Methods: A standardized protocol with evaluation of circumferential margins and the novel R1 definition were introduced into clinical routine in 2005. From a prospective database, patients undergoing pancreatoduodenectomy for pancreatic adenocarcinoma between 01/2006 and 12/2012 were identified. The rate of R0, R1 (≤1 mm clearance) and R1 (direct margin involvement) status and associated survival were assessed by uni- and multivariable analyses.
Results: Of 561 patients with pancreatic adenocarcinoma, 112 (20.0%) had R0, and 449 (80.0%) had R1 resections, including 123 (21.9%) R1 (≤1 mm) and 326 (58.1%) R1 (direct) resections. 438 (85.9%) patients received adjuvant chemotherapy. Margin status was significantly associated with survival. With R0, R1 (≤1 mm) and R1 (direct) status the median survival times and five-year survival rates were 41.6, 27.5 and 23.4 months; and 37.7, 30.1 and 20.3%, respectively (p < 0.0001). By multivariable analysis, margin status was confirmed to be independently associated with survival.
Conclusions: In the era of adjuvant chemotherapy the resection margin status remains an important independent determinant of post-resection survival. R0/R1 resection rates and associated survival vary significantly with the definitions used. An international consensus is urgently needed to achieve comparability with respect to studies and protocols on adjuvant therapy.
Prognostic Scoring System for Patients Who Present With Gastric Outlet Obstruction Due to Advanced Pancreatic Adenocarcinoma
T. Sugiura, Y. Okamura, T. Ito, Y. Yamamoto, R. Ashida, K. Uesaka. Division of Hepato-Biliary-Pancreatic Surgery, Shizuoka Cancer Center, Shizuoka, Japan.
Background: Gastroenterostomy and stent placement are the most commonly used as palliative treatments for patients with gastric outlet obstruction due to advanced pancreatic adenocarcinoma. Gastroenterostomy is considered as treatment of choice for patients in whom longer survival is expected, whereas stent placement is preferable for patients in whom relatively shorter survival is expected. However, the prognosis of such patients has not been fully evaluated.
Methods: The study population included patients who underwent gastroenterostomy or stent placement for gastric outlet obstruction due to advanced pancreatic adenocarcinoma between 2002 and 2012. Prognostic factors were evaluated by multivariate analyses. Factors that were significant by the multivariate analysis were given a prognostic score according to their hazard ratios (HR). Overall survival was calculated according to the total prognostic score.
Results: A total of 107 patients were enrolled. Eighty-three and 24 patients underwent gastroenterostomy and stent placement, respectively. The median survival time of the study population was 4.2 months. Univariate and multivariate analyses revealed neutrophil-lymphocyte ratio (NLR) of ≥ 4 (HR = 5.81, p < 0.001), presence of liver metastases (HR = 2.45, p < 0.001), and presence of cancer pain (HR = 2.83, p < 0.001) to be significant prognostic factors. Thereafter, NLR of ≥ 4, presence of liver metastases and presence of cancer pain were scored as 2, 1, and 1, respectively. The overall survival in patients with total prognostic scores of 0, 1, 2, 3 and 4, was 17.6, 8.3, 3.6, 3.4 and 1.6 months, respectively.
Conclusion: Our scoring system clearly demonstrated patient survival. Patients with scores of 0 or 1 were found to be favorable candidates for gastroenterostomy, while patients with scores of 2 to 4 were found to be candidates for stent placement.
Undifferentiated Pancreatic Carcinoma With Osteoclast-like Giant Cells Showing Tumor Thrombus Into the Main Pancreatic Duct: Report of a Case With Osteoid Formation
M. Sunahara, S. Ueki, Y. Ono, Y. Sakamoto, T. Sawano, T. Sato, H. Kasajima, M. Kurushima, S. Suzuki, J. Kimura. Department of Surgery, Institute of Gastroenterology, Hakodate Municipal Hospital, Hakodate, Japan.
Background: Undifferentiated pancreatic carcinoma with osteoclast-like giant cells is a rare tumor accounting for less than 1% of all exocrine pancreatic cancers and has a poor prognosis. The origin of this tumor, whether epithelial or mesenchymal, and a standard regimen for adjuvant chemotherapy are still unknown.
Methods: We report the case of an undifferentiated pancreatic carcinoma with osteoclast-like giant cells with tumor thrombus in the main pancreatic duct and focal osteoid formation. An 84-year-old woman was referred to our hospital for treatment a pancreatic tumor. She had been suffering from diabetes mellitus (type 2) for several years. Computed tomography scan showed a mass in the tail of the pancreas, measuring 48 mm in diameter. A distal pancreatectomy with splenectomy was performed and the regional lymph nodes were dissected.
Results: We could observe a massive intraductal tumor growth into the main pancreatic duct. Moreover, the tumor contained a focal area of osteoid formation. Specimen resection margins were free of tumor. The adjacent lymph nodes metastasis and vascular invasion by tumor cells were present. The postoperative course was uneventful. Adjuvant chemotherapy with S-1 was administered. She died of cerebral hemorrhage without metastasis for seven months after the surgery.
Conclusions: Foci of osteoid within undifferentiated pancreatic carcinomas with osteoclast-like giant cells have been described in only a few reports. This case pathologically presented unique features and may provide clues to the recognition of this rare tumor.
Pattern and Impact of Organ Failure on Human Acute Pancreatitis
P. Szatmary,1 W. Huang,1 T. Liu,1 D. de la Iglesia-Garcia,2 S. Parente,1 G. Primo,1 W. Greenhalf,1 R. Sutton.11NIHR Liverpool Pancreas Biomedical Research Unit, The Royal Liverpool University Hospital, Liverpool, United Kingdom; 2Department of Gastroenterology and Hepatology, University Hospital of Santiago de Compostela, Spain.
Background: Organ failure (OF) is a major cause of death in acute pancreatitis (AP) and critical component of outcome classifications; despite its significance, the pattern of OF lacks thorough description. This work sets out to describe the sequence and impact of failure of three major organ systems: respiratory, cardiovascular (CVS) and renal.
Methods: OF data was extracted from a prospectively maintained database of patients admitted within 72 h of onset of AP to a single unit in a 5 year period (2010 – 2015). Patients with pre-morbid critical OF, or at the extremes of age (<18, >85) were excluded. OF was defined as >2 on the sequential organ failure (SOFA) scoring system, or a deterioration of chronic, stable OF. Data were analyzed in IBM SPSS Statistics V21.0 software.
Results: 333 patients with AP were analyzed. Of these, 41 (12.3%) had transient (TOF; < 48 hours) and 30 (9.0%) persistent organ failure (POF). Respiratory failure (RF) was most prevalent, with TOF observed in 42 (12.6%) and POF in 29 (8.7%) patients. Both CVS and renal failure were never observed in isolation, but only in the presence of RF. The prevalence of CVS TOF was 1 (0.3%), CVS POF 12 (3.6%), renal TOF 7 (2.1%) and renal POF 8 (2.4%) patients. 40 (97.6%) of TOF and 28 (93.3%) of POF cases occurred within the first week of admission. Transient RF was mostly observed on admission and had little impact on mortality (1 death; 3.4%), except if it persisted beyond 48 h (6 deaths; 54. 5%; χ2 p = 0.001). Mortality was proportional to the number of failing organ systems, with 2 (3.7%), 2 (33.3%) and 8 (72.7%) deaths seen with 1, 2 and 3 failing organ systems respectively. CVS POF was the greatest predictor of mortality with 9 (75.0%) deaths, but was not seen in isolation.
Conclusions: While isolated early respiratory failure in AP has little impact on mortality if corrected within 48 h of admission, persistent respiratory failure precedes failure of other organ systems. While respiratory failure may result directly from the systemic inflammatory responses of AP, CVS and/or renal failure may also require hypoxia for induction.
Pattern of Infective Complications in Human Acute Pancreatitis
P. Szatmary,1 W. Huang,1 D. de la Iglesia-Garcia,2 S. Parente,1 G. Primo,1 W. Greenhalf,1 R. Sutton.11NIHR Liverpool Pancreas Biomedical Research Unit, Royal Liverpool University Hospital, Liverpool United Kingdom; 2Department of Gastroenterology and Hepatology, University Hospital of Santiago de Compostela, Spain.
Background: Acute pancreatitis (AP) is associated with bacterial translocation from gut and impairment of immunity. The role of infective complications (ICs) in AP remains a source of controversy, with opposing recommendations for prophylaxis in international guidelines. In this study we investigate the prevalence and impact of ICs in AP.
Methods: Microbial culture data (blood, pancreas, sputum and urine) collected during routine clinical care from adult patients admitted within 72 h of onset of AP to a single unit over 5 years (2010 – 2015) were extracted from a prospectively maintained database. Antibiotics were given only following positive blood or fluid culture. Data were analyzed in IBM SPSS Statistics V21.0 software.
Results: 333 patients with AP were analyzed. 34 (10.2%) had a positive culture, but there were 49 ICs (some multi-source, e.g. blood and pancreas). The most frequent source was urine (16; 47.1%); however positive blood (14; 41.2%), pancreas (11; 32.4%) and sputum (8; 23.5%) cultures were also common. Notably 4/14 (28.3%) of all bacteraemias were observed in patients classified as mild AP (revised Atlanta), making bacteraemia the commonest IC in this category (4/7; 57.1%). Gram negative, gut organisms were most frequent (24; 53.3%; median time to culture 5 days, IQR 2.25-15.75), followed by Gram positive (10; 22.2%; 7.5 days, IQR 4.5-11.75), anaerobic (7; 15.6%; 16 days, IQR 6–20) organisms and yeasts (4; 8.9%; 7 days, 3.25-32.5). Anaerobic organisms were isolated from necrotic pancreas in all but one case (6/7; 85.7%). ICs (including infected pancreatic necrosis) were not associated with increased mortality over severity-matched controls, but were associated with longer hospital stays (16.7 vs 41.4 days; paired T-test p = 0.10).
Conclusions: ICs are common in the context of AP, but with standard treatment (antibiotics +/−surgical drainage) not associated with excess mortality. ICs are associated with increased length of hospital stay, but no comment on causality can be made from these data. Bacteraemias with gut organisms occur early in AP, including in mild disease. Anaerobic organisms are only seen in the context of established pancreatic necrosis.
Circulating Histones Target Acinar Cells and Exacerbate Injury in Experimental Acute Pancreatitis
P. Szatmary,1,3 T. Liu,1,2 S. Abrams,2 L. Wen,1 W. Huang,1 G. Wang,2 C.H. Toh,2 D. Criddle,3 A. Tepikin,3 R. Sutton.11NIHR Liverpool Pancreas Biomedical Research Unit, 2Department of Blood Sciences, Royal Liverpool and Broadgreen University Hospital NHS Trust, Liverpool, United Kingdom; 3Dept. of Cellular and Molecular Physiology, University of Liverpool, Liverpool, United Kingdom.
Background: Acute pancreatitis (AP) is a common, debilitating inflammatory condition with significant morbidity and mortality. Cellular injury is accompanied by release of histones passively from acinar necrosis and actively from innate immune cells in experimental and clinical AP; circulating levels correlate with disease severity. We sought to determine the mechanisms by which histones influence the pathogenesis of AP.
Methods: Effects of extra-cellular histones on necrosis of isolated murine acinar cells were investigated measuring propidium iodide fluorescence every min for 90 min and binding mechanisms interrogated using inhibitor molecules. To determine the site of action of circulating histones in vivo, fluorescein isothianate (FITC) labelled histones (20mg/kg) were injected via tail vein into C57Bl6/J mice in experimental AP (cerulein μg/kg, 4 hourly injections) and epifluorescence of extracted vital organs measured. Effects of excess histones on histologic and biochemical markers of severity were assessed.
Results: Histones promoted necrosis of murine pancreatic acinar cells in a charge and concentration dependent manner in vitro, with rapid and complete necrosis observed at 200μg/ml. Using FITC-tagged histones in vitro demonstrated concentration of signal within acinar cell membranes and in vivo revealed signal within the pancreas only where there is concurrent pancreatic inflammation. Histone administration exacerbated pancreatic injury in experimental AP.
Conclusions: Histones exacerbate AP by inducing further pancreatic injury, targeting cell membranes in a charge and concentration dependent manner. Detoxification of circulating histones could be a viable strategy in the treatment of AP.
