Transient High Pressure in Pancreatic Ducts Promotes Inflammation and Alters Tight Junctions via Calcineurin Signaling in Mice.

BACKGROUND & AIMS: Pancreatitis after endoscopic retrograde cholangiopancreatography (PEP) is thought to be provoked by pancreatic ductal hypertension, via unknown mechanisms. We investigated the effects of hydrostatic pressures on the development of pancreatitis in mice. METHODS: We performed studies with Swiss Webster mice, B6129 mice (controls), and B6129 mice with disruption of the protein phosphatase 3, catalytic subunit, ßisoform gene (Cnab-/- mice). Acute pancreatitis was induced in mice by retrograde biliopancreatic ductal or intraductal infusion of saline with a constant hydrostatic pressure while the proximal common bile duct was clamped -these mice were used as a model of PEP. Some mice were given pancreatic infusions of adeno-associated virus 6-nuclear factor of activated T-cells-luciferase to monitor calcineurin activity or the calcineurin inhibitor FK506. Blood samples and pancreas were collected at 6 and 24 hours and analyzed by enzyme-linked immunosorbent assay, histology, immunohistochemistry, or fluorescence microscopy. Ca2+ signaling and mitochondrial permeability were measured in pancreatic acinar cells isolated 15 minutes after PEP induction. Ca2+-activated phosphatase calcineurin within the pancreas was tracked in vivo over 24 hours. RESULTS: Intraductal pressures of up to 130 mm Hg were observed in the previously reported model of PEP; we found that application of hydrostatic pressures of 100 and 150 mm Hg for 10 minutes consistently induced pancreatitis. Pancreatic tissues had markers of inflammation (increased levels of interleukin [IL] 6, IL1B, and tumor necrosis factor), activation of signal transducer and activator of transcription 3, increased serum amylase and IL6, and loss of tight junction integrity. Transiently high pressures dysregulated Ca2+ processing (reduced Ca2+ oscillations and an increased peak plateau Ca2+ signal) and reduced the mitochondrial membrane potential. We observed activation of pancreatic calcineurin in the pancreas in mice. Cnab-/- mice, which lack the catalytic subunit of calcineurin, and mice given FK506 did not develop pressure-induced pancreatic inflammation, edema, or loss of tight junction integrity. CONCLUSIONS: Transient high ductal pressure produces pancreatic inflammation and loss of tight junction integrity in a mouse model of PEP. These processes require calcineurin signaling. Calcineurin inhibitors might be used to prevent acute pancreatitis that results from obstruction.

Prognostic Value of Excision Repair Cross-Complementing Gene 1, Dihydropyrimidine Dehydrogenase, and Human Equilibrative Nucleotide Transporter 1 Expression and Their Implications for Adjuvant Treatment in Patients With Ampullary Carcinoma.

OBJECTIVES: The purpose of this study was to characterize the intratumoral expression profiles of excision repair cross-complementing gene 1 (ERCC1), dihydropyrimidine dehydrogenase (DPD), and human equilibrative nucleotide transporter 1 (hENT1) in ampullary carcinomas (ACs) to evaluate their prognostic values and better tailor adjuvant chemotherapy for individual patients with AC after surgery. METHODS: This study included 49 patients with AC who underwent a curative pancreaticoduodenectomy. Various clinicopathological factors, including ERCC1, DPD, and hENT1, were analyzed in relation to postoperative disease recurrence and the patients' survival. RESULTS: The median recurrence-free survival and overall survival were 24.5 months and 32.4 months, respectively. Multivariate Cox regression analysis of recurrence-free survival identified a DPD expression (hazard ratio [HR], 8.18; 95% confidence interval [CI], 2.00-34.8; P = 0.003) and combined ERCC1/DPD expression (HR, 134.8; 95% CI, 11.8-1920; P < 0.001) as independent predictors of disease recurrence. Multivariate Cox regression analysis of overall survival also identified a DPD expression (HR, 8.48; 95% CI, 1.71-46.3; P = 0.008) and combined ERCC1/ DPD expression (HR, 135.6; 95% CI, 11.8-1940; P < 0.001) as independent predictors of survival. CONCLUSIONS: The DPD and ERCC1 expression profile could potentially serve as a useful prognostic biomarker and therapeutic target for surgically resected patients with AC.

Mutational profiling of kinases in human tumours of pancreatic origin identifies candidate cancer genes in ductal and ampulla of vater carcinomas.

BACKGROUND: Protein kinases are key regulators of cellular processes (such as proliferation, apoptosis and invasion) that are often deregulated in human cancers. Accordingly, kinase genes have been the first to be systematically analyzed in human tumors leading to the discovery that many oncogenes correspond to mutated kinases. In most cases the genetic alterations translate in constitutively active kinase proteins, which are amenable of therapeutic targeting. Tumours of the pancreas are aggressive neoplasms for which no effective therapeutic strategy is currently available. METHODOLOGY/PRINCIPAL FINDINGS: We conducted a DNA-sequence analysis of a selected set of 35 kinase genes in a panel of 52 pancreatic exocrine neoplasms, including 36 pancreatic ductal adenocarcinoma, and 16 ampulla of Vater cancer. Among other changes we found somatic mutations in ATM, EGFR, EPHA3, EPHB2, and KIT, none of which was previously described in cancers. CONCLUSIONS/SIGNIFICANCE: Although the alterations identified require further experimental evaluation, the localization within defined protein domains indicates functional relevance for most of them. Some of the mutated genes, including the tyrosine kinases EPHA3 and EPHB2, are clearly amenable to pharmacological intervention and could represent novel therapeutic targets for these incurable cancers.

