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Journal of Gastroenterology and Hepatology (2012) 27 (Suppl. 4)

Basic Science Liver The erythroid signal in iron loading haemolytic anaemias reduces hepatic hepcidin expression despite increased BMP6 levels GREGORY ANDERSON, DAVID FRAZER, SARAH J WILKINS, ALISON BADRICK, DEEPAK DARSHAN Q I M R, Iron Metabolism Laboratory, Brisbane, QLD, Australia Background The liver-derived peptide hepcidin is an essential regulator of body iron homeostasis. Its expression can be stimulated by increased body iron levels or inflammation, and decreased by low iron or hypoxia. Hepcidin expression is also reduced in response to stimulated erythropoiesis, but the mechanism underlying this response is unknown. Aims Since the BMP (particularly BMP6)/SMAD signalling pathway plays a central role in regulating hepcidin expression in response to body iron levels, we investigated whether this pathway was involved in reducing hepcidin expression using two models of iron loading chronic haemolytic anaemia. Methods The two animal models used were (i) wild-type mice treated with graded doses (twice per week for 4 weeks) of the haemolytic agent phenylhydrazine (PHZ) and (ii) beta-thalassaemic (Hbbth3/+) mice. NonPHZ-treated wild-type mice, either with or without iron loading, were used as controls. Gene expression was measured by qPCR and protein levels by western blotting. Results Increasing doses of PHZ led to a haemolytic anaemia of increasing severity and a progressive increase in both hepatic iron and Bmp6 mRNA. However, Hamp1 (which encodes hepcidin) expression declined, consistent with the increased erythropoiesis. Wild-type mice loaded to the same level in the absence of haemolysis showed increases in both Hamp1 and Bmp6 expression, confirming that in the PHZ-treated mice the erythroid signal was overcoming the influence of the increased iron load. The increased Bmp6 expression in PHZ-treated mice did not lead to increased Smad1/5/8 phosphorylation, indicating that stimulated erythropoiesis decreases Bmp6-mediated Smad signalling. The erythroid signal also reduced the capacity of pSmad to induce hepcidin, as Hamp1 levels declined despite pSmad1/5/8 levels remaining unchanged. Similar results were seen in Hbbth3/+ mice, where increased hepatic iron was associated with elevated Bmp6, but Hamp1 levels declined despite the increased Bmp6 and unaltered Smad1/5/8 phosphorylation. In these models, candidate erythroid signals GDF15 and TWSG1 were not consistently associated with Hamp1 expression, nor were changes in the level of diferric transferrin, transferrin receptor 2 protein, MAPK signalling, or Smad pathway regulators/targets Tmprss6, Id1, Atoh8 and Smad7. Conclusion In conclusion, the reduction in hepcidin expression associated with stimulated erythropoiesis cannot be explained by alterations in Smad phosphorylation and occurs despite increased Bmp6 expression. Thus the erythroid regulator appears to modulate Bmp/Smad signalling downstream of Bmp6, although it could exert some of its effects through a Smad independent pathway.

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The role of receptor-mediated endocytosis of H-ferritin in induction of NFκB-dependent proinflammatory signalling in hepatic stellate cells ANITA BURGESS,1 STEVEN ARNOLD,1 AMBER GLANFIELD,1 DIEM M HOANG-LE,1 NATHAN SUBRAMANIAM,2 GRANT RAMM1 1 Hepatic Fibrosis Group, Queensland Institute of Medical Research, Brisbane, QLD, Australia, 2 Membrane Transport Group, Queensland Institute of Medical Research, Brisbane, QLD, Australia Background Hepatic stellate cells (HSCs) are responsible for collagen deposition leading to fibrosis following liver injury/inflammation. Serum levels of the acute phase protein ferritin are elevated in inflammation and act as an indicator of disease severity in chronic liver disease. We have previously demonstrated that H-subunit ferritin actually contributes to this process as a pro-inflammatory mediator in HSC biology via an ironindependent, NFκB-regulated signalling pathway, inducing the expression of cytokines e.g., IL-1β, IL-6, RANTES. Additionally, we have shown this does not involve an interaction between ferritin and the known low-affinity ferritin scavenging receptors TfR1, Scara5 and Tim-2. Aims To understand whether cell surface binding alone, or surface internalisation are required for ferritin-induced signalling we have used a number of lysosomotropic agents to further investigate signalling pathways elicited and examine the role of additional intermediates associated with the ERK/JNK/p38 pathways. Methods Primary rat HSCs were treated with 10 nM H-ferritin for 0–24 hrs. Western blotting with phospho-specific antibodies was used to determine phosphorylation status and induction of proteins involved in the NFκB and ERK/JNK/p38 pathways. Primary rat HSC were pre-treated with inhibitors of microtubule formation (colchicine), lysosomal acidification (chloroquine) and intracellular protein transport (monensin) prior to ferritin stimulation. qPCR was used to determine relative expression of NFκB-regulated transcripts. Results H-ferritin activated signalling intermediates associated with NFκB and the ERK/JNK/p38 pathways. Increased phosphorylation was observed for PI3K YXXM target motif (2.7 ± 0.3-fold at 2 hrs), PKC-Ζ (5.8 ± 1 at 2 hrs), IKKα/β (4.3 ± 1.5 at 2 hrs), p65/RelA (3.7 ± 0.4 at 30 min), ERK1/2 (3.5 ± 0.5 at 30 min), JNK1/SAPK (10.4 ± 1.6 at 15 min) and p38 (7.6 ± 1.8 at 15 min). Microtubule inhibition (colchicine) had no significant effect on ferritin-induced expression of NFκB-derived IL-1β suggesting that microtubule-dependent endocytosis is not necessary for signalling and that cell surface binding of ferritin may be required. Monensin treatment resulted in a 75% reduction in ferritin-induced IL-1β expression while chloroquine completely abolished IL-1β expression. Conclusion These results suggest that while ferritin uptake via microtubule-dependent pathways may not be necessary for ferritin-induced signalling, intracellular trafficking of either ferritin and/or ferritin-induced signalling intermediates are important for the induction of proinflammatory mediators of fibrogenesis. Further studies assessing the role of alternative microtubule-independent (i.e., caveolin-mediated) endocytotic pathways, and the localisation of H-ferritin on or within HSCs, will yield insight into the precise mechanisms of ferritin binding in HSC and proinflammatory signalling elicited in hepatic fibrogenesis.

Journal of Gastroenterology and Hepatology 2012; 27 (Suppl. 4): 2–16 © 2012 Journal of Gastroenterology and Hepatology Foundation and Wiley Publishing Asia Pty Ltd

Basic Science Liver

Interferon-stimulated gene expression in the liver of patients with chronic hepatitis B infection SWEE LIN G CHEN YI MEI, MARIO CONGIU, TIN NGUYEN, ROBERT CHEN, MARNO C RYAN, SALLY BELL, PAUL DESMOND, ALEX THOMPSON Department of Gastroenterology, St Vincent’s Hospital, Fitzroy, VIC, Australia Background The expression of interferon-stimulated genes (ISGs) in the liver of patients with genotype 1 chronic hepatitis C infection has been strongly associated with response to interferon-α (IFN)-based therapy, as well as host IL28B genotype. Chronic hepatitis B infection (CHB) is also IFN-responsive. There have been no data evaluating liver ISG expression in patients with CHB. Aims We have performed a retrospective analysis of the association between liver ISG expression and disease characteristics of CHB in a cohort of treatment-naïve patients. Methods This was a retrospective analysis of a well-characterised cohort of treatment-naïve CHB patients recruited at St Vincent’s Hospital, Melbourne. Whole liver specimens collected at the time of routine liver biopsy were stored in RNAlater. RNA was extracted from the liver tissue, complementary DNA was prepared, and the canonical ISGs Mx1 and ISG15 were measured by real-time PCR, normalized to housekeeping genes. The association between liver ISG expression and CHB disease characteristics including HBeAg status, serum HBV DNA level, serum ALT level, and liver histology (inflammatory activity and fibrosis stage) was tested. Treatment outcome data was not available. Statistical analysis was performed using SAS v9.2. Results The analysis included data from 47 patients. 19/47 (40%) were HBeAg-positive, 24/47 (51%) were male, median age was 42 years (IQR 28–49 years), and 42/47 (89%) were Asian; median ALT was 65 IU/mL (IQR 34–108 IU/mL), median serum HBV DNA level was 818,728 IU/ mL (IQR 38,098–1.75 × 107 IU/mL); and the distribution of liver fibrosis stage was F0 = 5/47 (11%), F1 = 20/47 (43%), F2 = 11/47 (23%), F3 = 4/47 (8%), F4 = 7/47 (15%). There was a positive correlation between liver Mx1 expression and serum HBV DNA level (r = 0.39, P = 0.0082). Liver Mx1 expression was also higher in patients with advanced fibrosis (F3–4 = 13.0 [9.7–57.9] vs. F0–2 = 9.0 [5.9–13.7], P = 0.065), (F3–4), P < 0.05. In a linear regression model, serum HBV DNA and liver fibrosis stage (F0–2 vs. F3–4) were independently associated with liver Mx1 expression. There was a trend for HBeAg-positive patients to have higher level expression of Mx1 but this was completely attenuated by adjustment for serum HBV DNA level. Similar data were observed for liver ISG15 expression. Conclusion The data suggest a role for liver ISG expression in the pathogenesis of CHB. Further evaluation is warranted.

Liver iron homeostasis is altered by colonic inflammation and dietary iron ANITA CHUA,1 DESIREE S HO,1 BORUT KLOPCIC,1 JOHN OLYNYK,2 IAN C LAWRANCE,1 DEBBIE TRINDER1 1 School of Medicine and Pharmacology, University of Western Australia, Fremantle, WA, Australia, 2 Fremantle Hospital, Fremantle, WA, Australia Background Patients with inflammatory bowel diseases (IBD) develop anaemia of inflammation (AI) due to disturbances in iron homeostasis that limits the availability of iron for erythropoiesis. Aims In this study, the effects of colonic inflammation and dietary iron levels on liver iron homeostasis were investigated in a mouse model of colitis.

Methods Colonic inflammation was induced by the administration of dextran sodium sulphate (DSS) to mice fed either an iron-supplemented (1%) or control iron (0.01%) diet. Liver and plasma iron concentrations as well as plasma transferrin saturation were measured biochemically. Liver gene expression was determined by real-time PCR and plasma IL-6 levels were measured by ELISA. Results DSS-induced colonic inflammation increased plasma IL-6 levels. Dietary iron supplementation further enhanced colonic inflammation and plasma IL-6 levels (p < 0.0001). Liver iron and plasma transferrin saturation were elevated in dietary iron-supplemented mice. Post-DSS treatment, liver iron levels increased (p < 0.01) and transferrin saturation decreased (p < 0.01) in mice fed the iron-supplemented and control iron diets, consistent with the presence of AI. Liver expression of the iron regulatory genes, hepcidin (Hamp1) and inhibitor of DNA binding 1 (Id1), was upregulated by dietary iron (p < 0.01) but unexpectedly downregulated by DSS treatment (p < 0.05). Smad7 gene expression was decreased in DSStreated mice and Bmp6 expression was increased by dietary iron supplementation (p < 0.001). Dietary iron supplementation decreased the gene expression of the iron importer transferrin receptor 1 (Tfr1), congruent with the iron-dependent regulation of Tfr1 and expression and further diminished by DSS treatment (p < 0.05). Gene expression of the iron importer Zip14 was increased (p < 0.05) whilst that of the iron exporter ferroportin 1A was decreased (p < 0.0001) with DSS treatment, consistent with the retention of iron by the liver. Conclusion The perturbations in iron homeostasis resulting from increased colonic inflammation observed in this study are consistent with AI. The regulatory pathways for the changes seen, however, are unclear. The lack of induction of Hamp1 expression by plasma IL-6 levels suggests that other regulatory signals may impede hepcidin induction by inflammation. A possible candidate is the erythroid signal as increased erythropoietic activity is known to be a strong negative regulator of hepcidin.

Kupffer cells induce liver progenitor cell proliferation in response to CDE diet-induced liver injury CARYN ELSEGOOD,1 JOHN OLYNYK,2 GEORGE YEOH3 1 Medicine (Fremantle Hospital), University of Western Australia, Crawley, WA, Australia, 2Gastoenterology, Fremantle Hospital, Fremantle, WA, Australia, 3School of Biochemistry and Chemistry, University of Western Australia, Crawley, WA, Australia Background We have previously shown that both resident Kupffer cell and infiltrating monocyte-derived macrophage numbers increase in the liver in response to injury induced by the CDE diet.(1) The Kupffer cells and infiltrating macrophages are located adjacent to the portal veins as early as two days after the mice are placed on the diet. As the increase in macrophage numbers precedes the increase in liver progenitor cell numbers and macrophages express the LPC mitogens, TNF and TWEAK, we hypothesised that macrophages contribute to LPC proliferation. Aims To investigate the role of Kupffer cells in liver progenitor cell proliferation in response to CDE diet-induced liver injury. Methods We examined the role of Kupffer cells in CDE diet-induced liver injury and LPC proliferation using clodronate liposomes to ablate Kupffer cells but not blood monocytes. Results We found that Kupffer cells were required for the induction of LPC proliferation in response to CDE diet-induced injury, as the number of LPCs did not increase in response to CDE diet-induced injury when the Kupffer cells were depleted either before or after the induction of injury. Importantly, clodronate liposomes were not directly toxic to LPCs in vitro. Kupffer cell depletion resulted in a reduction in expression of TNF, IL-6, and HGF, but not TWEAK, at the mRNA level in the liver. Kupffer cell depletion also delayed the recruitment of monocytes to the liver. Further,

Journal of Gastroenterology and Hepatology 2012; 27 (Suppl. 4): 2–16 © 2012 Journal of Gastroenterology and Hepatology Foundation and Wiley Publishing Asia Pty Ltd

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Basic Science Liver

Kupffer cell depletion did not affect liver damage, as judged by serum ALT levels, suggesting that the reduction in LPC numbers was not a result of reduced liver damage. This study also showed that the Kupffer cells did not appear to play a role in the recruitment of other immune cells, such as T and B lymphocytes, which may also provide LPC proliferative cytokines. Conclusion Our data indicates that Kupffer cells induce LPC proliferation as a result of influencing LPC proliferative cytokine production.

TLR4 expression and canonical and non-canonical NF-κB activation. We find that ceramide and sphingosine levels are altered and additionally that glucosylceramide is more elevated in ADN-HC fed mice than WT-HC. Importantly, this unique inflammatory and sphingolipid signature offers potential therapeutic avenues to limit NAFLD progression.

Reference 1. Viebahn, C. S., V. Benseler, L. E. Holz, C. L. Elsegood, M. Vo, P. Bertolino, R. Ganss, and G. C. Yeoh. 2010. Invading macrophages play a major role in the liver progenitor cell response to chronic liver injury. Journal of Hepatology 53:500–507.

Free cholesterol (FC) loading causes both apoptosis and necrosis to murine primary hepatocytes, while FC lipotoxicity predisposes hepatocytes to free fatty acid-mediated cellular injury LAY THENG GAN, DERRICK M VAN ROOYEN, DEBORAH HEYDET, VANESSA A BARN, GEOFFREY FARRELL Liver Research Group, ANU Medical School at The Canberra Hospital, Garran, ACT, Australia

The role of adiponectin and cholesterol in NAFLD progression SAEED ESMAILI,1 JACOB GEORGE,2 LIONEL HEBBARD2 1 Digestive Disease Research Institute, Tehran University of Medical Sciences, Tehran, Islamic Republic of Iran, 2Storr Liver Unit, Westmead Millennium Institute, Sydney, NSW, Australia Background Lipid toxicity and inflammation are important components in the transition of non-alcoholic fatty liver disease (NAFLD) to nonalcoholic steatohepatitis (NASH). Factors such as reduced adiponectin have been associated with hepatic steatosis and inflammation, and have also been shown to influence sphingolipid metabolism. Recent data suggests that the level of free cholesterol is associated with the severity of liver disease. In this study we examined the association of adiponectin and cholesterol to NAFLD progression. Aims To investigate the role of adiponectin and cholesterol in NAFLD progression. Methods Male C57BL/6 and Adiponectin KO (ADN) mice were fed normal chow (NC) or a high cholesterol (HC) diet containing 2% cholesterol and 0.5% cholate for 12 weeks. At 4 weeks, Glucose Tolerance Tests (GTT), Insulin Tolerance Tests (ITT), and Pyruvate Challenge Tests (PCT) were performed. At experiment end, serum and hepatic cholesterol, and free fatty acids were measured. Hepatic sphingolipid levels were determined using LC/MS. Liver tissues were examined by haematoxylin and eosin (H&E), sirius red, TUNEL and CD68 immunostaining. For in vitro analyses, rat kupffer and stellate cells were isolated and treated with acetylated LDL. qPCR and western blots of tissues and cells were undertaken. Results When fed the high cholesterol diet, both genotypes had similar increases in average liver/total body weight ratio and reduced fat pad weight. Wild-type mice had greater insulin sensitivity (p < 0.05). Biochemistry and histology showed that KO mice fed the HC (ADN-HC) diet had elevated hepatic total (148 vs. 92 mg/dl), and free cholesterol (99 vs. 80 mg/dl), greater fibrosis, more apoptosis and 2-fold more macrophages than WT fed HC (WT-HC). ADN-HC livers had increased gene expression for TNFα (10-fold), IL-1β (1.4-fold), CCL19 (6.0-fold,), CD3ε (2.0-fold), LTα (2.0-fold), CD40L (9.0-fold) and TLR4 (1.4 fold, all statistically significant), and through western analyses activation of the noncanonical NF-κB pathway, compared to WT-HC. LC/MS analysis of the liver showed a 2-fold increase in ceramide, a 2-fold decrease in sphingosine-1-P and a 1.5 fold increase in glucosylceramide levels in ADN-HC compared to WT-HC mice (all p < 0.01). In preliminary in vitro experiments we find that treatment of HSCs and Kupffer cells with acetylated LDL and LPS leads to increased expression of TLR4 and p100 processing to p52. Conclusion These data show that adiponectin null mice fed a high cholesterol diet develop robust hepatic inflammation, and have increased

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Background Hepatic accumulation of FC but not free fatty acids (FFA) distinguishes non-alcoholic steatohepatitis (NASH) from ‘non-NASH’ pathology in non-alcoholic fatty liver disease (NAFLD) [Puri, GE ‘08]. Further, hepatic FC but not FFA or triglycerides correlates with NASH severity in foz/foz mice [van Rooyen, GE ‘11]. While saturated palmitic acid [PA]-loading of primary hepatocytes has demonstrated lipotoxicity of this FFA, there are not similar in vitro experiments to test the hypothesis that FC contributes to lipotoxicity in NASH. Aims 1. To establish a method for loading primary hepatocytes with FC, determine whether such loading activates c-Jun N-terminal kinase (JNK) and causes apoptosis and necrosis, the pathways involved in lipotoxicity; 2. To test the hypothesis that FC accumulation sensitizes hepatocytes to saturated FFA-mediated lipotoxicity in interactive cytotoxicity. Methods Primary hepatocytes isolated from C57B6 female mice were cultured and exposed to 0–40 μM low density lipoprotein (LDL) with or without 0–850 μM of PA. After 24 h, FC content was assessed by filipin fluorescence and biochemical assay, viability was determined by: lactate dehydrogenase (LDH) leakage, apoptosis (Hoechst 33342) and necrosis (propidium iodide). Harvested cells and supernatant were immunoblotted for JNK activation and high-mobility-group-box-1 (HMGB1). Results We obtained a dose-dependent increase in murine hepatocyte FC incubated with LDL for 24 h, with apparent maximal content at 40 μM LDL. Increased hepatocyte FC was associated with JNK activation and release of HMGB1 into supernatant, consistent with the proposed lipotoxic role of FC in NASH. Concomitant with the increased FC content, there were significant increases (p < 0.05) in LDH release, apoptosis and necrosis indices that were clearly dose-related. Exposure of hepatocytes to PA produced similar, but more attenuated, JNK activation, HMGB1 efflux, LDH release, and increased apoptosis and necrosis. Primary hepatocytes pre-loaded with 20 μM of LDL, a dose selected for minimal direct toxicity, showed significant and substantial reductions in viability, particularly increased necrosis, when exposed to increasing concentration of PA. Conversely, primary hepatocytes pre-loaded with 250 μM of PA did not appear to exhibit increased susceptibility to increasing LDL loading over the consistent increase in apoptosis and necrosis caused by the same LDL concentrations added to naive hepatocytes. Conclusion FC is directly lipotoxic to primary hepatocytes by a process associated with JNK activation and release of HMGB1. While FC stimulates apoptosis, there is even more pronounced necrosis. Further, cholesterol-laden hepatocytes are more susceptible to PA lipotoxicity. These studies are the first to show FC-mediated lipotoxicity directly in hepatocytes, and reveal synergistic liver toxicity between cholesterol and FFA in which the role of FC is essential.

Journal of Gastroenterology and Hepatology 2012; 27 (Suppl. 4): 2–16 © 2012 Journal of Gastroenterology and Hepatology Foundation and Wiley Publishing Asia Pty Ltd

Basic Science Liver

Effects of exercise on fatty liver, adipose inflammation and obesity complications in high fat-fed wild type (WT) and hyperphagic foz/foz mice FAHRETTIN HACZEYNI, VANESSA A BARN, NARCISSUS TEOH, GEOFFREY FARRELL Medical School, The Australian National University, The Australian National University, Canberra, ACT, Australia Background Physical inactivity contributes to obesity complications. In type 2 diabetes and non-alcoholic fatty liver disease (NAFLD), exercise improves glycaemic control and liver indices, but its effect on adipose inflammation and liver histology are less clear. Aims We tested whether exercise delays onset of obesity and ameliorates adipose inflammation, steatosis and liver inflammation in murine models of NAFLD/NASH. Methods From weaning, male WT or Alms1−/− mutant (foz/foz) NOD. B10 mice fed either HF diet (SF03-020; Glen Forrest) or chow were caged in pairs until 24 wk-old (n > 6). Half the cages were fitted with exercise wheel (EW) (ASIFTB-PC; Able Scientific) and cycle computer (Bri2; Echowell). At 24 wks, blood, liver, subcutaneous white adipose tissue

(Sub WAT), mesenteric visceral adipose tissue (Mes VAT), and extensor digitalis longus muscle were removed. Results 20 wk-old mice with wheels often travelled 10–15 km in 24 h; obese mice were notably less active. EW provision reduced wt gain in HF-fed WT mice. In foz/foz mice fed either diet, exercise reduced (but did not normalise) wt gain for ∼16 wks, after which differences diminished. Exercise had no effect on liver size in WT mice, but caused a minor reduction in HF-fed foz/foz mice (9.5% body wt vs. 11%, P < 0.05) (Fig 1). Exercise decreased Sub WAT and Mes VAT mass in WT irrespective of diet, but increased WAT/VAT mass in foz/foz mice. Increased muscle mass in response to exercise was observed only in WT mice. In all 4 gp, fasting blood glucose (FBG) was lower (∼20%) in mice that exercised; effects on serum cholesterol were negligible. Exercise slightly decreased serum ALT in HF-fed WT and foz/foz mice (NS). In HF-fed WT mice, exercise abolished steatosis, which was mild-moderate in unexercised counterparts. Preliminary data did not reveal changes in steatosis, ballooning, inflammation or NAFLD activity score in foz/foz mice provided with EWs. Conversely, there were striking changes in both adipose compartments: adiponectin mRNA expression increased in exercised mice (Fig 2). Inflammation markers Cd68 (macrophages) and Cd11c (dendritic cell) transcripts were strikingly increased in HF-fed foz/ foz mice compared to other gp; values were nearly normalized by exercise.

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Conclusion Providing an EW prevents weight gain in WT male mice fed HF diet, decreases FBG and completely prevents steatosis. Exercise reduced (but failed to abolish) weight gain in foz/foz mice, and, despite improvement in FBG, exercise failed to prevent steatosis, liver injury and inflammation (NASH). Our preliminary studies on subcutaneous and

visceral adipose tissue compartments find no difference between them in inflammatory markers. Further, exercise prevents accumulation of dendritic cell and macrophage markers without conferring apparent benefits on development of NAFLD/NASH in these hyperphagic, appetite-driven mice.