Circulating Annexin II Predicts Rapid Recurrence After Surgery in Pancreatic Cancer
S. Takano,1 K. Sogawa,2 H. Yoshitomi,1 S. Kagawa,1 H. Shimizu,1 M. Ohtsuka,1 A. Kato,1 K. Furukawa,1 T. Takayashiki,1 S. Kuboki,1 D. Suzuki,1 N. Sakai,1 F. Nomura,2 M. Miyazaki.11Department of General Surgery, 2Department of Molecular Diagnosis, Chiba University, Chiba, Japan.
Introduction: We have previously identified Annexin II (ANX2) which is up-regulated in the gemcitabine-resistant pancreatic cancer (PDAC) cell line using proteomic approach, and associated with rapid recurrence in PDAC patients undergoing adjuvant therapy with gemcitabine after surgery. ANX2 has been also known as the molecule which promotes invasion, and recently considered as a serum biomarker for several tumors. Herein, we investigated whether the levels of ANX2 in serum have clinical relevance for disease recurrence and outcomes of PDAC patients.
Materials & Methods: A specific and sensitive ANX2 enzyme-linked immunosorbent assay (ELISA) was originally established to measure the levels of soluble ANX2 in serum. We compared pre- and postoperative ANX2 levels in sera obtained from PDAC patients who had curative pancreatectomy using the sandwich ELISA. The correlations between serum ANX2 levels and clinico-pathological characteristics, recurrence form after surgery, and prognosis in PDAC patients were analyzed, respectively.
Results: The levels of Anx2 were significantly higher in the preoperative serum than in the postoperative serum in PDAC patients (p<0.02; student-t test). Kaplan Meier analyses showed that the high level of Anx2 in preoperative serum significantly correlated with rapid recurrence (p<0.02; log-rank test) and showed the tendency of poorer prognosis (p<0.07; log-rank test). These results indicate that circulating ANX2 may accelerate cancer progression in PDAC, resulting in rapid recurrence and poor prognosis.
Conclusions: The level of Anx2 in serum may predict the clinical outcome of patients, leading to the development of a novel strategy for tailor-made treatment for pancreatic cancer.
Problems in the Diagnosis of Pancreatic Neuroendocrine Tumors (PNETs) in Clinical Practice
Y. Takayama,1 K. Shimizu,1 M. Yamamoto,2 T. Furukawa.31Department of Internal Medicine, 2Department of Surgery, Institute of Gastroenterology, 3Institute for Integrated Medical Sciences, Tokyo Women's Medical University, Tokyo, Japan.
Background: It is important to assess the function and determine the pathological classification of PNETs in order to make treatment decisions. We investigated problems in the diagnosis of PNETs in clinical practice.
Methods: 71 patients with a pathological or clinical diagnosis of PNET underwent pancreatectomy or biopsy. Variables, including clinical characteristics, surgical data, and pathological findings, were assessed.
Results: Resection was performed in 65 cases, US-FNA in 5 cases, and EUS-FNA in 22 cases. Based on the pathological findings the lesions were classified according to the WHO classification as NET G1 in 47 cases, NET G2 in 16 cases, and NEC in 1 case. There were 55 non-functional PNETs and 9 functional PNETs. The final diagnosis in the 7 patients who had diagnosed with PNET on the basis of symptoms and image findings without pathological findings was: lymph node metastasis by a MANEC, NET of the duodenum, pancreatic metastasis of renal cell carcinoma, pancreatic hemangioma, nesidioblastosis, lymphoplasmacytic sclerosing pancreatitis, and pancreatoblastoma. On the other hand, 16 patients whose clinical diagnosis had not been PNET because of atypical image findings or a small lesion, had misdiagnosed with other pancreatic lesions including pancreatic cancer.
Conclusion: In order to proper treatment decisions in patients with a PNET it is important to make a correct diagnosis of the site of origin the pancreas or other organ, pathological diagnosis if a PNET is suspected clinically, and diagnosis of the site of the primary lesion within the pancreas by detailed diagnostic imaging examination of the entire pancreas.
New Biomarkers for the Detection of Early-Stage and CA19-9-Low Pancreatic Cancer
H. Tang,1 K. Partyka,1 D. Kletter,2 P. Hsueh,1 Y. Huang,4 R.E. Brand,3 B.B. Haab.11Center for Cancer and Cell Biology, Van Andel Research Institute, Grand Rapids, MI; 2Palo Alto Research Center, Palo Alto, CA; 3Public Health Sciences Division, Fred Hutchinson Cancer Research Center, Seattle, WA; 4Division of Gastroenterology, University of Pittsburgh School of Medicine, Pittsburgh, PA. *Present address: Protein Metrics, Inc., San Carlos, CA.
We have found 4 new glycan biomarkers that show promise for identifying patients with early-stage pancreatic cancer or with low CA19-9, the current best biomarker for pancreatic cancer. The CA19-9 assay detects a glycan called sialyl-Lewis A, a sialylated and fucosylated quatrasaccharide. It is not elevated in about 25% of pancreatic cancer patients using a cutoff that gives a 25% false-positive rate. We hypothesized that the CA19-9-low patients have plasma elevations in glycans related to sialyl-Lewis A. We found that glycoforms of the proteins MUC5AC and MUC16 displaying a sialylated and non-fucoslated quatrasaccharide were elevated in 64% (70/109) and 51% (55/109) of pancreatic cancers, respectively, at cutoffs giving 1% (1/91) false-positive detection of patients with pancreatitis or benign biliary obstruction. We also found that glycoforms of MUC5AC decorated with sulfated and non-sulfated sialyl-Lewis X, an isomer of sialyl Lewis A, were elevated in 52% (57/109) of pancreatic cancer patients at a 1% false-positive cutoff. Each biomarker was elevated in CA19-9-low and early-stage cancers at rates similar to those observed in all patients, and the biomarkers were elevated in complementary groups of patients. In an expanded sample set in which a third of the cancer cases were early stage, a panel of markers achieved 85% sensitivity (133/156 cancers) and 90% specificity (144/160 controls), giving a total accuracy of 88%, whereas CA19-9 achieved 54% sensitivity (84/156) and 86% specificity (138/160 controls) for a total accuracy of 70%. The panel performed equally well for early-stage cancer as for late-stage cancer. The new biomarkers raise the prospect of achieving the performance necessary to enable the clinical detection and diagnosis of early-stage pancreatic cancer.
Clinical Observation of the Effect of Rhubarb Nasal Feeding and Mirabilite External Applying Combined With Western Medicine on the Treatment of Severe Acute Pancreatitis
Q.Q. Tang, M.Y. Fang. The First Affiliated Hospital of Anhui Medical University, Hefei, China.
Objective: To investigate the effects of rhubarb nasal feeding and mirabilite external therapy in the treatment of acute severe pancreatitis.
Methods: The diagnostic criteria was based on the Atlanta criteria revised in 2012. 60 patients were randomly divided into treatment group and control group, 30 cases for each group. The former was based on the western medicine treatment combined with rhubarb water feeding, pancreatic mirabilite external applying regional therapy, while the latter with pure western medicine comprehensive treatment.15g rhubarb was soak in 50ml boiling water until deep brown and then liquid temperature 38 to 40 degree for injection. After that the rhubarb water was injected through gastric tube and then clamping the tube for two hours, two times a day. Besides, 500g Mirabilite is arranged on the width of 20cm, 80cm long cylindrical bag for external pancreatic area and replace one time a day.
Results: Abdominal pain relief time, recovery time of blood amylase, hospitalization time was obviously different between the treatment group than the control group (P<0.05). Remission time of abdominal pain was 2.82±0.31 and 4.64±0.42, respectively. Recovery time of blood amylase was 9.20±091 and 14.40±1.19, respectively. In addition, the average length of hospitalization was 17.04±1.21 and 25.96±1.31, respectively.
Conclusion: Combination of traditional Chinese and western medicine on severe acute pancreatitis is worthy of application.
Solid-Pseudopapillary Neoplasm (SPN): Aspects of a Rare Disease
C. Tjaden, U. Hinz, T. Kehayova, M.W. Büchler, T. Hackert. Department of Surgery, University of Heidelberg, Heidelberg, Germany.
Background: SPNs (solid pseudopapillary neoplasms) were first described 1959 by Frantz and 1970 by Hamoudi. 1996 the WHO classified them as borderline tumors of the exocrine pancreas. In most cases they can be distinguished from other pancreatic neoplasms by characteristic morphological features in CT or MRI scans. Malignancy is seldom, and even in case of metastasis or recurrence prognosis is well. Aim of the study was to analyses patient parameters and tumor characteristics as well as outcomes of all patients who underwent pancreatic resection for SPN in a single center.
Method: Data of all patients who underwent a pancreatic resection for SPN at the Department of Surgery, University of Heidelberg, Germany, between 01/2004 and 12/2014 were prospectively collected and analyzed, regarding demographic and relevant clinical parameters as far as long term follow up. Our data were partially compared with a recent review about all case reports and series, which were published in English language 2000–2012.
Results: Since SPN was first described in our hospital in 2004, 32 SPN patients underwent surgery. Most of them were female (n=26; 81%), median age at diagnosis was 33.6 years. 18 patients (56%) showed symptoms possibly related to SPN (abdominal pain, jaundice, weight loss). The preoperative CT/MRI was suspicious for SPN in 14 patients (42%). The mean tumor size was 6.9 cm, located mostly in pancreas corpus or tail. R0 resection was performed in 31 patients (94%), positive lymph nodes (N1) were found in 3.2%. Follow up was reported for 18 patients (55%), with a mean observation time of 22 months. Three women (12%) developed recurrences (1 local, 1 liver metastases, 1 widespread) after a median interval of 22 months. These results were comparable to other publications.
Conclusion: SPN is a risingly observed cystic tumor, mostly seen in young women, with a malignant potential of 10-15%. Therapy of choice is surgical resection for both primary and recurrent tumor. Therefore, regular follow up should be recommended.
Preoperative Neutrophil to Lymphocyte Ratio Predict Overall Survival After Macroscopic Curative Resection for Pancreatic Cancer
Y. Toyoki, K. Ishido, D. Kudo, T. Wakiya, Y. Wakasa, D. Kasai, K. Hakamada. Department of Gastroenterological Surgery, Hirosaki University Graduate School of Medicine, Japan.
Background & Aim: Several papers have shown that preoperative neutrophil to lymphocyte ratio (NLR) is associated with survival in a variety of tumors. In this study, we evaluated the prognostic significance of NLR in our patients with pancreatic ductal adenocarcinoma.
Methods: January 2004 to December 2012, 146 patients with pancreatic cancer underwent macroscopic curative resection at our institute. In the present study, we retrospectively analyzed 50 patients with stage I or II or III pancreatic cancer. Analysis of overall survival (OS) was performed using Kaplan-Meier and log-rank tests and the Cox proportional hazards regression model.
Results: In univariate analysis, NLR, CA19-9, tumor location, operative bleeding volume, and operative time were prognostic factors for OS. In multivariate analysis, Increased NLR was an independent prognostic factor for OS (hazard ratio 0.334, 95 % confidence interval 0.117-0.956, p = 0.041).
Conclusion: The preoperative NLR is a simple and useful potential biomarker for OS in patients with stage I or II or III pancreatic cancer after macroscopic curative resection.
Multidisciplinary Approach to Disconnected Pancreatic Duct Syndrome (DPDS) in Acute Pancreatitis (AP): A Single Tertiary Center Experience
G. Trikudanathan, E. Aby, R. Attam, S.K. Amateau, S. Mallery, M.L. Freeman, M.A. Arain. GI Division, Department of Medicine, University of Minnesota, Minneapolis, MN.
Background: DPDS is defined as a complete disruption of the main pancreatic duct with imaging evidence of viable pancreatic tissue upstream that results in a persistent pancreatic fistula or peri-pancreatic fluid collections refractory to conservative management. Previous reports are limited to less than 50 pts. We report a large series from a center offering the entire spectrum of advanced interventions for DPDS.
Aim: To describe the management and outcomes of patients (pts) with DPDS in necrotizing pancreatitis.
Methods: Data were extracted from a prospectively maintained database of pts with NP. Pts were managed by a multidisciplinary team with interventions as indicated utilizing a step up approach based on endoscopic +\- percutaneous catheter drainage (PCD) as first line treatment, with surgery reserved for failures, except pts referred from other institutions with PCD or surgery as the primary approach. Clinical outcomes and management approach are reported.
