Targeted next-generation sequencing identifies distinct clinicopathologic and molecular entities of intraductal papillary neoplasms of the bile duct.

Intraductal papillary neoplasm of the bile duct (IPNB) is a mass-forming neoplasm in the bile duct considered to be the biliary counterpart of pancreatic intraductal papillary mucinous neoplasm (IPMN). By its cell lineage, IPNB can be classified into gastric, intestinal, pancreatobiliary, and oncocytic types. Recently, a group of Japanese and Korean pathologists proposed that IPNB be classified into two types, with type 1, being the histological counterpart of IPMN and type 2, having a more complex histological architecture. We used targeted next-generation sequencing to study the molecular change of 37 IPNBs and identified frequent mutations of KRAS (49%), GNAS (32%), RNF43 (24%), APC (24%), TP53 (24%), and CTNNB1 (11%) in IPNBs. Intestinal-type IPNB was associated with KRAS, GNAS, and RNF43 mutations. Japan-Korea consensus type 1 was associated with KRAS and GNAS mutations. All four IPNBs with CTNNB1 mutations were of pancreatobiliary type and located in the extrahepatic bile duct. A hierarchical analysis identified three distinct groups within IPNB: group 1 was Japan-Korea consensus type 1 tumors with macroscopic mucin, old age, and frequent KRAS, GNAS, and RNF43 mutations. Group 2 was Japan-Korea consensus type 2 with intestinal differentiation and frequent KRAS mutation but rare GNAS mutation, MUC2 expression, and macroscopic mucin. Group 3 was characterized by CTNNB1 mutation, extrahepatic location, lack of expression of intestinal markers, Japan-Korea consensus type 2, and lack of mutations in KRAS, APC, RNF43, and GNAS. Our results indicated that IPNB is a heterogeneous disease and that the activation of Ras-mitogen-activated protein kinase (MAPK), Wnt/ß-catenin, and G-protein-coupled receptor (GPCR)-cAMP signaling is the main oncogenic mechanism of IPNB.

Deletion of cadherin-17 enhances intestinal permeability and susceptibility to intestinal tumour formation.

Cadherin-17 is an adhesion molecule expressed specifically in intestinal epithelial cells. It is frequently underexpressed in human colorectal cancer. The physiological function of cadherin-17 and its role in tumourigenesis have not yet been determined. We used the transcription activator-like effector nuclease technique to generate a Cdh17 knockout (KO) mouse model. Intestinal tissues were analysed with histological, immunohistochemical and ultrastructural methods. Colitis was induced by oral administration of dextran sulphate sodium (DSS), and, to study effects on intestinal tumourigenesis, mice were given azoxymethane (AOM) and DSS to induce colitis-associated cancer. Cdh17 KO mice were viable and fertile. The histology of their small and large intestines was similar to that of wild-type mice. The junctional architecture of the intestinal epithelium was preserved. The loss of cadherin-17 resulted in increased permeability and susceptibility to DSS-induced colitis. The AOM/DSS model demonstrated that Cdh17 KO enhanced tumour formation and progression in the intestine. Increased nuclear translocation of Yap1, but not of ß-catenin, was identified in the tumours of Cdh17 KO mice. In conclusion, cadherin-17 plays a crucial role in intestinal homeostasis by limiting the permeability of the intestinal epithelium. Cadherin-17 is also a tumour suppressor for intestinal epithelia. Copyright © 2018 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.

Microinsertions in PRKACA cause activation of the protein kinase A pathway in cardiac myxoma.

Cardiac myxoma is the most common cardiac tumour. Most lesions occur sporadically, but occasional lesions develop in patients with Carney complex, a syndrome characterized by cardiac myxoma, spotty pigmentation, and endocrine overactivity. Two-thirds of patients with Carney complex harbour germline mutations in PRKAR1A, which encodes the type I regulatory subunit of protein kinase A (PKA). Most studies have not found a mutation in PRKAR1A in sporadic cardiac myxoma cases. Recent studies identified frequent mutations in PRKACA, which encodes the catalytic subunit of PKA, in cortisol-secreting adrenocortical adenoma cases. To determine whether the PRKACA mutation is involved in the tumourigenesis of cardiac myxoma, we performed Sanger sequencing of 41 specimens of sporadic cardiac myxoma to test for the presence of mutations in the coding regions and intron-exon boundaries of PRKACA. Mutations were identified in four cases (9.7%). In contrast to the point mutations identified in adrenocortical adenoma, all mutations were in-frame microinsertions of 18-33 bp clustered in exons 7 and 8. The mutated PRKACA proteins lost their ability to bind to PRKAR1A, and thereby lead to constitutive activation of the PKA pathway. Together with previous reports of PRKAR1A mutations in syndromic cardiac myxoma, our study demonstrates the importance of the PKA pathway in the tumourigenesis of cardiac myxoma. Copyright © 2017 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.