Adiponectin mediates hepatic stellate cell invasion through TIMP-1 and CD63 regulation of FAK activity LIONEL HEBBARD, MEHDI RAMEZANI-MOGHADAM, JIANHUA WANG, JACOB GEORGE Storr Liver Unit, Westmead Millennium Institute, University of Sydney, Westmead, NSW, Australia

promote their apoptosis in vitro. We find that adiponectin through AMPK stimulates HSCs to release the matrix metalloproteinase inhibitor, TIMP-1. Traditionally, TIMP-1 has been associated with the progression of liver fibrosis. Thus, the induction of TIMP-1 by adiponectin would appear to be counter-intuitive to its published anti-fibrotic effects. TIMP-1 has been recently reported to inhibit focal adhesion kinase (FAK) phosphorylation, a marker of decreased cellular invasion and migration. Additionally, CD63 has been reported to be a novel binding partner for TIMP-1. Thus, the aim of this study was to investigate whether ADN stimulated TIMP-1 is associated with altered FAK activity in rat HSCs and in an in vivo model of liver fibrosis.

Background Adiponectin (ADN) is an adipocytokine that inhibits hepatic stellate cell (HSCs) proliferation, migration and invasion, and can

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Journal of Gastroenterology and Hepatology 2012; 27 (Suppl. 4): 2–16 © 2012 Journal of Gastroenterology and Hepatology Foundation and Wiley Publishing Asia Pty Ltd

Basic Science Liver

Aims To characterise the adiponectin-TIMP-1 relationship and determine its role on HSC function. Methods Activated primary rat HSCs were treated with ADN for between 4 and 48 hours. TIMP-1 protein levels were determined by ELISA. Cells were treated with ADN, CD63 or TIMP-1 blocking antibodies and TIMP-1 recombinant protein. p-FAK levels were determined by western blot. HSC invasion was assessed with a modified migration assay using Boyden chambers coated with rat-tail collagen I. Wild type mice were injected with carbon tetrachloride for 8 weeks, followed by tail vein injections of ADN or vehicle for 24 hours and qPCR and western blot analyses performed. Serum from a cohort of NASH patients with low (10 μg/ml) ADN was tested for circulating TIMP-1 protein by ELISA and statistical analyses undertaken. Results ADN treatment of HSCs for 4 to 48 hours resulted in a ∼3-fold increase in TIMP-1 levels. ADN treatment of activated HSCs resulted in a 50% reduction in invasion and a 3-fold reduction in p-FAK protein. Concurrent treatment of activated HSCs with ADN and TIMP-1 or CD63 blocking antibodies reversed the inhibitory effects of ADN on HSC invasion and upregulated p-FAK. Recombinant TIMP-1 protein treatment reduced FAK phosphorylation in HSCs. Western blot analysis of p-FAK from the livers of mice treated with ADN was significantly lower than vehicle treated, and a positive correlation between ADN and TIMP-1 (Spearman’s rho, R = 0.71) was observed in the serum from the NASH cohort. Conclusion The treatment of activated HSCs with ADN and TIMP-1 reduces the phosphorylation of FAK and suggests a mechanism through which ADN regulates HSC invasion. Our data also demonstrate that adiponectin induced TIMP-1 through CD63 is partially responsible for mediating hepatic stellate cell FAK activity. These findings have relevant effects in vivo and the ADN-TIMP-1 association is apparent in humans. Our study suggests a new mechanistic role for adiponectin and TIMP-1 in liver fibrosis.

The progression of NAFLD to NASH in a mouse model of Hfe−/−-associated steatohepatitis is attenuated by co-administration of curcumin and vitamin E MANDY L HERITAGE,1 ASHLEY S WILKINSON,1 LESLEY A JASKOWSKI,1 LAURENCE J BRITTON,1 TERRENCE C TAN,1 KIM BRIDLE,1 ANDREW CLOUSTON,2 GREGORY ANDERSON,3 LINDA M FLETCHER,2 GRAEME A MACDONALD,2 NATHAN SUBRAMANIAM,3 DARRELL H CRAWFORD1 1 School of Medicine, University of Queensland, Gallipoli Medical research centre, Greenslopes Private Hospital, Brisbane, QLD, Australia, 2Gastroenterology and Hepatology, The Princess Alexandra Hospital, Brisbane, QLD, Australia, 3Cell and Molecular Biology, The Queensland Institute of Medical Research, Brisbane, QLD, Australia Background There is sufficient evidence to implicate iron and/or HFE mutations as having an important role in the progression of liver disease in subjects with NAFLD. Previous work in our laboratory has shown that Hfe−/− mice fed a high-fat, high-carbohydrate diet (HF/HCD) develop severe steatohepatitis whereas wild-type mice only develop steatosis. The progression of NAFLD to NASH in this model involves alterations in lipid signalling, inflammatory and oxidative stress pathways with an accelerated progression of injury to fibrosis. Aims This study aims to investigate the effects of single and combination treatment of the putative anti-inflammatory and anti-oxidant agents,

curcumin and vitamin E in comparison to removal of the HF/HCD in a mouse model of Hfe−/−-associated steatohepatitis. Methods Hfe−/− mice on a C57BL/6J background (8 weeks old, n = 80) were fed a HF/HCD for 10 weeks and then for a further 10 weeks fed either HF/HCD (n = 9) or chow (n = 9) (CH) or treatment groups (n = 9) as follows: HF/HCD + 1% curcumin (CU); HF/HCD + 1.5% Vitamin E (VE); HF/HCD + 1% curcumin + 1.5% Vitamin E (CUVE). Statistical analysis was performed using one-way ANOVA. Results The Hfe−/− mice fed a HF/HCD developed severe steatohepatitis as indicated by widespread macro- and micro-vesicular fat deposits, hepatocyte ballooning, Mallory’s hyaline and increased inflammatory cell infiltrates. Those mice switched to chow (CH) or treated with combination CUVE diet showed a significant reduction of both macro- and microvesicular fat deposits and inflammatory cell infiltrates whereas single CU or VE treatment did not. There was evidence of fibrosis in the mice fed a HF/HCD for 20 weeks as assessed by Pico Sirius red staining which was significantly reduced in CH and CUVE groups. There was a reduction in the expression of a panel of fibrosis related genes, α-Sma, Pdgfr, Mmp2, Timp1 and Timp2 for the CH group but not CUVE group compared to HF/ HCD alone. However, there was a reduction in expression of Tgfβ1 and Mcp-1 in CH and CUVE treated mice compared to HF/HCD. The lipogenesis genes, Srebp-1c and Fas were significantly elevated in CUVE treated mice compared to HF/HCD controls as was Cpt1 expression. Pparα and adiponectin receptor 2 expression were increased in CUVE treated mice. HF/HCD-fed mice had a significantly elevated GSH/GSSG ratio indicative of oxidative stress which was completely suppressed in both CH and CUVE treated mice. A concurrent increase in catalase activity in both CU and CUVE-fed mice implies an anti-oxidant effect of curcumin. Conclusion Hfe−/− mice fed a HF/HCD for 20 weeks develop severe steatohepatitis; the progression to steatohepatitis was attenuated by feeding a combination treatment regime of curcumin and vitamin E or by removal of the HF/HCD altogether. The effect of combination treatment appears to be mediated by reduced oxidative stress and altered inflammatory and fibrogenic responses.

Human amniotic epithelial cell conditioned media reduces hepatic stellate cell activation and collagen partially through increased apoptosis ALEXANDER HODGE,1 DINUSHKA LOURENSZ,1 VIJESH VAGHJIANI,2 URSULA MANUELPILLAI,2 WILLIAM SIEVERT1 1 Centre for Inflammatory Disease, Monash Medical Centre, Monash University, Melbourne, VIC, Australia, 2 Monash Institute of Medical Research, Monash University, Melbourne, VIC, Australia Background Hepatic fibrosis is the driving force behind the progression of chronic liver disease and the key effector is the hepatic stellate cell (HSC). Cell therapy with human amniotic epithelial cells (hAEC) has been shown to ameliorate fibrosis in murine models. Aims This study investigated whether factors secreted by hAEC reduce fibrosis by inhibiting HSC and whether soluble HLA-G (sHLA-G) is partly responsible. Methods The effect of conditioned media (CM) from hAEC was studied on LX2 cells, a human HSC cell line. Confluent HSC were treated with CM for 48 hours and mRNA expression of alpha smooth muscle actin (α-SMA) and platelet derived growth factor receptor (βPDGF-R) assessed by RT-PCR. Protein levels of transforming growth factor beta1 (TGF-β1) in the supernatant were measured by ELISA. Intracellular collagen production and supernatant collagen content were determined by 3H-proline

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incorporation and Sircol collagen assay respectively. HSC apoptosis was determined by FACS for Annexin V and proliferation by BrdU incorporation. Expression of genes responsible for collagen degradation; matrix metalloproteinases 2 and 9 (MMP2, MMP9) and tissue inhibitor of metalloproteinase 2 (TIMP2) were determined by RT-PCR. Anti-HLA-G neutralizing antibody was added to CM experiments to block the effects of sHLA-G in the hAEC CM. Results HSC treated with hAEC CM displayed reduced activation markers; α-SMA and βPDGF-R mRNA expression were reduced by 56% (P = 0.0023) and 12% (P = 0.045), respectively. TGF-β1 protein levels were reduced by 59% (P = 0.0009). In separate experiments in which TGF-β was added to HSC, hAEC CM ameliorated α-SMA and βPDGF-R mRNA expression to levels similar to controls (P < 0.001). Intracellular collagen production was reduced by 54% (P < 0.0001) in hAEC CM treated cells. Addition of hAEC CM to HSC cultures reduced collagen in the supernatant to levels below that in control media (P = 0.0025). Expression of MMP2 and MMP9 increased two-fold compared to controls (P = 0.0015; P = 0.0004) while TIMP2 was reduced by a third (P = 0.0024). Proliferation of HSC in sub-confluent cultures was reduced by 12% (P = 0.0047) in cells treated with hAEC CM. There was a 2.34 fold increase in early apoptosis in HSC treated with hAEC CM. Antibody blocking of sHLA-G in hAEC CM resulted in a partial reversal of diminished collagen production and TGF-β1 suppression (P < 0.05). Conclusion hAEC CM treatment of activated hepatic stellate cells results in the suppression of markers of activation, proliferation and collagen production, likely through induction of HSC apoptosis. In addition active collagen degradation is enhanced by increasing MMP and decreasing TIMP gene expression. hAEC produce soluble factors, including sHLA-G, that inhibit human hepatic stellate cell collagen expression and therefore may be effective in ameliorating liver fibrosis.

Cyclosporine and tacrolimus have different inhibitory effects on Toll-like receptor signalling post liver transplantation JESSICA HOWELL,1 ROHIT SAWHNEY,1 NARELLE A SKINNER,2 DILIP RATNAM,2 PETER ANGUS,1 PAUL GOW,1 KUMAR VISVANATHAN2 1 Gastroenterology, Austin Hospital, IVANHOE, VIC, Australia, 2Innate Immune Laboratory, Monash Medical Centre, Clayton, VIC, Australia Background Hepatitis C (HCV) recurrence post liver transplant follows a more aggressive course than pre-transplantation and the mechanisms remain poorly understood. Toll-like receptors (TLRs) are critical to innate immune antiviral responses. The interplay between TLR function and immunosuppressive drugs and how this may affect recurrence of diseases such as HCV infection post transplant is unknown. Aims To determine the relationship between serum calcineurin inhibitor levels and TLR function post liver transplantation. Methods Peripheral blood mononuclear cells (PBMCs) from 83 HCV post liver transplant patients, 53 non-HCV post transplant controls (matched for sex, race and time post transplant) and 10 healthy controls were stimulated with TLR specific ligands LPS (TLR4), P3C (TLR2), PIC (TLR3), R848 (TLR7/8) and CpG (TLR9) for 24 hrs. Production of interleukin 6 (IL-6), tumour necrosis factor (TNF) and interferon alpha (IFNα) was then measured using ELISA. Flow cytometry was used to determine intracellular cytokine production by monocytes, NK, NKT and T cells. Results were compared between groups using Mann-Whitney and Spearman correlation. Results Cyclosporine levels correlated with reduced TLR3-induced IFNγ production by NK cells (NK cells p = 0.011, CD56bright NK cells p = 0.017) and T cells (p = 0.049). Cyclosporine levels also correlated with reduced TLR3-induced monocyte IL-6 (p = 0.028) and TNF (p = 0.070) 8

production. Cyclosporine level correlated positively with T cell frequency (p = 0.040). In contrast, tacrolimus levels correlated inversely with TLR4-induced PBMC IL-6 production (p = 0.030), TLR3-induced TNF production by PBMCs (p = 0.027) and TLR7/8-induced NK cell TNF (NK cells p = 0.009, CD56dim NK cells p = 0.013). Tacrolimus levels correlated inversely with NKT cell frequency (p = 0.032). Conclusion NK cells and monocytes have impaired TLR3-induced cytokine production that inversely correlated with increasing cyclosporine level. In contrast, tacrolimus levels correlated inversely with TLR4induced IL-6 by PBMCs, TLR3-induced TNF from monocytes, and TLR7/8 induced TNF from NK cells. These data demonstrate important relationships between TLR function and calcineurin inhibitor levels that may contribute to disease recurrence post liver transplant, including aggressive HCV recurrence. Importantly, our data also suggest that tacrolimus and cyclosporine have different effects on innate immune signalling, which may influence choice of therapy post liver transplantation in different clinical contexts.

NK cell marker expression is dysregulated post liver transplantation and correlates with HCV viral load JESSICA HOWELL,1 ROHIT SAWHNEY,1 NARELLE A SKINNER,2 DILIP RATNAM,2 PAUL GOW,1 PETER ANGUS,1 KUMAR VISVANATHAN2 1 Gastroenterology, Austin Hospital, IVANHOE, VIC, Australia, 2Innate Immune Laboratory, Monash Medical Centre, Clayton, VIC, Australia Background Hepatitis C (HCV) recurrence post liver transplantation is universal, with a subgroup developing rapid hepatic fibrosis. Natural killer (NK) cells are critical to anti-viral immunity and the role of NK cell function in HCV rapid fibrosis post transplant is unknown. Aims This study determines NK cell marker expression in HCV patients post liver transplant and the relationship to HCV viral load and graft fibrosis progression. Methods Peripheral blood mononuclear cells (PBMCs) from 35 HCV patients post transplant, 10 non-HCV patients post transplant (matched for sex, age and time post transplant) and 8 healthy controls were labelled for CD3 and CD56, activation markers CD69 and NKG2D, degranulation marker CD107a, inhibitory KIR 2DL2/2DL3 (CD158b) and baseline intracellular TNFα and IFNγ and analysed by flow cytometry. Rate of fibrosis progression was calculated using post transplant liver biopsies graded by Metavir scoring (F0–4; R = fibrosis stage/year post transplant). Rapid fibrosis was defined as > 0.4 units/yr, dichotomized about the median observed rate (0.4). Results Both HCV and non-HCV patients post transplant had reduced NK cell CD158b (NK cells p = 0.037, CD56bright NK cells p = 0.0081) and NKG2D expression (NK cells p = 0.030, CD56dim NK cells p = 0.003) compared with healthy controls. NK cell NKG2D expression inversely correlated with HCV viral load (p = 0.012, r = −0.57). HCV NK CD56dim cells had greater CD69 expression compared with post transplant controls (p = 0.043) and CD69 expression correlated with both TNF and IFNγ production (TNF p = 0.043, r = 0.43; IFNγ p = 0.004, r = 0.58). HCV NK cells produced greater TNFα (NK CD56 dim p = 0.005, NK CD56bright p = 0.044) and IFNγ (NK CD56dim p = 0.008, NK CD56bright p = 0.016) at baseline compared with non-HCV post transplant patients. There was no relationship between NK cell marker expression and HCV fibrosis progression post transplant. Conclusion Our data suggest that impaired NK CD56dim NKG2D expression post transplant may contribute to poor immune control of HCV viraemia, whilst reduced NK cell inhibitory CD158b expression contributes to greater NK-mediated pro-inflammatory cytokine production in

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HCV post transplant. However, the absence of a direct relationship between HCV rapid fibrosis progression and NK cell marker expression suggests other additional factors are important for fibrosis progression.

Liver disease alters the glycosylation of proteins AIMEI LEE,1 MAJA N CHRISTIANSEN,2 NICOLLE H PACKER,2 EMILIA PRAKOSO,1 ROBERT CHENG,3 SUSAN V MCLENNAN,4 NICHOLAS SHACKEL3 1 Liver Injury and Cancer, Centenary Institute of Cancer Medicine and Cell Biology, Camperdown, NSW, Australia, 2Department of Chemistry and Biomolecular Sciences, Macquarie University, Macquarie Park, NSW, Australia, 3AW Morrow Gastroenterology and Liver Centre, Royal Prince Alfred Hospital, Camperdown, NSW, Australia, 4Sydney Medical School, The University of Sydney, Camperdown, NSW, Australia Background Glycosylation is one of the most important post-translational modifications where carbohydrates are enzymatically attached to proteins. These carbohydrates, referred to as glycans, play an important role in protein structure and function such as cell adhesion, immune responses and inflammatory processes. Alterations to the protein glycosylation pathway are well established in tumour formation and metastasis, as well as other diseases. Aims To determine if changes in membrane protein nitrogen atom (N) linked-glycosylation is associated with different pathologies of the liver. Methods Liver tissue samples were obtained from non-diseased donors and patients with either autoimmune hepatitis (AIH) or primary biliary cirrhosis (PBC). The global N-glycan profile of the cell membrane proteins was analysed. Cytosolic and membrane proteins were separated by ultracentrifugation, followed by Triton X-114 phase partitioning to further enrich for membrane proteins. N-glycans were released from the different protein fractions by peptide: N-glycosidase F (PNGaseF) treatment. The released glycans were subsequently analysed by liquid chromatographyelectrospray ionization tandem mass spectrometry (LC-ESI-MS/MS). Results The glycosylation profile of the diseased liver tissue exhibited significant differences in specific glycan structures compared to the nondiseased donor tissue. There were significantly more hybrid N-glycans in both the AIH and PBC liver tissue compared to the donor, whilst the donor liver tissue contained more complex N-glycans than the diseased tissue. Conclusion These changes suggest that damage to the liver leads to an alteration in the protein glycosylation pathway, with N-linked glycosylation terminating in the Golgi apparatus before complex type glycoproteins can be made.

Advanced glycosylation endproducts in nonalcoholic steatohepatitis: a second hit that drives hepatic stellate cell activation? CHRISTOPHER LEUNG,1 CHANDANA HERATH,1 JIA ZHIYUAN,1 MICHELLE GOODWIN,1 KAI YAN MAK,1 JOSEPHINE M FORBES,2 PETER ANGUS1 1 The RAS and AGE Laboratory, Department of Medicine, Austin Health, The University of Melbourne, Heidelberg, VIC, Australia, 2Glycation and Diabetes Complications Laboratory, Mater Medical Research Institute, South Brisbane, QLD, Australia Background Advanced glycosylation endproducts (AGEs) are compounds present in large amounts in the Western diet. Their production is

also increased in diabetes where they contribute to tissue injury via activation of RAGE (receptor for AGEs) and generation of oxidative stress. AGEs are implicated in non-alcoholic fatty liver disease (NAFLD), the most common liver disease worldwide. However, it is unclear whether AGEs contribute to NASH via direct effects on hepatic stellate stellate (HSCs), the primary effector cells for liver fibrosis. Aims We aimed to determine the role of the AGE/RAGE axis in HSCs from healthy livers and livers with non-alcoholic steatohepatitis (NASH). Methods 10 week old Sprague Dawley rats were randomised into 2 groups. 1) NASH induced by the methionine choline deficient (MCD) diet and 2) a methionine choline replete (MCR) diet as control. After 12 weeks, primary HSCs were isolated via portal vein perfusion, lysis and Nycodenz separation. Preactivated HSCs (from MCD animals) and healthy HSCs (from MCR animals) were treated with BSA vehicle (100 μg/mL) and AGE-BSA (100 μg/mL). Two additional groups had AGE-BSA with NADPH oxidase inhibitor, diphenyleneiodonium (DPI) or RAGE antibody. Cell proliferation was measured via a bromodeoxyuridine chromogenic assay and reactive oxygen species (ROS) generation was determined using a fluorescence-based assay. Cells were also collected for determination of gene expression. Results HSCs from NASH livers had greater ROS generation, interleukin-6 (IL-6) and alpha-smooth muscle actin (aSMA) expression than HSCs from healthy livers (p < 0.05). HSCs from healthy livers did not respond to AGEs. However, in HSCs from animals with NASH, AGEs markedly increased cell proliferation (p = 0.01), ROS production (p = 0.02) and gene expression of monocyte chemoattractant protein-1 (MCP1), IL-6 and aSMA (p < 0.05). These effects were abrogated by both RAGE blockade and NADPH oxidase inhibition with DPI (p < 0.05). Conclusion The finding that AGEs only affect HSCs from diseased livers helps explain why previous studies have produced conflicting results regarding the role of the AGE/RAGE axis in HSC activation. They also suggest that AGEs may provide a ‘second hit’ that drives progression of simple steatosis to fibrosis in NAFLD.

Hepatitis C, cyclosporine and mycophenolate mofetil induce apoptosis in primary mouse hepatocytes EU JIN LIM,1 RUTH CHIN,2 PETER ANGUS,1 JOSEPH TORRESI3 1 Liver Transplant Unit, Austin Hospital, Heidelberg, VIC, Australia, 2Medicine, University of Melbourne, Austin Health, Heidelberg, VIC, Australia, 3Infectious Diseases, Austin Hospital, Heidelberg, VIC, Australia Background Severe recurrent hepatitis C (HCV) disease post-liver transplantation results in rapidly progressive liver fibrosis, leading to cirrhosis and liver failure. The mechanism responsible for this remains unclear. Cyclosporine (CyA) and mycophenolate mofetil (MMF) are commonly used for immunosuppression post-liver transplant for HCV disease. We previously demonstrated that both HCV infection and immunosuppressants induced apoptosis in an immortalised human hepatocyte line. Aims We now investigate the effects of HCV and immunosuppressants in primary mouse hepatocytes (PMoH). Methods The effects of HCV infection, CyA and MMF, alone and in combination were investigated. PMoH harvested from 12-week-old wildtype C57BL/6 mice were exposed to viral constructs made using the AdEasy system and contain either green fluorescent protein alone (rAdGFP) or with HCV constructs expressing the structural (rAdHCVCoreE1E2) or non-structural (rAdHCV-NS3-5B) proteins, in the presence or absence of CyA and/or MMF at physiologically relevant concentrations. Treated cells were evaluated at 48 hours and compared to mock. PMoH apoptosis was evaluated using Western immunoblots performed on cell

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lysates probed for cleaved caspase 3 and cleaved PARP. Experiments were done in triplicate. Results CyA at 1 mcg/mL had no effect on cleaved caspase 3 or cleaved PARP in PMoH compared to mock. In contrast, MMF at 5 mcg/mL reduced cleaved caspase 3 by 1.9 fold and reduced cleaved PARP by 2.9 fold. The combination of CyA and MMF resulted in an increase by 2.8 fold in cleaved PARP, but had no effect on cleaved caspase 3. rAdGFP infection did not significantly increase cleaved caspase 3 and only increased cleaved PARP by 1.6 fold, but in infection with rAdHCVCoreE1E2, rAdHCV-NS3-5B and co-infection, cleaved caspase 3 was increased by 2.4, 2.7 and 3.2 fold, and cleaved PARP was increased by 2.6, 2.7 and 2.7 fold respectively. Addition of CyA to HCV infections increased cleaved caspase 3 by 2.8, 2.6, and 2.7 fold, and cleaved PARP by 6.3, 6.4, and 6.7 fold respectively. Addition of MMF to respective HCV infections increased cleaved caspase 3 by 2.0, 2.2, and 1.9 fold, and cleaved PARP by 3.5, 4.5 and 4.5 fold. Addition of both CyA and MMF to various HCV infections increased cleaved caspase 3 by 7.6, 8.7 and 9.1 fold, and cleaved PARP by 8.3, 10.4, and 10.3 fold. Conclusion At physiologically relevant doses, MMF by itself reduced apoptosis and CyA by itself had no effect on apoptosis in PMoH. However, the combination of CyA and MMF increased PMoH apoptosis. Infection with the various HCV constructs also increased PMoH apoptosis. Addition of CyA and/or MMF to HCV infection further increased apoptosis. These results show that CyA and MMF increases apoptosis in primary hepatocytes infected with HCV and may contribute to the accelerated progression of liver disease seen in HCV recurrence post-liver transplantation.