Results: 170 pts were admitted for NP in the study period (2009-2014). 65 pts (38.2%) had DPDS [Males = 43, median age 55 (range 19 - 85)]. Indications for intervention were infection 35 (53.8%), gastric outlet obstruction in 13 (20%), persistent symptomatic WON in 17 (26.1%) and DPDS in 2 (3.1%). Mean duration from initial onset to intervention was 69 days (13- 159 days) and to diagnosis of DPDS was 114 days. The site of PD disconnection was head 1 (1.5%), neck 29 (44.6%), body 30 (46.1%) and tail 5 (7.7%). DPDS was diagnosed at time of initial intervention in 36 pts (55.3%). 62/65 pts had endoscopic transmural stenting for WON; cystgastrostomy (CG) in 47 (73.8%), cystduodenostomy (CD) in 2 (3.1%) and both CG and CD in 13 (20%). 6 had EUS-guided and 2 percutaneous/endoscopic rendezvous to reestablish enteric drainage of the disconnected duct; of these 5 had surgery (distal or Whipple).61.3% of DPDS pts remained asymptomatic in spite of no transmural stent, whereas 50% of patients remained symptomatic in spite of transmural stents.
Conclusion: DPDS occurs in approximately 40% of pts with necrotizing pancreatitis. Development of complications from DPDS does not appear to be related to presence or absence of permanent transmural stents. This is the largest series to date with long term follow up demonstrating the success of the multi-disciplinary approach.
Prevalence and In-Hospital Mortality Trends of Clostridium Difficile Among Patients Admitted With Acute Pancreatitis: A Nationwide Study of Hospitalized Patients in the United States
G. Trikudanathan, S. Munigala, M.A. Arain, M.L. Freeman. GI Division, Department of Medicine, University of Minnesota, Minneapolis, MN.
Background: Acute pancreatitis (AP) is the most common gastroenterology discharge diagnosis in USA. Clostridium difficile-associated disease (CDAD) is associated with antibiotic use and hospitalization, both of which frequently occur in AP. Currently, there is no data on CDAD in AP patients who are hospitalized.
Aim: To estimate the prevalence of CDAD in AP hospitalized patients and to examine their impact on the in-hospital length of stay/ mortality and health-care utilization.
Methods: We used the National Hospital Discharge Survey (NHDS) from 1991- 2010 to calculate the prevalence of CDAD for primary diagnosis of Acute Pancreatitis (AP). All primary AP discharges (ICD9 code 577.0) were identified. Among discharges with primary AP, secondary diagnosis code (ICD 9 code 008.45) for CDAD was noted and was grouped into patients with and without CDAD infections. Patient characteristics (age group, sex, and race), length of stay (in days), in-hospital mortality and presence/absence of complications were noted. A trend test using linear regression analysis was performed to check for the prevalence of CDAD over time.
Results: Of the 3,907,519 hospitalized patients with primary AP in the study period, 11,778 (0.3%) had CDAD. AP patients with CDAD were more likely to be male (55.7% vs. 51.2%, p< 0.0001), white (68.0% vs. 56.6%, p< 0.0001) and older (40.5% vs. 27.1%, p< 0.0001). AP patients with CDAD were hospitalized for longer duration than patients without CDAD (Mean 19 days vs. 6 days, p < 0.05) and were more likely to be transferred to a short/long-term care facility compared to patients without CDAD (22.7% vs. 7.5%, p< 0.0001). There was no significant difference in in-hospital deaths between the two groups. From 2002-2005, there was a trend towards increasing prevalence of CDAD (0.5-0.7%) and continued to remain on a higher percentage compared to the initial years (p=0.0046).
Conclusions: Increase in CDAD has been noted in the past decade in patients with AP. CDAD in AP resulted in increased morbidity (prolonged hospital stay and increased transfer to short term/long term facility. Antibiotic therapy has been routinely used in patients with severe AP. Increased prevalence of CDAD might be a reflection of injudicious use of antibiotics in AP patients.
Branched Duct Intraductal Papillary Mucinous Neoplasm (BD-IPMN) of Pancreas in Solid Organ Transplant Patients: A Single Tertiary Center Experience
G. Trikudanathan, U. Barlass, M.A. Arain, S.K. Amateau, R. Attam, M.L. Freeman, S. Mallery. GI Division, Department of Medicine, University of Minnesota, Minneapolis, MN.
Background: Incidentally detected BD-IPMN represents a significant concern for transplant recipients because of their uncertain malignant potential especially under immunosuppression. There is very limited data regarding the risk of progression of BD-IPMN in patients undergoing solid-organ transplant.
Aim: To determine the prevalence of BD-IPMN in solid organ transplant recipients and describe their clinical course.
Methods: Consecutive adult patients undergoing solid-organ transplant (liver/kidney/pancreas/heart/lung) between January 2004-July 2014 were identified from our database. Patients with presumed BD-IPMN identified on imaging (MRI, CT and/ or EUS) either prior to/after solid-organ transplant were included. Patients were excluded if they had high-risk cyst characteristics at initial diagnosis or follow-up of < 6 months. Demographics, relevant information regarding transplant including indication/ immunosuppression and clinical/imaging characteristics of cyst were extracted. Progression of the BD-IPMN was defined as development of ‘high-risk stigmata' or ‘worrisome' features (Fukuoka guidelines).
Results: 3151 patients underwent solid organ transplant during the study period. 264 patients underwent imaging. 41/264 (15.6%) were identified to have possible BD-IPMN. 27 were males, Median age was 64 years (range 37- 78 years). 18 underwent liver, 15 kidney, 5 combined liver/kidney, 2 pancreas and 1 heart transplant with a mean duration of immunosuppression exposure of 44.3 months. Mean duration of clinical-follow up was for a period of 45 months (7-114 months) and mean duration of imaging follow up was 29 months (6- 83 months). 1/41 (2.4%) with a BD-IPMN, after 68 months of follow up was noted to have pancreatic adenocarcinoma. 2.4% of patients developed ‘worrisome feature' (PD dilation to 5 mm) and 7.3% of patients had cyst enlargement to >3 cm without ‘high risk stigmata' and are under surveillance without resection. Among the transplant recipients, all-cause mortality was 9.75% in patients with BD-IPMN and 10.6% in patients without BD-IPMN. None died from pancreatic cancer.
Conclusions: All-cause mortality of BD-IPMN patients did not differ significantly from those without BD-IPMN in solid organ transplant patients. Therefore the presence of BD-IPMN should not preclude patient from undergoing transplant and following transplant, they should undergo surveillance similar to the immunocompetent patients.
Colipase (CLPS) Mediates Adipocyte Death and Inflammation: An In-Vitro Model of Fat Necrosis (FN)
R.N. Trivedi, K. Patel, P. Noel, A. Singh, C. de Oliveira, V.P. Singh. Department of Medicine, Mayo Clinic, Scottsdale, AZ.
Background: FN is an integral part of the Balthazar, Schroder and revised Atlanta criteria for classification of severe acute pancreatitis (SAP). In several studies, we have shown FN to result in SAP by excessive lipolysis of visceral triglycerides resulting in free fatty acid (FFA) release and acute lipotoxicity. However the mechanisms underlying this are unknown. To understand FN during SAP better, we analyzed whether FN contained the components, and the mechanisms resulting in FN.
Methods: Fat pads from ob/ob control mice and those with SAP were western blotted (WB) for adipocyte triglyceride lipase (ATGL), hormone sensitive lipase (HSL), pancreatic triglyceride lipase (PTL), pancreatic lipase related protein-2(PLRP2) and CLPS and pancreatic lipase activity was measured in these. PTL, alone, or with CLPS was overexpressed in 3T3-L1 cells and adipocyte necro-apoptosis (active caspase-3, LDH leakage, propidium iodide [PI] uptake, ATP levels), lipolysis (glycerol, FFA release), inflammation (IL-6, MCP-1 secretion by Luminex) were measured in the presence of the ATGL inhibitor Atglistatin. Primary adipocytes were stimulated by isoproterenol (Iso 10μM) or co-cultured with pancreatic acini in the presence of Atglistatin or the pancreatic lipase inhibitor orlistat (50μM each). Values are reported as means and a p-value <0.05 is considered significant.
Results: Pancreatic lipase activity and CLPS, PTL and PLRP2 but not ATGL or HSL amounts increased in FN. Atglistatin unlike orlistat inhibited Iso regulated lipolysis (0.5 vs 0.3 mM,p=0.02). Conversely orlistat but not Atglistatin inhibited lipolysis in the acinar-adipocyte co-culture (1.07 vs 0.09 mM, p<0.01). Exogenous CLPS (0.5 μg/ml) significantly increased glycerol(control 0.05 vs 0.5 mM), FFA generation (control 86.6 vs 472 μM), LDH leakage (control 7.5% vs 41.5%) and reduced ATP levels(control 100% vs 10.5%) vs. PTL alone in 3T3-L1 cells. IL-6 and MCP-1 increased significantly (IL6 282.2 vs. 22.8 pg/ml in control; MCP1 429 vs. 203 pg/ml in control) when only when both CLPS and PTL were present. All these cell death and inflammatory phenomena were inhibited by orlistat.
Conclusions: Colipase and pancreatic lipases are present in SAP associated FN. Colipase worsens FN independent of ATGL and HSL by enhancing adipocyte triglyceride lipolysis and inflammatory mediator generation mediated by pancreatic lipases.
Very Early Onset Acute Recurrent and Chronic Pancreatitis are Associated With PRSS1 or CTRC Mutations
A. Uc,1 M. Giefer,2 M.E. Lowe,3 S. Werlin,4 on behalf of INSPPIRE Consortium. 1University of Iowa Children’s Hospital, Iowa City, IA; 2Seattle Children’s Hospital, Seattle, WA; 3Children Hospital of Pittsburgh, Pittsburgh, PA; 4Medical College of Wisconsin, Milwaukee, WI.
Background & Aims: Genetic mutations and obstructive factors are common in pediatric acute recurrent pancreatitis (ARP) and chronic pancreatitis (CP). We investigated whether pancreatitis that starts at a very young age (<6 y/o) has unique risk factors and disease course.
Methods: Demographic and clinical information on children with ARP or CP, <20 years of age at the time of enrollment into INSPPIRE (INternational Study Group of Pediatric Pancreatitis: In Search for a CuRE) study were collected at 15 centers. A cross-sectional study was performed comparing the differences between those first diagnosed at age <6 y/o and those diagnosed at age >6 y/o using Fisher’s exact test for categorical variables, Wilcoxon rank-sum test for ordinal/continuous variables.
Results: Of 263 children with ARP or CP, 105 (40%) were younger than 6 y/o at the time of their first acute pancreatitis (AP) attack. The diagnosis of CP was not different between the age groups (46% for both <6 y/o and >6 y/o; p=0.98). Younger children were more likely to have mutations in the cationic trypsinogen (PRSS1) or chymotrypsin C (CTRC) genes and a family history of AP or CP (p<0.01). Older children were more likely to be Hispanic (p<0.05), have medication history or autoimmune disease (p=0.036 and p=0.005 respectively). Pancreas divisum did not differ between the age groups, however there was a significant association of pancreas divisum with SPINK1 mutations in the older onset group (Odds ratio 7.33; 95% CI: 1.23, 50.89; p=0.013), but not in younger onset group (Odds ratio 0.68; 95% CI: 0.01, 6.56; p=1.0). Disease burden, with significantly more constant pain, lifelong emergency room visits, hospitalizations and missed school days was higher in the older age group (p<0.05).
Conclusions: Very early onset ARP or CP are more strongly associated with PRSS1 and CTRC mutations compared to the older population, and older children with pancreas divisum are more likely to have SPINK1 mutation. Disease burden is higher in older children. Future studies will investigate whether the disease course, response to therapy and/or outcomes are different in children diagnosed with pancreatitis at a very young age.
The Difference in Mechanisms of Neutrophil Infiltration between Type 1 and Type2 Autoimmune Pancreatitis
K. Uchida,1 T. Mitsuyama,1 M. Yanagawa,1 H. Miyoshi,1 T. Ikeura,1 M. Shimatani,1 T. Fukui,1 M. Takaoka,1 A. Nishio,1 N. Mizuno,2 K. Notohara,3 G. Zamboni,4 L. Frulloni,5 T. Shimosegawa,6 K. Okazaki.11Department of Gastroenterology and Hepatology, Kansai Medical University, Hirakata, Japan; 2Department of Gastroenterology, Aichi Cancer Center Hospital, Nagoya, Japan; 3Department of Pathology, Kurashiki Central Hospital, Kurashiki, Japan; 4Department of Pathology, University of Verona, Verona, Italy; 5Department of Medicine, University of Verona, Verona, Italy; 6Division of Gastroenterology, Tohoku University Graduate School of Medicine, Sendai, Japan.