Complementary roles of ß-catenin and glutamine synthetase immunostaining in diagnosis of chemotherapy-treated and untreated hepatoblastoma.

BACKGROUND/PURPOSE: This study aimed to evaluate the expression of ß-catenin and its downstream target glutamine synthetase (GS) in hepatoblastoma (HB), and to evaluate the use of these two markers for diagnosing HB. METHODS: Eighteen untreated HBs and 22 HBs resected after neoadjuvant chemotherapy were analyzed using ß-catenin and GS immunostaining. RESULTS: We detected nuclear ß-catenin immunostaining in nearly all untreated HBs, including in fetal and embryonal epithelial components and in mesenchymal elements. We also observed diffuse GS expression in the epithelial component; however, it was frequently absent in embryonal and mesenchymal areas. In HBs resected after neoadjuvant chemotherapy, we recognized four histological patterns: fetal, hepatocellular-carcinoma-like, clear-cell, and normal-liver-like. All these patterns displayed diffuse GS expression. Fetal pattern showed diffuse nuclear ß-catenin immunostaining. Nuclear ß-catenin immunostaining was weak in the hepatocellular-carcinoma-like and clear-cell patterns. In normal-liver-like area, ß-catenin expression was only located in the cell membrane. CONCLUSION: The results suggest that nuclear ß-catenin expression and diffuse GS immunostaining are the hallmarks of HB. Although epithelial and mesenchymal components of HB display nuclear ß-catenin staining, this expression is attenuated following chemotherapy-induced cell maturation. GS immunostaining is especially useful for the assessment of section margins after neoadjuvant chemotherapy.

Tumor size is a major determinant of prognosis of resected stage I hepatocellular carcinoma.

PURPOSE: The current American Joint Committee on Cancer (AJCC) staging system for hepatocellular carcinoma (HCC) includes all solitary tumors without vascular invasion as stage I, regardless of tumor size. The aim of this study is to determine the prognostic significance of tumor size in stage I HCC patients. METHODS: A total of 230 stage I primary HCCs were selected retrospectively. Based on univariate and multivariate analyses, clinical and pathological factors correlated with 5-year disease-free survival (DFS) and 5-year overall survival (OS) were determined. RESULTS: Univariate and multivariate analyses showed significant correlations of low serum α-fetoprotein levels (≤20 ng/ml), small tumor size (≤3 cm), wide resection margin (≥ 1 cm), and absence of cirrhotic liver with better DFS, while smaller tumor size, and wide resection margin with better OS. Of all the parameters, tumor size is the most statistically significant markers for DFS and OS. Interestingly, liver cirrhosis exerted prognostic significance in patients with small-size tumors, while resection margin exerted prognostic significance in patients with large-size tumors. CONCLUSIONS: Our results indicated that tumor size is the most important determinant of DFS and OS in resected primary stage I HCC patients. Therefore, we advocate redefining solitary tumors of ≤3 cm as T1a disease and tumors >3 cm as T1b disease. This stratification of stage I HCC patients could aid in the determination of prognosis and the development of superior protocols for patient management. However, further analysis of big registry cohorts is needed to establish a common consensus.

The roles of dopamine receptor and adrenoreceptor on the inhibition of gastric emptying and intestinal transit by amphetamine in male rats.

Amphetamine, a central nervous stimulant, acts on the central nervous system (CNS) by increasing levels of norepinephrine, serotonin and dopamine (DA) in the brain. It stimulates the colonic motility and gastrointestinal (GI) function via the brain-gut connections as well as diminishes appetite to control bodyweight, but the mechanism between amphetamine and GI motility was unclear. We investigated the role of DA receptor and adrenoreceptor in the inhibitory effects of amphetamine on GI motility in male rats. After fasting overnight, the male rats received an intraperitoneal (i.p.) injection of amphetamine (3 mg/ml/kg) 15 min after i.p. injection of different antagonists for dopamine receptors and adrenoreceptors. Gastric emptying and intestinal transit were assessed 15 min after intragastric instillation of a test meal containing radioactive Na25¹CrO4 and 10% charcoal. Haloperidol, a DA receptor antagonist, acting exactly as the selective D2 receptor antagonist, eticlopride, completely prevented amphetamineinduced inhibition of gastric emptying and intestinal transit in male rats. The selective D1 receptor antagonist, SCH 23390, significantly attenuated the amphetamine-induced inhibition of gastric emptying and intestinal transit in male rats. Both selective and nonselective α adrenoreceptor antagonist, phentolamine, prazosin and yohimbine, did not reverse the inhibitory effects of amphetamine on gastric emptying and intestinal transit. Propanolol, a ß adrenoreceptor antagonist, partially attenuated the amphetamine-induced inhibition of gastric emptying but did not significantly attenuate intestinal transit. These results suggest that amphetamine-induced inhibition of gastric emptying and intestinal transit is mediated by an action on dopaminergic mechanism and adrenoreceptor with little involvement. These findings may clarify the influence of central nervous stimulant on GI tract and may provide the appropriate medication in CNS.