Hepatitis C, cyclosporine, and mycophenolate mofetil enhance cell death in human liver cells, with partial reversal by inhibitors of apoptosis and necroptosis EU JIN LIM,1 RUTH CHIN,2 PETER ANGUS,1 JOSEPH TORRESI3 1 Liver Transplant Unit, Austin Hospital, Heidelberg, VIC, Australia, 2Medicine, University of Melbourne, Austin Health, Heidelberg, VIC, Australia, 3Infectious Diseases, Austin Hospital, Heidelberg, VIC, Australia Background Severe recurrent hepatitis C (HCV) post-liver transplantation results in rapidly progressive liver fibrosis, but the mechanism responsible remains unclear. We previously showed that both HCV and cyclosporine (CyA) reduced hepatocyte viability and enhanced apoptosis. Mycophenolate mofetil (MMF) is also used with CyA for liver transplant immunosuppression. Aims To investigate the effect of MMF and CyA on HCV-induced hepatocyte apoptosis. To determine the effect of apoptosis and necroptosis inhibitors on this system. Methods Huh7 cells were exposed to physiologically relevant doses of MMF and CyA, and/or viral constructs made using the AdEasy system, containing either green fluorescent protein alone (rAdGFP), or with HCV constructs expressing the structural (rAdHCV-CoreE1E2) or non-structural (rAdHCV-NS3–5B) proteins ± TNF-α. Treated cells were evaluated at set time points up to 72 hours and compared to mock. Pan-caspase inhibitor Q-VD-Oph (Q-VD) and RIP-kinase inhibitor necrostatin-1 (Nec-1) were used to inhibit apoptosis and necroptosis respectively. Cell viability was evaluated using crystal violet assays. Cell apoptosis was evaluated using Western immunoblots performed on cell lysates probed for cleaved PARP. Experiments were done in triplicate. Results Infection of Huh7 cells with both HCV constructs reduced cell viability by 1.6 fold compared to mock, but cell viability was improved by 1.5 and 1.4 fold with the addition of Q-VD and Nec-1 respectively. HCV infection with TNF-α reduced cell viability by 2.7 fold, but Q-VD and Nec-1 improved this by 2.2 and 1.7 fold respectively. CyA at 1 mcg/

10

mL had no effect on cell viability, but MMF at 5 mcg/mL increased cell viability by 1.2 fold (p = 0.03). However, the combination of CyA and MMF reduced cell viability by 1.9 fold (p < 0.01). Addition of CyA and MMF to infection with rAdHCV-CoreE1E2, rAdHCV-NS3–5B and both viral constructs further reduced cell viability by 2.2, 2.6 and 2.2 fold compared to respectively infections alone. Infection with both HCV constructs increased cleaved PARP by 3.3 fold, but cleaved PARP was reduced 27 fold by Q-VD. HCV infection with TNF-α increased cleaved PARP by 4.2 fold, but Q-VD reduced this by 26 fold (p < 0.01). Nec-1 had no effect on cleaved PARP. CyA and/or MMF alone did not increase cleaved PARP compared to mock. However, addition of CyA and MMF to infection with rAdHCV-CoreE1E2, rAdHCV-NS3–5B and both increased cleaved PARP by 1.4, 1.7 and 1.5 fold compared to respective infections alone. Conclusion HCV infection ± TNF-α reduces cell viability and increases apoptosis. Addition of CyA and MMF to HCV further worsens cell viability and apoptosis, perhaps contributing to accelerated liver disease progression in post-transplant HCV recurrence. Apoptosis and necroptosis inhibition both partially improved cell viability, suggesting that cell death in infected cells may occur by both apoptosis and necroptosis.

Application of next-generation sequencing technology to diagnosis of genetic iron disorders CAMERON MCDONALD,1 DANIEL WALLACE,1 DARRELL H CRAWFORD,2 NATHAN SUBRAMANIAM1 1 Membrane Transport Laboratory, Queensland Institute of Medical Research, Brisbane, QLD, Australia, 2School of Medicine, University of Queensland, Brisbane, QLD, Australia Background Most disorders of primary iron overload and iron-refractory anaemia have a genetic origin. Hereditary haemochromatosis (HH) due to mutations in the HFE gene affects around 1 in 200 people of northern European descent. Mutations in a number of other genes are collectively termed non-HFE HH and are being increasingly identified in non-European populations. Our group has identified and characterised a number of novel mutations associated with genetic iron disorders in the Asia-Pacific region. Aims This project aims to develop and apply Next Generation Sequencing technology to the identification and diagnosis of genetic iron disorders in a systematic fashion. This will enable rapid and comprehensive analysis for a fixed number of whole genes implicated in iron disorders. Ultimately, we intend to establish an International Referral Centre for Iron Disorders where these analyses are performed routinely. Methods The Ion Torrent Personal Genome Machine is the first sequencing platform to utilise post-light technology. Sequencing is performed directly on silicon chips which reduces the run and analysis processing time from weeks to a single day whilst the scalability of these chips allows matching of the assay size with sample numbers to maintain the assays scale of economy. This technology will be combined with a custom designed amplification approach to provide high coverage sequencing of 11 core iron regulatory genes including full coding, UTRs, intron-exon boundaries, and promoter regions. These genes cover known causative genes of both iron overload and anaemia including HFE, HJV, Hepcidin, TFR2, Ferroportin, FTL, FTH1, TFR1, TF and TMPRSS6. A further 20 genes with potential regulatory effects will also be sequenced. Results This combination of technology will allow rapid throughput, high coverage sequencing of a comprehensive panel of iron overload and anaemia-related genes. The technology for this facility is now in place, with clinical sample processing underway shortly. The sequencing process takes approximately 2 days from sample to report generation, with the capacity to multiplex several patients per cycle. Data derived from patient

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samples using this facility will be presented. The Iron Disorders Referral Centre will initially aim to meet the costs of testing patient samples on a collaborative basis with clinicians. Conclusion This centre will provide a valuable resource for both clinicians and researchers within the Asia-Pacific region, and eventually worldwide. The comprehensive gene panel eliminates the individual candidate genes approach, significantly increasing the probability of identifying causative mutations in genetic iron disorders. The sequencing and clinical data will also be compiled into a database, building an un-paralleled resource for the identification of modifiers of iron status, disease penetrance and the natural history of these disorders.

CD147 dependant intrahepatic immune and cancer cell aggregation; a novel mechanism of liver injury ALISON J MORGAN,1 ANNETTE E MACZUREK,1 SARAH N CALABRO,1 GEOFFREY MCCAUGHAN,2 FIONA J WARNER,1 SUSAN V MCLENNAN,3 NICHOLAS SHACKEL1 1 Centenary Institute, Centenary Institute, Newtown, NSW, Australia, 2A W Morrow Gastroenterology and Liver Centre, Royal Prince Alfred Hospital, Sydney, NSW, Australia, 3Medicine, University of Sydney, Sydney, NSW, Australia Background Chronic liver injury is typically characterised by inflammation, progressive fibrosis and resultant regenerative responses. Intrahepatic inflammatory responses drive almost all types of liver injury with resultant fibrosis, eventual cirrhosis and sequelae including hepatocellular carcinoma (HCC). CD147 is an abundant membrane receptor. We have previously demonstrated CD147 within the liver to regulates hepatocyte matrix metalloproteinase (MMP) expression and inflammatory/immune cell chemotaxis. Our previous striking novel finding was the identification of CD147 dependant immune cell aggregation. Aims The role of CD147 in pathogenesis of intrahepatic inflammation and liver cancer cell aggregation was investigated. Methods Mouse models of progressive liver injury were induced in C57BL/6 and BALB/c mice by either thioacetamide (TAA) or IP injection of CCl4. In-vitro interventions consisted of α-CD147 mAb (RL73.2) and an isotype control. Liver leukocytes and the HCC cell line Hepa 1–6 were analysed by eight-coloured flow cytometry analysis as well as being assessed in-vitro for aggregation using a BD Pathway Analysis. Confocal immunofluorescence was performed on frozen fixed injured mouse and human liver tissue as well as HCC. Results We have shown that CD147 (expressed on CD4+, CD8+, NK, macrophages, B-cells and macrophages) increased in liver leukocytes during progressive injury. Further, we have made the novel & important observation that CD45+ cells form aggregates in progressive human liver injury irrespective of cause of injury. Anti-CD147 intervention leads to a reduction in immune cell aggregation as well as reduction in serum transaminases (C57Bl6 & BALB/c, CCl4 & TAA. No significant change in the total number of CD45+ cells infiltrating the liver. Aggregates that diminish with α-CD147 intervention in injury contain macrophages/Kupffer cells (F4/80) and B220 cells. Further, CD147 −/− animals show a decrease in the number of immune aggregates compared to wild type mice with equivalent injury (CCl4 and TAA). In-vitro analysis of both inflammatory and HCC cell lines showed significantly less aggregation with the addition of α-CD147 mAb (p < 0.05). Conclusion This study has demonstrated that with progressive inflammation associated liver injury and HCC that liver leukocytes and HCC cells undergo aggregation and that with inflammation this directly

contributes to the magnitude of the injury. Specifically, B-cells (B220/ CD19), macrophages/Kupffer cells (F4/80), neutrophils (GR-1) and Hepa 1–6 cells are dependent on CD147 interaction for their intrahepatic aggregation. This study is the first to demonstrate a completely novel mechanism of intrahepatic immune and HCC cell aggregation, which directly impacts the magnitude of intrahepatic injury.

Genetic background influences fibrotic severity of NASH in foz/foz mice via factors modulated by metabolic determinants AUVRO R MRIDHA,1 CLAIRE LARTER,1 MATTHEW M YEH,2 TORI NGUYEN,1 DEBORAH HEYDET,1 NICHOLAS SHACKEL,3 SUSAN V MCLENNAN,4 NARCISSUS TEOH,1 GEOFFREY FARRELL1 1 Australian National University Medical School at The Canberra Hospital, Garran, ACT, Australia, 2 Department of Pathology, University of Washington, Seattle, WA, 3Centenary Institute, Camperdown, NSW, Australia, 4Department of Endocrinology, Royal Prince Alfred Hospital, Sydney, NSW, Australia Background Genetic factors influence progression of fibrosis in NASH, such as via PNPLA3 polymorphisms in humans. We have previously reported that foz/foz mice on HF-Diet develop obesity, diabetes, dyslipidaemia, hepatic steatosis and inflammation (NASH). However, there are major differences in liver fibrosis between both male and female BALB/c and C57BL/6 (C57) foz/foz mice, even though both strains are obese and develop NASH. Aims In this study, we investigated how strain differences influence liver fibrosis in equally obese mice. Methods Mice from BALB/c and C57 background were fed HF diet 6 mo; steatohepatitis severity, markers of metabolic syndrome and fibrosis were measured. Protein and mRNA levels were assessed by Western Immunoblot and RT-qPCR. Tissue protein localisation was by immunofluorescence staining, MMPs-2 and −9 by zymography. Total MMP activities were localised by in-situ zymography and quantified using a fluorimetric assay. Results After 6 mo HF-feeding, foz/foz were similarly obese, irrespective of strain (and gender). BALB/c mice showed lower ALT (120 U/L vs C57 > 500 U/L), lower blood glucose, lower insulin, smaller liver and higher serum adiponectin compared to C57. Picro Sirius Red staining clearly demonstrated fibrosis was more severe in C57 mice, but was absent in Balb/c mice. Collagen 1 mRNA was 4.5-fold higher in C57 mice compared to Balb/c mice, and α-SMA expression was also increased in C57 mice. Although hepatic TGF-β levels were similar between strains, mRNA levels of CTGF were increased in C57 mice. MMP activity was localised in a pattern similar to fibrosis in C57 mice, but not in BALB/c. Increased expression of CD147 localised around hepatocytes was also clearly evident only in the C57 mice. In addition, C57 foz/foz mice on HF diet showed increased MMP-2 (3fold), MMP-9 (3fold) and total MMP activity (1.5fold) compared to WT-Chow mice. These parameters were not affected significantly by foz/foz-Chow or WT-HF diet. Conclusion The results from this study show that although obesity develops independently of background strain in foz/foz mice, fibrosis develops in a strain-specific manner, most likely via metabolic effects (hyperinsulinaemia, hyperglycaemia) that influence CTGF and MMP expression and function.

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Oestradiol and progesterone attenuate diethylnitrosamine-induced liver carcinogenesis in association with restitution of G1/S checkpoint control SHARON POK, VANESSA A BARN, GEOFFREY FARRELL, NARCISSUS TEOH Liver Laboratory, ANU Medical School at The Canberra Hospital, Garran, ACT, Australia Background We have previously shown that cyclin E and its low molecular weight (LMW) isoforms facilitate dysplastic hepatocytes to bypass G1/S checkpoint, and enhance proliferative drive in diethylnitrosamine (DEN)-induced hepatocellular carcinoma (HCC) in male mice (JGH 2011:26[S4]:3). While male predominance of both clinical and experimental HCC is well known, the role of sex hormones in hepatocarcinogenesis remains poorly defined. Aims To determine the gender differences and effect of oestradiol/progesterone (E/P) and testosterone in HCC development. Methods Sex hormonal manipulation studies using orchidectomy (ORC) and ovariectomy (OV) with E/P or testosterone implants were performed (Table 1) in DEN-injected (10 mg/kg ip, age day 15) C57BL/6 adult mice. Dysplastic liver and HCCs at 6, 9 and 12 mth were analysed for cyclin E, its kinase activity and effect on proliferation, tumour suppressors p53 and p21 by immunoblotting, radioimmunoassay, immunohistochemistry and immunoprecipitation. Results DEN-treated intact female mice displayed a marked reduction in dysplastic nodules (DNs) compared to intact males at 3, 6 mth, while HCC incidence, number and size of tumours were significantly lower in females. In intact female mice, cyclin E (native and LMW isoforms) protein expression and kinase activity were decreased compared to males at 6–12 mth, with concomitant reduction in hepatocyte proliferation by PCNA and cyclin D1 expression. G1/S checkpoint control was operative in DEN-female liver, evident by associated robust p53-mediated apoptosis and functional p21 inhibition of cdk2 and cdk4. ORC-DEN male mice resulted in a loss of cyclin E LMW isoforms compared to intact males, diminution of cyclin E kinase activity and pRb expression, induction of p53-mediated apoptosis in dysplastic liver, leading to a decrease in number of DNs by 6 mth. These anti-proliferative and pro-apoptotic effects were magnified by E/P replacement in ORC-DEN males. In contrast, testosterone replacement in OV-female mice restored LMW cyclin E expression, and these mice, like intact males, exhibited accelerated hepatocarcinogenesis compared to intact DEN-female animals. Conclusion Testosterone is the key driver of cyclin E aberrant (LMW) expression after DEN injection. In intact female mice or E/P implanted castrated male or female mice, E/P and lack of testosterone are associated with suppression of cyclin E LMW isoforms, functional G1/S checkpoint control and induction of p53-mediated cell death in pre-neoplastic hepatocytes. These hormonal factors are the basis for the delayed development of DNs and HCC in intact female mice and gonadectomised male mice, compared with intact males. Table 1. Males Surgery ORC ORC ORC Sham Females Surgery OV OV OV Sham

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Allocation of mice for hormonal manipulation study

Pellet implant None E/P Testosterone E/P

Age at termination (mths) 3, 6 (n = 5 each) 3, 6 (n = 5 each) 3, 6 (n = 5 each) 3, 6 (n = 5 each)

Pellet implant None Testosterone E/P Testosterone

Age at termination (mths) 6, 9 (n = 5 each) 6, 9 (n = 5 each) 6, 9 (n = 5 each) 6, 9 (n = 5 each)

TWEAK and Fn14 in human liver and the effect of cytokines and immunosuppression EMILIA PRAKOSO, DAVID BOWEN, GEOFFREY MCCAUGHAN, NICHOLAS SHACKEL Centenary Institute, Royal Prince Alfred Hospital, The University of Sydney, Sydney, NSW, Australia Background TWEAK (TNF-associated weak inducer of apoptosis), acting through its receptor Fn14, has been found as a novel mitogen for oval or hepatic progenitor cells in mice. The role of TWEAK in human liver injury is still not fully understood. In Hepatitis C Virus (HCV) recurrence post transplant, we have previously demonstrated an intense ductular reaction and have shown that this structure has unique phenotypes of hepatic progenitor cells. Aims In this study, we wished to examine the expression of TWEAK and Fn14 in HCV-associated liver injury pre and post transplant. Additionally, we sought to explore the role of TWEAK and Fn14 in human liver injury and regeneration by assessing their expression in human liver cells and analysing the in vitro effect of cytokines and immunosuppression on TWEAK and Fn14 expression, as well as the effect of TWEAK, cytokines and immunosuppression on liver cell proliferation. Methods Hepatocyte (PH5CH8), cholangiocyte (H69), and hepatic stellate cell (LX-2) lines were treated for 48 hours with IFN-γ, TNF-α, TGF-β, IL-6, steroid, cyclosporine, tacrolimus or azathioprine. qRT-PCR was used to assess TWEAK and Fn14 expression in cell lines post treatment, and in donor and HCV livers pre and post transplant. ELISA was utilised to assess TWEAK levels in cell cultures supernatant post treatment. Liver cell proliferation was assessed using alamarBlue® following treatment with TWEAK, cytokines and immunosuppressants. Results TWEAK and Fn14 were significantly upregulated in HCV livers pre transplant compared to donor livers (p ≤ 0.005), and TWEAK was also overexpressed in HCV livers with hepatocellular carcinoma (p ≤ 0.005); these findings however were not evident in the post transplant setting. In-vitro, TWEAK and Fn14 were expressed by PH5CH8, H69 and LX-2 cells, and expression was downregulated by cytokines and most immunosuppressants. However, Fn14 was significantly upregulated in PH5CH8 and H69 with high dose azathioprine (p ≤ 0.005). TWEAK was also detected in the culture supernatant of the three cell lines, and levels were reduced post treatment in H69 and LX-2 but increased significantly in PH5CH8 post low dose TNF-α (p ≤ 0.005). Moderate dose TWEAK increased PH5CH8 and LX-2 proliferation (p ≤ 0.005) while other cytokines and immunosuppression had a negative effect on cell proliferation. Conclusion TWEAK and Fn14 are present in human liver and cell lines, and generally are downregulated by cytokines and immunosuppression. Importantly, TWEAK and Fn14 are upregulated in HCV-associated liver injury pre transplant but not post transplant. This suggests that different cytokine profiles and immunosuppression may contribute to downregulation of TWEAK and Fn14, affecting liver regeneration post transplant for HCV liver injury.

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Deferasirox does not exhibit antifibrotic activity when administered to Mdr2−/− mice AMY SOBBE,1 KIM BRIDLE,1 CLOE ERIKA DE GUZMAN,1 NISHREEN SANTRAMPURWALA,1 NATHAN SUBRAMANIAM,2 DARRELL H CRAWFORD1 1 School of Medicine University of Queensland, Gallipoli Medical Research Foundation Greenslopes Private Hospital, Brisbane, QLD, Australia, 2Membrane Transport Laboratory, Queensland Institute of Medical Research, Brisbane, QLD, Australia Background Development of antifibrotic treatments is one of the most important challenges in contemporary hepatology. While many agents have shown effective antifibrotic properties in animal models, there has been little translation to clinical use. Evidence from a recent study in β-thalassaemia patients indicated that treatment with deferasirox reversed or stabilized liver fibrosis independent of its iron chelating properties1. Aims In this study we investigated the use of deferasirox in both cell and animal models to better understand any potential antifibrotic effects. Methods A human stellate cell line was treated with 5 μM or 50 μM deferasirox (Exjade) for up to 120 hours. Real time PCR was used to assess changes in the expression of fibrogenic genes. Three week old Mdr2−/− mice received oral deferasirox for 4 weeks (30 mg/kg/day). Liver tissue was collected for assessment of fibrosis. Results In stellate cells treated with 50 μM deferasirox for 12 hours α1(I)procollagen expression was decreased by 25%. Maximal reductions (10-fold) were seen with 24–120 hours of treatment. Similarly, αSMA expression was also significantly lower (45%, 71%, 88%, 89%, 81% and 77% reductions at 12, 24, 48, 72, 96 and 120 hours respectively). However, TGF-β mRNA expression was not different in treated vs. untreated cells. We also saw alterations in matrix remodelling genes, specifically decreased expression of MMP2 and TIMP2. Treatment with 5 μM deferasirox altered gene expression in the same pattern, however changes were of a smaller magnitude and occurred later in the time course. We also noted morphological changes in the cells after 48 hours of treatment with 50 μM deferasirox. These morphological changes were rapidly reversible upon removal of the drug. Despite these changes in cell culture, there was no significant difference in hepatic hydroxyproline content in Mdr2−/− mice following deferasirox administration (vehicle: 395 ± 27 μg/g vs. deferasirox: 421 ± 33 μg/g). Similarly, there were no reductions in the expression of key fibrogenic genes including α1(I)procollagen, Tgf-β, αSma, PdgfrB, Mcp-1 and Timp-1. Conclusion Despite inducing reductions in α1(I)procollagen and αSMA expression and alterations in matrix degradation genes in the stellate cell line, deferasirox did not exhibit antifibrotic activity in Mdr2−/− mice. Given the positive outcomes seen in human trials, it may be appropriate to study deferasirox in other animal models of fibrosis and/or for a longer duration of therapy. Reference 1. Deugnier Y. et al. Gastroenterology 2011;141:1202–1211.

Iron toxicity impairs autophagy and promotes ER stress in obesity and alcohol-induced fibrosing steatohepatitis TERRENCE C TAN,1 DARRELL H CRAWFORD,1 LESLEY A JASKOWSKI,1 THERESE L MURPHY,1 NISHREEN SANTRAMPURWALA,1 MANDY L HERITAGE,1 NATHAN SUBRAMANIAM,3 ANDREW CLOUSTON,1 DENIS I CRANE,5 GREGORY ANDERSON,4 LINDA M FLETCHER2 1 School of Medicine, University of Queensland, South Brisbane, QLD, Australia, 2Department of Gastroenterology, Princess Alexandra Hospital, Brisbane, QLD, Australia, 3Membrane Transport Laboratory, Queensland Institute of Medical Research, Herston, QLD, Australia, 4Iron Metabolism Laboratory, Queensland Institute of Medical Research, Herston, QLD, Australia, 5School of Biomolecular and Physical Sciences, Griffith University, Nathan, QLD, Australia Background Iron overload is a co-factor in aggravating liver injury induced by alcohol and obesity but the underlying mechanisms remain unclear. Aims We investigated the mechanisms of co-toxicity in a mouse model of fibrosing steatohepatitis caused by alcohol, a high-fat diet (HFD) and iron overload. Methods Wild-type (WT) and the iron-loaded Hfe-null (Hfe−/−) mice were fed either chow or a HFD with or without ethanol (20%) in the drinking water for eight weeks. Results WT mice developed mild steatohepatitis with combined ethanol and HFD. However, severe steatohepatitis and advanced fibrosis with portal-to-portal tract linkage fibrosis occurred in the similarly-fed Hfe−/− mice. This was associated with obesity, hypertriglyceridaemia, hepatic iron overload (2-fold over mean WT values), a 7-fold increase in serum ALT, Tnf-α mRNA induction and upregulation of pro-apoptotic (Bax and Bcl2) profibrogenic (Col1a1, Col3a1, Col4a1, Tgf-β1 and α-Sma) and matrix turnover (Mmp-2, Timp-1 and Timp-2) genes. Fibrotic livers also displayed increased intrahepatic hypoxia, adiponectin resistance, impaired AMPK signalling and ER stress as evident by increased CHOP, PERK and p-ERK1/2 protein levels but impaired unfolded protein response (decreased IRE1α, XBP1 and p-JNK). A reduction in p-Akt and PTEN also suggested insulin resistance in the fibrotic livers, while increased p62 and reduced Atg3, Atg7 and beclin-1 protein levels suggest defective autophagy. Conclusion Iron toxicity promotes ER stress and impaired autophagy in alcohol and obesity-induced liver injury. This may have important implications for the treatment of hepatic fibrosis.