Aim: One of the histopathological characteristics of type 2 autoimmune pancreatitis (AIP) is granulocyte epithelial lesions. To clarify pathogenesis of type 2 AIP, we investigated mechanism of neutrophil infiltration in type 1 and type 2 AIP.
Method: We recruited 10 cases of alcoholic chronic pancreatitis (ACP), 10 cases of type1 AIP and 12 cases of type 2 AIP. We analyzed the expression of Granulocyte Chemotactic Protein (GCP)-2 or IL-8 using immunohistochemistry. The staining intensity is scored from 0 (negative) to 3 (strong).
Results: The number of neutrophils around the interlobular pancreatic ducts was significantly higher in type 2 AIP (15.16, interquartile range (IQR) 9.74-18.41) than in ACP (2.66, IQR 1.33-4.33; P<0.05) and type 1 AIP (3.16, IQR 2.74-4.57; P=0.0082). There was no significant difference in the number of neutrophils around the intralobular pancreatic ducts among ACP (1.16, IQR 0.33-3.41), type 1 AIP (3.16, IQR 0.74-5.5) and type 2 AIP (3.00, IQR 1.08-7.91). The score of GCP-2 in the interlobular pancreatic duct epithelia was significantly higher in type 2 AIP (1.5, IQR 0.25-2) than in ACP (0, IQR 0-0; P<0.05 and type 1 AIP (0, IQR 0-0; P<0.05). There was no significant difference in the score of IL-8 in the interlobular pancreatic duct epithelia among ACP (0, IQR 0-0), type 1 AIP (1, IQR 0-1.75) and type 2 AIP (0.5, IQR 0-1).
Conclusions: In type 2 AIP, GCP-2 have an important roll in neutrophil infiltration around interlobular pancreatic duct.
Selective Arterial Secretagogue Injection Test (SASI Test) in 15 Insulinoma Patients
K. Ueda,1 L. Lee,1 T. Fujiyama,1 Y. Tachibana,1 M. Masami,1 K. Yasunaga,1 K. Kawabe,1 H. Honda,2 M. Nakamura,3 Y. Oda,4 T. Ito.11Department of Medicine and Bioregulatory Science, 2Department of Clinical Radiology, 3Department of Surgery and Oncology, 4Department of Anatomic Pathology, Graduate School of Medical Sciences, Kyushu University, Fukuoka, Japan.
Background: The SASI test is considered indispensable for an accurate localization of insulinoma. However, the optimal timing of evaluation (0–120 or 0–60 sec after drug administration) has not been determined. Further, evaluation is often difficult when several arteries test positive.
Objective: We evaluated the localization by using the SASI test retrospectively.
Subjects and methods: SASI test results were available for 15 patients with histologically proven insulinoma from Oct 2000 to Nov 2014. For the SASI test, calcium gluconate (0.025 mEq/kg) was injected through the gastroduodenal, superior mesenteric, and splenic arteries. Blood insulin levels were measured before and at 30, 60, 90, 120, and 180 sec. Arteries were considered positive when the level was twice as high as the pre-test value.
Results: The mean age and BMI were 51.5 (20–75) years and 22.2 (16.5–28.3), respectively; 13 subjects were women (86.7%). All subjects tested positive in the 48-h fasting test. The mean tumor size was 13.9 (9.0–27.0) mm. In the SASI test at 0–120 and 0–60 sec, the respective findings were as follows: positive predictive value, 72.7% and 65.0%; false positive rate, 46.7% and 38.1%; and rate of multiple positive arteries, 60.0% and 40.0%. When the artery with the largest change was taken as the dominant artery, the localization diagnosis rate was 86.7% at 0–120 sec and 73.3% at 0–60 sec. The localization diagnosis rate was 73.3% for CT, 64.3% for MRI, 66.7% for angiography, and 85.7% for EUS.
Conclusion: We found that the best localization diagnosis rate was obtained by using the SASI test at 0–120 sec. It was appropriate that the artery with the largest change was taken as the dominant artery when several arteries test positive.
Direct Chemical Inhibition of Nuclear Factor κB only Ameliorates Local Pancreatic but not Remote Lung Inflammation via Up-regulating Pancreatitis Associated Protein 1
C. Ulrich,* Z. Yuan,* J. George, U. Barlass, S. Garg, A. Sareen, A.K. Dixit, R.K. Dawra, A.K. Saluja. Division of Basic and Translational Research, Department of Surgery, University of Minnesota Medical School, Minneapolis, MN. *Equal contribution.
Background & Aims: Acute pancreatitis is both a local and systemic disease. The role of NFкB on acute pancreatitis is still controversial. NFкB inhibitors used in past reports are indirect inhibitors.
Methods: Three direct inhibitors: withaferin A (WA) targeting NEMO, wedelolactone (WL) acting on IKK complex, and BAY 11-7085 (BAY) binding IкBα, were used to determine their effects on acute pancreatitis induced by supermaximal cerulein. Acinar cell death, tissue damage, neutrophil infiltration, and early intra-acinar activation of the transcription factor nuclear factor кB (NFкB), were compared among treated and untreated groups. Lung inflammation and injury was assessed by histology, flow cytometry and TUNEL assay respectively.
Results: Inhibition of NFкB activation was confirmed by immunoblot of IкBα and electrophoretic mobility shift assay (EMSA). All three inhibitors significantly attenuated pancreas necrosis and inflammation. Lung inflammation, represented by H&E staining, myeloperoxidase activity, immunohistochemistry, flow cytometry and TUNEL positive cells didn’t improve after treatment with all three inhibitors. All the inhibitors increased expression of pancreatitis associated protein 1 (PAP1) in the pancreas.
Conclusion: Direct chemical inhibition of NFкB activation can only attenuate local pancreatic but not remote lung inflammation associated with acute pancreatitis. Increased inflammation in the lung could be associated with up-regulation of pancreatitis associated protein 1.
Role of Mitochondria Dynamics During Selective Autophagy Induced by Acute Pancreatitis
V. Vanasco, D. Grasso, S. Alvarez, M.I. Vaccaro. Institute of Biochemistry and Molecular Medicine (UBA-CONICET), School of Pharmacy and Biochemistry, University of Buenos Aires, Buenos Aires, Argentina.
We have previously demonstrated the selective autophagic pathway zymophagy as an early protective mechanism in acute pancreatitis (AP). Zymophagy impairment during AP, promotes cell damage and probably the progression of the disease. ATP and other molecules necessary in cellular stress processes are provided by mitochondria. Thus, a normal mitochondrial bioenergetics would be necessary for zymophagy in response to the disease. The aim of this study is to analyze the mitochondrial dynamics and function during selective autophagy induced by acute pancreatitis. Female Sprague-Dawley rats (45 days old) were ip injected with cerulein (CAE) 50 mg/kg in 1h intervals. Treated groups studied: CAE 1, animals sacrificed 1 hour after first injection; CAE 3, animals sacrificed after 3h treatment; CAE 24 and CAE 48, animals injected with 7 doses and sacrificed at 24 and 48 h respectively. Control groups (CG) were injected with vehicle. Zymophagy induced by AP was confirmed by VMP1 expression, Trypsinogen/LC3 and Trypsinogen/Lamp2 colocalizations as well as p62 degradation. Between 1 and 24 hours of experimental pancreatitis, mitochondrial O2 consumption and ATP production decreased by 35% and 70% respectively (CG: 40 ± 5 ng-atO/min.mg protein; 140 ± 18 nmol ATP/ min.mg.protein, P < 0.01). CAE48 shows control values in both parameters. On the other hand, OPA1 expression (mitochondrial fusion protein) is significantly decreased after 1 h of pancreatitis, but it increases in a time course towards 48 hours. Moreover, no expression of DRP1 (mitochondrial fission protein) is observed during the first 24 hours of pancreatitis. Our results show mitochondrial dysfunction during experimental pancreatitis, which recovers after 48 hours. Data also show the increment of OPA1 suggesting mitochondria elongation, which avoids degradation as a mitochondria preservation mechanism during acute pancreatitis induced zymophagy.
Defective Lysosomal Hydrolase Trafficking Causes Spontaneous Pancreatitis
E.T. Vegh,1,2 E.M. Lotshaw,1 N. Shalbueva,1 O.A. Mareninova,1 J.M. Elperin,1 S.W. French,3 P. Hegyi,2 Z. Rakonczay,2 I. Gukovsky,1 A.S. Gukovskaya.11VA Greater Los Angeles Healthcare System and University of California at Los Angeles, CA; 2First Department of Medicine, University of Szeged, Hungary; 3Harbor-UCLA Medical Center, Torrance, CA.
Background & Aims: Experimental pancreatitis is associated with impaired processing, activation, and subcellular distribution of cathepsin B (CatB) and other lysosomal hydrolases. Here we investigate the role of impaired cathepsin trafficking and lysosomal dysfunction in pancreatitis, using mice deficient in Gnptab, the gene coding for a key enzyme mediating addition of mannose 6-phosphate (M6P) onto hydrolases necessary for their delivery to lysosomes.
Methods: We measured parameters of lysosomal and mitochondrial functions, autophagy, and pancreatitis responses in Gnptab-/- mice of various age, using immunoblot and immunofluorescence analyses, electron microscopy, and enzymatic assays.
Results: Cathepsin trafficking and processing is defective in Gnptab-/- pancreas, causing impaired lysosomal proteolytic activity and autophagic flux. In particular, defective CatB processing is manifest by a dramatic accumulation of CatB intermediate form and decrease in its mature form. The lysosomal/autophagic dysfunction causes mitochondrial failure, manifest by decreased mitochondrial membrane potential and ATP synthase activity, and mitochondrial fragmentation. Pancreas of Gnptab deficient mice shows decreased amylase content, greatly elevated trypsin activity, macrophage and neutrophil infiltration, stellate cell activation (α-SMA upregulation), fibrosis, and acinar cell death – key pathologic responses of pancreatitis. Importantly, Gnptab deficiency has no effect on liver histology, autophagy, and mitochondrial functions.
Conclusions: Block of M6P-mediated hydrolase trafficking to lysosomes causes spontaneous pancreatitis. The results indicate a critical role for defective cathepsin trafficking and lysosomal/autophagic dysfunction in the pathogenesis of pancreatitis.
Insights Into the Biochemical Behavior of Full length Human HP1γ, an Epigenetic Regulator Downstream of KRAS
G. Velez, M. Lin, W.A. Faubion, T. Christensen, G. Lomberk, R. Urrutia. Epigenetics and Chromatin Dynamics Laboratory, Translational Epigenomics Program (CIM), GIH Division, Department of Medicine, Biophysics, Biochemistry and Molecular Biology, Mayo Clinic, Rochester, MN.
Epigenetic mechanisms working downstream of oncogenes are critical for establishing the development and pathobiological behavior of PDAC. We have found that HP1γ not only is upregulated in human PDAC and the p48-Cre/LSD-KrasG12D PDAC mouse model, but functions as an epigenetic regulator downstream from KRAS to promote pancreatic cell growth by regulating expression of proliferative gene networks. These characteristics make it an ideal target for developing small drugs for both mechanistic experimentation and potential therapies. While high-resolution structures of the two globular regions of HP1γ, the chromo- and chromoshadow domains, have been solved, little is currently known about the conformational behavior of the full-length protein. Consequently, here we use threading, homology-based molecular modeling, and molecular dynamics simulations, to develop models that allow us to infer properties of full length HP1γ, at an atomic resolution level. HP1γ is revealed as an elongated molecule in which three Intrinsically Disorder Regions (IDRs, 1, 2, and 3) endow this protein with dynamic flexibility, intermolecular recognition properties, and the ability to integrate signals from various intracellular pathways often implicated in PDAC. The ability of the three IDRs for integrating cell signals is demonstrated by combined Linear Motif Analyses showing that posttranslational modifications can generate a histone mimetic sequence within the IDR2 of HP1γ, which when bound by the chromodomain can lead to an autoinhibited state. Combined, this data underscores the importance of IDRs 1,2 and 3 in defining the structural and dynamic properties of HP1γ, discoveries that have both mechanistic and potentially biomedical relevance, including to aid in the development of future PDAC therapies.
Outcomes in Acute Pancreatitis: A Systematic Review of the Medical Literature
D. Verma, B.U. Wu. Department of Gastroenterology, Kaiser Permanente, Los Angeles, CA.
Background: There has been a lack of progress in developing new treatment in acute pancreatitis (AP). We hypothesized that one potential barrier to new drug development has been a lack of well-established outcome to assess treatment effect.
Objective: To summarize previous outcome parameters used in randomized clinical trials of therapy in AP for the purpose of informing future clinical trial design.