A radioimmunoassay for rat ghrelin: evaluation of method and effects of nonylphenol on ghrelin secretion in force-fed young rats.

Antiserum YJC 13-31 against the rat ghrelin conjugated to bovine serum albumin (BSA) was produced in the rabbit and a double antibody radioimmunoassay (RIA) for ghrelin has been developed. Characterization results of this antiserum revealed no cross-reaction with human growth hormone and somatostatin. Weak cross-reactions with insulin (0.1%), rat growth hormone (0.1%) and glucagon (0.3%) were observed, which scarcely interfered the assay system. The sensitivity of this RIA was 5 pg per assay tube. With the rat serum samples, the within-assay precision was 7.1% and the between-assay precision was 12.3%. The RIA was also available to detect the ghrelin in rat tissue extracts with good parallelism to the rat ghrelin standard. In application, the serum ghrelin and corticosterone levels in weaned rats were measured by RIA. Gavage of saline was sufficient to raise serum ghrelin from 2.6 +/- 0.18 to 6.7 +/- 0.7 ng/ml (P < 0.01). Gavage with nonylphenol (NP) suppressed the elevation of serum ghrelin levels in a dose-dependent manner. Besides, gavages of saline elevated the serum levels of corticosterone from 108.8 +/- 13.5 to 188.7 +/- 23.5 ng/ml (P < 0.01) but the elevation effects of corticosterone from gavages were overcome by NP in the low dose of 50 mg/kg. It can be speculated that ingestion of NP is harmful to young animals during growth and environmental adaptation.

Propofol inhibits endogenous formyl peptide-induced neutrophil activation and alleviates lung injury.

Critically ill patients have a high risk of sepsis. Various studies have demonstrated that propofol has anti-inflammatory effects that may benefit critically ill patients who require anesthesia. However, the mechanism and therapeutic effect remain incompletely understood. Our previous data suggest that propofol can act as a formyl peptide receptor 1 (FPR1) antagonist. Here, we hypothesize that propofol mitigates sepsis-induced acute lung injury (ALI) by inhibiting mitochondria-derived N-formyl peptide-mediated neutrophil activation. Oxidative stress caused by activated neutrophils is involved in the pathogenesis of ALI. In human neutrophils, propofol competitively reduced the release of superoxide and associated reactive oxygen species induced by fMMYALF, a human mitochondria-derived N-formyl peptide, suggesting that propofol effectively suppresses neutrophilic oxidative stress. In addition, propofol significantly inhibited fMMYALF-induced elastase release, chemotaxis, calcium mobilization, and phosphorylation of protein kinase B and mitogen-activated protein kinases. These results indicate that propofol suppresses neutrophil activation by blocking the interaction between endogenous N-formyl peptide and its receptor, FPR1, thus inhibiting downstream signaling. Furthermore, propofol alleviated alveolar wall disruption, edematous changes, and neutrophil infiltration in lipopolysaccharide-induced ALI in mice. Noticeably, propofol improved the survival of sepsis mice. This study indicates that the anti-neutrophil effects of propofol may benefit critically ill septic patients.

Expression of hypoxic marker carbonic anhydrase IX predicts poor prognosis in resectable hepatocellular carcinoma.

Carbonic anhydrase IX (CA-IX), a hypoxia marker, correlates with tumor progression in a variety of human cancers. However, the role of CA-IX in hepatocellular carcinomas (HCCs) remains largely unknown. We examined the expression of 277 unifocal, resectable, primary HCC tumors using immunohistochemistry. The CA-IX protein was expressed in 110 of the 227 (48.5%) HCC tumors. The expression of CA-IX correlated with younger age (P = 0.0446), female sex (P = 0.0049), high serum α-fetoprotein levels (P<1x10-6), larger tumor size (P = 0.0031), high tumor grade P<1x10-6) and high tumor stage (P = 1.5x10-6). Patients with HCC tumors that expressed CA-IX were more likely to have lower 5-year disease-free survival (DFS; P = 0.0001) and 5-year overall survival (OS; P<1x10-6). The multivariate analysis indicated that CA-IX expression was an independent predictor for high tumor stage (P = 0.0047) and DFS (P = 0.0456), and a borderline predictor for OS (P = 0.0762). Furthermore, CA-IX expression predicted poor DFS and OS in patients with high tumor stage (P = 0.0004 and P<1x10-6, respectively). Interestingly, CA-IX expression might contribute to the worse prognosis of female patients with advanced HCCs. Our study indicates the expression of the CA-IX protein is a crucial predictor of poor prognosis in resectable HCC, and it is also an unfavorable prognostic predictor in HCC patients with high tumor stage.