Microparticles mediate liver ischemiareperfusion injury and are the therapeutic targets of the annexin V homodimer, diannexin NARCISSUS TEOH,1 HUSSAM AJAMIEH,1 KEVIN CROFT,2 TREVOR A MORI,2 ANTHONY C ALLISON,3 GEOFFREY FARRELL1 1 Liver Research Group, Australian National University Medical School at The Canberra Hospital, Canberra, ACT, Australia, 2Medicine and Pharmacology, University of Western Australia, Perth, WA, Australia, 3 Alavita Inc, San Francisco, CA Background Ischemia-reperfusion injury (IRI) can cause hepatic failure after liver surgery or transplantation. Damage originates with sinusoidal endothelial cells (SECs), which by unknown processes recruit/activate

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Kupffer cells (KCs) and platelets leading to microcirculatory impairment. We hypothesized that injured SECs release microparticles (MP) whose pro-inflammatory effects are a critical link in IRI pathogenesis. Aims To elucidate the pathogenic significance of MPs in contributing to the microvascular inflammatory and pro-thrombotic response in hepatic IRI. Methods We subjected mice to 60 min partial hepatic ischemia and 15 min–24 hr reperfusion, measured circulating MPs, and determined their protein and lipid content. We utilized in vitro approaches in primary hepatocytes to determine mechanisms for MP production and clarify the biological properties of MPs. The effects of an annexin V-homodimer, Diannexin on post-ischemic MP production and function were characterized. Results Circulating MPs released at 15–30 min post-ischemic reperfusion exhibited markers of SECs, platelets, natural killer-T and CD8 cells, followed later (4 hr) by macrophages. MPs contained F2-isoprostanes, biomarkers of oxidative damage to membrane lipids. TNFα infusion accentuated MP formation, while Diannexin substantially reduced MP release and prevented IRI. Hypoxia-reoxygenation (used to simulate IRI in vitro) generated MPs from primary hepatocytes via oxidative stress (H2O2) and Ca2+-dependent processes, accentuated by TNFα. MPs isolated from the circulation after hepatic IRI were added to cultured hepatocytes. Such MPs were engulfed by hepatocytes in vitro and injured them by a process involving mitochondrial membrane permeability transition. MPs also activated platelets and induced migration of inflammatory cells in vitro. The pro-inflammatory properties of MPs involved NF-κB, JNK activation, E-selectin, P-selectin, ICAM-1 and VCAM expression; these processes were blocked by ‘coating’ MPs with Diannexin. Conclusion MPs generated during hepatic IRI are mainly derived from SECs, lymphocytes and platelets. These MPs may be taken up by hepatocytes to activate signalling pathways that mediate inflammation and hepatocyte injury in IRI. Diannexin prevents MP formation and abrogate their pro-inflammatory consequences.

The secretion of cytokines has a greater influence on the severity of acute hepatitis than the survival of infiltrating CD8 T cells MICHELLE VO,1 LAUREN HOLZ,1 VOLKER BENSELER,1 CLAIRE MCGUFFOG,1 ANDREAS STRASSER,2 PHILIPPE BOUILLET,2 ROBYN STARR,3 DOUG HILTON,2 GEOFFREY W MCCAUGHAN,1 DAVID G BOWEN,1 PATRICK BERTOLINO1 1 Centenary Institute, Sydney, NSW, Australia, 2Walter Eliza Hall Institute (WEHI), Melbourne, NSW, Australia, 3 St Vincent’s Institute, Melbourne, NSW, Australia Background Acute hepatitis is often mediated by CD8 T cells that directly kill hepatocytes or secrete hepatotoxic cytokines. Although the mechanisms by which cytotoxic CD8 T cells mediate liver damage are known, the parameters that regulate CD8 T cell effector function during acute hepatitis are not well understood. Aims To determine the intrinsic T cell parameters that regulate CD8 T cell induced acute hepatitis. Methods We used a well-characterized mouse model of immune mediated hepatitis in which acute severe, but self-limiting, liver injury was induced by the transfer of T cell receptor (TCR) transgenic (Tg) CD8 T cells into Tg recipient mice expressing the antigen recognized by these T cells in the liver. Acute hepatitis mediated by Tg T cells was assessed by measuring serum alanine transaminase (ALT) levels and histology. To investigate whether cytokine regulation and apoptosis of CD8 T cells were critical in limiting the acute damage induced by cytotoxic T lymphocytes (CTLs), we performed transfer experiments using TCR Tg T

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cells deficient for either suppressor of cytokine signalling-1 (SOCS-1) or the pro-apoptotic molecule Bim. Results Bim−/− Tg T cells accumulated in the livers of recipient mice at 6-fold higher levels compared to wild-type (WT) Tg cells. Despite this substantial accumulation, the outcome of hepatitis remained unchanged, neither more prolonged nor severe. These findings suggest that, although T cells died in a Bim-dependent manner, T cell survival was not a critical parameter in limiting liver damage. In contrast, SOCS-1−/− Tg T cells induced more severe hepatitis compared to their WT counterparts, as indicated by significantly elevated ALT levels. SOCS-1−/− Tg T cells isolated from the liver displayed upregulated IL-2Rα chain, required to form the high affinity IL-2R that is necessary for IL-2 induced CD8 T cell survival and proliferation. Upregulated IL-2Rα was associated with higher IFN-γ production, CTL activity and proliferation rates, consistent with enhanced effector function. Together, these data support a critical role for cytokines in CTL function during acute hepatitis. Conclusion Our findings demonstrate that the propensity of CD8 T cells to mediate acute hepatitis is determined by the quality, rather than the number of CTLs infiltrating the liver. In the long term however, both Bim and SOCS deficiency led to the accumulation of CD8 T cells that were unable to cause chronic liver damage. These results reveal the existence of mechanisms able to silence potentially autoreactive T cells surviving acute hepatitis. Such mechanisms might explain why immune infiltrates do not always correlate with ALT levels in patients chronically infected with HBV/HCV or with other immune mediated liver conditions such as autoimmune hepatitis.

The role of adiponectin in hepatocellular carcinoma: adiponectin loss is associated with a more aggressive phenotype SARAH L WALKER, NIKITA DEO, JACOB GEORGE, LIONEL HEBBARD Storr Liver Unit, Westmead Millennium Institute, Westmead, NSW, Australia Background Hepatocellular carcinoma (HCC) is the fastest rising incident cancer in Australia. Most HCCs arise in cirrhotic livers but obesity is an independent risk factor for its development. In addition, obese patients have higher death rates from liver cancer. The mechanisms for this association are unknown. Adiponectin, a protein secreted by adipocytes, is reduced in obesity and low levels are associated with many common forms of cancer. Aims The aim of our study was to investigate the role of adiponectin in a mouse model of HCC and its mechanism of action. Methods Hepatocellular carcinoma was induced in adiponectin knockout (ko) and wild type mice using diethylnitrosamine (DEN). At monthly intervals from 3 to 9 months after injection, mice were euthanased, blood collected, livers dissected and weighed. Tumour incidence (>0.5 mm) and dimensions were measured and tumour volume estimated. Paraffin embedded livers were sectioned and stained. Protein extracts from liver and tumour tissue were subjected to immunoblotting to examine signal transduction pathways. Total tissue mRNA was extracted to assess expression of key inflammatory mediators. Primary mouse tumour cells were cultured. Adiponectin ko and wild type derived HCC cells were characterised with regards to cell signalling and ability to form tumour spheres. Primary cultured HCC cells were subcutaneously injected into wild type mice and growth characteristics studied. The cells were treated with mTOR inhibitors: rapamycin, PP242 and BEZ235. Results Following DEN treatment, male adiponectin knockout (ko) mice developed significantly larger liver tumours. This was reflected in both greater calculated tumour volume (207 ± 463 mm3, n = 21 vs 30.2 ± 70.2 mm3, n = 22; p = 0.049) and liver: body weight ratio (5.5 ± 1.3%, n

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= 21 vs 4.5 ± 0.47%, n = 22; p = 0.0008). These tumours displayed distinct histological features with steatosis and eosinophilic inclusions. There were no differences in tumour inflammatory markers. Immunoblotting revealed increased activation of the Akt-mTOR pathway in adiponectin ko tumour compared to wild type. Treatment of primary cultured HCC cells with mTOR inhibitors revealed that adiponectin ko tumour cells required higher doses of all inhibitors to reduce proliferation. Primary cultured adiponectin ko HCC cells formed larger tumour spheres and larger subcutaneous tumours at 28 days post-injection into wild type mice, suggesting a more aggressive phenotype (121 ± 157 mm3, n = 12 vs 35.0 ± 35.5 mm3, n = 12; p = 0.043). Conclusion This study demonstrates that adiponectin loss is associated with larger and more aggressive liver tumours with distinct histological and signalling changes. These tumours display up-regulation of the AktmTOR pathway, which has been shown to be associated with a poor prognosis in human HCC. We propose that obesity and the associated reduced adiponectin may cause a more aggressive tumour phenotype in human HCC.

Conclusion These results suggest that Hfe and Tfr2 are involved in the iron-induced upregulation of Bmp6 and hepcidin. This study also highlights the role of Hfe and Tfr2 in the differential handling of iron administered by different routes and distributed in different cell types. Hfe and Tfr2 do not appear to be required for non-parenchymal iron-induced upregulation of Bmp6, but are important for hepatocyte iron-induced upregulation of Bmp6. Hfe and Tfr2 are required for the correct response of hepcidin to increased levels of Bmp6.

A central role for HFE and transferrin receptor 2 in the non-parenchymal cell-derived BMP6mediated regulation of hepcidin DANIEL WALLACE, CAMERON MCDONALD, LESA OSTINI, NATHAN SUBRAMANIAM Queensland Institute of Medical Research, Brisbane, QLD, Australia

Curcumin and vitamin E combination treatment reduces the severity of disease in a diet-induced mouse model of NAFLD ASHLEY S WILKINSON,1 MANDY L HERITAGE,1 LESLEY A JASKOWSKI,1 LAURENCE J BRITTON,1 TERRENCE C TAN,1 ANDREW CLOUSTON,1 KIM BRIDLE,1 GRAEME A MACDONALD,3 GREGORY ANDERSON,2 LINDA M FLETCHER,3 NATHAN SUBRAMANIAM,2 DARRELL H CRAWFORD1 1 School of Medicine, University of Queensland, Gallipoli Medical research centre, Greenslopes Private Hospital, Brisbane, QLD, Australia, 2Cell and Molecular Biology, The Queensland Institute of Medical Research, Brisbane, QLD, Australia, 3Gastroenterology and Hepatology, Princess Alexandra Hospital, Brisbane, QLD, Australia

Background The liver-expressed peptide hepcidin plays a central role in the regulation of iron homeostasis and the pathogenesis of iron overload disorders such as hereditary haemochromatosis (HH) and iron deficiency disorders such as anaemia. BMP/SMAD signalling has emerged as a major pathway responsible for the upregulation of hepcidin in response to iron. BMP6 expression is upregulated by iron in the liver and signals through the SMAD pathway to increase hepcidin expression. Elevated levels of hepcidin then decrease iron absorption and recycling by reducing the cell surface expression of the iron exporter ferroportin. The HH associated proteins HFE and TFR2 are also involved in hepcidin regulation, but their precise roles are still unclear. Aims The aims of the study were to examine the response of the hepcidin regulatory pathway to different iron challenges in the presence or absence of Hfe and Tfr2. Methods Wild-type and Hfe−/−/Tfr2−/− mice on a C57BL6 background were challenged with iron either via dietary or parenteral routes. Dietary iron was administered by feeding mice with a high iron diet (2% carbonyl iron) from weaning for 2 weeks; control mice were fed an equivalent diet containing 0.0075% iron. Parenteral iron was administered via a single injection of iron-dextran (0.3 mg/g) four days prior to sacrifice; control mice were injected with saline. All mice were sacrificed at 5 weeks of age and blood and tissues taken for analysis. Serum and tissues iron indices were measured and the expression of iron-associated genes and proteins in the livers of mice were assessed by real-time PCR or western blotting. Results While dietary iron resulted in exclusive hepatocyte iron loading with a predominantly periportal distribution in the liver, parenteral iron accumulated predominantly in macrophages and Kupffer cells. The response of the hepcidin regulatory pathway to iron challenge was blunted in the Hfe−/−/Tfr2−/− mice, with differential responses of hepcidin and Bmp6 to the route of iron administration. In Hfe−/−/Tfr2−/−, the Bmp6 response was blunted in iron fed mice, but not in mice injected with iron. Smad signalling was unaffected relative to Bmp6 in all animals, however, whilst Hfe−/−/Tfr2−/− mice fed iron showed mildly reduced hepcidin upregulation relative to pSmad, iron-dextran injected mice showed significantly impaired regulation of hepcidin by pSmad.

Background Nonalcoholic fatty liver disease (NAFLD) is a common cause of chronic liver disease and end stage liver failure. Therapies to reduce the inflammatory and oxidative pathogenic mechanisms involved in the progression of NAFLD have limited efficacy. Aims This study aims to replicate the clinical setting of NAFLD by feeding mice a high-fat, high-carbohydrate diet (HF/HCD) and investigating the effects of the putative anti-inflammatory and anti-oxidant agents, curcumin and vitamin E in comparison to removal of the HF/HCD. Methods Wild-type C57BL/6J mice (8 weeks old, n = 80) were fed a HF/HCD for 10 weeks and then for a further 10 weeks fed either a HF/ HCD (n = 10) or chow (n = 9) (CH) or treated (n = 10) as follows: HF/ HCD + 1% curcumin (CU); HF/HCD + 1.5% Vitamin E (VE); HF/HCD + 1% curcumin + 1.5% Vitamin E (CUVE). Statistical analysis was performed using one-way ANOVA. Results The mice fed a HF/HCD for 20 weeks developed widespread macro- and micro-vesicular fat deposits. Those mice switched to chow (CH) had a significant reduction of both macro- and micro-vesicular fat deposits, with the liver returning to virtually normal histology. Treatment groups CU or VE alone showed reduced fat deposits; however, the combination treatment, CUVE, resulted in a greater reduction of both macroand micro-vesicular fat deposits and the degree of change was similar to the CH group. There was no evidence of fibrosis in the mice fed a HF/ HCD for 20 weeks as assessed by Pico Sirius Red staining and no upregulation of a panel of fibrosis-related genes apart from Timp-1 expression which was elevated in HF/HCD and suppressed in all treatment groups. Genes involved in fatty acid synthesis, (Srebp-1c, Fas and Acc1) were suppressed in the HF/HCD mice and significantly elevated in CH, CU, VE and CUVE groups. Mice fed CU and CUVE showed an upregulation in Cpt1 which suggests an increase in mitochondrial uptake of fatty acids. There was also an upregulation of PPARα and adiponectin receptor 2 in these mice which suggests an increase in β-oxidation pathways. Mitochondrial biogenesis genes (Nrf-1 and Tfb2) were suppressed in HF/ HCD fed mice and upregulated in CH, CU, VE and CUVE fed mice. There was no change in catalase activity, reduced/oxidized glutathione ratio for all groups of mice. Glutathione peroxidase activity was increased in VE mice compared to HF/HCD controls.

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Conclusion Treatment of mice with a HF/HCD for 20 weeks resulted in moderate to severe steatosis which was completely reversed by removing the HF/HCD. Combination treatment of curcumin and vitamin E reduced steatosis in this mouse model to a similar extent as removing the injurious HF/HCD and the alterations in lipid signalling and the adiponectin insulinsensitising system are possible pathogenic mechanisms that need to be further explored.

Notch and IL6 signalling pathways interact in hepatocellular carcinoma GEORGE WILSON, JACOB GEORGE, KENNY HU, AIPING TIAN, LIANG QIAO Storr Liver Unit, Millennium Institute, Westmead, NSW, Australia Background Inflammation mediated through IL-6 plays a key role in driving hepatocellular carcinoma (HCC) tumorigenesis. Notch signalling pathway is a key regulatory pathway that mediates tumorigenesis in a variety of solid tumours. Aims This study explores the interaction between IL6 and Notch in HCC and the functional significance of the interaction. Methods Immunohistochemistry and western blots of IL-6 and Notch signalling components were undertaken on HCC tumour biopsies. C57 and IL6 knockout mice were injected with diethylnitrosamine (DEN) at 100 mg/kg and harvested at 12 h, 24 h, 36 h, 48 h, 72 h and 96 h after injection. HCC cell lines SNU182, SNU423 and HUH7 were transfected with an IL-6 overexpression plasmid. The three HCC cell lines were also treated with the Notch inhibitor RO4929097. Gene expression of HCC cell lines was determined using quantitative real time PCR (qPCR), luciferase reporter plasmids and western blots. Colony formation assays, BrDU cell proliferation assays and annexin V apoptosis assays were used to determine the effect of IL6 and Notch signalling on tumorigenesis. Results Western blots of HCC tumour biopsies showed a strong correlation between IL-6 and Notch signalling. Immunohistochemistry showed both IL-6 and Notch signalling components were predominantly expressed within the hepatocytes of HCC tumours. Western blots on livers harvested from mice after DEN injection demonstrated that DEN-induced IL-6 up regulation resulted in an up regulation of Notch signalling components in C57 mice. Conversely in IL-6 knockout mice there was no upregulation of Notch signalling components after DEN injection. In vitro, qPCR and luciferase reporter assays on HCC cells transfected with IL-6 overexpression plasmid showed an upregulation of Notch signalling components. The interaction between IL-6 and Notch signalling affected apoptosis. Treatment of HCC cell lines with the Notch inhibitor RO4929097 increased apoptosis, reduced cell proliferation and decreased colony formation. Importantly, treatment with RO4929097 abated the increased tumorigenesis in the three HCC cell lines treated with the IL-6 overexpression plasmid. Conclusion Our data shows a positive interaction between IL6 and Notch signalling in HCC. The interaction between IL6 and Notch appears to affect HCC tumorigenesis behaviour in vitro.

Characterisation of ARL6IP5 in hepatitis C induced liver cancer GEORGE WILSON, JACOB GEORGE, KENNY HU, LIANG QIAO Storr Liver Unit, Westmead Millennium Institute, Westmead, NSW, Australia Background Hepatitis C virus (HCV) infection is the major etiological agent causing HCC. We recently identified a novel gene ADPribosylation-like factor 6 interacting protein 5 (ARL6IP5) that was sig-

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nificantly upregulated during HCV infection. ARL6IP5 plays a role in tumorigenesis in a variety of solid cancers and haematological malignancies. Aims This study explores the role ARL6IP5 plays in HCC tumorigenesis. Methods Gene expression of ARL6IP5 in human liver tissue and HCC cell lines were determined using western blotting, immunohistochemistry and quantitative real time PCR (qPCR). To induce HCC in mice, C57 mice were injected with diethylnitrosamine at 14 days of age. Livers were then harvested 9 months post injection and separated into tumour and normal tissue. HCC cell line HUH7 was infected with JFH1 strain of HCV. HCC cell lines SNU423, HUH7 HCV+ and HUH7 HCV− cells were transfected with siRNA targeting ARL6IP5. PCNA Cell proliferation and colony formation assays were undertaken to determine the effect of ARL6IP5 on HCC tumorigenesis. Results Western blotting, immunohistochemistry and qPCR of HCC tumour biopsies and adjacent normal tissues showed higher expression of ARL6IP5 in HCC tissues. The expression of ARL6IP5 was 5-fold greater in HCV+ HCC tissues relative to HCV− HCC tissues. Western blotting of mice HCC tumours showed increased expression of ARL6IP5 compared to normal tissues. In vitro, qPCR and western blotting confirmed HCV specific upregulation of ARL6IP5 with HCV infected HUH7 cells showing increased ARL6IP5 expression relative to non infected HUH7 cells. Treatment of HCC cell lines SNU423, HUH7 HCV+ and HUH7 HCV− cells with ARL6IP5 siRNA lead to reduced cell proliferation. ARL6IP5 siRNA also reduced colony formation in SNU423. Conclusion ARL6IP5 is upregulated in HCC and its expression is further increased by HCV infection of HCC tissues. ARL6IP5 plays a role in promoting HCC tumorigenesis.

Efficacy of liver cancer stem cell markers in isolating and characterising liver cancer stem cells GEORGE WILSON, JACOB GEORGE, KENNY HU, AIPING TIAN, LIANG QIAO Storr Liver Unit, Westmead Millennium Institute, Westmead, NSW, Australia Background Recent evidence suggests a subset of HCC tumours are derived from liver cancer stem cells (LCSCs). In order to effectively isolate and characterise LCSCs, markers that are specific to LCSCs are needed. Aims Our study tests the efficacy of the most widely utilised stem cell markers to isolate and characterise LCSCs. Methods Immunohistochemistry and western blots of LCSC markers (CK19, CD133, ALDH, CD90, CD44, Epcam, AFP) were undertaken on HCC tumour biopsies and adjacent normal tissues. Magnetic bead separation was used to separate cells from cell lines HUH7, SNU423, SNU182 and PLC/PRF/5 into subpopulations based on the expression of LCSC markers. Flow cytometry was undertaken on the total cell population, the marker enriched and marker depleted subpopulations to confirm marker enrichment. Tumour sphere assays were then undertaken on the marker enriched and depleted fractions to detect the presence of stem cells. Results Western blots of human biopsies showed no significant difference in stem cell marker expression between tumour and adjacent normal tissues. Tumour sphere assays on stem cell enriched and depleted subpopulations showed not all enriched fractions showed an increase in stem cells. Immunohistochemistry analysis of HCC tumour biopsies and flow cytometry analysis of HCC cell lines showed LCSC derived from different patients and cell lines express unique combinations of LCSC markers. Conclusion Our data shows the most widely used LCSC markers are not specific to LCSCs. We have also shown LCSCs express phenotype specific combinations of LCSC markers. This finding suggests there are subtypes of LCSCs.

Journal of Gastroenterology and Hepatology 2012; 27 (Suppl. 4): 2–16 © 2012 Journal of Gastroenterology and Hepatology Foundation and Wiley Publishing Asia Pty Ltd

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Emu oil maintains intestinal goblet cell numbers and reduces acute inflammation in a rat model of chemotherapy-induced mucositis SUZANNE M ABIMOSLEH,1 CUONG TRAN,2 ZAHRA LOTFOLLAHI,1 ELAINE M PENASCOZA,1 DAMIANO G STOMACI,1 GORDON HOWARTH3 1 Discipline of Physiology, School of Medical Sciences, The University of Adelaide, Adelaide, SA, Australia, 2 Gastroenterology Department, The Women’s and Children’s Hospital, North Adelaide, SA, Australia, 3 School of Animal and Veterinary Sciences, The University of Adelaide, Roseworthy, SA, Australia Background Mucositis, a serious side-effect of cancer chemotherapy, is characterised by intestinal inflammation and ulceration. We have previously demonstrated1 that Emu Oil (EO; extracted from Emu adipose tissue) administered during the initial phase of experimentally-induced mucositis, improved intestinal repair and decreased inflammation. Aims A mechanism of EO action was sought by determining the number of protective neutral mucin-secreting goblet cells (GC) in the mucosa, indicative of intestinal barrier function and determining levels of acute inflammation during the recovery phase of chemotherapy-induced mucositis in rats. Methods Female Dark Agouti rats (n = 8/group) were gavaged with water (1 ml), Olive Oil (OO; 1 ml) or EO (1 ml) once daily and injected with 5-Fluorouracil (5-FU; 150 mg/kg) or saline, on day 5. Rats were euthanized on days 8, 9, 10 and 11 for tissue collection. Jejunal and ileal GC were stained with Periodic acid-Schiff reagent for neutral mucins. Furthermore, acute inflammation was assessed in the jejunum and ileum using a colorimetric assay for myeloperoxidase (MPO), an enzyme found in the intracellular granules of neutrophils. p < 0.05 was considered significant. Results 5-FU decreased GC numbers by 35% (jejunum) and 53% (ileum) on day 8; and by 45% (jejunum) on day 9, compared with healthy controls (p < 0.05). However, no such differences were observed on days 9 (ileum only), 10 or 11. Amongst 5-FU-injected groups, both OO and EO resulted in a greater number of ileal GC on day 10, by 75% and 53%, respectively, compared with 5-FU controls (p < 0.05). However, on day 11, only EO increased ileal GC numbers compared to 5-FU controls (53%; p < 0.05). EO did not significantly affect jejunal GC numbers in normal or 5-FU-injected rats. MPO levels were greater in the jejunum of 5-FU-injected rats on days 8 (403 ± 60 U/g) and 9 (150 ± 28 U/g) compared with healthy controls (107 ± 20 U/g and 63 ± 17 U/g respectively; p < 0.05), which was normalised by day 10. Amongst 5-FU-injected groups, both OO and EO resulted in decreased levels of acute inflammation in the jejunum on days 8 (223 ± 39 U/g and 155 ± 14 U/g respectively) and 9 (44 ± 10 U/g and 25 ± 9 U/g respectively) compared with 5-FU controls (p < 0.05). Moreover, on day 8, MPO levels were greater in the ileum of 5-FU-injected rats (424 ± 86 U/g) compared with healthy controls (55 ± 7 U/g; p < 0.05), however, there were no differences between these groups on subsequent days. Only EO decreased acute inflammation on day 8 in the ileum of 5-FU-injected rats (191 ± 12 U/g) compared with 5-FU controls (p < 0.05). Both OO and EO did not significantly affect basal levels of MPO in healthy rats in either the jejunum or ileum. Conclusion Following chemotherapy, Emu Oil maintained ileal barrier function and decreased myeloperoxidase levels in the jejunum and ileum of rats, suggesting therapeutic potential in mucositis.