Methods: We conducted a review of the published literature. We searched PubMed and Cochrane databases from 1996 to May 2014 looking at randomized control trials assessing therapy in AP. Our inclusion criteria were randomized control trials, English language, use of human subjects, and studies that evaluated the effect of therapy. Exclusion criteria was preventative studies either primary e.g. post ERCP pancreatitis or secondary e.g. prevention of recurrent AP due to gallstones or alcohol. Primary Outcomes of each study were analyzed and the Jadad Scale was used for quality assessment.
Results: Our initial search yielded 345 abstracts. Total number of studies after inclusion and exclusion was 61, with a Jadad Scale average of 3.2. Most trials (52%) studied patients with severe AP. The most frequently evaluated interventions were the effect of antibiotics in prevention of infection (15%), effect of enteral nutrition vs. parenteral nutrition (13%) and effect of Glutamine therapy (6%). The most common primary outcome was mortality (16%). Others were organ failure (15%), pancreatic infections (13%) and SIRS (10%). In the control arms the mortality rate ranged from 8.3% to 32%. Jadad Scale scores were variable and did not improve with more recent studies.
Conclusion: Most studies focused on relatively few interventions in AP. Mortality was the most common primary outcome measure. However based on the observed mortality in the control arms many of these studies were underpowered based on a priori sample size calculations. Adequately powered studies assessing treatment effect on additional outcomes are needed.
Role of AMPK in the Inhibitory Effects of Metformin and Berberine in Human Pancreatic Cancer Cells
J. Wang, L.L. Han, J. Sinnett-Smith, S. Young, E. Rozengurt. Division of Digestive Diseases, Department of Medicine, CURE: Digestive Diseases Research Center, David Geffen School of Medicine, University of California at Los Angeles, CA.
Background: The precise role of AMP-activated protein kinase (AMPK) in the proliferation and survival of cancer cells has become controversial. Specifically, it remains unclear whether AMPK activation suppresses cancer cell proliferation (tumor suppressive function) or enhances cancer cell survival under conditions of metabolic stress (tumor promoter function). Here we examined these alternative models using human pancreatic ductal adenocarcinoma (PDAC) cells treated with metformin or berberine.
Results: Treatment of PDAC cells (PANC-1, MiaPaCa-2) with increasing doses of metformin or the isoquinoline alkaloid berberine decreased mitochondrial membrane potential, reduced intracellular ATP and stimulated AMPK activation, as scored by the phosphorylation of the AMPK substrates acetyl-CoA carboxylase (ACC) at Ser79 and Raptor at Ser792. Metformin and berberine inhibited mTORC1 signaling (S6K phosphorylation at Thr389 and S6 phosphorylation at Ser240), ERK activation and DNA synthesis in PDAC cells. To determine the role of AMPK in these effects, we used short interfering RNA (siRNA) to knockdown the protein expression of α1 and α2 catalytic subunits of AMPK and prevent phosphorylation of ACC at Ser79 or Raptor at Ser792. Knockdown of AMPK blunted the inhibitory effects produced by low doses of either metformin or berberine on the stimulation of mTORC1, ERK and DNA synthesis in PDAC cells. In sharp contrast, knockdown of AMPK did not prevent the inhibitory effects of either metformin or berberine when added at higher concentrations.
Conclusion: We propose that metformin and berberine, at low concentrations, inhibit mitogenic signaling in PDAC cells through an AMPK-dependent pathway but act via AMPK-independent pathways in the same cells when added at higher doses. This conclusion provides a plausible explanation for apparently contradictory reports on the role of AMPK in the mechanism of action of berberine and metformin in other model systems and emphasizes the need of using detailed dose–response studies to define the anticancer mechanisms of action of agents that produce metabolic stress via inhibition of mitochondrial function.
Clinical Outcomes of Combined Necrotizing Pancreatitis Versus Extrapancreatic Necrosis Alone
M. Wang, A. Wei, Z. Zhang, W. Hu. Department of Pancreatic Surgery, West China Hospital, Chengdu, Sichuan, China.
Introduction: Extrapancreatic necrosis (EPN) alone without the pancreatic parenchyma necrosis has been regarded as a separate entity of acute necrotizing pancreatitis (ANP). However, data regarding the prognostic significance of EPN is quite limited, and the surgical outcomes of patients with EPN alone are not well elucidated. The aim of this study was to explore the differences of outcomes between patients with EPN alone and patients with both the pancreatic parenchyma and extrapancreatic necrosis (combined necrosis).
Methods: From January 2009 to December 2013, a total of 334 patients with ANP received interventions in West China Hospital, China were respectively included. The patients were divided into group 1 (n = 285), in which necrosis involved both the pancreatic parenchyma and extrapancreatic tissue, and group 2 (n = 49), in which necrosis involved only extrapancreatic necrosis. The demographic characteristics, the incidence of organ failure, infected necrosis and persistent SIRS in the first week of onset, CT severe index, intervention types, as well as postoperative stay, ICU utility, and complications were collected and compared between the two groups.
Results: Compared with those in group 1, patients in group 2 suffered from less persistent SIRS in the first week of onset (12/24.5%vs. 145/50.9%; P = 0.001), persistent organ failure (6/12.2% vs. 95/33.3%; P = 0.003), persistent multiple organ failure (3/6.1 % vs. 67/23.5%; P = 0.006), and bacteremia (5/10.2% vs. 107/37.5%; P = 0.000). The intervention types were significantly different between the two groups (P = 0.000), open necrosectomy was performed in 174/61.1% and 8/16.3% patients in group 1 and 2 respectively, while percutaneous catheter drainage (PCD) was performed in 73/25.6% and 29/59.2% patients in the two groups. More patients in group 1 were diagnosed with infected necrosis (145/50.1% vs. 10/20.4%; P = 0.000), and required to be sent to ICU for postoperative further care (221/77.5% vs. 23/46.9%; P = 0.000). Postoperative stay was longer in group 1 (median: 43 vs. 26.5 days; P = 0.000). Residual necrotic tissue or abscess was the most common postoperative complication in both groups. The mortality was higher in group 1 (52/18.2% vs. 1/2.1%; P = 0.004).
Conclusion: EPN alone should be a separate category of ANP because it has less severe course. PCD should be the first choice for patients with EPN alone when interventions were required.
Novel Patient-Derived CTC-Xenograft Models for the Study of Pancreatic Cancer Biology, Metastasis and Therapy
R.X. Wang,1 C.Y. Chu,1 N.N. Nissen,1 M.S. Lewis,2 N. Palanisamy,3 M. Edderkaoui,1 A. Annamalai,1 S. Lewis,1 H.E. Zhau,1 S.J. Pandol,1,2 L.W.K. Chung.11Cedars-Sinai Medical Center, Los Angeles, CA; 2VA Greater Los Angeles Health System, Los Angeles, CA; 3Henry Ford Health System, Detroit, MI.
Background and Objective: Pancreatic cancer (PaCa) often spreads rapidly to form metastatic tumors. We expanded respectively circulating tumor cells (CTCs) and stromal cells from liquid and tissue biopsies as source materials to examine the pathobiology and therapeutic responses of PaCa patients.
Methods: Peripheral blood mononuclear and pancreatic stromal cells were isolated respectively from the whole blood and PaCa tumor biopsies from the same patient. Ex vivo expansion of CTCs and stromal cells was performed by standardized outgrowth protocols and characterized. The tumorigenicity of CTCs and their responses to pancreatic stromal fibroblasts were assessed. Therapeutic responses of CTCs and CTC-derived patient xenografts (CTC-PDXs) were evaluated.
Results: We have successfully expanded 9 candidate CTC cultures from 42 patients. CTCs grew as spherical organoids with high proliferative activity, which was not observed in healthy donor blood cultures. CTC-PDX tumors expressed PaCa markers and exhibited local invasive and metastatic behaviors when inoculated orthotopically into the pancreas or intraosseously but not subcutaneously. CTC growth was enhanced by stromal conditioned media and inhibited by conventional and novel anti-tumor agents of dye-gemcitabine conjugate and CSME-357.
Discussion and Conclusion: For the first time in this feasibility study, we succeeded in establishing CTC-PDX models using PaCa liquid biopsy. CTC-PDX tumors have malignant features and can be used as a model for the study of PaCa metastasis to facilitate personalized oncological research on PaCa diagnosis, treatment and prognosis.
Predictors of Early Complications in Acute Necrotizing Pancreatitis Patients With Invasive Intervention
A. Wei, Q. Guo, M. Wang, W. Hu. Department of Pancreatic Surgery, West China Hospital, Chengdu, China.
Aim: This study identified the possible predictors of early complications after the initial intervention in acute necrotizing pancreatitis.
Background: Few studies focused on elucidating the pathophysiology of acute pancreatitis and identifying the predictors of complications arising after intervention.
Methods: We collected the medical records of 334 patients whose initial intervention was performed in our center. Complications associated with predictors were analyzed.
Results: The postoperative mortality rate was 16% (53/334). 31% of patients were successfully treated with percutaneous catheter drainage alone. The rate of intra-abdominal bleeding, colonic fistula, and progressive infection were 15% (50/334), 20% (68/334), and 26% (87/334), respectively. Marshall score upon admission (OR = 1.723, 95% CI = 1.526–2.995, p = 0.046), multiple organ failure (MOF) (OR = 2.031, 95%CI = 3.410–10.054, p = 0.038), preoperative respiratory infection (OR = 4.304, 95%CI = 2.085–8.884, p < 0.0001), and preoperative sepsis (OR = 11.165, 95% CI = 5.384–23.153, p < 0.0001) were the predictors of postoperative progressive infection. Single organ failure (OR = 6.289, 95%CI = 1.652–23.948, p = 0.007), SIRS upon admission (OR = 2.927, 95% CI = 1.102–7.776, p = 0.031) and CRP level upon admission (OR = 1.005, 95%CI =1.001–1.009, p = 0.010) were the risk factors of postoperative colonic fistula. Moreover, preoperative Marshall score (OR = 1.560, 95%CI = 1.003–2.426, p = 0.048), single organ failure (OR = 5.287, 95% CI = 1.524–7.369, p = 0.031), MOF (OR = 4.327, 95% CI = 1.325–6.548, p = 0.041), preoperative sepsis (OR = 1.227, 95% CI = 1.089–1.578, p = 0.002), and preoperative SIRS (OR = 1.294, 95% CI = 1.086–2.002, p = 0.050) were the risk factors of postoperative intra-abdominal bleeding.
Conclusions: Intra-abdominal bleeding, colonic fistula and progressive infection are the three major early postoperative complications in patients with acute necrotizing pancreatitis. Marshall score, organ failures, preoperative respiratory infection, sepsis, preoperative SIRS, and CRP level upon admission played a role in postoperative complications. Closer attention must be extended to patients prone to postoperative complications, and immediate treatment of these conditions must be performed to prevent the occurrence of complications.
Surgical Treatment Choice for Pancreatic Ductal Stone: An Institutional Experience
J. Wei, X. Liu, W. Xu, Z. Lu, J. Chen, F. Guo, K. Jiang, C. Dai, Y. Miao. Pancreas Center, The First Affiliated Hospital of Nanjing Medical University, Nanjing, China.
Objectives: To analyze the classification and individualized surgery procedure of pancreatic ductal stone (PDS).
Methods: The clinic records of 47 patients with PDS treated in pancreatic center of Nanjing Medical University from January 2009 to December 2014 were analyzed retrospectively. All the patients were diagnosed by preoperative imaging.
Results: This study enrolled 47 patients, 37 males and 10 female with average age 52 ± 13 years, and 17 patients had a history of alcoholism. The most common symptoms were epigastric pain in 41 patients, diarrhea in 9 patients, and obstructive jaundice in 5 patients. All the 47 patients were complicated with chronic pancreatitis. 17 patients had the complication of diabetes, 10 bile stones, 4 pancreatic head carcinoma, 1 pancreatic body and tail carcinoma, 3 pancreatic pseudocyst, 1 pancreatic neuroendocrine tumor, and 1 pancreas divisum. There were 23 type I PDS, 1 type II PDS, 1 type III PDS, and 22 type IV PDS in this group. In 47 cases, of which 7 cases underwent PD procedure, 5 underwent PPPD procedure, 7 underwent Bern procedure, 2 underwent Frey procedure, 19 underwent Partington procedure, 1 cases associated with pancreatic duct carcinoma underwent totally pancreatic resection, 1 cases with pancreatic body and tail cancer underwent Partington and pancreatic body and tail resection procedure, 1 case with pancreatic pseudocyst underwent pancreatic cystojejunostomy, and 1 under CBDE only. There was no postoperative death and 10 patients (21.3%) had postoperative complications.