The expression of matrix metalloproteinases in intrahepatic cholangiocarcinoma, hilar (Klatskin tumor), middle and distal extrahepatic cholangiocarcinoma, gallbladder cancer, and ampullary carcinoma: role of matrix metalloproteinases in tumor progression and prognosis.

BACKGROUND/AIMS: Carcinomas of the biliary tree are rare tumors of the gastrointestinal tract, with an increasing incidence in recent years. Biliary neoplasms are classified into intra- and extrahepatic cholangiocarcinoma (Klatskin tumor, middle and distal extrahepatic tumors), gallbladder adenocarcinoma, and ampullary carcinoma. We aimed to determine the expression profile of matrix metalloproteinase (MMP)-2, MMP-9 and MMP-14 in the biliary neoplasms classified according to their localization and the relation with the prognosis. METHODS: Ten gallbladder adenocarcinoma, 8 distal bile duct carcinomas (distal cholangiocarcinoma), 8 Klatskin tumors, 8 intrahepatic cholangiocarcinomas and 10 ampullary carcinomas were included in the study. The immunohistochemical expression of MMP-2, MMP-9 and MMP-14 was detected in the nontumoral, metaplastic, dysplastic and tumoral epithelia. The tumor differentiation, angiolymphatic and perineural invasion of the tumor, and presence of lymph node and distant metastasis were determined. Survey of the patients was noted from the patient follow-up data. RESULTS: The nontumoral epithelia of the gallbladder, intrahepatic ducts, and Klatskin tumor did not express MMP-2. MMP-2 expression was detected in the distal part of the biliary ducts, in 75% (6/18) of cases and in the nontumoral epithelia of the ampullary region in 50% (5/10) of cases. The metaplastic and dysplastic epithelia were positively stained in all of the gallbladder adenocarcinoma, distal cholangiocarcinoma and ampullary tumors. In the intrahepatic cholangiocarcinoma, the hepatocytes were positively stained but the infiltrative tumors were spared. Klatskin tumors were also not stained with MMP-2. The gallbladder adenocarcinoma, distal cholangiocarcinoma and ampullary carcinomas expressed MMP-2 in 30%, 37% and 40% of the cases, respectively. MMP-9 and MMP-14 were expressed in normal, metaplastic, and dysplastic epithelium and tumoral cells in all of the cases of the groups. Expressions of MMPs were higher in subjects with neural invasion, but there was no correlation between MMP expression and tumor differentiation or angiolymphatic invasion. CONCLUSIONS: When tumors of the biliary system are divided as intrahepatic and extrahepatic cholangiocarcinomas, MMP-2 expression was present in the extrahepatic cholangiocarcinomas including gallbladder carcinomas. Like the intrahepatic cholangiocarcinoma, Klatskin tumors also did not express MMP-2. This can be related with its characteristic growth pattern. MMP-9 and MMP- 14 were present in metaplasia, dysplasia carcinoma sequence in all of the bile tract tumors, suggesting that MMPs play an important role in carcinogenesis. The higher expression of the MMPs with neural invasion suggests the significant role of those tumors in the invasion activity.

COX-2 expression of ampullary carcinoma: correlation with different histotypes and clinicopathological parameters.

Epidemiological studies suggest that regular intake of nonsteroidal anti-inflammatory drugs (NSAIDs) are associated with reduced incidence of gastrointestinal cancer. Several lines of evidence indicate that the antineoplastic effect of NSAIDs is attributable to COX-2 inhibition. The aim of our study was to assess COX-2 expression in a series of primary untreated ampullary carcinomas and its possible correlation with clinicopathological parameters. In the present study, 45 surgical specimens of invasive ampullary carcinomas were histologically classified into pancreaticobiliary, intestinal, and unusual types. COX-2 expression by immunohistochemical method was analyzed. High COX-2 expression was detected in 35 (77.8%) ampullary carcinomas. Among these, 20/21 (95.2%) were classified as intestinal, 9/18 (50%) pancreaticobiliary, and 6/6 (100%) unusual type. A significant statistical difference in terms of COX-2 expression was found between pancreaticobiliary vs intestinal type (P=0.002). Furthermore, a negative significant statistical correlation was found between T factor and COX-2 expression (P=0.047). The different COX-2 expression among histopathological types supports the concept of histogenetical difference of ampullary carcinomas. Furthermore, the high rate of COX-2 expression in the intestinal subtype of ampullary carcinoma may represent the rational for a histotype-tailored therapy targeting COX-2.

Expression of cyclooxygenase-2 and its correlation with clinicopathologic factors of ampulla of vater cancer.