Reference 1. Lindsay RJ, Geier MS, Yazbeck R, Butler RN, Howarth GS. Orally administered emu oil decreases acute inflammation and alters selected small intestinal parameters in a rat model of mucositis. Br J Nutr. 2010;104(4):513–9.

Attenuated immune response to Helicobacter infection in Winnie mouse model of spontaneous colitis SOPHIA P ANG,1 MHAIRI BAIRD,1 IDIT ZIV,1 MATTEW C COOK,2 DOUGLAS R TAUPIN1 1 Cancer Research, The Canberra Hospital, Garran, ACT, Australia, 2Immunology Research, The Canberra Hospital, Garran, ACT, Australia Background Epidemiological studies demonstrate a consistent negative association between the prevalence of Helicobacter infection and inflammatory bowel disease (IBD) and this is often attributed to a protective effect of chronic Helicobacter infection. The effect of IBD on the immune response elicited by Helicobacter Spp. is not known. Aims We characterised gastric pathology and immune response elicited by Helicobacter felis (H. felis) in a mouse model of spontaneous colitis. Methods Wild type (n = 38) and Winnie (n = 74) (bearing a mis-sense mutation in the Muc2 gene causing protein misfolding, ER stress and colitis) mice in C57Bl/6 background were infected with H. felis or BHI (sham infection) for up to 12 months. qPCR was used to determine RNA levels of IFNγ (Th1 cytokine), FOXP3 (Treg marker) and Th17 signature genes (RORC, IL17a, IL17f, Tgfb, Ccr6) in gastric mucosa. Activation of ER stress effectors GRP78 and CHOP in gastric mucosa were also assessed. To determine whether the immune dysregulation was secondary to colitis or to an intrinsic immune defect in Winnie, bone marrow chimeras of Winnie and wild type donors and wild type recipients were created. Results Wild type C57Bl/6 mice showed significant increase in IFNγ RNA level (P = 0.02) and marked increase of Treg and Th17 responses at 6 months post-infection. Levels of IFNγ remained elevated while Treg and Th17 responses diminished at 12 months post-infection in wild type mice. In contrast, Winnie mice exhibited attenuated immune response throughout the course of H. felis infection. Wild type mice made chimeric for Winnie bone marrow exhibited wild type immune responses to H. felis. Despite the lack of immune response in Winnie mice against H. felis infection, the ER stress effector CHOP was upregulated in both mouse models throughout the course of infection. Conclusion An attenuated immune response to gastric Helicobacter infection is seen in the Winnie model of IBD and arises secondary to the colitis phenotype rather than an intrinsic immune defect. These results indicate that (i) active IBD may alter the immune response to pathogens at remote sites and (ii) the outcome of chronic Helicobacter infection may be determined by co-morbid inflammatory conditions. References 1. Luther J, Dave M, Higgins PD, et al. Association between Helicobacter pylori infection and inflammatory bowel disease: a meta-analysis and systematic review of the literature. Inflamm Bowel Dis. 2010;16(6):1077–84. 2. Heazlewood CK, Cook MC, Eri R, et al. Aberrant mucin assembly in mice causes endoplasmic reticulum stress and spontaneous inflammation resembling ulcerative colitis. PLoS Med. 2008;5(3):e54.

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Chromosomal instability in BRAF mutant, microsatellite stable colorectal cancers CATHERINE E BOND,1 AARTI UMAPATHY,1 RON L BUTTENSHAW,1 LEESA F WOCKNER,2 DEREK J NANCARROW,3 LEANNE WALLACE,4 GRANT W MONTGOMERY,4 BARBARA LEGGETT,1 VICKI WHITEHALL1 1 Conjoint Gastroenterology Laboratory, Queensland Institute of Medical Research, Brisbane, QLD, Australia, 2Statistics Unit, Queensland Institute of Medical Research, Brisbane, QLD, Australia, 3 Oncogenomics Laboratory, Queensland Institute of Medical Research, Brisbane, QLD, Australia, 4Molecular Epidemiology Laboratory, Queensland Institute of Medical Research, Brisbane, QLD, Australia Background BRAF mutant colorectal cancers derive from serrated precursor lesions and progress via the ‘serrated neoplastic pathway’(1). Approximately half of all BRAF mutant cancers lose mismatch repair activity and accumulate microsatellite instability (MSI)(2). These BRAF mutant/MSI cancers have been well described and confer an excellent patient prognosis. By contrast, the remaining BRAF mutant cancers that are microsatellite stable (MSS), have not been well characterized and are associated with a very poor patient outcome(3). The molecular mechanisms underlying this are not well understood. BRAF wild type cancers are typically MSS, and progress from conventional adenomas to cancer via the ‘traditional pathway’, which involves acquisition of well described molecular events such as chromosomal instability (CIN)(4). CIN is linked to a worse prognosis in these cancers; whilst BRAF mutant/MSI cancers are diploid(5,6). Aims We aimed to further characterize the aggressive BRAF mutant/ MSS cancers of the serrated pathway, and hypothesized that CIN may be frequent in this cancer type. Methods Presence of CIN was investigated in 60 BRAF mutant/MSS (BRAFmut/MSS), and 90 BRAF wild type/MSS (BRAFwt/MSS) using loss of heterozygosity (LOH) analysis encompassing chromosomal regions 5q, 8p, 17p and 18q. Genome-wide SNP arrays (Illumina CytoSNP-12) were typed on a subset of MSS cancers (30 BRAFmut/MSS, 18 BRAFwt/ MSS), and compared to 30 BRAFmutant/MSI cancers. Results LOH analysis demonstrated comparably high rates of CIN in the MSS subgroups (72% BRAFmut/MSS, 82% BRAFwt/MSS). In BRAFmut/ MSS cancers, CIN correlated with advanced stage and worse survival at specific chromosomal regions. SNP arrays confirmed similarly frequent rates of CIN in both MSS cohorts, with 60% BRAFmut/MSS and 62% BRAFwt/MSS chromosomal arms affected. BRAFmut/MSI cancers had a low degree of CIN (13%). Of the regions with copy number variation, loss events were predominant over gain in both MSS cohorts (BRAFmut/MSS 533/736, 72%; BRAFwt/MSS 279/389, 72%). BRAFwt/MSS cancers mostly demonstrated whole chromosomal arm loss events (185/279, 66%), whereas BRAFmut/MSS cancers showed primarily regional loss events (336/533, 63%) (p < 0.0001). BRAFmut/MSS cancers had significantly greater rates of loss at 17q, 6p and 6q compared to BRAFwt/MSS cancers (p = 0.002, p = 0.009, p = 0.04 respectively). Conclusion This study has identified a novel form of genetic instability associated with BRAFmut/MSS cancers primarily involving regional rather than whole arm deletions. High rates of loss were identified in regions not typically implicated in colorectal cancer, which may contain putative tumour suppressor genes associated with the aggressiveness of this cancer type. References 1 Leggett and Whitehall. Gastroenterology. 2010,138;2088. 2 Kane et al. Canc Res. 1997,57;808. 3 Samowitz et al. Canc Res. 2005,65;6063.

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4 Fearon and Vogelstein. Cell. 1990,61;759. 5 Sinicrope et al. Gastroenterology. 2006,131;729. 6 Watanabe et al. J Clin Oncol. 2012, Apr 30.

Hypoxic regulation of β1 integrin during mucosal wound healing CIARAN CHANEY,1 KYRA MINAHAN,1 SEAN MATEER,1 GAVIN NOLAN,1 NICHOLAS TALLEY,2 SIMON KEELY1 1 School of Biomedical Sciences & Pharmacy, University of Newcastle, Newcastle, NSW, Australia, 2 Faculty of Health, University of Newcastle, Callaghan, NSW, Australia Background Crohn’s disease (CD) is characterised by repeated wounding and inflammation of the intestinal mucosa. During mucosal inflammation, changes in metabolic activity and vascular tissue damage, lead to a reduction in tissue-oxygen tension (hypoxia), in which healing processes such as angiogenesis, cell migration and re-epithelialisation occur. Previous work has shown that induction of hypoxia-inducible factor (HIF), a global transcriptional regulator of the hypoxic response, is protective in murine models of colitis. This appears to involve increased epithelial barrier function and restitution. Little is known about the role of HIF in mucosal wound healing. In a series of in vitro assays we identified induction of integrin β1 (ITGB1) during hypoxia. As integrins play critical roles in wound healing through, both epithelial proliferation and extracellular matrix (ECM) binding, we hypothesised that HIF promotes epithelial wound healing through ITGB1-dependent signalling. Aims Our aims were to 1) investigate the role of HIF in epithelial ITGB1 induction during hypoxia. 2) confirm a functional role for HIF induced β1-integrin 3) characterise ITGB1 expression in mucosal inflammation using a chemically induced (TNBS) murine model of colitis. Methods SKCO intestinal epithelial monolayers were subjected to normoxia, (p02 157 torr) or hypoxia (p02 20 torr) for 12 hr and examined for ITGB1 expression by qPCR. SKCOs were transfected with ITGB1Luciferease promoter constructs, with and without a functional HIF binding site and examined for hypoxia-response. Anti-β1 integrin antibodies were employed to examine whether induced β1-integrin influenced collagen binding and contraction in hypoxia. Colitis was induced in C57/ Blk6 mice by rectal instillation of Trinitrobenzene sulfonic acid (TNBS). Disease severity was monitored over 7 days. Mice were euthanised and colonic epithelial cells, harvested for mRNA and protein for examination by qPCR and western blot. Results Hypoxia led to a 2-fold induction in ITGB1 after 12 hours. ITGB1-promoter transfected cells demonstrated a 7-fold increase in promoter activity in response to hypoxia, which was lost in the absence of a functional HIF binding site. Incubation with anti-α2β1 integrin, led to 25% less epithelial-collagen interaction when compared to untreated cells, as measured by collagen contraction. TNBS colitis led to a significant increase in colonic epithelial ITGB1 transcript and protein (7-fold increase). A clear correlation (R2 = 0.71) between ITGB1 expression and disease severity (weight loss & colon shortening) was observed in colitic animals. Conclusion These data suggest that ITGB1 is regulated by HIF and that hypoxia leads to expression of functional collagen-binding β1-integrin. Further, ITGB1 is increased in epithelial cells during hypoxia concurrent with mucosal inflammation and may function as a mediator of epithelial wound healing. References 1. Karrasch et al. Z Gastroenterol (2009);47:1221–9. 2. Robinson et al. Gastroenterology (2008);134:145–55. 3. Karhausen et al. J Clin Invest (2004);114:1098–106. 4. Olson. Dig Dis Sci 38 (1993);388–95.

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Primary HIV-1 infection (PHI) is associated with reduced CD4 cell counts in terminal ileum biopsies, but not other gut biopsy sites MARK DANTA,1 JOHN ZAUNDERS,2 MICHELLE R BAILEY,3 KATHERINE MARKS,2 NABILA SEDDIKI,3 SEAN EMERY,3 JOHN M MURRAY,4 DAVID A COOPER,3 KERSTEN KOELSCH,3 ANTHONY D KELLEHER3 1 Clinical School, St Vincent’s Hospital, Sydney, NSW, Australia, 2HIV, St Vincents Centre for Applied Medical Research, Darlinghurst, NSW, Australia, 3Kirby Institute, UNSW, Sydney, NSW, Australia, 4Dept of Mathematics, UNSW, Sydney, NSW, Australia Background CD4 T lymphocytes in gut-associated lymphoid tissue (GALT) are believed to be particularly susceptible to cytopathic HIV-1 infection due to a heightened level of activation and increased expression of the co-receptor CCR5. Several studies have suggested a massive depletion during primary HIV-1 infection (PHI), based on a reduced proportion of CD4 cells in gut biopsies, compared to blood. This could also be due to a relative increase in CD8+ T lymphocytes in response to the acute viral infection, and no studies have accurately counted CD4 cells in biopsies. Aims To perform accurate CD4 cell counts in gut biopsies obtained during PHI Methods 7 patients, diagnosed with PHI, and 8 patients with chronic HIV-1 infection (CHI) were enrolled in the PINT trial of open label therapy with integrase inhibitor, Raltegravir, plus Truvada. All underwent endoscopy and colonoscopy with ten pinch biopsies taken from five sites: rectum, left colon (LC), right colon (RC), terminal ileum (TI) and duodenum before therapy and at 52 weeks. 12 healthy adult volunteers underwent colonoscopy with 10 biopsies from 3 sites. Single cell suspensions were prepared by digestion with collagenase type III and pushed through 70 μm nylon sieves. CD4+ cells were identified by flow cytometry, by first gating lymphocytes as CD45+/Epcam-negative, and then the total recovered CD4 cell count was calculated for each cell suspension using TruCount tubes. Results for patient groups were compared by MannWhitney test. Results The number of CD4 cells recovered from TI biopsies from HIVnegative volunteers was quite variable (median: 233,000; interquartile range: 88,704–583,508), but was significantly higher compared to TI biopsies from PHI subjects (median: 65,664; IQR 18,065–88,625; p = 0.023) and much higher than TI biopsies during CHI (28,595; IQR 19,111– 84,621; p = 0.003). However, CD4 cell counts for LC from HIV-negative controls (68,838; IQR: 44,827–196,930) were not significantly different to PHI (76,679; IQR: 45,680–104,691), but slightly higher than CHI (32,575; IQR 5,884–71,022; p = 0.063). Similarly, CD4 cell counts for RC from HIV-negative controls (124,301; IQR: 45,298–271,064) were not significantly different to PHI (150,920; IQR: 76,465–205,928), but much higher than CHI (19,148; IQR 8,568–69,213; p = 0.003). After 52 weeks of therapy, there was a trend to increased CD4 cell counts in biopsies for CHI subjects (p = 0.11). Conclusion PHI does not lead to widespread, dramatic changes in CD4 cell counts in GALT, unlike CHI where low counts were observed in all tissues. However, there was a significant reduction in CD4 cell counts for terminal ileum during PHI, and current work aims to better define this deficit in terms of CD4 subsets and function.

Spinal cord signalling in a mouse model of inflammatory bowel disease KRISTEN E FARRELL, SIMON KEELY, BRETT A GRAHAM, KYRA MINAHAN, JESSICA MADDEN, ROBERT CALLISTER School of Biomedical Sciences & Pharmacy, University of Newcastle, Newcastle, NSW, Australia Background Chronic pain is commonly experienced by patients with inflammatory bowel disease (IBD) during periods of exacerbation. However, active inflammation can correlate poorly with pain in IBD, as up to 20% of sufferers experience pain during remission. Interestingly, peripheral hypersensitivity (i.e. a reduced rectal sensory threshold for pain in response to colorectal distension: CRD) in IBD is often accompanied by referred pain to a cutaneous region. For example, in mice with TNBS colitis, a reduced threshold for pain in response to CRD may be coupled with increased hindpaw sensitivity to heat. Thus, in the presence of IBD, areas of the skin also become sensitised, and are more likely to transmit nociceptive (pain) signals. This suggests remodelling of CNS neural circuits that transmit nociceptive information. The first region of the CNS that receives afferent input from the gut is the dorsal horn (DH) of the spinal cord. In the DH, afferent signals are subject to processing by excitatory and inhibitory interneurons, and by descending projections from brainstem centres. Despite this, afferent signalling from the gut to the spinal cord is relatively uncharacterised in both normal and pathological states. Thus, any remodelling or functional changes facilitating neural hypersensitivity are yet to be examined. Aims Here, we aim to investigate changes in nociceptive signal processing within spinal DH neurons using a mouse model of TNBS colitis. We performed immunohistochemical analysis of various ‘pain related’ protein expression. Methods Acute TNBS colitis was induced in C57Bl/6 mice (both sexes). As a positive control for pain related neuropeptides we also induced neuropathic pain using chronic constriction injury (CCI) (loose ligation of the sciatic nerve with 3 ligatures). Sham animals undergo surgery without ligation. The expressions of Substance P, Iba1, GFAP were assessed in the spinal DH using immunohistochemistry. Substance P is a peptide neurotransmitter in pain pathways, while Iba1 and GFAP label microglia and astrocytes, respectively. Activation of these glial populations has been implicated in the development of chronic pain, but remain uninvestigated in the TNBS model of IBD. Here in a preliminary set of experiments we compared TNBS/control with CCI/sham animals (n = 2 for all). Results Immunohistochemical analysis of the DH detected a substantial up-regulation of substance P expression in the superficial laminae of the spinal DH in both TNBS and CCI animals (vs. ethanol/sham controls). In contrast, the expression of Iba1 and GFAP were similar in TNBS colitis and sham controls, whereas Iba1 was up regulated in CCI animals. Conclusion Together, these data suggest that, at the time point studied, nociceptive pathways are activated in animals with TNBS colitis and CCI, while glia, which are implicated in long term remodelling of nociceptive circuits, are only activated during CCI.

Tumour microenvironment differs between murine sporadic and inflammation-associated colorectal tumorigenesis SHUYI K FU, FRANCES LLOYD, CYNTHIA FORREST, BORUT KLOPCIC, IAN C LAWRANCE University of Western Australia, Fremantle, WA, Australia Background Colorectal cancer (CRC) is common but human sporadic and colitis-associated CRC (CAC) have different pathogeneses which may contribute to differences in tumour progression. The animal models used

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to study colon cancer are essential in furthering understanding of the disease and also in developing and advancing medical treatment. Aims The aim of this study was to identify differences in gene expression levels and inflammatory cell infiltration of tumours from mice models of sporadic and CAC. Methods Mice used in this study are on a FVB background. Sporadic CRC tumorigenesis was mimicked by giving 6 weekly intraperitoneal (IP) injections of azoxymethane (AOM). CAC was modelled by a single AOM IP injection prior to two cycles of 7 days of dextran sodium sulphate (DSS) in the drinking water followed by 14 days of plain water. Tumours of similar size were harvested from both models at weeks 13 (AOM-alone) and 8 (AOM/DSS) respectively and processed for further analysis. Histological and immunofluorescent (IF) assessment of CD4, CD8a, F4/80, and Ly6G was performed on tumours. Whole-genome microarray (Illumina BeadChip, MouseRef 8 v2) was used to compare gene expression in tumours from both models. Gene ontology analysis was performed using the Database for Annotation, Visualisation and Integrated Discovery. Results The majority of tumours were polypoid and all were dysplastic. Unlike AOM/DSS, the AOM-alone protocol induced high-grade dysplasic polyps (21% [4/19] vs 0% [0/9]) and 2 flat lesions one with invading CRC. Microarray analysis identified that 665 genes were differentially expressed between the tumours (p < 0.05), 398 had increased expression levels in the AOM-alone tumours and included genes associated with cytokine activity, cell division, and the regulation of apoptosis. 267 genes were reduced and were associated with immune response, the lysosome and positive regulation of the immune system. IF assessment of the inflammatory cell infiltrates showed the presence of CD4-positive, CD8a-positive, F4/80-positive and Ly6G-positive cells in all tumours without any significant differences in the cell numbers between the models. Conclusion AOM and AOM/DSS tumours are histologically different with marked gene-expression differences suggesting a different pathogenesis. All AOM-alone tumours displayed inflammatory cell infiltration suggesting a crucial role for the immune cells in the progression of tumours in both models once tumorigenesis is initiated. Further work is required to confirm histological differences between AOM and AOM/DSS tumours.

Somatic mutations in pancreatic ductal adenocarcinoma using amplicon sequencing from formalin fixed paraffin embedded tissue JEREMY HUMPHRIS, JIANG TAO, DAVID CHANG, AMBER JOHNS, MARK J COWLEY, MARINA PAJIC, MARC JONES, SKYE SIMPSON, SCOTT MEAD, ANDREW BIANKIN Kinghorn Cancer Centre/Garvan Institute of Medical Research, Darlinghurst, NSW, Australia Background INTRODUCTION: Cancer believed to be a disease of the genome that develops due to underlying genetic and epigenetic changes. Understanding these changes can allow identification of actionable (druggable) mutations enabling tailoring of therapy for an individual patient, a central concept in personalised medicine. Formalin-fixed paraffin embedded tissues offer a rich resource for retrospective analysis but the processing of the tissue often results in fragmented and cross-linked DNA. Recent developments in Next Generation Sequencing technologies now allows for the rapid high-throughput sequencing of short amplicons (amplicon sequencing) overcoming many of the problems with FFPE DNA. Aims Here we report on our initial experience of targeted amplicon sequencing in PC for actionable mutations. Methods METHODS: DNA was extracted from tumour and normal FFPE blocks using Qiagen Allprep DNA/RNA kit. The extracted DNA was quantified using the Qubit fluorometer and dsDNA HS assay kit. Using 20 ng of DNA a library was constructed of 190 amplicons which were then sequenced using the Ion Ampliseq Cancer Panel v1 and the Ion

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Torrent Personal Genome Machine (PGM) sequencer. The target genes include whole-exon coverage of KRAS, BRAF and EGFR and ‘hot-spots’ in 43 other oncogenes and tumour suppressor genes. Results RESULTS: We compared the results of amplicon sequencing with whole-exome sequencing (SOLID 4 system, Applied Biosystems) from matched FFPE and snap frozen tumour tissue respectively. Amplicon sequencing was able to detect all single-nucleotide variants in the target regions seen in the fresh tissue. DNA from a further 100+ tumours has been extracted and entered the sequencing pipeline and is currently undergoing analysis. Conclusion CONCLUSIONS: Amplicon sequencing of DNA from FFPE tissue is feasible and shows good correlation with findings in snap-frozen tissue. Amplicon sequencing has potential clinical utility in identifying actionable tumour genomic alterations. We will present our final analysis of targeted sequencing from over 100 pancreatic adenocarcinomas.

Indigenous Australians (IA) have a distinctive gut microbial profile compared to patient’s with inflammatory bowel disease (IBD) and non indigenous controls GURU IYNGKARAN,1 CHRIS MCSWEENEY,2 SEUNGHA KANG,2 SURESH SIVANESAN,1 MARK MORRISON,2 FINLAY MACRAE1 1 Colorectal Medicine and Genetics, Royal Melbourne Hospital, Parkville, VIC, Australia, 2Livestock industries, CSIRO, Brisbane, QLD, Australia Background Inflammatory bowel disease (IBD) is more common in developed nations and urban as opposed to rural communities. The incidence of IBD in IAs, who traditionally live in rural communities, is not well documented but reported to be exceedingly rare. Australian health surveys of IAs reveal a high level of childhood gastroenteritis but no cases of IBD (1). Similarly low rates of IBD were noted in Indigenous Canadians living in Manitoba (2). The hygiene hypothesis suggests that the increase in chronic inflammatory disorders in developed countries is a result of defective regulation of the immune system due to diminished exposure to a broader diversity of environmental microorganisms. We hypothesise that IA’s have a different gut microbial profile as a result of widespread exposure to environmental microorganisms in infancy, which may account for their low incidence of IBD. Aims To determine the gut tissue adherent microbiota in patient’s with IBD, non Indigenous controls and the Indigenous population. Methods Tissue samples from the terminal ileum (TI) and colon (Col) were obtained by colonoscopy from IA’s, non Indigenous healthy normals (HN), patients with active Crohns (CD) and Ulcerative Colitis (UC). Custom phylogenetic microarrays based on 16S rRNA gene sequences from gastrointestinal bacteria were employed to detect microbial diversity. Differences revealed by the microarray in relative abundance of microbial populations between patient groups were verified using Correspondence Analysis (CA) and Between group analysis (BGA) from ‘R package’ with ade4 and made4 library. Type and degree of inflammation was confirmed histologically. Results 43 patients were recruited (10 IA, 14 CD, 11 UC, 8 HN). There were significant differences in the microbial profiles between groups (CD, UC, HN and IA) within the colon and TI (p = 0.001) using BGA. Patients with active CD had similar microbial profiles irrespective of the type of therapy as did patients with active UC. No significant differences in the microbial profiles between inflamed and non-inflamed tissue were noted in the terminal ileum (p = 0.764). Conclusion There is a difference in the microbial profiles between patients with active CD, active UC, healthy normals and IAs. The gut

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microbial profile of IAs, a population which lives in rural Australia, is distinctly different which suggests that there may be an environmental influence on gut microbiota. This potentially may have an impact on bowel diseases such as IBD. More samples are being collected to confirm this finding.