Conclusions: The treatment of PDS is rather difficult, and individualized surgery procedure according to classification of stones is more accepted than before. Relief obstruction of pancreatic and bile duct, removing of the cause, complete stone clearance, and unobstructed drainage and preservation of pancreatic functions are the principle of surgery.
Pancreatic Insulinoma: Diagnosis and Surgical Management of 33 Cases
J. Wei,1 X. Liu,1 W. Xu,1 K. Jiang,1 C. Dai,1 Z. Zhang,2 Y. Miao.11Pancreas Center, the First Affiliated Hospital of Nanjing Medical University, Nanjing, China; 2Department of Pathology, the First Affiliated Hospital of Nanjing Medical University, Nanjing, China.
Objectives: To evaluate diagnosis and surgical management for insulinoma and draw conclusions for surgical strategy.
Methods: A retrospective analysis was performed of patients underwent surgery for insulinoma at a tertiary center from 2009 to 2013. Demographic details, main presentations, preoperative diagnosis and localization, surgery strategy, postoperative management, and fellow-up were assessed.
Results: 33 patients with pathologically confirmed insulinoma were included in this study. The median time from the first onset of symptoms to diagnosis were 52 months (range: 15 days-50 years). All cases presented with Whipple’s triad (100%) and were transferred from endocrinology or psychiatric department. All patients had the fasting insulin to glucose ratio higher than 0.33(100%). The preoperative detection rates of trans-abdominal ultrasound, CT, MRI and EUS were 22% (2/9), 72% (23/32), 75% (9/12) and 80% (4/5), respectively. Intraoperative manual palpation detected 33 case (100%). 30 cases (91%) had solitary tumors, 3 cases (9%) had synchronous double primary tumors. Enucleation was performed in 58% (19/33) cases, segmental pancreatic resection in 39% (13/33) cases and enucleation plus segmental pancreatic resection in 3% (1/33) cases. Pancreatic fistula occurred in 14 patients (42%), fistula combined hemorrhage in 1 patient (3%), delayed gastric emptying in 2 patients (6%). There was no mortality. During a median follow-up of 32 moths, 31 patients were without symptoms recurrence and 2 patients lost to follow-up.
Conclusions: Whipple’s triad and insulin release index can be the main basis for the diagnosis. Surgical resection can achieve disease-free survival with low morbidity and mortality. Compared with segmental pancreatic resection, enucleation showed significant benefits in terms of surgical hemorrhage and operation time.
Serum Trypsinogen Levels As a Biomarker of Pancreatic Exocrine Insufficiency (PEI) In Chronic Pancreatitis (CP): Results From the NAPS2 Study
D.C. Whitcomb, B. Sandhu, C. Wilcox, S. Alkaade, M. Anderson, D. Yadav. for the NAPS2 Consortium, Pittsburgh, PA.
Aim: To evaluate performance of serum trypsinogen as a biomarker for PEI.
Background: A key feature of CP is progressive loss of acinar cells resulting in PEI and maldigestion. Measuring pancreatic exocrine function remains challenging, while determining PEI requires additional consideration of diet and intestinal function. We hypothesize that serum trypsinogen levels are a useful biomarker of pancreatic exocrine function in CP.
Methods: In CP patients and controls enrolled in the North American Pancreatitis Study II, serum trypsinogen levels were measured in collaboration with a commercial laboratory. Plasma samples were collected under strict standard operating procedures and processed frozen in aliquots to limit freeze-thaw damage. Physicians indicated whether a patient had PEI based on clinical symptoms or formal testing.
Results: Trypsinogen concentrations in controls ranged from <5-181 ng/ml and were normally distributed (mean 35 ± 11); in CP patients they ranged from <5-1904 ng/ml and were non-normally distributed. After excluding outliers (value >100 - 4 controls, 24 patients) and missing information on PEI (n = 10), 288 CP cases and 262 controls were included in the analysis. Trypsinogen levels in CP patients were significantly lower (p < 0.001) - levels were low normal (>1 and ≤2 sd below control mean) in 22.2% and low (>2 SD below control mean) in 35.1% CP patients. PEI was present in 47% patients. The sensitivity, specificity, PPV and NPV for trypsinogen to detect PEI was 47%, 75%, 62% and 62% when using trypsinogen value of >2 sd below control mean as a cut-off for PEI, and, 69%, 53%, 56% and 67% when using values of >1 and ≤2 sd below control mean as a cut-off for PEI.
Conclusions: Serum trypsinogen levels are significantly lower in CP patients when compared with controls, and perform modestly as a biomarker for PEI. However, since PEI also reflects diet and intestinal factors, further research is warranted.
Epigenetic Regulation of Mitotic Cell Division in Pancreatic Cancer Cells by a Novel EGF-RAF-MEK-ERK-Aurora B-HP1α Pathway
M. Williams, A. Mathison, J. Willenborg, G. Lomberk, R. Urrutia. Epigenetics and Chromatin Dynamics Laboratory, Translational Epigenomics Program (CIM), GIH Division, Department of Medicine, Biophysics, Biochemistry and Molecular Biology, Mayo Clinic, Rochester, MN.
Epigenetic regulator proteins are the ultimate effectors of oncogenic pathways. Heterochromatin Protein 1 α (HP1α), is one of the best-studied epigenetic regulators, which functions as a histone code reader of the di- and tri-methylK9-H3 marks in organisms ranging from yeast to human. We are investigating the function of this protein in pancreatic cancer, where it is found overexpressed. Here, we report for the first time that activation of linked oncogenic pathways, namely the RAF-MEK-ERK cascade, ultimately activates Aurora kinase B, which in turn phosphorylates HP1α at serine 92. This phosphorylation event occurs within an Aurora consensus site located within the linker region of this protein. Using double-thymidine block in HeLa cells, we define that the phosphorylation of HP1α takes place during mitosis, concordant with the time that the phosphorylated serine10 mark is deposited on Histone H3. Congruently, the phosphorylated form of this protein localizes to the mitotic apparatus. Experiments employing overexpression vectors, pharmacological inhibitors, dominant negative proteins, and siRNA-based knockdown of Aurora B confirm that this kinase is responsible for the observed phosphorylation. Immunofluorescence analysis demonstrates colocalization of HP1α and Aurora B beginning at prophase, peaking at metaphase and subsequently decreasing at anaphase. Affinity protein purification combined with proteomic experiments reveals that HP1α associates with critical components of the centromere-kinetochore complex, which support a role for this pathway in the maintenance of chromosome structure and condensation. Combined, these results demonstrate that the epigenetic regulator HP1α is a bona fide effector of one of the most important oncogenic pathways for pancreatic cancer.
Increased Neuroendocrine Heterogeneity in Pancreatic Ductal Adenocarcinoma is Associated With More Aggressive Biology
P.J. Worth, A.S. Farrell, J.M. Link, Z.P. Jenny, E.W. Gilbert, B.C. Sheppard, R.C. Sears. The Brenden-Colson Center for Pancreatic Care, Oregon Health & Science University, Portland, OR.
Background: Resistance and heterogeneity make the treatment of pancreatic cancer (PDAc) challenging. Recent work from our lab in a novel murine model of PDAc suggests that increased neuroendocrine (NE) cells within PDAc is associated with a more heterogeneous and plastic tumor. These cells co-stain for the NE marker synaptophysin and ductal cytokeratins. These scattered NE cells have been described in few prior series. We hypothesize that increased NE cells in PDAc is associated with worse biology and outcomes.
Methods: We selected tumors from chemo-naïve patients who had surgery for PDAc, controlling for age, grade, PNI, LVSI, venous involvement, stage, and smoking. Mortality from surgical complications or patients found to have metastasis at pathology were excluded. All patients underwent adjuvant chemotherapy and had standard follow-up imaging. Tissue was stained for synaptophysin and cytokeratin 8 & 18 and graded on number of co-positive cells normalized to the number of tumor cells per section. Specimens were divided into high (≥5%) or low (<5%) co-positivity for analysis. Disease free (DF) and overall survival (OS) intervals were calculated.
Results: Sixteen patients were analyzed; ten were found to have low co-positivity, six with high. High co-positivity was associated with decreased DF survival (median days to recur 98.5 [60–237] vs 395 [233–475], p = 0.01). Median OS was similar for high and low co-positivity (424 days [327–480] vs 502 days [326–754], p = 0.233).
Conclusions: Patients with high levels of neuroendocrine cells in PDAc have shorter DFS. Though OS is similar, this may reflect the tendency to receive second line therapies, which could not be controlled for. While the significance of these cells is unknown, we are investigating the role this phenotypic heterogeneity may play in biology and resistance to therapy.
Analysis of Surgical Management and Pathological Features of Pancreatic Neuroendocrine Neoplasms
J. Wu, C. Dai, K. Jiang, W. Gao, Q. Li, W. Xu, J. Wei, J. Chen, F. Guo, Z. Lu, Y. Miao. Pancreas Center, The First Affiliated Hospital of Nanjing Medical University, Nanjing, China.
Background: Pancreatic neuroendocrine neoplasms (pNENs) are relatively rare in pancreatic tumors, but the incidence is increasing in recent years. Surgical treatment is still the only chance for cure despite a variety of medications proven to be effective in tumor control.
Methods: A total of 123 consecutive pNENs patients who received surgical treatment in our institution were analyzed retrospectively. Clinical data were collected from Mar. 2006 to May 2014. All patients were histologically confirmed as primary pNENs and were reviewed for epidemiological characteristics, clinical manifestation, perioperative parameters, pathological features, postoperative course and survival data.
Results: All 123 patients (74 female patients with mean age of 48.3 years) underwent operation with curative intent. Sixty-two in 123 cases were non-functional tumors. Tumor size ranged from 0.3 to 18.0 cm (mean 2.93 cm). Postoperative complication rate was 55.3%, with pancreatic fistula in 60 patients (27 in grade A, 28 grade B and 5 grade C), intraabdominal fluid collection in 12 patients, bleeding in 4 patients (2 patients received re-operation, 1 patient died), delayed gastric emptying in 3 patients and biliary fistula in 1 patient. Tumor grade was evaluated in 74 cases (49 in G1, 22 G2 and 3 G3). For TNM staging, stage I, II, III and IV were 106,13,0 and 4, respectively. The final survival analysis included 87 patients. The 1-year, 3-year and 5-year survival rates were 95.1%, 87.8% and 84.0%. TNM staging, WHO classification and distant metastasis were shown to be risk factors for prognosis of pNENs.
Conclusion: Surgical resection with regional lymphadenectomy is expected to be curative treatment for most resectable pNENs, while enucleation is appropriate for most insulinomas. TNM staging, WHO classification and distant metastasis are independent risk factors for worse prognosis of pNENs.
The Endoscopic Diagnosis and Treatment of Mass-type Autoimmune Pancreatitis
B. Xiao, K. Jiang, J. Wu, W. Gao, J. Chen, J. Wei, F. Guo, Z. Lu, Y. Miao. Pancreas Center, The First Affiliated Hospital of Nanjing Medical University, Nanjing, China.
Background: Autoimmune pancreatitis (AIP) is characterized by lymphoplasmacytic infiltration and fibrosis. About 1/3 of AIP cases show a low-density mass on imaging study, which makes it a real challenge to differentiate AIP from pancreatic cancer. In recent years, advances in endoscopy technology have improved our diagnosis and treatment ability for AIP.
Aim: To evaluate the value of endoscopy in diagnosis and management of mass-type AIP.
Methods: There were 6 patients with pancreas mass diagnoses as AIP during the last 3 years (2012–2015). We analyzed every case retrospectively to evaluate the role of EUS in diagnosis and treatment in mass-type AIP. Diagnosis of AIP was based on radiology, cytology/histology, serology, and response to steroids treatment. Patients were followed-up from 3 month to 3 years.
Results: All six patients (4 males) were diagnosed as AIP. Main symptoms were abdominal pain (6/6) and obstructive jaundice (4/6). All patients undertook contrast enhanced computed tomography or magnetic resonance imaging, which showed pancreas mass in pancreas head (5/6) or body (1/6). Only 1 patient underwent surgery whose histology revealed no malignancy. Histology and cytology analysis were performed in all remaining cases with samples taken under endoscopic ultrasound (EUS) guidance. Desmoplasia and plasma cell infiltration were observed. IgG4 was positive in immunohistochemical staining and serology. Endoscopic Retrograde Cholangiopancreatographies (ERCPs) were performed to relieve jaundice in 4 patients. Five patients had a significant respond to steroids. During follow-up, EUS was utilized to evaluate the mass and ERCP for replacement of biliary stent. Masses in five cases who have undertaken steroids therapy diminished to vary extent.
