Pan-ErbB inhibitor CI 1033 on pancreatic cancer therapy

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Abstracts / Pancreatology 13 (2013) e1–e94

P214. The development of fibrosis in a novel model of chronic pancreatitis is mediated by complement factor C5 M. Sendler 1, G. Beyer 1, V. Kauschke 1, T. Wartmann 2, F. Ulrich Weiss 1, W. Halangk 2, M.M. Lerch 1, J. Mayerle. 1. 1 Department of Medicine A, University Medicine, Ernst-Moritz-ArndtUniversity Greifswald, Germany 2 Division of Experimental Surgery, Otto-von-Guericke-University Magdeburg, Germany Aim of the study: Chronic pancreatitis is a progressive inflammatory disorder in which the pancreatic parenchyma is destroyed and replaced by fibrous tissue. We studied the role of complement factor C5/C5a, a strong chemo-attractant for neutrophils and macrophages, for the necrosis-fibrosis-sequence in chronic pancreatitis is studied here. Material and methods: Chronic pancreatitis was induced by ligation of the pancreatic duct of mice and a single i.p. injection of caerulein (10mg/ kgBW) 2 days after surgery in C5-/- and C5+/+ mice (B10.D2-H2dH2T18cHco ⁄ o2Sn (C5)) or by 7 repetitive injections of caerulein twice a week over 10 weeks. Acute pancreatitis was induced by 7 repetitive injections of caerulein. Pancreatic tissue was examined at day 3, 7 and 21 after ligation. Pancreatic fibrosis was evaluated by histological scoring of Goldner stained tissue and characterisation of the cellular components. Local and systemic damage markers of pancreatitis were monitored. Results: All animals developed acute pancreatitis with increased levels of serum amylase and lipase 3d after ligation of the pancreatic duct and progressive pancreatic necrosis until day 7. Necrotic tissue was replaced by a strong fibrotic reaction at 21d. In C5 deleted animals we did not detect a significant difference in severity of acute pancreatitis, but we found a significantly reduced fibrosis reaction associated with a decreased number of M2 macrophages compared to littermates in the chronic model. Conclusion: C5a contributes to the progression of chronic pancreatitis and the formation of fibrosis. This effect is independent of the severity of acute pancreatitis. Whether C5a directly stimulates fibroblasts or stellate cells or whether the effect is indirectly mediated by increased infiltration of inflammatory cells needs to be clarified.

P215. Improved long term survival after extended pancreatic cancer operations: A single-institution experience H. Seppänen 1, A. Juuti 1, H. Mustonen 1, C. Haapamäki 1, S. Nordling 2, M. Carpelan-Holmström 1, J. Sirén 1, C. Haglund 1, T. Kiviluoto 1. 1

Department of Surgery, Helsinki University Central Hospital, Helsinki, Finland 2 Department of Pathology, Helsinki University Central Hospital, Helsinki, Finland Background: The poor survival results in the 1990’s for PDAC have demanded development of the treatment of pancreatic cancer. The aim of our study was to evaluate the outcomes of pancreatic operations in general and extended operations on pancreatic cancer in our hospital at the era of centralization. Material and methods: We identified from a database all patients undergoing pancreatic operation in the Helsinki University Hospital 20002009 retrospectively. The operations were performed in an extended manner to those with malignant disease. Distal gastrectomy as well as extended lymphadenectomy (D2-D3) was performed. Extended resection included right side omentectomy, excision of the right cranial peritoneal leaf revealing caval vein and the renal veins, excision of the lymph nodes of the hepaticoduodenal ligament and excision of the aorto-caval notch as well as around the origin of the superior mesenteric artery. Results: There were 546 patients undergoing a pancreatic operation during the ten year time period. 389 patients underwent pancreaticoduodenectomy, 103 distal pancreatic resection, 19 total pancreatectomy and 40 other operation. Histological diagnosis was pancreatic ductal adenocarsinoma in 194, invasive IPMN in 26 and papillary

adenocarcinoma in 44 patients. Of the PDAC patients 100 (52 %) received adjuvant therapy. Overall survival rates for patients radically operated for PDAC (n¼194) were for one year 71 %, three years 33 % and for five years 18 %. 151 (78 %) had R0 resection and 67 (34 %) were free of the lymph node metastases. For the lymph node negative and R0 resected patients the survival rates were 79 %, 47 % and 31 % respectively. Conclusions: When the treatment of pancreatic cancer was concentrated to a multidisciplinary team with extended cancer surgery the long term survival was increased in PDAC.