Difference in gut microbiota between the groups (p = 0.001) Ca_con = Caucasian controls Indi = Indigenous CD = Crohns Disease UC = Ulcerative colitis References 1. The Health and Welfare of Australia’s Aboriginal and Torres Strait Islander Peoples 2008. Australian Bureau of Statistics. 2. Bernstein, C et al. The Epidemiology of IBD in Canada. A population based study. American Journal of Gastroenterology 2006;101:1559–68.

The safety and efficacy of physician directed nurse administered balanced propofol sedation (PHD NAPS) MAHESH JAYANNA, RAJVINDER SINGH, JONATHAN FERNANDO, SANDEEP S SETHI, KASIA REDEL, SHARON DRUMMOND, ANGELINE ASHBY, PHIL CARRINGTON, WILLIAM TAM, BIJU GEORGE, NEVILLE GREEN, GARRY NIND Gastroenterology, Lyell McEwin Hospital, Elizabeth Vale, SA, Australia Background Endoscopist Directed Propofol Sedation remains controversial. We embarked on a study to prospectively assess the safety and efficacy of a Physician Directed Nurse Administered balanced Propofol Sedation programme. Aims To assess the safety and efficacy of a Physician Directed Nurse Administered balanced Propofol Sedation programme. Methods Outpatients and stable inpatients (ASA 1 & 2, BMI ≤ 35) presenting with any indication for endoscopy, colonoscopy or both were prospectively recruited. Basic demographic data, total sedation, intra and post procedural complications and recovery times were documented. Propofol sedation was administered by a Nurse Sedationist under the direction of an Endoscopist. All Endoscopists had Advanced Life Support certification, underwent an accreditation exercise on Propofol Sedation by

the institutions’ Anaesthetic Department every 2 years and had deep sedation privileges by the Accreditation and Privileges Committee of the hospital. All NS’s undergo a rigorous 1 year preparatory programme, which includes a 3 month NS course and a 9 month Anaesthesia and Recovery course. They are required to have ALS certification and undergo 6 monthly evaluations by an Anaesthetist observing a Sedation list and need to perform a minimum of 250 procedures/year to maintain accreditation. All patients were triaged by Gastroenterologists and were seen again by the NS’s on the day of the procedure. Depth of sedation was aimed for moderate to deep sedation. Patients were initially administered Midazolam at a dose of 1–2 mg followed by a slow bolus induction dose of Propofol of 0.3–0.75 mg/kg and top up doses of 10–20 mg. Results A total of 910 patients with a mean age of 53.2 years (range: 16–87) with 416 endoscopies, 381 colonoscopies and 113 combined procedures have been performed to date. The average ASA score was 1.7. The mean Propofol and Midazolam administered per patient were 205.59 mg (range: 40–660) and 1.1 mg (range: 0.5–5.5) respectively. The average time for an endoscopy, colonoscopy and combined procedure were 11.9 mins, 23.2 mins and 31.5 mins respectively. The mean recovery time was 23.4 minutes which was defined as the length of time from the end of procedure to the time taken to achieve a sedation score of 0 (awake and alert) following which patients were discharged from the endoscopy unit. Three patients developed intra procedural hypotension, 2 of which resolved with IV fluid administration. One procedure was aborted due to persistent recurrent hypotension after re-administration of Propofol. Another patient developed a brief episode of paroxysmal AF post procedurally which resolved with no subsequent sequelae. Conclusion Contrary to recommended guidelines, PhD NAPS is safe, effective and is associated with rapid patient recovery when a strict patient selection criterion is used and appropriate training and accreditation measures are undertaken.

Epithelial HIF-1α is critical to the protective action of prolyl hydroxylase inhibition in a murine model of inflammatory bowel disease SIMON KEELY,1 ALAN W BAIRD,2 SEAN COLGAN3 1 School of Biomedical Sciences & Pharmacy, University of Newcastle, NEWCASTLE, NSW, Australia, 2 School of Veterinary Medicine, University College Dublin, Dublin, Ireland, 3Mucosal Inflammation Program, University of Colorado, Denver, CO Background Inflammatory bowel diseases (IBD), are characterised by a repeated wounding of the mucosa and loss of the intestinal epithelium, leading to the disruption of mucosal barrier integrity, inflammation and bacteraemia. Due to vascular damage and increased cellular oxygen demand, the inflamed mucosa is a predominantly hypoxic tissue. Infiltrating mucosal immune cells are required to adapt to and function within these reduced oxygen tensions. The extent to which hypoxia influences immune cell function at the inflamed mucosa is not well characterised. The stabilisation of hypoxia-inducible factor (HIF); a global, oxygen-sensitive transcription factor, through pharmacological prolyl hydroxylase inhibition (PHDi) has been shown to reduce disease severity in murine models of colitis. This mucosal protection is multifactorial, and roles for compensatory epithelial barrier pathways and the promotion of restitution and wound healing have been demonstrated. However, little is known regarding the effect of PHDi on immune cell function or their relative importance to barrier function in PHDi protection during mucosal inflammation. Aims We hypothesized that PHDi modulates immune cell responses to colitic bacteraemia We aimed to investigate 1) the effect of PHDi on immune cell function. 2) the relative contribution of epithelial and innate cells to PHDi protection from colitis.

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Methods TNBS colitis animals were treated with AKB-4924, a HIF-1 isoform-predominant PHD inhibitor. Core temperature and disease progression was recorded over 7 days. Isolated leukocytes were treated with LPS and PHDi and examined for nuclear HIF stabilisation and cytokine expression. Phagocytosis was assayed by the uptake of FITC-LPS. The importance of epithelial HIF-1α was assessed in epithelial targeted HIF-1α KO mice. Intestinal barrier function was assessed by measuring the flux of paracellular markers across intestinal tissue. Serum was assayed for LPS and inflammatory cytokines. Epithelial markers for barrier protection and wound healing were examined by qPCR. Results TNBS colitis led to pyrexia (+1.6 ± 0.4°C), followed by hypothermia (+1.9 ± 0.6°C). PHDi-treated animals had significantly reduced disease activity compared to vehicle and did not become hypothermic. PHDi augmented epithelial barrier function and repair inducing CD73, ITF and ITGB1 and reducing serum endotoxin (50-fold) during colitis. PHDi significantly reduced serum pyrogens, IL-1β (2-fold), IL-6 (17-fold) and TNF-α (3-fold). PHDi-treatment of LPS-stimulated neutrophils induced HIF stabilisation. PHDi-treated neutrophils exhibited increased phagocytotic capacity and reduced pyrogen expression. PHDi treatment offered no protection against colitis in epithelial HIF-1α KO mice. Conclusion Taken together, these results suggest that PHDi enhances the innate response against bacteraemia but that epithelial HIF-1α is critical for the mucosal protection against colitis. References 1) Karrasch et al., Zeitschrift fur Gastroenterologie 47, 1221 (2009). 2) Nizet et al., Nat Rev Immunol 9, 609 (2009). 3) Cummins et al., Gastroenterology 134, 156 (2008). 4) Robinson et al., Gastroenterology 134, 145 (2008). 5) Hams et al., Shock 36, 295 (2011). 6) Ward et al., FASEB J 25, 535 (2011). 7) Keely et al., FASEB J 23, 1338 (2009). 8) Tambuwala et al., Gastroenterology 139, 2093 (2010).

Altered gut flora following small bowel resection as revealed by high throughput DNA sequencing SUSAN LAPTHORNE,1 PRUE PEREIRA-FANTINI,1 FIONA FOUHY,2 SARAH L THOMAS,1 GUINEVA WILSON,1 NICOLE L DELLIOS,1 MICHELLE SCURR,1 PAUL D COTTER,2 JULIE BINES3 1 Murdoch Children’s Research Institute, Parkville, VIC, Australia, 2Teagasc Food Research Centre, Fermoy, Ireland, 3University of Melbourne, Parkville, VIC, Australia Background Following massive small bowel resection (SBR) the luminal environment is altered, which contributes to clinical manifestations of short bowel syndrome (SBS) such as malabsorption, mucosal inflammation, bacterial overgrowth and bacteraemia. Until recently, the ability to accurately quantitate and identify changes in individual bacterial species following SBR has been limited. Aims The aim of this study was to use high throughout DNA sequencing to accurately characterise the changes in microflora following SBR and determine the effect of these changes on mucosal inflammation using a pre-clinical piglet model of SBS in children. Methods Piglets underwent a 75% small bowel resection (SBR), shamoperation (sham), or no operation (NOC). The profile of the bacterial community was identified by pyrosequencing in colonic content collected 2- and 6-weeks post-surgery. The gene expression of the pro-inflammatory cytokines interleukin (IL)-1β, IL-6, IL-8, IL-18 and tumour necrosis factor (TNF)-α was assessed in the terminal ileum and colon at each time-point by real-time PCR.

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Results Following SBR, there were distinct alterations in the diversity of microflora and significant changes in specific bacterial species compared to the sham and NOC groups. There was no difference in total bacterial load. At the phylum level, there were significant increases in Bacteroidetes and Fusobacteria in the SBR group (p < 0.01). There were significant increases in the proportion of Veillonellaceae and Fusobacteriaceae families, while Prevotellaceae, Peptostreptococcaceae, Peptococcaceae and Ruminococcaceae were decreased (p < 0.05). At the genus level there were significant increases in Megasphaera, Acidaminococcus and Fusobacterium (p < 0.05). There were decreases in the proportion of Clostridium, and the Peptococcus genus was obliterated by the surgery. No increase in the proportion of Lactobacillus was observed. An increase in the gene expression of pro-inflammatory cytokines IL-8, IL-1β, IL-18 and TNF-α was observed in the colon at 6 weeks post-surgery (p < 0.05 and p < 0.01). An increased expression of IL-18 was also observed in the terminal ileum (p < 0.01). Conclusion Massive SBR was associated with a decrease in overall bacterial diversity. We observed a decrease in important commensal genera such as clostridia and peptococci, coupled with an increase in known inflammatory genera such as fusobacteria and acidaminococci. Contrary to previous studies, there was no increase in the proportion of lactobacilli following SBR. This may reflect the more comprehensive identification of previously unidentified bacterial content. Pro-inflammatory cytokines were increased in the colon, supporting the hypothesis that the colon is impacted by proximal SBR and may play an important role in defining clinical outcome.

Dysregulation of innate immune signalling in ulcerative colitis IAN C LAWRANCE,1 ANGELA C CHEW,1 MERI K TULIC,2 DOMINIC MALLON,3 GRAHAM RADFORD-SMITH4 1 Centre for Inflammatory Bowel Diseases and School of Medicine and Pharmacology, Fremantle Hospital and UWA, Fremantle, WA, Australia, 2School of Paediatrics and Child Health, University of Western Australia, Perth, WA, Australia, 3Department of Immunology, Fremantle Hospital, Fremantle, WA, Australia, 4Department of Gastroenterology, Royal Brisbane and Women’s Hospital, Brisbane, QLD, Australia Background Ulcerative colitis (UC) is a chronic inflammatory condition thought to result from inappropriate activation of the innate immune system. Dysregulation of intestinal mucosal immunity results in overproduction of inflammatory cytokines and trafficking of effector leukocytes into the bowel leading to inflammation. Toll-like receptors (TLRs) are key regulators of immune tolerance and promote tumour necrosis factor alpha (TNFα), and other inflammatory cytokines. Aims The aim of this study was to better understand the pathophysiology of UC, by investigating differences between UC patients who remitted and those who did not respond to anti-TNFα therapy. Methods Peripheral Blood Mononuclear Cells (PBMCs) were isolated from 10 controls (C), 10 UC responders (R) and 10 UC non-responders (NR) and the cell subtypes characterised by flow cytometry (FACS). PMBC cytokine expression levels were measured with and without specific TLR activation by Luminex® Xmap multiplexing technology. TLR2, −4, −9 and CD14 PBMC receptor levels were assessed and IRAK4, Iκβα and NFκB measured following specific TLR activation by FACS. Results Characterisation of PBMC cell populations demonstrated no significant differences in monocyte, natural killer cell, myeloid dendritic cell and T cell (naïve, helper, regulatory, cytotoxic and memory) numbers

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between Rs and NRs (p > 0.05), confirming that any differences in response to anti-TNFα treatment is not a result of the cell populations but due to dysregulation of TLR signalling. Basal PBMC expression in Rs was lower than NRs for TNFα (p = 0.002), IL-1β (p = 0.003) and IL-10 (p = 0.02). Rs production of TNFα, however, was significantly higher than in NRs following specific TLR2 (p = 0.02), −4 (p = 0.04) and −9 (p = 0.0002) stimulation. Similar findings were observed for IL-1β and IL-10 expression, but no differences were observed between Rs and NRs for IL-6. No differences in TLR2, −4, −9 and CD14 PBMC receptor expression were detected, but NRs had less Iκβα and a reduced ability to phosphorylate NFκB compared to Rs which paralleled the cytokine production profile. Conclusion The findings suggest that TLR function, in particular, may play a significant role in a patient’s ability to respond to TNF therapy. With further investigation into the specific mechanisms driving these differences in TLR function, these findings could be used to subtype UC patients and predict response to anti-TNFα medications allowing for more targeted and cost effective therapy.

SPARC modifies colonic tissue healing and inflammation by regulating collagen and MMP expression IAN C LAWRANCE,1 BORUT KLOPCIC,2 YOKE-LENG NG,2 SHUYI K FU,2 FRANCES LLOYD2 1 Medicine and Pharmacology, University of Western Australia, Fremantle, WA, Australia, 2Centre for Inflammatory Bowel Diseases, Fremantle Hospital, Fremantle, WA, Australia Background Secreted Protein Acidic and Rich in Cysteine (SPARC) is a matricellular protein expressed during tissue repair. It binds to, and interacts with, collagen and regulates matrix metalloproteinase (MMP) expression. Aims The aim was to determine if SPARC modifies intestinal healing. Methods Wild-type (WT) and SPARC-null (KO) 29/SvJ × C57/BL6 mice received 7 days of 3% dextran sodium sulphate (DSS) in the drinking water and were sacrificed on days 7, 14, 21 and 35. Colonic tissue was assessed histologically, RNA isolated and the collagen bundles examined by transmission electronmicroscopy (TE). Colonic myofibroblasts were isolated from WT and KO mice, stimulated with phorbol 12-myristate 13-acetate (PMA) and the RNA isolated. Expression of collagen (Col)1α1, Col3α1, MMP13, MMP3, TIMP1, TIMP2, TGFβ1 and TGFβ3 in colonic tissue and the fibroblasts were analysed by real time PCR. Results By day 35, KO colonic mucosa had completely regenerated unlike WT mice and at days 14, 21 and 35 WT mice had higher histological inflammation and damage. At day 7, when intestinal inflammation was maximal, sirius red staining was greater in the DSS-treated animals compared to controls (KO vs con p < 0.01) with no differences at other time points. Col1α1, Col3α1 MMP13 and MMP3 expression was reduced in DSS-treated WT colons at day 7 and these were significantly lower than in KO colons (p < 0.01). No differences were observed at other time points. TIMP1, TIMP2 and TGFβ1, TGFβ2 were not different at any time point. The collagen fibre diameters by TE in KO colons were smaller than WTs (40 nm vs 30 nm P < 0.0001) suggesting that SPARC modifies collagen bundling. PMA reduced Col1α1 and Col3α1 (p < 0.05) expression and increased MMP13 (p < 0.01) and TIMP1 (p < 0.05) expression in the isolated fibroblasts. PMA had no effect on MMP3, TIMP2, TGFβ1 and TGFβ2 expression. Conclusion SPARC appears to modify tissue healing by regulating collagen expression, bundling and its degradation by MMPs resulting in faster tissue turnover and a faster healing rate. Differences between the WT and KO fibroblasts, however, did not account for these changes suggesting that interaction between cells is important. SPARC modifies colonic tissue healing and inflammation by regulating collagen and MMP expression.

SPARC enhances intestinal inflammation in dextran sodium sulphate-induced murine colitis IAN C LAWRANCE,1 YOKE-LENG NG,2 BORUT KLOPCIC,2 FRANCES LLOYD2 1 Centre for Inflammatory Bowel diseases, Fremantle Hospital, UWA, Fremantle, WA, Australia, 2Medicine and Pharmacology, University of Western Australia, Fremantle, WA, Australia Background Secreted Protein Acidic and Rich in Cysteine (SPARC) is a matricellular protein expressed during tissue repair and regulates cell proliferation and migration. Aims The aim was to determine if SPARC modifies intestinal inflammation. Methods Wild-type (WT) and SPARC-null (KO) 129/SvJ × C57/BL6 mice received 7 days of 3% dextran sodium sulphate (DSS) in the drinking water and were sacrificed on days 7, 14, 21 and 35. Inflammation was assessed endoscopically, clinically and histologically. Expression levels of IL1β, IL6, TNFα, MCP1, MIG, RANTES and TGFβ1 were assessed as was the cellular inflammatory infiltrate by the presence of CD3, CD4, CD25, FoxP3 by flow cytometry and CD11b, CD68 and Ly6G by immunofluorescence. Results Maximal histological inflammation was at day 7, but KO animals had lower endoscopic inflammatory scores at day 6 and each time point (days 6, 20 p < 0.05, day 34 p < 0.001), suffered less weight loss, diarrhoea, faecal blood and had lower spleen to body weight ratios at day 7 (p < 0.01) and 21 (p < 0.05) consistent with less colonic and systemic inflammation. In KO colons at day 7 there was less IL1β (p < 0.05) and MIG (p < 0.05) expression while IL6, TNFα levels were numerically lower but were not significant. RANTES was reduced at day 21 (p < 0.05). TGFβ1 levels were higher in KO colons that was significant at day 21 (p < 0.05). Flow cytometry identified a greater percentage of FoxP3 regulatory T cells in KO spleens (p < 0.01 days 21, 35) and in the draining lymph nodes (p < 0.05 day 14). KO colons also had less CD68+ macrophages at days 21 (p < 0.05) and 35 (p < 0.01) and were lower at days 7 and 14 but these differences were not significant. The number of CD11 macrophages and Ly6G+ neutrophils were significantly less in KO colons at day 35 (p < 0.05). Conclusion DSS induces less colonic and systemic inflammation in SPARC KO than WT mice and the inflammation appears to resolve faster. This may be secondary to increased levels of regulatory T cells and increased TGFβ1 levels which may inhibit effector cells, cytokine and chemokine expression and aid in the more rapid resolution of inflammation and restoration of the intestinal mucosa.

Phosphatidylcholine regulates intestinal inflammation and modifies in vivo and in vitro extracellular matrix regulation IAN C LAWRANCE, ANGELA C CHEW, FRANCES LLOYD Centre of Inflammatory Bowel Diseases and School of Medicine & Pharmacology, Fremantle Hospital and UWA, Fremantle, WA, Australia Background Ulcerative colitis (UC) is associated with a lack of phosphatidylcholines (PC) and this may produce a defective mucosal barrier and allow for the translocation of bacteria into the mucosa promoting inflammation. Aims This study aims to investigate the effects of PC on inflammation and extracellular matrix (ECM) regulation in the murine model of 2,4,6-Trinitrobenzene Sulfonic Acid (TNBS)-induced intestinal inflammation and fibrosis, and ECM gene expression in human intestinal fibroblasts.

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Methods C57bl6 mice received TNBS enemas weekly for 2 or 6 weeks, ± PC (200 mg/kg/day/orally), ± indomethacin (Indo) (0.2 mg/kg/day/ orally), or a combination of PC/Indo. Inflammation and fibrosis was assessed histologically. The human colonic fibroblast cell line, CCD-18co, was stimulated with PMA (10 ng/μl) for 24 hrs prior to treatment Indo (50 μM) or with two isoforms of PC, dilineoyl-PC (DLPC) (50 μM) and linoleoyl-palmitoyl-PC (LPPC) (50 μM) ± Indo. mRNA was isolated and collagen 1 (Col1), MMP-1, TIMP-1 and TGF-β1 expression assessed by RT-qPCR. Results At 2 weeks compared to TNBS alone, mice treated with Indo/ TNBS had significantly more inflammation and fibrosis (p = 0.004 and 0.00004), those receiving PC/TNBS had significantly less inflammation (p = 0.009), while those treated with PC/Indo/TNBS had less inflammation and fibrosis (p = 0.001 and 0.03). At 6 weeks compared to TNBS alone, inflammation and fibrosis was not different in the Indo/TNBS and PC/ Indo/TNBS-treated animals, but PC/TNBS-treated animals had less inflammation (p = 0.01) and fibrosis (p = 0.006). The addition of PC to Indo/TNBS at 2 weeks significantly decreased inflammation and fibrosis compared to Indo/TNBS alone (p = 0.03 and 0.00001), however at 6 weeks, this addition had no effect on Indo/TNBS-induced inflammation or fibrosis. In CCD-18co cells the addition of DLPC increased MMP-1 and TIMP-1 mRNA expression (p = 0.03 and 0.01), with no effect on Col1 or TGF-β1 suggesting that DLPC may decrease ECM deposition. By contrast, LPPC increased Col1 (p = 0.0008) and TIMP-1 (p = 0.03) and decreased MMP-1 expression (p = 0.003) with no effect on TGF-β1 suggesting that LPPC may increase ECM deposition. DLPC/Indo and LPPC/ Indo treatment mirrored DLPC and LPPC treatment alone. Conclusion The findings demonstrate that PC can reduce inflammation and fibrosis but may require a higher dose to completely abolish the effects of Indo/TNBS in vivo. The PC isoform appears to be important in vitro as DLPC may decrease and LPPC increase ECM deposition by isolated fibroblasts.

Prostaglandin E2 regulates intestinal inflammation and modifies in vivo and in vitro extracellular matrix regulation IAN C LAWRANCE, ANGELA C CHEW, FRANCES LLOYD Centre of Inflammatory Bowel Diseases and School of Medicine & Pharmacology, Fremantle Hospital and UWA, Fremantle, WA, Australia Background The chronic intestinal inflammation observed in the inflammatory bowel diseases (IBD) can induce fibrosis and stricture formation by sustained dysregulation of the extracellular matrix (ECM). Prostaglandin E2 (PGE2) is an anti-fibrotic lipid mediator derived from the metabolism of arachidonic acid by cyclooxygenase, which has been reported to be associated with IBD. Understanding the role PGE2 plays in the regulation of ECM deposition and degradation is crucial in determining the severity of scar tissue formation following chronic inflammation. Aims The aim of this study was to investigate the effects of PGE2 on inflammation and ECM regulation in vivo using the murine model of 2,4,6-Trinitrobenzene Sulfonic Acid (TNBS)-induced intestinal inflammation and fibrosis and ECM gene expression in vitro using a human colonic fibroblast cell line. Methods C57bl6 mice received TNBS enemas weekly for 2 or 6 weeks, ± Indo (0.2 mg/kg/day/orally), ± PGE2 (10 mg/kg/day/orally) or a combination of Indo/PGE2. Inflammation and fibrosis was assessed histologically. The human colonic fibroblast cell line, CCD-18co, was stimulated with PMA (10 ng/μl) for 24 hrs prior to treatment with Indo (50 μM), PGE2 (50 μM) or a combination of Indo/PGE2. mRNA was isolated and collagen 1 (Col1), MMP-1, TIMP-1 and TGF-β1 expression levels assessed by RT-qPCR.

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Results At 2 weeks compared to TNBS alone, mice treated with Indo/ TNBS had significantly more inflammation and fibrosis (p = 0.004 and 0.00004), while those receiving PGE2/TNBS had significantly less inflammation (p < 0.05) and fibrosis (p < 0.05). At 6 weeks compared to TNBS alone, inflammation and fibrosis was not different in the Indo/TNBStreated animals, but PGE2/TNBS treatment had less inflammation (p < 0.05) and fibrosis (p < 0.05). No differences were observed between PGE2/ Indo/TNBS and PGE2/TNBS treated animals, however, inflammation and fibrosis were significantly decreased when compared to Indo/TNBStreated animals at 2 (p = 0.00001 and 0.009; p = 0.0001 and 0.00001 respectively) and 6 (p = 0.029 and 0.01; p = 0.021 and 0.002 respectively) weeks. In CCD-18co cells the addition of Indo significantly increased Col1 (p = 0.00005), TIMP-1 (p = 0.02) and TGF-β1 (p = 0.006) and decreased MMP-1 (p = 0.04) mRNA expression compared to PMA treatment alone suggesting that Indo can increase ECM deposition. The addition of PGE2 (±Indo) reduced Col1 (p < 0.05) and increased MMP-1 (p < 0.05) expression compared to PMA and Indo alone with no significant changes in TIMP-1 and TGF-β1 mRNA levels suggesting that PGE2 decreases ECM deposition Conclusion The findings demonstrate that Indo induces inflammation and fibrosis in the TNBS murine model of colitis and fibrosis, which can be overcome with the addition of PGE2. These findings were supported by the in vitro effects of PGE2 on cultured colonic fibroblasts suggesting that PGE2 may be a potential therapy for the prevention of intestinal fibrosis.