Conclusion: To distinguish mass-type AIP from pancreatic cancer is a thorny problem, while endoscopy could play an important role in diagnosis and management of this disease.
Resveratrol Inhibits Hypoxia Induced PSC Activation and Pancreatic Cancer Cell Viability and Invasion
Q. Xu, W. Duan, J. Lei, Q. Ma. Department of Hepatobiliary Surgery, First Affiliated Hospital of Medical College, Xi’an Jiaotong University, Xi’an, China.
Background: Resveratrol (RSV), a natural polyphenolic compound detected in grapes, berries, and peanuts, possesses a wide spectrum of pharmacological properties, including antioxidant, anti-inflammation and anti-tumor activities. Tumor microenvironment plays a key role in tumor progression. Pancreatic stellate cells (PSCs) as a novel and important member of tumor microenvironment, has gained much more focus lately. Solid tumors often experience low oxygen tension environments. Tumor hypoxia is associated with enhanced tumor invasion, angiogenesis, and distant metastasis. In this study, we investigated the role of RSV in hypoxia-driven PCa progression.
Methods: Human PSCs were isolated from normal pancreas tissues. The purity of the PSCs was assessed by morphology, oil red O staining, and immunofluorescence of a-smooth muscle actin. qRT-PCR and ELISA were performed to quantify the expression of IL-6, VEGF-A and SDF-1. Metastasis and invasion experiment were performed to measure the ability to undergo EMT and to invade through a reconstituted matrigel barrier.
Results: We observed that hypoxia increased PSC activation; PSCs cultured under hypoxic conditions exhibit higher levels of IL-6, VEGF-A and SDF-1 transcription and secretion; these factors are known to be involved in modulating the response of tumor cells to activated PSCs. Hypoxia was mildly active in promoting the invasiveness of PCa cells. In addition, hypoxia Conditional media (CM) from PSCs significantly decreased the E-cadherin level and increased the vimentin level of PCa cells, which indicates that hypoxia can drive PCa cells to undergo EMT. Exposure of PSCs to hypoxia during their activation enhanced their ability to affect PCa motility and invasiveness. Conditional media (CM) from PSCs significantly decreased the E-cadherin and increased the vimentin of PCa cells under hypoxic conditions, which indicates that CM from PSCs can drive PCa cells to undergo EMT. Moreover, treatment with the RSV or down-regulated expression of HIF1 with siRNA, abrogated the hypoxia-induced effect on PSCs and PCa cells.
Conclusion: These findings suggest that PSCs are sensitive to hypoxia, which enhances their promotion of PCa invasiveness. RSV has a protective effect against hypoxia in the pancreatic tumor-stromal interaction. RSV might be a potential anticancer agent for the treatment of PCa.
Smoking Worsens the Fibrosis of Alcoholic Chronic Pancreatitis via Activation of Pancreatic Stellate Cells
Z. Xu,1,2 S. Pothula,1,2 S.J. Pandol,3 R.C. Pirola,1,2 J.S. Wilson,1,2 M.V. Apte.1,21Pancreatic Research Group, SWS Clinical School, UNSW, 2Ingham Institute, Sydney, Australia; 3Cedars Sinai Medical Center, Los Angeles, CA.
Background: Epidemiological studies indicate that smoking accelerates the progression of alcoholic chronic pancreatitis (AP) as evidenced by earlier development of calcification and fibrosis. However, the mechanisms mediating these effects are unknown. Activated pancreatic stellate cells (PSCs) are the key cells responsible for producing the fibrosis of AP. We have recently shown that PSCs express nicotinic acetylcholine receptors (nAChRs) that bind the ligands nicotine and nicotine-derived nitrosamine ketone, NNK. Thus we hypothesize that smoke compounds in combination with ethanol, activate PSCs leading to enhanced fibrosis of the gland.
Aims: To assess 1) the effects of cigarette smoke exposure on the extent of necroinflammation and fibrosis in a rat model of AP; and 2) the in vitro effects of smoke compounds ± alcohol on rat PSC proliferation in the presence and absence of the nAChR antagonist, mecamylamine.
Methods:1) SD rats fed Lieber-DeCarli alcohol diets for 11 weeks were challenged with LPS (3 mg/kg; weekly IV inj) or saline in weeks 9, 10, 11. Rats were either housed conventionally (exposed to room air), or in a custom-designed smoke exposure chamber and from week 5 to week 11, exposed to cigarette smoke, at a level equivalent to heavy smoking (25 cigarettes/day). Hence, there were 4 groups (n = 3 rats/group): Alcohol + saline (A); Alcohol + LPS (AL); Alcohol + smoke + saline (AS); and Alcohol + smoke + LPS (ASL). H&E sections scored for injury (edema, acinar necrosis, inflammation); Sirius Red sections assessed morphometrically for collagen deposition. 2) Rat PSCs were exposed to cigarette smoke extract (CSE, 40 μg/ml) or NNK (100 mM) ± ethanol (E, 10 mM) for 48 hours in the presence and absence of mecamylamine (500 μM), and cell proliferation assessed.
Results: Expressed as mean ± SEM 1) Histology scores: A 3.43 ± 0.3; AS 5.27 ± 0.4; AL 7.56 ± 0.2**; ASL 10.23 ± 0.9** #; **p < 0.01 vs A; #p < 0.05 vs AS & AL. Fibrosis scores: A 1.68 ± 0.1; AS 1.67 ± 0.4; AL 2.74 ± 0.3; ASL 3.58 ± 0.5**; **p < 0.01 vs A & AS. 2) CSE and NNK, alone and in combination with ethanol significantly increased PSC proliferation (% of control - CSE 128.9 ± 1.5*; NNK 127.6 ± 1.6*; CSE + E 130.2 ± 2.3*; NNK + E 119.3 ± 4.0 *p < 0.05 vs control n = 4/group). This induction was abolished in the presence of mecamylamine.
Conclusions: 1) Smoke compounds significantly worsened pancreatic necroinflammation and fibrosis in a rat model of AP. The inductive effects of ethanol and smoking on PSC proliferation were inhibited by the nAChR antagonist mecamylamine, suggesting that nAChRs may mediate PSC responses to smoke compounds.
Implications: Smoking-related acceleration of alcoholic pancreatitis may be mediated by activation of PSCs upon exposure to alcohol and smoke compounds.
Primary Pre-adipocytes Stimulate DNA Synthesis of Pancreatic Intraepithelial Neoplasia in the KrasG12D Mouse Model
M. Xu, H.-H. Chang, A. Moro, X. Jung, A. Stark, A. Schmidt, O.J. Hines, G. Eibl. Department of Surgery, University of California, Los Angeles, Los Angeles, CA.
Introduction: A high fat, high calorie diet (HFCD) is closely associated with obesity, which increases the incidence of multiple types of cancer. Our previous work has shown that the HFCD leads to weight gain, inflammation of the visceral adipose tissue and pancreas, and acceleration of pancreatic neoplasia in KrasG12D (KC) mice, suggesting that visceral obesity may play an active, rather than a permissive, role in pancreatic cancer development. In this project we aimed to investigate the effects of visceral fat on the proliferation of pancreatic intraepithelial neoplasms (PanINs), precursors of pancreatic cancer.
Methods: We isolated primary pre-adipocytes from visceral fat depots of wildtype (WT) and KC mice fed either the control diet (CD) or HFCD. PanIN cells were isolated from KC mice. We co-cultured PanIN cells with primary pre-adipocytes for 24 hours, and evaluated DNA synthesis by the thymidine incorporation assay as a measure of cell proliferation.
Results: We found that pre-adipocytes from WT and KC on CD had similar effect in stimulating PanIN proliferation (p > 0.05). However, pre-adipocytes from KC mice on HFCD stimulated PanIN proliferation by 1.7 to 3.4-fold compared those from KC mice on CD (p < 0.05).
Conclusions: Our results showed that visceral pre-adipocytes are capable of stimulating proliferation of PanIN cells. Importantly, pre-adipocytes from mice fed a HFCD have a more robust effect on PanIN proliferation than pre-adipocytes from mice on a CD. Our results indicate a potentially important promoting effect of visceral adipose tissue on PanIN cell growth in the KC mouse model. This finding could constitute an important mechanism, by which diet-induced obesity can accelerate pancreatic cancer development.
Use of Lifetime Drinking History (LDH) to Stratify Alcohol Exposure in Chronic Pancreatitis (CP) Patients
D. Yadav, B. Sandhu, S. Al-Kaade, Y. Tian, N. Guda, A. Gelrud, R. Brand, A. Slivka, C. Wilcox, D. Whitcomb. for the NAPS2-consortium, Pittsburgh, PA.
Aim: To categorize CP patients based on lifetime drinking habits for possible risk stratification.
Background: Data are scarce data on cumulative alcohol consumption and drinking habits in CP patients. We hypothesized that CP patients have a wide spectrum of drinking habits that can be organized into specific patterns.
Methods: The NAPS2-CV Study prospectively enrolled a validation cohort of CP patients from 2008–2012. At 5 out of 13 NAPS centers, a trained nurse coordinator administered a modified Skinner's LDH questionnaire to patients. Cluster analyses were used to define drinking trajectories prior to CP diagnosis. In a sub-analysis, drinking trajectories in patients ≥50 years were compared with published data on population controls.
Results: Of 261 Caucasian CP patients (54% male, mean age 50 ± 15.2 yrs) enrolled from the 5 sites, 44 (17%) were lifetime abstainers. Of 217 (83%) ever drinkers, 193 (89%) completed LDH. In 54% ever drinkers, enrolling physician considered alcohol as CP etiology. Prior to CP diagnosis, median daily average was 1.97 drinks, drink day average was 5.1 drinks, drinking duration was 25 years and cumulative alcohol exposure was 2.6 kg/kg body weight. Cluster analysis classified lifetime drinking habits of all patients into 3 trajectories of exposure based on daily average (low 19.2%, intermediate 49.4%, high 31.3%) and drink day average (low 14.5%, intermediate 53.9% intermediate, high 31.5%). Analysis limited to patients ≥50 yrs revealed 2 trajectories based on daily average (low 74.4%, high 25.6%) and 3 categories on drink day average (low 30.3%, intermediate 59.7%, high 10%), which differed distinctly from control population.
Conclusions: Cumulative alcohol exposure and drinking patterns of CP patients differ from population controls, and can be classified into distinct trajectories. These data will be used to classify patients for more complex gene x environment studies and model development.
Induction of R201C Mutant Gαs Facilitates Murine Pancreatic Tumorigenesis in Cooperation With G12D Mutant Kras
H. Yamaguchi,1 G. Dangol,1 B. Ghosh,1 J. S. Gutkind,2 A. Maitra.11Sheikh Ahmed Center for Pancreatic Cancer Research, University of Texas, MD Anderson Cancer Center, Houston, TX; 2Oral and Pharyngeal Cancer Branch, National Institute of Dental and Craniofacial Research, National Institute of Health, Bethesda, MD.
Background: The alpha subunit of the stimulatory G protein (Gαs) encoded by GNAS regulates cyclic AMP production and plays an important role in signal transductions upstream of multiple signaling pathways. Recently, recurrent mutations in codon 201 of GNAS, which resulted in constitutively active Gαs, were reported in intraductal papillary mucinous neoplasm (IPMN) of the pancreas. The role of R201C Gαs in tumorigenesis of IPMN largely remains to be elucidated.
Methods: We generated a new mouse model in which expression of R201C Gαs was inducible by tetracycline (Tet) in exocrine pancreas conditionally under Cre-mediated recombination. Mice harboring reverse Tet transactivator (Linker) flanked by Lox-STOP-Lox (LSL) site and R201C Gnas with Tet responsive element (TetGnas R201C) were crossed with LSL-Kras G12D; p48-Cre (KC) mice to generate Linker; TetGnas R201C; p48-Cre with or without LSL-Kras G12D (LGKC or LGC) mice.
Results: Six to eight-week old LGKC mice treated with doxycycline (Dox) showed enlarged pancreas in one week, increased body weight by ascites, and macroscopic or ultrasound-detectable cystic lesions after two weeks of treatment. Histologically, Dox treatment induced massive acinar-ductal metaplasia (ADM), an initial step of murine pancreatic tumorigenesis, and extensive acinar loss with fibrosis and inflammation. Dilated ductal lesions generated by ADM merged and formed cystic lesions. Age-matched LGKC mice without Dox treatment showed only KC-compatible focal PanIN lesions. In LGC mice, weight of pancreas was slightly increased but there was no change in tissue architecture after three weeks of Dox treatment.