P216. Anti-inflammatory macrophage induced MMP9 and ADAM8 expression increases invasion rate of pancreatic cancer cells H. Seppänen 1, H. Mustonen 1, S. Vainionpää 1, Z.H. Shen 1, H. Repo 2, E. Kemppainen 1, P. Puolakkainen 1. 1

Department of Surgery, Helsinki University Central Hospital, Finland Department of Bacteriology and Immunology, University of Helsinki, The Hartmann Institute, Helsinki, Finland Introduction: Patients with chronic pancreatitis with local inflammation have high risk for pancreatic cancer. The aim of this study is to examine the role of the inflammatory cells in the invasion of pancreatic cancer cells, focusing on the involvement of a disintegrin and metalloproteinase 8 (ADAM8) and matrix metalloproteinase 9 (MMP9) proteins. Invasion is known to be associated with MMP9 expression and on the other hand ADAM8 expression is associated with worse survival of pancreatic cancer patients. Methods: Monocytes from healthy donors were differentiated into macrophages by GM-CSF incubation. Pancreatic adenocarcinoma cells (PANC-1) were cultured either alone or with differentiated macrophages in Matrigel. The cancer cell migration rate in Matrigel was measured by imaging fluorescently stained cells for 24h. After invasion, cells were sorted in CD14 positive/negative macrophages and the cancer cells with magnetic separation. The expression of ADAM8 and MMP9 were measured by the real-time PCR. In two series, siRNA technique was used to reduce either ADAM8 or MMP9 expression in the cancer cells. Results: The co-culture with macrophages increased cancer cell migration rate in Matrigel and, increased ADAM8 and MMP9 mRNA expression in the cancer cells. Reduction of ADAM8 expression with siRNA in the cancer cells decreased macrophage induced migration rate of the cancer cells from 11.7+/-0.3 mm/h to 9.0+/-0.2 mm/h (p<0.01), and reduction of MMP9 expression, decreased the migration rate to 10.1+/- 0.2 mm/h (p<0.01). Conclusion: Anti-inflammatory macrophages increase pancreatic cancer cell migration rate in basement membrane matrix by inducing ADAM8 and MMP9 expression in cancer cells, thereby possibly enhancing the invasiveness of cancer. 2

P217. Pan-ErbB inhibitor CI 1033 on pancreatic cancer therapy P. Seshacharyulu 1, M.P. Ponnusamy 1, I. Lakshmanan 1, S.K. Batra 1. 1

Department of Biochemistry and Molecular Biology, University of Nebraska Medical Center, Omaha, NE, USA Aim: To evaluate the role of EGFR family member specific inhibitor CI 1033 in inhibition of MUC4 mediated therapeutic resistance in PC cells. Background: Pancreatic cancer (PC) is the fourth leading cause of cancer related deaths in the US. HER1/EGFR and HER2 are overexpressed in PC and are associated with poor prognosis. Studies on EGFR family members and inhibitors have shown that these inhibitors can effect PC progression. Our studies have shown that HER2/ErbB2, an EGFR family member is stabilized by MUC4, a transmembrane mucin, which also affects its downstream signaling. Further, we elucidated the role of MUC4 in imparting therapeutic resistance in PC against Gemcitabine. CI 1033 is a universal inhibitor of all the EGFR family members.

Abstracts / Pancreatology 13 (2013) e1–e94

Method: The CD18/HPAF and Capan-1 PC cells were treated with CI 1033. Western blot analysis was performed to investigate the effect of CI 1033 on the activation and expression of EGFR family members and their downstream signaling targets. Co-immunolocalisation was also performed to analyze the correlation between the expression of EGFR family proteins and MUC4 on treatment with CI 1033. Real time PCR and immunoblotting was performed to analyze the effect of CI 1033 on MUC4 expression. Results: We observed decreased phosphorylation status of EGFR and HER-2 along with loss of total EGFR, HER2, HER3 and MUC4 levels on CI 1033 treatment. Similarly, the EGFR downstream signaling targets, such as pERK and pAKT and cyclin D1 and cyclin A also decreased on CI 1033 treatment. Additionally, treatment with CI 1033 led to significant induction of apoptosis, decreased proliferation and migration. Finally, mechanisms for MUC4 down regulation were confirmed by following expression of transcription factor Stat1. Conclusion: Our results demonstrate that CI 1033 is an attractive drug to inhibit the growth of human PC cells as it directly and indirectly affects EGFR family member downstream signaling and MUC4 downregulation, respectively.

P218. The impact of diabetes on acute pancreatitis M. Shafiei 1, B. Depczynski 2, H.D. Russell 1, M.V. Apte 3, J.S. Wilson 4, J. Descallar 5.

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Background: Severe acute pancreatitis (AP) and critical illness (CI) is associated with multiple organ dysfunction syndrome (MODS), most often including cardiac and pulmonary dysfunction. The ‘gut-lymph hypothesis’ states that the release of toxic factors from the intestine into mesenteric lymph (ML) contributes to the development of MODS. The aim of this study was to determine the effect of AP conditioned ML on cardiac function and whether external drainage of ML would protect the heart. Methods: The first experiment used 3 groups of 8 Wistar rats: normal, taurocholate induced AP with or without thoracic duct (TD) ligation and external drainage of ML. After 6hrs the hearts were removed for ex vivo functional measurements, including cardiac output, ventricular contractility (+dP/dt), and relaxation (-dP/dt). In a second experiment ML from normal rats and those with established taurocholate AP was infused into ex vivo perfused working normal rat hearts to assess cardiac function. Results: Cardiac dysfunction was found in rats with established AP and no lymphatic intervention compared with the control group (p<0.05). Strikingly this dysfunction did not occur in rats with TD ligation and external drainage of ML. In the second experiment infusion of AP conditioned ML resulted in an immediate, significant and similar reduction of cardiac output (p<0.05), cardiac contractility (p<0.05) and relaxation (p<0.05) compared with the infusion of normal lymph. Conclusions: AP conditioned ML causes significant cardiac dysfunction which can be prevented by TD ligation and external drainage of ML. This cardio-depressant effect of ML is likely to be important in other CI's. Further research is required to study the impact of conditioned ML on the function of other vital organs in MODS.