Effect of high red meat intake and resistant starch in humans on risk factors for colorectal cancer RICHARD LE LEU,1 MICHAEL CONLON,1 JEAN WINTER,2 KAREN HUMPHREYS,2 MICHAEL MICHAEL,2 YING HU,2 ANTHONY BIRD,1 DAVID TOPPING,1 GRAEME YOUNG2 1 Food and Nutritional Sciences, CSIRO, Adelaide, SA, Australia, 2Flinders Centre for Innovation in Cancer, Flinders University, Adelaide, SA, Australia Background High consumption of red meat is associated with an increased risk of colorectal cancer (CRC). In rodents red meat induces pro-mutagenic adducts ‘O6-Methyl-2-deoxyguanosine’ (O6MeG) in the colon, and these adducts are reduced with Resistant Starch (RS). Aims This study examines the effect of high red meat and RS on O6MeG and putative biomarkers for CRC risk in humans. Methods The study was a randomised cross over design with two 4-week interventions of either high red meat (300 g/day) or high red meat plus butyrylated RS (40 g/day) with a washout period of 4-weeks between each intervention. Rectal biopsies were collected and analysed for O6MeG adducts and mi-RNAs from the miR-17-92 cluster (which are over expressed in CRC and potentially oncogenic), while faeces were collected for measures of short chain fatty acids (SCFAs), phenol & p-cresol. Results Red meat increased O6MeG load (P < 0.001) and the expression of miR-17-92 miRNAs (P < 0.05) within the rectal biopsy compared to that of their habitual entry diet, whereas co-consumption of RS was able to normalise both O6MeG load and miR-17-92 miRNAs to that of entry levels. In the faeces, total SCFA as well as butyrate concentrations were significantly increased in the RS intervention (P < 0.001), whereas the protein fermentation products phenol (P < 0.05) and p-cresol (P < 0.01) were significantly decreased with RS supplementation. Conclusion Our findings indicate that a high red meat intake is associated with potentially damaging changes in the colorectal epithelium which may be associated with an increase risk of colorectal cancer. Butyrylated resistant starch supplementation appears to have a beneficial effect in the large bowel most likely attributed to increased butyrate production and

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was able to ameliorate some of the negative effects of the high red meat diet. Reference 1. Winter J, Nyskohus L, Young GP, Hu Y, Conlon MA, Bird AR, Topping DL & Le Leu RK (2011) Inhibition by resistant starch of red meatinduced promutagenic adducts in mouse colon. Cancer Prev Res (Phila) 4, 1920–1928.

Development of pulmonary inflammation and altered lung function in a mouse model of colitis SEAN MATEER, PHILIP HANSBRO, GAVIN NOLAN, CIARAN CHANEY, KYRA MINAHAN, SIMON KEELY School of Biomedical Sciences & Pharmacy, University of Newcastle, NEWCASTLE, NSW, Australia Background Pulmonary manifestations of Inflammatory Bowel Disease (IBD) were first reported in 1976(1) and numerous clinical studies have since highlighted the involvement of the respiratory tract in IBD. In a study of 52 IBD patients, abnormal lung computed tomography was observed in 50% of sufferers(2). Three separate studies of randomly selected IBD patients showed incidence rates of pulmonary involvement to be 44–50%(3). These pulmonary symptoms manifest as interstitial lung disease, peribronchial inflammation, increased numbers of alveolar lymphocytes and a reduction in the diffusion capacity of the lung(4), suggesting that pulmonary involvement is an underappreciated aspect of IBD. Left untreated these symptoms may progress to irreversible lung damage. While there is a clear link between inflammatory diseases in the intestinal and pulmonary systems in the clinical literature, there have been no basic research studies to investigate the mechanisms of this inflammatory cross talk. Aims In this study we aimed to examine whether intestinal inflammation leads to respiratory involvement (pulmonary inflammation and changes in lung function) in a chemically induced (TNBS) model of colitis. Methods Colitis was induced in C57/Blk6 mice by rectal instillation of Trinitrobenzene sulfonic acid (TNBS). Disease severity was monitored over 9 days. Airways responsiveness was analysed in vivo by animal ventilar flexivent. Bronchoavelor lavage fluid (BALf) counts and differential stains were conducted to quantify the level and phenotype of immune cells. Protein, serum and lavage samples were analysed to determine the level of lung cytokines in TNBS mice. Gene expression analysis was conducted by qPCR. Results TNBS mice had increased lung inflammatory score and destruction compared to control within 8 days of induction of colitis. This inflammation was accompanied by increases in MPO activity in lung homogenate tissue. Brocho-alveolar lavage fluid (BALf) counts increased two-fold in TNBS mice over control and differential stains showed an influx of alveolar macrophages. Given the evidence of inflammation in the lungs of TNBS colitic mice, we screened lung homogenate for inflammatory mediators by qPCR and ultra-sensitive ELISA. We observed increased levels of MIP-1α, IL-1β and KC at the mRNA and protein level. The lung function of TNBS animals showed significant change in transpose airway resistance, compliance, elastance and inertance compared to controls. Conclusion These data suggest that intestinal inflammation in TNBS colitis leads to immune-mediated damage to lung tissue. Changes in compliance and resistance indicate that the lungs ability to expand and recoil has been affected. This may lead to prolonged lung expiration and subsequent airflow limitation. Our data suggest that this immune damage is macrophage mediated and further studies will define the mechanisms behind this macrophage recruitment.

References 1. Unexplained bronchopulmonary disease with inflammatory bowel disease, Kraft SC, Earle RH, Roesler M, Esterly JR. Arch Intern Med. 1976;136(4):454. 2. Pulmonary function tests, high-resolution computed tomography findings and inflammatory bowel disease. Tunc B, Filik L, Bilgic F, Arda K, Ulker A. Acta Gastroenterol Belg. 2006;69(3):255 3. Thoracic manifestations of inflammatory bowel disease. Black H, Mendoza M, et al. Chest 2007;131:524–32. 4. The lung in inflammatory bowel disease, Camus P, Piard F, Ashcroft T, Gal AA, Colby TV. Medicine (Baltimore). 1993;72(3):151.

Transcriptomic responses of Bacteroides vulgatus PC510 to faecal water additions to complex medium MARK MORRISON,1 STANISLAS MONDOT,1 ELINE S KLAASSENS,2 PARAIC O CUIV1 1 CSIRO Preventative Health Flagship Research Program, CSIRO Animal, Food and Health Sciences, Saint Lucia, QLD, Australia, 2School of Molecular Bioscience, University of Sydney, Sydney, NSW, Australia Background Bacteroides vulgatus is a member of the ‘core’ microbiota of the healthy human gut; but can also be recovered in high abundance from IBD patients. Some strains appear capable of inducing pro-inflammatory cytokines and B. vulgatus-specific antibodies have been identified in Crohn’s disease (CD) subjects. We have isolated a new strain of B. vulgatus (PC510) and genome sequence analysis has revealed that the strain possesses a putative lipoprotein and transporter system highly similar to proteins encoded by a CD-gut derived metagenomic fosmid clone shown to modulate NF-κB DNA transcription factor activity in an HT-29-based cell line, by Lakhdari et al (2010). Aims To characterize the gene expression (transcriptomic) profile of B. vulgatus PC510 in response to its growth environment; to identify and understand what biotic and/or abiotic factors may affect the growth and/ or immunomodulatory properties of this bacterium. Methods Strain PC510 was cultured in a complex medium; or medium supplemented with human faecal waters prepared from either healthy subjects, or patients suffering from ulcerative colitis. Total RNA was extracted, enriched for mRNA, and reverse transcribed to cDNA. These libraries were subjected to next generation sequencing and the mean size of each dataset was almost 9 million paired-end reads. Read datasets were mapped to the PC510 genome using BWA. SAMTOOLS was used to extract/and quality trim the mapped data. Statistical analysis was performed using R program and edgeR package. Results There were no substantial differences in the growth patterns of strain PC510 with the different media, suggesting the metabolic capacity of the strain was not greatly altered in response to the complex growth environments used. Indeed, no substantial differences in the expression of housekeeping genes were apparent from the transcriptomic analysis. However, genes encoding functions coordinating cell wall, membrane, and envelope biogenesis were affected when the cultures were supplemented with both types of faecal waters. Lipoprotein genes were also more highly expressed in cultures supplemented with the faecal water prepared from UC patients; as well as some genes encoding unknown function(s), underlining potential new functionalities. Conclusion The transcriptomic results reveal that while the growth rate of B. vulgatus PC510 might not be dramatically altered by faecal water components, the bacterium does show some substantive changes in behaviour, especially with respect to cell wall assembly and decoration of its extracellular surface. Considering that strain PC510 and a metagenomic clone bearing a presumptive B. vulgatus lipoprotein have already been

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shown to attenuate host epithelial cell signalling via NF-κB; the strain appears to be a good model for genetic dissection of the microbial signals that may contribute to the attenuation of gut inflammatory responses. Reference Lakhdari O, Cultrone A, Tap J, Gloux K, Bernard F, Ehrlich SD, Lefèvre F, Doré J, Blottière HM (2010). Functional metagenomics: a high throughput screening method to decipher microbiota-driven NF-κB modulation in the human gut. PLoS ONE 30:5. pii: e13092.

Hypoxia-inducible factor-1α promotes mucosal protection in colitis via the IL-12 signalling pathway GAVIN NOLAN,1 KYRA MINAHAN,1 KRISTEN E FARRELL,1 CIARAN CHANEY,1 SEAN MATEER,1 NICHOLAS TALLEY,2 SIMON KEELY1 1 School of Biomedical Sciences & Pharmacy, University of Newcastle, NEWCASTLE, NSW, Australia, 2 Faculty of Health, University of Newcastle, Callaghan, NSW, Australia Background Crohn’s disease (CD) is a chronic, inflammatory, T-cell mediated disease. The IL-12 family of cytokines are pro-inflammatory heterodimeric signalling molecules pivotal to the induction of the Th1/ Th17 response that drives CD inflammation. The IL-12p40 subunit dimerises with IL-12p35 or IL23p19 to form IL-12p70 or IL-23 respectively. IL-12p40 may also be secreted as a homodimer (IL-12p80) which antagonises IL-12p70 and IL-23 inflammatory signalling. Vascular damage and infiltrating immune cells in the inflamed mucosa reduce oxygen tensions (hypoxia) leading to the stabilisation of hypoxia inducible factor, HIF (a global regulator for tissue adaption to hypoxia). Pharmacological stabilisation of HIF-1α through the inhibition of prolyl hydroxlases (PHD) is protective against murine colitis. In these models we observed a significant increase in IL-12p40 gene (IL12B) expression with PHD inhibitor treatment and identified a canonical HIF binding site on the IL-12p40 promoter. We hypothesised that HIF regulates IL-12 signalling in colitis, promoting anti-inflammatory pathways and resolution of disease. Aims We aimed to characterise the transcriptional regulation of the IL-12 family of cytokines by HIF and to determine whether IL-12p80 offers mucosal protection in a chemically induced (TNBS) murine model of colitis. Methods The IL12B promoter was cloned into a luciferase reporter and transfected into U937 human monocyte cells. Cells were subjected to normoxia, hypoxia or treated with AKB4924 (a HIF-1 isoform-predominant PhD inhibitor). TNBS colitis was induced in C57/Blk6 mice. Animals were treated every second day with either AKB-4924 or recombinant IL-12p80 (i.p.). Mice were monitored over 7 days to assess disease progression. IL-12 cytokines (IL-12p70, IL-12p40, IL-23p19) were examined in colon tissue and serum by qPCR and ELISA. Results IL-12p40 was significantly increased (2.5 fold) in mice treated with PHDi while both IL-12p70 (4-fold) and IL-23 (2-fold) levels were significantly reduced over vehicle. In addition, PHDi treated animals showed a significant increase in IL-12B transcript levels, concurrent with decreased IL-12A and IL-23A. Cells transfected with a IL-12B-luciferase reporter construct led to a significant 4 fold induction in luciferase over 6 hours upon exposure to hypoxia or PHDi treatment. In addition, animals treated with rIL-12p80 demonstrated an accelerated recovery from TNBS induced weight loss and disease activity. Conclusion Here we showed that pharmacological HIF stabilisation leads to an increase in IL-12p40 transcript. and protein levels, while decreasing p35 and p19 levels in TNBS colitis, possibly promoting p40 homodimer secretion. Exogenous IL-12p40 homodimer offers mucosal protection in colitis. These results suggest that HIF may shift the balance

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of IL-12 signalling from a pro- to anti-inflammatory pathway through selective induction of IL-12p40. References 1. Fuss I.J. et al. Both IL-12p70 and IL-23 Are Synthesized During Active Crohn ‘s Disease and Are Down-regulated by Treatment with Anti-IL-12 p40 Monoclonal Antibody. Inflamm Bowel Dis 2006:12:9. 2. Gately M. et al. Interleukin-12 antagonist activity of mouse interleukin-12 p40 homodimer in vitro and in vivo. Ann N Y Acad Sci 1996:795:1. 3. Karhausen J. and V.H. Haase. Inflammatory Hypoxia Perspective Role of Hypoxia-Inducible Factor. Cell Cycle 2005:4:256. 4. Robinson A. et al. Mucosal protection by hypoxia-inducible factor prolyl hydroxylase inhibition. Gastroenterology 2008:134:145. 5. Shimozato O. et al. The secreted form of the p40 subunit of interleukin (IL)-12 inhibits IL-23 functions and abrogates IL-23mediated antitumour effects. Cytokine 2005:2:22.

High-throughput sequencing analysis of gut microbiota pre- and post-colonoscopy CLAIRE L O’BRIEN,1 FLORIAN GRIMPEN,2 GWEN E ALLISON,3 PAUL PAVLI2 1 Medical School, The Australian National University, Canberra, ACT, Australia, 2Gastroenterology & Hepatology Unit, The Canberra Hospital, Canberra, ACT, Australia, 3Research School of Biology, The Australian National University, Canberra, ACT, Australia Background The gut microbiota is important in maintaining human health. Numerous factors have the potential to alter the microbiota’s composition. Changes in gut microbiota may contribute to or exacerbate a range of diseases, such as the inflammatory bowel diseases. Aims The aim of this study was to investigate the composition of the faecal microbiota pre- and post-colonoscopy. Methods Fifteen subjects undergoing colonoscopy consumed a bowel preparation comprised of 10 mg bisacodyl and 2 L polyethylene glycol. The microbiota of five stool samples; two collected pre-colonoscopy, and three collected post-colonoscopy were evaluated. Two samples were taken 3–6 months apart from 5 healthy controls who did not undergo bowel preparation. Universal primers targeting the V1-V3 region of the 16S rRNA gene were used to amplify all samples analysed using denaturing gradient gel electrophoresis (DGGE). High throughput sequencing (HTS) was conducted on two pre-colonoscopy and one post-colonoscopy sample from ten patients with varying DGGE results. HTS samples were amplified using primers targeting the V1-V3 region of the 16S rRNA gene. DGGE samples were analysed using BioNumerics, HTS samples using Mothur. Results The DGGE analyses showed a small number of patients had short-term changes, but no patients exhibited significant long-term changes. The HTS results indicate that variation between pre- and postcolonoscopy samples from the same patient, with respect to the presence/ absence of OTUs, was no greater than the variation observed between control samples. Variation in the relative abundance of OTUs was similar for pre- and post-colonoscopy samples from the same patient, and overall not significantly different from controls. However, variation in the relative abundance of OTUs appeared to be greater in ulcerative colitis (UC) patients (n = 3) than other patients and controls. Conclusion Our results suggest that a standard bowel preparation does not have a lasting effect on the composition of the intestinal microbiota, although it may have an impact short-term in some people. The relative abundance of OTUs may vary more in UC patients, and the impact of colonoscopy preparation on the microbiota of inflammatory bowel disease patients should be assessed.

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Patterns of participation over multiple rounds of faecal immunochemical test-based screening for colorectal cancer JOANNE M OSBORNE,1 AMY DUNCAN,3 CARLENE WILSON,1 DEBORAH TURNBULL,3 INGRID FLIGHT,4 STEPHEN R COLE,2 GRAEME YOUNG1 1 Flinders Centre for Innovation in Cancer, Flinders University of South Australia, Bedford Park, SA, Australia, 2Bowel Health Service, Repatriation General Hospital, Daw Park, SA, Australia, 3School of Psychology, University of Adelaide, Adelaide, SA, Australia, 4CSIRO Preventive Health Flagship, CSIRO, Adelaide, SA, Australia Background Some people are seemingly inconsistent in their participation in biennial cancer screening with faecal immunochemical testing (FIT). The reasons for this inconsistency are unclear. The current study examines the extent to which this inconsistency may reflect participation in other CRC screening and diagnostic regimens. Aims To determine the extent to which estimates of participation rates based on return of FIT kits is confounded by participation in other screening offers. Methods A baseline survey detailing participants’ demographic information (age, gender, marital status, education, health insurance, country of birth), prior experience with CRC screening, and attitudes toward screening was mailed to 4000 South Australians. Survey respondents were offered 3 rounds of annual FIT screening using OC-Sensor (Eiken Chemical Co.). Participant behaviour at each of the 3 rounds was recorded as 1) Study FIT-participant, 2) Self-reported participant (e.g. notified BHS that their screening status was up-to-date), or 3) Non-participant (did not participate in the Study and was not up-to-date). Participatory behaviour across the 3 rounds was recorded as Stable (e.g. Always or Never) or Changeable. Results Survey response was 48.2% (n = 1928). On a round by round basis, study FIT participation was 58.5%, 66.6%, and 73% respectively. At each round, 7%, 13.7% and 5.6% of participants self-reported they were ‘overscreeners’ (i.e. participated despite being up to date having done another test or recent colonoscopy). The proportion of study non-participants classified as up-to-date with other CRC screening was 31.9%, 12.5% and 12.7% in each round respectively. Over three rounds, 43% of the survey-respondents consistently returned FITs offered through the study. When self-report of participation in nonstudy screening behaviour was included, participation rate was estimated at 60.3%. Consistent participants, defined in either way, were significantly more likely to be older, married and no longer working full time. No significant differences were found between the categories for gender, country of birth or language spoken at home. Conclusion Population FIT-based colorectal cancer screening programs which do not incorporate participation external to the program will underestimate population screening rates. This is particularly salient in an Australian context with the recent extension of a nationally funded CRC FIT-based screening program. People who are older, married and no longer working full time are most likely to sustain participation in a colorectal cancer screening program.

Regional characteristics and proposed mechanisms of mucosal adaptation in short bowel syndrome SIMON J PROCTOR,3 WILLIAM ROEDIGER,2 ADRIAN G CUMMINS1 1 Gastroenterology and Hepatology, The Queen Elizabeth Hospital, Woodville South, SA, Australia, 2 Department of Surgery, The Queen Elizabeth Hospital, Woodville South, SA, Australia, 3Medicine, University of Adelaide, Adelaide, SA, Australia Background Regional specific differences in the extent of the adaptive response of the small intestine to short bowel syndrome are not adequately defined. Short bowel syndrome occurs during a state of reduced intestinal surface area, usually induced after extensive bowel resections or in preterm infants. Intestinal failure ensues, and recovery is reliant upon an adaptive response, without which intravenous nutrition is required. The adaptive response involves compensatory increases in primary and secondary mucosal surface area brought about by increased villous area, crypt area and crypt number. The β-catenin signalling pathways are important in promoting crypt fission during intestinal growth and could do the same during adaptation. Aims The aims were to compare adaptation in the jejunal and ileal remnants and to ascertain if β-catenin signalling was involved. Methods Short bowel syndrome was induced in 20 week old HoodedWistar rats. Stereology was used to measure primary and secondary surface area pre and post-resection. A tissue microdissection technique was used to investigate intestinal morphometry, crypt fission and hyperplasia. Crypt hyperplasia was confirmed using immunohistochemistry against Ki67 and phospho-Histone H3. Changes in positional expression of β-catenin signalling protein phospho-β-catenin (Ser552) were examined in crypt cells. Results Post-operatively, resected rats lost a mean of 20.6 grams more weight than controls (p = 0.036). Stereology revealed an approximate increase of 25% in secondary surface area in the 7 days following resection. Villous area increased in the short bowel group versus the control group at one week, but only in the ileum (p = 0.019). Crypt area increased in the jejunum (p = 0.0006) and the ileum (p < 0.0001). The percentage of crypt fission increased (jejunum p = 0.018, ileum p = 0.012), as did mitotic count (p = 0.013, p = 0.0005) and the apoptotic count per crypt (p = 0.014) in the ileum. Immunostaining confirmed these results, found proliferative crypt cell expansion and demonstrated increased numbers of cells undergoing active β-catenin signalling (p = 0.031) in the ileum. A frequency distribution of phospho-β-catenin positive crypt cells revealed an increase in the stem cell region. Conclusion Thus, adaptation occurred by both increased crypt fission and hyperplasia, driven by stem or progenitor cell division. B-catenin signalling may have been involved. Adaptation was most evident in the ileum rather than jejunal remnants. Although there was increased jejunal crypt fission and crypt hyperplasia, this did not result in jejunal villous hypeplasia. Results of this study may provide insights into new treatment options for patients with short bowel syndrome.

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Microbial fingerprinting detects unique bacterial communities in the faecal microbiota of rats with experimentally-induced colitis VALERIA A TOROK,1 ASHIS K SAMANTA,2 NIGEL J PERCY,1 SUZANNE M ABIMOSLEH,3 GORDON HOWARTH4 1 Plant and Soil Health, South Australian Research and Development Institute, Urrbrae, SA, Australia, 2 National Institute of Animal Nutrition and Physiology, Bangalore, India, 3Discipline of Physiology, School of Medical Sciences, The University of Adelaide, Adelaide, SA, Australia, 4School of Animal and Veterinary Sciences, The University of Adelaide, Roseworthy, SA, Australia Background An abnormal composition of the gut microbiota is believed to be associated with the pathogenesis of inflammatory bowel disease (IBD). Aims We utilized terminal restriction fragment length polymorphism (T-RFLP) analysis to quantify faecal bacterial communities from rats with experimental colitis. Methods Male Sprague Dawley rats (n = 10/group) ingested 2% dextran sulfate sodium (DSS) or water for up to 7 days. Rats were killed and colonic tissues collected for histological analysis. Results Damage severity score in the distal colon was significantly greater (P < 0.001) following DSS consumption compared to controls. T-RFLP faecal bacterial profiles generated with either MspI or CfoI revealed a significant difference (P < 0.001) in community composition between healthy and colitic rats, with bacterial composition in healthy rats more variable than in rats with colitis. Operational taxonomic units (OTU: taxonomically related groups of bacteria) associated with either the healthy or colitic state were identified. OTU (116, 226, 360, and 948; CfoI) and (118 and 188; MspI) were strongly associated with untreated healthy rats, while OTU (94, 98, 174, and 384; CfoI) and (94 and 914; MspI) were predominantly associated with colitic rats. Phylogenetic OTU assignment suggested that Bacteroidales and Lactobacillus sp. were primarily associated with colitic and healthy rats, respectively. Conclusion Faecal bacterial profiling is a rapid, sensitive and non-invasive tool for detecting and identifying changes in gut microbiota associated with colitis. Restoring microbial homeostasis by targeting colitis-associated OTU through specific microbiological interventions could form the basis of novel therapeutic strategies for IBD.