Targeted next-generation sequencing of cancer genes identified frequent TP53 and ATRX mutations in leiomyosarcoma.

Leiomyosarcoma is an aggressive soft tissue sarcoma with poor patient survival. The genetic changes of leiomyosarcoma remain to be discovered. In this study, we analyzed the genetic changes of 44 cancer-related genes by using next-generation sequencing in 54 leiomyosarcomas. We identified TP53 mutations in 19 of the 54 tumors (35%) and ATRX mutations in 9 of the 54 tumors (17%). The TP53-mutated leiomyosarcomas were limited to female patients (P = 0.006). All but 2 of the TP53-mutated leiomyosarcomas were located in the uterus (n = 11) or retroperitoneum (n = 6). The ATRX mutations were associated with poorly differentiated leiomyosarcomas (P = 0.028) and the presence of tumor necrosis (P = 0.015). Kaplan-Meier survival analysis showed that patients with ATRX-mutated leiomyosarcomas had worse overall survival than did patients with ATRX-wild-type leiomyosarcomas. All of the ATRX-mutated leiomyosarcomas showed the alternative lengthening of telomere phenotype. The ATRX mutations did not correlate with ATRX protein expression, as detected using immunohistochemistry. In conclusion, we identified loss of function of the p53 and ATRX pathways being the main mechanisms for leiomyosarcomas. The molecular mechanisms may provide new opportunities to treat these aggressive neoplasms.

Effects of juice from Morinda citrifolia (Noni) on gastric emptying in male rats.

The effects of juice from Morinda citrifolia (noni) on gastric emptying, gastrointestinal transit, and plasma level of cholecystokinin (CCK) in rats were studied. Male rats were given noni by gavage at levels of 0.25, 1, or 4 ml/kg once per day for one or 7 days. The rats in the control group were given water, while the rats in the experimental group were fasted overnight before measurement of gastrointestinal motility. Gastrointestinal motility was assessed in rats 15 min after intragastric instillation of a test meal containing charcoal (10%) and Na251CrO4 (0.5 microCi/ml). Gastric emptying was determined by measuring the amount of radiolabeled chromium contained in the small intestine as a percentage of the initial amount received. Then, gastrointestinal transit was evaluated by calculating the geometric center of distribution of the radiolabeled marker. Finally, blood samples were collected for measurement of CCK by radioimmunoassay. The administration of noni at 0.25 ml/kg, but not at 1 ml/kg and 4 ml/kg, for 1 day significantly inhibited gastric emptying. In contrast, gastric emptying was significantly inhibited by oral noni (0.25, 1, or 4 ml/kg) for 7 days. Intraperitoneal injection of lorglumide (5 or 10 mg/kg), a selective CCK1 receptor antagonist, effectively attenuated the noni-induced inhibition of gastric emptying. The intestinal transit and body weight, food intake, water intake, urine volume as well as feces weight were not altered by the administration of noni either acutely or chronically, but the administration of oral noni (1 ml/kg) for 7 days increased the level of plasma CCK in male rats. These results suggest that oral noni inhibits gastric emptying in male rats via a mechanism involving stimulation of CCK secretion and CCK1 receptor activation.

Human chorionic gonadotropin regulates gastric emptying in ovariectomized rats.

Prolongation of gastrointestinal transit resulting in nausea and vomiting in pregnancy (NVP) is the most common phenomenon during the first trimester of pregnancy. Increased human chorionic gonadotropin (hCG) concentration during the first trimester is the most likely cause of NVP. The aim of this study was to investigate the effect of hCG on gastrointestinal transit and plasma concentrations of cholecystokinin (CCK) in ovariectomized (Ovx) rats. I.p. injection of hCG was used to evaluate the dose effect of hCG on gastrointestinal transit in Ovx rats. The CCK antagonist lorglumide was used to clarify the role of CCK in regulating gastrointestinal transit. Gastrointestinal transit was assessed 15 min after intragastric gavage of a mixture of 10% charcoal and Na(2)(51)CrO(4) (0.5 µCi/ml). After i.p. administration of hCG, gastric emptying was inhibited in Ovx rats, but intestinal transit was not affected. Plasma CCK concentrations were increased in a dose-dependent manner after hCG treatment, and gastric emptying showed a significant negative correlation with CCK concentrations (P=0.01, r(2)=-0.5104). Peripheral administration (i.p.) of lorglumide, a selective CCK(1) receptor antagonist, attenuated the hCG-induced inhibition of gastric emptying in Ovx rats, whereas central administration via the i.c.v. route did not. hCG treatment of Ovx rats inhibits gastric emptying in a dose-dependent manner via a peripheral mechanism of CCK hypersecretion and activation of CCK(1) receptors.