1

Endocrinology, Liverpool Hospital, Liverpool, NSW, Australia Endocrinology, Prince of Wales Hospital, Randwick, NSW, Australia 3 School of Medical Sciences, University of New South Wales, Randwick, NSW, Australia 4 Gastroenterology, Liverpool Hospital, Liverpool, NSW, Australia 5 Ingham Institute for Applied Medical Research, Liverpool, NSW, Australia Diabetes mellitus (DM) may be associated with an increased risk of acute pancreatitis (AP). It is unknown if the underlying aetiology of AP differs between those with and those free of DM. The aim of this study was to determine whether there is any difference in the cause, severity of AP, length of stay, or the occurrence of local versus systemic complications between those with DM versus those free of DM, admitted with AP. This was a cross-sectional study based on review of medical record of adult patients admitted to a single hospital with acute pancreatitis, 2008 - 2009. There were 252 admissions for AP during the study period. Diabetes was a co-morbidity in 58 of 252 presentations. Mean duration of diabetes was 9 years (range 0-28 years). The cause of AP did not differ between those with DM or free of DM. The in-hospital mortality rate was 6.9% for those with diabetes, and 3.1% for those free of diabetes. The length of stay was not different in those with diabetes as compared to those free of diabetes (11.3+/-11.5 days as compared to 9.2+/-12.2 days). Mean IMRE score was higher in those with DM, even if glucose was excluded from the score but there was no difference in the mean CT severity score. The occurrence of systemic inflammatory response syndrome, sepsis or septic shock (SIRS) was more frequent in those with DM (but there was no significant difference in HbA1c between patients with SIRS and patients without SIRS); and in smokers. Our series demonstrates that presence of DM is associated with increased risk of SIRS in patients admitted with AP. 2

P219. Acute pancreatitis conditioned mesenteric lymph causes preventable cardiac dysfunction S.T. Shanbhag 1, B. Choong 2, R. Premkumar 2, L. Phang 2, M. Petrov 1, J.A. Windsor 1, 2, A.J. Phillips 1, 2. 1 Pancreas Research Group, Department of Surgery, Auckland, New Zealand 2 Applied Surgery and Metabolism Laboratory, School of Biological Sciences, University of Auckland, Auckland, New Zealand

P220. The pancreatic tumor microenvironment comprises discreet domains and is regulated by crosstalk between PSCs and tissue macrophages C. Shi 1, R. Chaturvedi 2, F. Revetta 1, K.T. Wilson 2, M.K. Washington 1, A.L. Means 3, 4. 1

Department of Pathology, Vanderbilt University, Nashville, TN, USA Department of Medicine, Vanderbilt University, Nashville, TN, USA 3 Department of Surgery, Vanderbilt University, Nashville, TN, USA 4 Department of Cell and Developmental Biology, Vanderbilt University, Nashville, TN, USA We explored previously unrecognized heterogeneity in the human pancreatic cancer tumor microenvironment (TME), extensively cataloguing spatial relationships between tumor epithelium and stromal cells, and also explored functional interactions among stromal cells. We found that the pancreatic cancer TME comprises distinct patterns surrounding benign and malignant lesions which differ from the patterns observed in chronic pancreatitis. Early PanINs are enclosed in a thin layer of myofibroblasts immediately juxtaposed to the epithelium. This layer thickens in advanced PanINs, culminating in extensive, less coordinated pools of myofibroblasts around invasive lesions. Thick capsules of well-organized collagen largely devoid of periostin surround PanIN lesions, suggesting that this matrix undergoes little remodeling. However, invasive lesions showed less organized collagen and high amounts of periostin suggesting that active remodeling was occurring. Blood vessels immediately adjacent to invasive lesions were not reduced. Rather, areas farther removed from the tumor epithelium displaying well-organized matrix and aligned myofibroblasts provided a barrier to blood vessels and leukocytes. We developed a novel in vitro co-culture system to understand how pancreatic fibrosis is regulated. Pancreatic stellate cells (PSCs), which are progenitors of myofibroblasts, produced chemokines that attracted macrophages, and macrophages produced cytokines capable of activating PSCs. Co-culture dramatically upregulated many of these cytokines, suggesting a feed-forward loop between PSCs and macrophages for accelerating and maintaining the fibrotic microenvironment. In summary, the pancreatic tumor microenvironment is a complex mix with discreet cell and matrix components that is regulated by interactions between inflammatory and fibrotic cell types. 2