There has been no report for the expression of cyclooxygenase-2 (COX-2) and its clinicopathologic and biologic significance in ampulla of Vater cancer. This study was aimed for the clarification of COX-2 expression and its biologic roles in ampulla of Vater cancer. Fourty-six patients with ampulla of Vater cancer were enrolled and their COX-2 expression and clinicopathologic features were analyzed. The median age of patients was 60 yr and the mean duration of follow-up was 35 months (range: 14-82 months). Immunohistochemical stainings for COX-2, Ki-67, CD34 and TUNEL staining were performed. The immunoreactive COX-2 expression was present in 24 (52.2%) patients of ampulla of Vater cancer and mainly localized in cytosolic and perinuclear region. There was no significant difference in the length of survival between COX-2 postive and negative group (p=0.9420 by Log Rank test). Also, there were no significant differences of proliferation index (p=0.326), apoptotic index (p=0.764) and microvessel density (p=0.135) between COX-2 positive and negative group. Initial pTNM stage (p=0.0028 by Log Rank test) and blood transfusion over 4 pints during operation (p=0.0254 by Log Rank test) were independent prognostic factor in patients with ampulla of Vater cancer. It is suggested that immunoreactivity of COX-2 is not correlated with clinicopathologic and biologic features of ampulla of Vater cancer.

Expression of Cyclooxygenase-2 and Its Correlation with Clinicopathologic Factors of Ampulla of Vater Cancer

There has been no report for the expression of cyclooxygenase-2 (COX-2) and its clinicopathologic and biologic significance in ampulla of Vater cancer. This study was aimed for the clarification of COX-2 expression and its biologic roles in ampulla of Vater cancer. Fourty-six patients with ampulla of Vater cancer were enrolled and their COX-2 expression and clinicopathologic features were analyzed. The median age of patients was 60 yr and the mean duration of follow-up was 35 months (range: 14-82 months). Immunohistochemical stainings for COX-2, Ki-67, CD34 and TUNEL staining were performed. The immunoreactive COX-2 expression was present in 24 (52.2%) patients of ampulla of Vater cancer and mainly localized in cytosolic and perinuclear region. There was no significant difference in the length of survival between COX-2 postive and negative group (p=0.9420 by Log Rank test). Also, there were no significant differences of proliferation index (p=0.326), apoptotic index (p=0.764) and microvessel density (p=0.135) between COX-2 positive and negative group. Initial pTNM stage (p=0.0028 by Log Rank test) and blood transfusion over 4 pints during operation (p=0.0254 by Log Rank test) were independent prognostic factor in patients with ampulla of Vater cancer. It is suggested that immunoreactivity of COX-2 is not correlated with clinicopathologic and biologic features of ampulla of Vater cancer.

Expression of thymidylate synthase in cancer of the ampulla of Vater.

The clinical and therapeutic significance of thymidylate synthase (TS) in cancers of the ampulla of Vater have not yet been reported. We immunohistochemically evaluated TS expression in 33 ampullary cancers using an anti-TS antibody. TS expression, clinicopathologic variables, and survival rates were examined and the correlations between these parameters were identified. Fifteen patients were found to express high levels of TS (high TS group), while 18 patients expressed low levels of TS (low TS group). No significant difference was found between TS expression and clinicopathologic factors. Univariate and multivariate analysis revealed that lymph node metastasis and pancreatic invasion are important variables for independently predicting post-operative survival. Although TS expression was not identified as an important factor for postoperative survival, recurrent cases in patients with chemotherapy existed only in the high TS group. In the present study, it was found that TS expression itself in cancers of the ampulla of Vater has no impact in predicting the prognosis of ampullary cancers, but a chemotherapeutic benefit of evaluating TS expression may exist.

Long-term prophylactic administration of octreotide reduces the rise in serum amylase after endoscopic procedures on Vater's papilla.

The pancreas commonly reacts to endoscopic papillosphincterotomy (EST) with a rise in serum amylase, and acute pancreatitis may also develop. The long-acting somatostatin analogue octreotide has recently been proposed for prevention of colangiopancreatography (ERCP)/EST-induced pancreatic reaction. Therefore, we tested the prophylactic effects of a subcutaneous 3-day administration of octreotide to 60 consecutive patients undergoing ERCP and EST. They were randomly allocated to receive either 200 micrograms octreotide t.i.d. for 3 days (30 cases) or placebo (control group, 30 cases) before the procedure. On the day of the examination, serum amylase levels were determined at baseline and 2, 4, 8, and 24 h thereafter. In the patients as a whole, the increases were statistically significant at 4 h (p < 0.01) and 8 h (p < 0.01). Epigastric pain occurred in 2 patients in the octreotide group and in 13 control subjects (p < 0.001). Even in some patients who had had previous episodes of relapsing pancreatitis, the rise in serum amylase was significantly lower in the octreotide group than in the control group at 4 h (p < 0.01), 8 h (p = 0.05), and 24 h (p = 0.05). Our data suggest that 3 days of prophylactic treatment with octreotide is effective for reducing the rise in serum amylase after EST/ERCP and could be proposed for patients with relapsing pancreatitis and other risk conditions before the Vater's papilla manipulation.