Conclusion: Induction of the R201C Gαs accelerated ADM in corporation with G12D Kras and developed cystic lesions.
Adverse Effect of Re-elevation of Postoperative Inflammatory Reaction on Recurrence Free Survival Time of Patients Undergoing Potentially Curative Resection for Distal Pancreatic Cancer
S. Yamazoe, R. Amano, K. Kimura, G. Ohira, K. Nishio, K. Miura, M. Ohira, K. Hirakawa. Department of Surgical Oncology (First Department of Surgery), Osaka City University, Graduate School of Medicine, Osaka, Japan.
Background: In recent years, it has been reported that postoperative complications is one of the poor prognostic factor in a variety of cancers.
Aim: The purpose of this retrospective study is to analyze the effect of postoperative inflammatory reaction (POIR) on outcome of patients undergoing potentially curative resection for distal pancreatic cancer.
Methods: From January 2000 and June 2013, all patients who underwent potentially curative resection for distal pancreatic cancer at our institution were enrolled. In recurrent cases, survival was analyzed according to clinicopathological variables. Re-elevation of POIR was defined as serum C-reactive protein (CRP) level rise again over 20 mg/l or more in acute phase (within two weeks after surgery).
Results: Distal pancreatectomy was performed in 58 cases and 38 cases have relapsed. Median recurrence-free survival time (RFS) was 6.95 months and RFS rates were 36.8%, 10.5%, 2.6% at 1, 3, and 5 –years, respectively. On univariate analysis, male, histopathological grade 3 or 4, transfusion, neutrophil-lymphocyte ratio > 3 and re-elevation of POIR were significantly associated with survival. On multivariate analysis, re-elevation of POIR was identified as an independent prognostic factor.
Discussion: In this study, postoperative complication was not associated with survival. It has been reported that the inflammatory response to tumor cells, reflected by an elevated plasma CRP level, results in a tumor microenvironment enriched with proinflammatory cytokines, angiogenic and lymphogenic factors and chemokines that promote tumor growth, angiogenesis and metastasis.
Conclusion: Re-elevation of POIR might predict poor outcome in patients undergoing potentially curative resection for distal pancreatic cancer.
Thrombin-Dependent Pathways Drive Pancreatic Ductal Adenocarcinoma Disease
Y. Yang,1 C.L. Rewerts,2 M.J. Flick,2 S.F. Konieczny.11Department of Biological Science & the Purdue Center for Cancer Research, Purdue University, West Lafayette, IN; 2Division of Experimental Hematology & Cancer Biology, Cincinnati Children’s Hospital, Cincinnati, OH.
Background: Pancreatic ductal adenocarcinoma (PDAC) is the fourth leading cause of cancer death in the U.S. with an overall survival rate of less than 7%. The extreme poor survival is associated with the aggressive metastatic nature of PDAC, making it essential to explore new targets that drive tumor aggressiveness and invasiveness. Here, we demonstrate that PDAC disease is closely tied to activation of the coagulation cascade, which is among the most profound of the reactive changes that occur following pancreatic cancer tumorigenesis.
Method: Protease-Activated Receptor (PAR-1) and Tissue Factor (TF) expression were suppressed by shRNA or CRISPR-Cas9 technology in a mouse PDAC cell line (KPC2) that initially was derived from a PDAC tumor harboring mutated Tp53R172H and KrasG12D alleles. In vitro cell growth was evaluated in these genetically modified lines and in allograft studies where the cell lines were transplanted into either wild type or GEM mice harboring genetic defects in genes involved in the coagulation cascade. Tumor growth and lung metastasis were determined.
Results: shRNA knockdown of Par-1 and TF in the KPC2 lines produced similar in vitro growth rates when compared to the parental KPC2 cells. However, in allograft studies, both Par-1 and TF shRNA knockdown lines produced dramatically reduced primary tumor growth and metastasis in WT mice. Interestingly, parental KPC2 cell tumor growth and metastasis was also dramatically repressed when the cells were transplanted into Prothrombinlow mice. A similar reduction in tumor burden was obtained when KPC2 cells were transplanted into Fibrinogen null animals.
Conclusion: Our results demonstrate that PDAC aggressiveness and invasiveness are directly affected by tumor-derived Par-1 and TF expression as well as by host-derived fibrinogen and prothrombin expression.
Extracellular Matrix Modulation Therapy of Pancreatic Cancer by 4-methylumbelliferone and 5-fluorouracil
E. Yoshida, D. Kudo, H. Nagase, A. Suto, T. Wakiya, K. Ishido, Y. Toyoki, K. Hakamada. Gastroenterological Surgery, Hirosaki University, Graduate School of Medicine, Hirosaki, Aomori, Japan.
Purpose: Pancreatic cancer responds poorly to most chemotherapeutic agents. Therefore, a new therapy for advanced pancreatic cancer has been expected. Hyaluronan (HA) is a major component of extracellular matrix (ECM). Several studies have reported the importance of HA rich ECM as a biological barrier against chemotherapeutic agents. If depletion of HA around the cancer cells by MU enhances cytotoxic effect of 5-FU, MU can provide a new approach for the treatment of pancreatic cancer.
Methods: we analyzed a concentration of 5-FU for the dosage of MU by HPLC in vitro and examined change of the antitumor effect by combination therapy of MU and 5-FU both of in vitro and in vivo.
Results: MU plus 5-FU treatment inhibited cell proliferation higher than 5-FU alone. 0.01 mM 5-FU inhibited cell proliferation 37.7%. 0.01 mM 5-FU + 0.5 mM MU inhibited cell proliferation 57.4%. MU enhanced intracellular concentration of 5-FU as much as 47.3 % compared to the control. In mice, tumor size treated with 5-FU and MU group was significantly decreased and survival time of the 5-FU plus MU group was significantly prolonged (p < 0.05). MU did not enhanced side effect of 5-FU.
Conclusion: Combination therapy with MU and 5-FU is effective for pancreatic cancer.
Arterial Resection Without Reconstruction Following Preoperative Arterial Embolization for Locally Advanced Pancreatic Head Cancer With Arterial Invasion
H. Yoshitomi, N. Sakai, H. Shimizu, M. Ohtsuka, A. Kato, K. Furukawa, T. Takayashiki, S. Takano, S. Kuboki, D. Suzuki, S. Kagawa, M. Miyazaki. Department of General Surgery, Chiba University, Chiba, Japan.
Background: The locally advanced pancreatic cancer with arterial invasion is usually considered as contra-indication of surgical resection, due to high morbidity and mortality of arterial resection, especially due to the obstruction of reconstructed artery.
Aim: To assess the effectiveness of combined arterial resection without reconstruction following preoperative arterial embolization for locally advanced pancreatic cancer with arterial invasion.
Methods: We analyzed 11 cases which underwent pancreaticoduodenectomy (n = 7) or total pancreatectomy (n = 4) with combined hepatic artery resection without reconstruction for pancreatic head cancer invading hepatic artery in Chiba University Hospital between 2008 and 2013. In all cases, no arterial reconstruction was performed.
Results: Median age was 67 (40–74) years old and M:F was 7:4. In all cases, preoperative coiling of PHA and/or CHA was performed and collateral hepatic arterial flow was confirmed by contrast-enhanced CT. Combined portal vein resection and reconstruction was performed in 9 cases. Median operative time was 522 (331–573) min and median blood loss was 2160 (1315–19865) ml. Post-operative serum AST/ALT level was elevated to 1273/967 IU/L (average) at day 1 but rapidly decreased to 99/75 IU/L at day7. Post-operative liver abscess was found in 1 case and percutaneous drainage was performed. Clavien-Dindo IIIa post-operative complication was found in 4 cases. No perioperative mortality was observed. R0 resection was achieved in 6 cases.
Conclusion: Pancreatic resection combined with hepatic arterial resection without reconstruction following preoperative coil embolization can be performed safely. This method also achieved high R0 rate with 55%. The more precise indication should be assessed according to the prognosis.
Activating Transcription Factor 3 Promotes De-differentiation of Acinar Cells During Cerulein-Induced Pancreatitis in Mice
C.C. Young,1,2,4 C.L. Pin.1,2,3,4Departments of 1Paediatrics, 2Physiology and Pharmacology, and 3Oncology, University of Western Ontario, 4Children's Health Research Institute, London, Ontario, Canada.
Background & Rationale: Activating Transcription Factor 3 (ATF3) is a downstream mediator of the Unfolded Protein Response (UPR), a signaling pathway activated upon induction of pancreatitis. ATF3 is expressed in acinar cells only after injury, and we have identified ATF3 target genes in acinar cell differentiation. However, it is unclear how ATF3’s link to acinar differentiation affects pancreatitis progression and severity. The goals of this study were to assess the severity of cerulein-induced pancreatitis (CIP) in mice containing a targeted deletion of Atf3 (Atf3−/−) and determine if acinar cell differentiation is compromised during regeneration.
Methods & Results: Acute CIP was induced in Atf3−/− and congenic C57Bl/6 mice, and tissue and blood collected 4 hours to 7 days following initial injection. Immunofluorescence analysis (IF) for markers of acinar cell differentiation (MIST1, Cx32, NR5A2) and pancreatic progenitors (SOX9, PDX1) was performed. Serum amylase and histology revealed increased severity of CIP in Atf3−/− mice based on enhanced serum amylase and tissue necrosis at early time points (8–32 hrs). IF showed maintained differentiation in Atf3−/− mice relative to WT mice, while progenitor cell markers normally activated by CIP were not increased in Atf3−/− mice. Histological analysis revealed reduced acinar-to-ductal metaplasia (ADM) in Atf3−/− mice at later time points in CIP (72 hr).
Conclusion: Our findings suggest ATF3 promotes a loss of the acinar cell phenotype during CIP that is protective against tissue damage. A reduction of ADM in Atf3−/− tissue suggests limited regeneration in the absence of ATF3. However, since ADM is believed to be an initiating event in PanIN formation, it is possible that the protective actions of ATF3 in CIP may increase the potential for pancreatic adenocarcinoma.
Cadmium Induced Transformation Leads to Pancreatic Cancer
W. Yu,1 Y. Ma,1 R.K. Srivastava,1,* S. Shankar.1,2,*1Kansas City VA Medical Center, Kansas City, MO; 2Department of Pathology, School of Medicine, University of Missouri-Kansas City, Kansas City, MO; *Equal contributors.
Persistent and cumulative human exposures to toxic metals lead to various health concerns and chronic illnesses. Cadmium is a heavy metal of considerable occupational and environmental concern and cadmium exposure has been shown to have an epidemiological association with pancreatic cancer. Our current focus was to determine the biology of cadmium induced cancer of the pancreas. In this pursuit we have demonstrated that cadmium metal is carcinogenic and can induce transformation and reprogramming of normal pancreatic epithelial cells. Interestingly, our data also reveal that chronic exposure of normal pancreatic ductal epithelial HPNE cells to low levels of Cd+2 induce cellular transformation and also induce the expression of a transcription factor SATB2 which is oncogenic in pancreatic cancer. During cadmium-induced cell transformation, cells gain the phenotype of cancer stem cells (CSCs) / progenitor cells and express pluripotency maintaining factors. This suggests that overexpression of SATB2 may induce cellular transformation. Using ChIP assay, we have demonstrated that SATB2 can directly bind to Bcl-2, Nanog, Bsp, Klf4, Myc, Xiap, and Hoxa2, and regulate the expression of EMT genes, pluripotency maintaining factors and drug resistance genes. Based on these observations, we conclude that the SATB2, induced by cadmium exposure or through over-expression studies, play a role in cell transformation of human pancreatic normal ductal epithelial cells. To evaluate the human health risks associated with chronic exposure to cadmium we develop a mechanistic understanding of the cadmium induced human pancreatic normal ductal epithelial cell transformation. The results shown set the stage that cadmium toxicity enhances pancreatic carcinogenesis by up-regulating SATB2 which may thus be a potential target for development of targeted therapeutic interventions.
Integrin-linked Kinase Pathway Plays an Important Role in Initiating Acute Pancreatitis
Z. Yuan, J. George, C. Ulrich, U. Barlass, S. Garg, A. Sareen, A.K. Dixit, R.K. Dawra, A.K. Saluja. Division of Basic and Translational Research, Department of Surgery, University of Minnesota Medical School, Minneapolis, MN.