DNA methylation signatures in colorectal cancer VICKI WHITEHALL,1 TROY DUMENIL,1 ANDREAS SCHERER,2 SONIA GRECO,1 ARNE MOULD,1 GLEN BOYLE,1 BARBARA LEGGETT1 1 Department of Cell and Molecular Biology, Queensland Institute of Medical Research, Brisbane, QLD, Australia, 2Australian Genome Research Facility, Melbourne, VIC, Australia Background Colorectal cancer is a heterogeneous disease, where clinically relevant subgroups can be recognised based on shared molecular alterations. A significant subgroup displays the CpG Island Methylator Phenotype (CIMP), which is defined by high levels of DNA methylation at specific CpG islands and is highly correlated with oncogenic BRAF mutation and microsatellite instability (MSI). The spectrum of genes that are hypermethylated and transcriptionally silenced as a result of CIMP is not well defined. Aims To further explore cancer subgroups based on DNA methylation changes, we characterised a large cohort of colorectal cancers for multiple

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molecular features including CIMP, and examined genome-wide DNA methylation and expression signatures in a selected subset of these cancers. Methods A consecutive series of 785 colorectal cancers was characterised for CIMP (103/784, 13% CIMP-High), microsatellite instability (109/784, 14% MSI), mutation of BRAF (124/784, 16% mutant), KRAS (272/784, 35% mutant), p53 (280/727, 39% mutant), PIK3CA (108/784, 14% mutant) and methylation by quantitative MethyLight of MLH1 (69/774, 9%), MGMT (204/779, 26%) and p16 (158/774, 20%). Molecular alterations were correlated with clinical features including age, sex, site and tumour stage. Methylated DNA immunoprecipitation (MeDIP) was used to isolate methylated sequences from 14 CIMP-High and 12 CIMPNegative cancers and adjacent normal samples, before application to 400 K custom DNA methylation microarrays (Agilent Technologies). Data was validated on the same samples using the Illumina HumanMethylation450 BeadChip arrays and compared to genome-wide expression data using Illumina HumanHT-12 BeadChip arrays. Results In unsupervised hierarchical cluster analysis using the most variable 1308 probes (chosen based on a standard deviation of signal intensity > 1.0), the data clearly dichotomised into CIMP-positive/BRAF mutant and CIMP-negative/BRAF wild type groups. Interestingly, even in the CIMP-negative samples, a unique methylation signature segregated with KRAS mutation. We identified 1892 genes specifically hypermethylated in CIMP-positive cancers. Of these, 39 were either members of the canonical Wnt signalling pathway, or involved in regulation of this pathway. Data will also be presented from our current DNA methylation array study of 250 consecutive colorectal cancers. Conclusion These data support the hypothesis that distinct methylation signatures characterise colorectal cancer subgroups, and that these signatures synergise with activation of the MAPKinase signalling pathway through mutation of either the BRAF or KRAS oncogenes. Candidate CIMP target genes identified have provided insight into the biology of these cancers. Reference 1. Leggett B and Whitehall V (2010) Role of the serrated pathway in colorectal carcinogenesis. Gastroenterology 138: 2088–100.

Enrichment of epithelial intermediate filament proteins in Crohn’s disease sera associated with disease behaviour phenotype and disease activity YUNKI YAU,1 RUPERT W LEONG,2 VALERIE C WASINGER1 1 Bioanalytical Mass Spectrometry Facility, The University of New South Wales, Sydney, NSW, Australia, 2Gastroenterology and Liver Services, Concord Hospital, Sydney, NSW, Australia Background The manifestation of Crohn’s disease (CD) is dynamic and variable. Although disease progression has proven to correlate with duration of affliction, the pathologies of the different behavioural phenotypes are poorly understood and difficult to predict at a clinical level. Proteomics is the study of body proteins, and proteins are the direct mediators of metabolic and systemic functions. Identification of plasma protein biomarkers may not only assist in the identification of pathogenic factors of CD, but also in monitoring and prediction of behavioural complications. Aims The purpose of this study was to investigate whether a particular functional annotation would significantly associate with disease behaviour in the low-mass serum proteome of CD subjects. Methods In total, 16 inflammatory, 9 stricturing, and 9 fistulizing sera were analysed from consecutively recruited CD subjects from inflammatory bowel disease clinics. Phenotyping was performed according to the Montreal Classification by IBD gastroenterologists. Sera was partitioned

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using in-solution electrophoresis on an MF10 (NuSep) and low mass (≤25 kDa) proteins were analysed by C18 stage tip followed by liquid chromatography/tandem mass spectrometry on an LTQ Orbitrap (Thermo Scientific). Principal component analysis (PCA) was used to categorize proteins based on abundance correlation with CD behaviour phenotype. Pathway mapping was performed using cluster analysis software (DAVID) on protein species that exhibited CD behaviour-characteristic abundance patterns. EASE score (modified Fisher Exact P-Value) was used to determine degree of enrichment in the categorized protein groups. Results A total of 231 proteins exhibited abundance patterns that correlated with worsening CD (P ≤ 0.05). 135 clustered into 8 primary categories of known molecular processes. Intermediate filament and epithelial cell differentiation proteins made up 13% of highly enriched proteins in fistulizing CD (P ≤ 0.001). Amongst these, Catalase (P ≤ 0.001) and Actin beta/gamma 1 (P ≤ 0.001) were mapped to pathways of microbial metabolism and bacterial invasion of intestinal epithelial cells, respectively.

Conclusion A low-mass serum proteome overrepresented with filamentrelated species is associated with fistulizing CD. Such a systemic profile may correlate with known increased permeability and shedding of the CD intestinal barrier. Proteins that measure microbial metabolism and bacterial invasion of intestinal epithelial cells may be relevant biomarkers of disease. References 1. Oshitani N, Watanabe K, Nakamura S, Fujiwara Y, Higuchi K, Arakawa T. Dislocation of tight junction proteins without F-actin disruption in inactive Crohn’s disease. Int J Mol Med. 2005;15(3):407–10. 2. Huang da W, Sherman BT, Lempicki RA. Systematic and integrative analysis of large gene lists using DAVID bioinformatics resources. Nat Protoc. 2009;4(1):44–57. 3. Kanehisa Laboratories (1995–2012) Kyoto Encyclopedia of Genes and Genomes. Viewed May 31 2012.

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Endoscopy Lower CT diagnosis of colitis and endoscopic correlation ZAID S ARDALAN, SUJIEVVAN CHANDRAN, ADAM TESTRO Austin Health, Heidelberg, VIC, Australia Background An increasing reliance on computer tomography (CT) as a primary investigation for patients with abdominal complaints has lead to increasing referrals for endoscopic investigation of radiologically diagnosed colonic thickening or colitis. Aims We seek to investigate whether there is a correlation between a CT diagnosis of colitis and endoscopic pathology in our patient population. Methods A retrospective review was performed on the Austin Health radiology database for the CT diagnosis of colitis from 1st January 2008 to 31st December 2011. The endoscopy, radiology, pathology reports and case notes for each patient were then reviewed. Results 57 patients were identified who underwent a CT with both oral and IV contrast and subsequently had an endoscopy for the indication of CT diagnosed colitis. Mean age was 54 years with a male to female ratio of 1:1.44. 37% of patients presented with abdominal pain alone followed by, 23% acute diarrhoeal illness, 18% with rectal bleeding/abdominal pain and 14% with chronic diarrhoea. 14/57 (25%) patients had a faecal specimen collected prior to the CT being performed. The mean time from CT to colonoscopy was 7.5 days. Endoscopic follow up was complete (colonoscopy) in 41 patients with 16 patients undergoing a flexible sigmoidoscopy. 37 pts (65%) were found to have endoscopic features consistent with colitis compared to 11 pts (16%) with a normal endoscopy and a further 9 pts (18%) with polyps/colonic carcinoma (9 polyps/2 cancers). The distribution of the colitis at endoscopy correlated with CT in 29/37 (78%) of patients. Of the patients with histologically confirmed colitis, 45% were defined as acute with 55% having changes of chronicity. Conclusion Our series is the largest of its kind to date and found that the 65% of our patients with CT changes consistent with colitis had corroborative endoscopic findings. However given that approximately 45% of patients had acute causes of colitis such as infective gastroenteritis brings into question the appropriateness of CT scanning in patients with abdominal complaints. This is further highlighted by the fact that only 25% of our patient population had a faecal specimen result prior to the CT being performed.

patients up to 2011. This data included tumour type and stage. The individual colonoscopy records were reviewed by two independent reviewers to confirm the endoscopists’ diagnosis or strong suspicion of CRC. Mortality data was derived from the Ministry of Health records (range of follow-up from 5–12 years). Patients with IBD, carcinoids, anal SCC and appendiceal tumours were excluded. Results 32,313 colonoscopy reports were collated. 1299 patients were diagnosed with CRC; 19 patients had two CRC’s. Data from the earliest CRC diagnosis and the colonscopy preceding the diagnosis were reviewed. 136 cancers were diagnosed after colonoscopy and not suspected at time of procedure (9.8% of all first CRC’s. For 0–3 years post-colonoscopy this was 49 (3.8%). 14 were diagnosed in the first 6 months. Patient risk factors for increased risk of CRCAC compared with CRC diagnosed at time of colonoscopy were age > 70 and previous finding of polyp. Presentation with bleeding decreased risk while social deprivation scale not significant. Procedural risk factors for increased risk were presence of diverticular disease, procedure date before 2002; factors not significant were experience of endoscopist and use of anti-spasmodic. There was a trend to more R-sided lesions. CRCACs were more likely to have advanced staging and lower survival (5-year survival 36% compared 56%). The majority of incomplete procedures were followed by appropriate investigations (80%). 56% of colonoscopy procedures that had subsequent cancer found had a finding of polyps. Conclusion CRC after colonoscopy is a significant clinical problem particularly given the lower survival for these patients. Awareness of risk factors may help reduce the risk. CRCAC is an important audit tool and should be assessed over time to encourage improved practice to reduce this risk. This appears to be an issue for endoscopists of all degrees of experience.

Colorectal cancer after colonoscopy: risk factors and survival data compared with cancer diagnosed at time of colonoscopy NATHAN S ATKINSON,1 ALAN G FRASER,2 GEETA GALA,3 GREG D GAMBLE,2 MARK LANE1 1 Gastroenterology Department, Auckland Hospital, Auckland, New Zealand, 2Department of Medicine, University of Auckland, Auckland, New Zealand, 3 Northern Cancer Network, Auckland, New Zealand Background Colorectal cancer (CRC) diagnosed after a colonoscopy (CRCAC) may be due to a missed cancer or advanced polyp, incomplete resection of a polyp or rapid progression to cancer. Reported rates of cancer after colonoscopy have varied widely but this does seem to be a significant problem that could be altered with improved quality of colonoscopy. Aims Many previous studies have not reviewed the endoscopy reports and have assumed that cancers diagnosed 6 months after colonoscopy are ‘missed’. Survival data comparing CRCAC with CRC diagnosed at time of colonoscopy has not been previously reported. The aim of this study was to determine local rates of CRCAC. Methods All colonoscopy records from all 3 public hospitals in the Auckland region were collated from 1996–2005. Records were merged with the National Cancer Registry identifying all CRC diagnosed in these

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Improving the experience of colonoscopy – the answer is not just more propofol – a report on an 8 year clinical improvement project PETER BAMPTON,1 SANDFORD JAYNE,2 BEV DRAPER2 1 Gastroenterology, Flinders Medical Centre, Bedford Park, SA, Australia, 2Surgical and Specialty Services, Flinders Medical Centre, Bedford Park, SA, Australia Background A desire to increase patient comfort in endoscopy has seen an increasing use of anaesthetic support, which further adds to the cost of the procedure, yet there is evidence to suggest that optimised standard sedation can give good patient tolerance of colonoscopy. Aims To improve comfort for patients undergoing colonoscopy in the Endoscopy Unit at Flinders Medical Centre through a multi-step clinical improvement project. Methods In 2004 we commenced clinical practice improvement (CPI) project that involved a series of interventions and assessment using standard CPI methodology. All patients were asked to rate their discomfort on a score of 0–10, with patients rating 0 and 1 having minimal discomfort, and 9 and 10 unacceptable discomfort, with data being prospectively collected on the endoscopy unit recovery spreadsheet. Length of time in recovery, dosage of sedation (midazolam and fentanyl) used was also recorded in a quarterly fashion. The following interventions were introduced in a step wise fashion: collection and distribution of baseline data, improved patient education on the nature of sedation, providing individual data in comparison to peers on a quarterly basis, education of medical staff in resuscitation management and administering conscious sedation, introduction of CO2 as insufflating gas, identification of patient factors predicting likely poor tolerance of procedure and booking these patients on the anaesthetic lists (15% of total lists) and facilitated learning from proceduralists with good patient comfort ratings. Results The percentage of patients describing no or minimal discomfort increased from a baseline of 57% in 2004 to 79% in 2011, with improvement being accrued with each of the interventions introduced. Triage of ‘at risk’ patients to anaesthetic lists only contributed to 3% of this improvement and 85% of colonoscopies continue to be performed using standard sedation only. The dose of midazolam and fentanyl used initially increased, but is now decreasing despite ongoing improvement in sedation outcomes, there was no increase in flumazenil or naloxone use. The number of patients having unacceptable discomfort has decreased from 6.9% to 3.1% over this period. Average recovery time remained stable with an average between 89 and 94 minutes in each measured quarter. Conclusion Patient comfort in colonoscopy can be improved through optimisation of standard sedation avoiding the cost implications of universal anaesthetic support.

resection. Endoscopic recognition of the MTS allows prompt defect closure and may avoid surgery. Aims To assess the impact of routine inspection for a TS/MTS on the rate of clinically significant perforation after colonic ER. Methods Data from a prospective, multi-centre cohort of ER for colonic LSTs ≥20 mm was analysed (June 2008-November 2011). ER protocol was amended in January 2010 to include routine assessment for the TS/ MTS. Outcomes were compared before and after this date. Clinical and endoscopic data was collected including presence/management of TS/ MTS, rates of clinically significant colonic perforation (defined as requiring hospitalisation ≥48 hours or surgery) and intervention required. If a MTS was detected endoscopic closure was attempted. Patients were discharged the same day if well, pain free and had no free intraperitoneal gas. Results ER was performed on 877 lesions, mean size 35.2 mm. 510 were performed after January 2010 with close assessment of the ER defect. Lesion size, morphology and distribution was similar in both groups (table 1). Prior to January 2010 the TS was noted 9 times (2.5%) and after this 16 times (3.2%), p = 0.6. All patients with a TS had intraprocedural clip closure of the MP defect. Prior to January 2010 clinically significant perforation occurred in 4 patients (1.1%). In one patient a TS was recognised and endoscopic closure performed. This patient was managed conservatively, the rest required surgery. After this time there was 1 clinically significant perforation (0.2%, p = 0.08). No TS was noted. This patient was managed conservatively. In total the TS was identified 25 times. but in these the rate of clinically significant perforation was 4%. Fewer number of pieces to achieve resection (2.6 vs 4.3, p = 0.02) and the presence of submucosal invasion (17% vs 5.7%, p = 0.03) were predictors of a TS. Conclusion There is a trend towards reduction in clinically significant perforation with close assessment of the ER defect and resected specimen for a TS/MTS. Early recognition of the TS facilitates early endoscopic intervention and ameliorates the frequency and severity of clinical sequelae. The presence of submucosal invasion and resection in fewer pieces are associated with the occurrence of a TS. Table 1.

Patient and lesion characteristics and outcomes

Number of lesions Lesion size (mean) Right colon Submucosal fibrosis Submucosal invasion Target sign Clinically significant perforation

Jun 08–Dec 09

Jan 10–Nov 11

p

368 35.0 218 (59.4%) 62 (17.3%) 24 (6.6%) 9 (2.5%) 4 (1.1%)

510 35.3 300 (59.2%) 97 (19.2%) 27 (5.6%) 16 (3.2%) 1 (0.2%)

0.8 0.9 0.5 0.5 0.6 0.08

Recognition of the target sign reduces clinically significant perforation during endoscopic resection of large colonic lesions: a prospective, multi-centre cohort study MILAN S BASSAN,1 BRONTE A HOLT,1 STEPHEN WILLIAMS,1 ALAN MOSS,1 MICHAEL SWAN,1 REBECCA SONSON,1 GREGOR J BROWN,2 SIMON A ZANATI,3 LUKE HOURIGAN,4 MICHAEL BOURKE1 1 Gastroenterology and Hepatology, Westmead Hospital, Wentworthville, NSW, Australia, 2The Alfred Hospital, Melbourne, VIC, Australia, 3Western Health, Melbourne, VIC, Australia, 4Princess Alexandra Hospital, Brisbane, QLD, Australia Background Colonic endoscopic resection (ER) carries a small but significant risk of perforation. The ‘target sign’ (TS) and corresponding ‘mirror target sign’ (MTS) indicates focal muscularis propria (MP)

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Divergent understanding of health professionals regarding correct subsequent management of an asymptomatic patient with a positive faecal occult blood test and negative colonoscopy with resultant cost implications LAUREN BESWICK,1 ALVIN Y TING,1 FINLAY MACRAE,2 DAMIAN DOWLING,1 CHRISTOPHER HAIR1 1 Department of Gastroenterology, Barwon Health, Geelong, VIC, Australia, 2Department of Colorectal Medicine and Genetics, Royal Melbourne Hospital, Melbourne, VIC, Australia Background The National Bowel Cancer Screening Program (NBCSP) was implemented in Australia in 2006 and involves screening with faecal occult blood test (FOBT) in all individuals aged 50–65. Between August 2006 and January 2010, 967,537 FOBT were returned as part of the Government program and 64,700 (6.7%) were positive. Of those positive FOBT investigated with colonoscopy, colorectal carcinoma (CRC) was found in 3.5% and CRC, adenoma or polyp in 56% [1]. The immunogenicity of haemoglobin is lost through small bowel digestion, so the NBCSP test is specific for lower GI bleeding. It has been shown previously that gastroscopy has no role in the further investigation of a positive FOBT after a normal colonoscopy [2]. Aims We aimed to assess the current practice after a negative colonoscopy for positive FOBT. Methods A questionnaire was sent out to a range of medical practitioners including general practitioners, colorectal surgeons and gastroenterologists within Victoria. The questionnaire asked what the next step of management would be in a 55-year-old asymptomatic male with no family history of CRC, a positive FOBT and subsequent normal colonoscopy. Using the survey data and the known cost of each potential subsequent investigation, we calculated the potential cost of inappropriate investigation following positive FOBT tests within the NBCSP. Results After a normal colonoscopy for a positive FOBT, 65.6% of general practitioners, 19.5% of gastroenterologists and 14.2% of colorectal surgeons would recommend further investigation with either repeat FOBT, gastroscopy, or capsule endoscopy. Calculations based on the survey results and known cost of additional investigations suggest that during the initial 3.5 years of the NBCSP the cost associated with inappropriate investigation following positive FOBTs may have been as high as 25 million dollars [1]. Conclusion The current study reveals a poor understanding amongst clinicians of the significance of a normal colonoscopy after a positive FOBT. Given that the considerable potential negative health economic impact of inappropriate decision making and subsequent resource utilization this limited study warrants further evaluation on a much larger scale.

Table 1.

Endoscopic outcomes for CT diagnosed colonic wall thickening RAY BOYAPATI, ANN FARRELL, KUSHANI JAYASINGHE, MICHAEL SWAN, GREGORY MOORE Gastroenterology, Southern Health, Clayton, VIC, Australia Background The finding of colonic bowel wall thickening (BWT) seen on abdominal Computerised Tomography (CT) scans is common but the significance is unknown. Although recommendations on further investigations are lacking, colonoscopy is frequently used to further evaluate the finding. It is unclear what the yield of colonoscopy is in this setting. Aims To correlate BWT seen on CT scans with subsequent colonoscopy and identify the yield in performing colonoscopy in this setting. Methods A retrospective analysis was performed at two large hospitals in Melbourne, Australia by searching the radiological database for all patients with colonic bowel wall thickening found on CT scan reports from March 2006 to February 2012. Correlation with institutional endoscopy database and outside endoscopy reports was performed. Patients who had no case notes available and those with known pre-existing bowel pathology were excluded. Comparison of yields was analysed with Fisher’s exact test. Results 114 patients with BWT on CT scan and no pre-existing bowel pathology were identified. 63 patients (55.3%) had isolated BWT in one segment of the bowel. 41 patients (36%) underwent colonoscopy; in all cases, the area of BWT found on CT was reached. Of the 73 patients who did not undergo colonoscopy, 7 died shortly after CT scan and 21 went straight to operation, mainly for an acute abdomen. Of those undergoing colonoscopy, 6 patients (14.6%) were found to have a malignancy at the site of BWT, 12 (29.3%) had diverticulosis or diverticulitis, 3 (7.3%) had inflammatory bowel disease, 2 (4.9%) had polyps and 1 (2.4%) had pseudomembranous colitis. 17 patients (41.5%) had normal findings at the site of BWT although two of these patients had pathology elsewhere in the colon. There was no difference in the yield of colonoscopy for segmental versus extensive BWT (68.2% vs 56.3%, p = 0.72). Conclusion Bowel wall thickening on CT scan is an important radiological finding. Clinical and endoscopic correlation is warranted as clinically significant conditions are often present.

Recommended outcome after normal colonoscopy following positive FOBT

General Practitioners Gastroenterologists Colorectal Surgeons General Physicians Gastroenterology Registrars General Medical Registrars

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References 1. Menzies Centre for Health Policy. http://www.menzieshealthpolicy. edu.au/other_tops/pdfs_pubs/Australia-needs-a-bowel-cancerscreening-program.pdf 2. Levi Z, Vilkin A, Niv Y. Esophago-gastro-duodenoscopy is not indicated in patients with positive immunochemical test and nonexplanatory colonoscopy. Eur J Gastroenterol Hepatol. 2010 Dec; 22(12): 1431–4.

Invited

Response rates

No further treatment

Gastroscopy

Capsule

Repeat FOBT

100 50 36 43 46 23

61% 82% 78% 56% 72% 65%

34.4% 80.5% 82.2% 45.8% 79% 53.3%

41% 7.3% 7.1% 37.5% 12% 33.3%

3.3% 0 0 4.2% 0 6.7%

21.3% 12.2% 7.1% 12.5% 6 6.7%

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Risk factors, management and outcomes of clinically significant bleeding after wide field endoscopic mucosal resection of large colonic lesions NICHOLAS G BURGESS,1 ANDREW METZ,1 STEPHEN WILLIAMS,1 RAJVINDER SINGH,2 LUKE HOURIGAN,3 GREGOR J BROWN,4 SIMON A ZANATI,5 JOSHUA BUTT,6 MICHAEL BOURKE1 1 Gastroenterology and Hepatology, Westmead Hospital, Sydney, NSW, Australia, 2Gastroenterology and Hepatology, Lyell McEwin Hospital, Adelaide, SA, Australia, 3Gastroenterology and Hepatology, Princess Alexandra Hospital and Greenslopes Private Hospital, Brisbane, QLD, Australia, 4Gastroenterology and Hepatology, The Alfred and Epworth Hospitals, Melbourne, VIC, Australia, 5Gastroenterology and Hepatology, The Alfred and Western Hospitals, Melbourne, VIC, Australia, 6Gastroenterology and Hepatology, The Alfred Hospital, Melbourne, VIC, Australia Background Wide Field Endoscopic Mucosal Resection (WF-EMR) for large colonic lesions is a safe and cost effective outpatient treatment. Clinically significant (defined as requiring hospital admission) post EMR bleeding (CSPEB) following an uneventful procedure occurs in 7% and may be serious. Few studies have examined risk factors for CSPEB following WF-EMR and guidelines on predicting and managing bleeding do not exist. Aims We aimed to examine CSPEB risk factors and outcomes in a large cohort and propose a protocol for management. Methods Prospective multi-centre data for WF-EMR of large sessile colorectal polyps or LSTs ≥20 mm (June 2008–March 2012: ClinicalTrials. gov NCT01368289) was analysed. Data collection included patient and lesion characteristics, procedural outcomes, complications and scheduled follow up at 14 days, 4 and 12 months. Resection technique and peri procedural care was standardised. Bleeding cases underwent standardised chart review. Bleeding was managed according to an established strategy. Results WF-EMR was performed on 1014 lesions (mean size 35 mm, right colon 54.2%) in 951 patients (mean age 68 years). 60 patients had CSPEB (6.0%), 25 (42%) presenting within 24 hours, 17 (28%) at 24–48 hours and 18 (30%) at > 48 hours. 10 patients (17%) had haemodynamic instability at presentation and 9 patients (15%) required transfusion. Mean inpatient stay was 2.4 nights. Right colon location predicted bleeding on univariate analysis (RR 2.1, p 0.006). Age, lesion size, comorbidities, antiplatelet and anticoagulant use did not predict bleeding. 35 patients (58%) were managed conservatively. The remainder (25) underwent colonoscopy: endoscopic haemostasis was applied to 16 lesions in 15 patients (Clip 13 (81%); Thermal 5 (31%); Combination 1 (6.3%)). 10 patients (38%) did not require any form of endoscopic haemostasis and did not rebleed. One patient required angiographic embolization for rebleeding after initial clip haemostasis. One patient had surgery for perforation from endoscopic clips. There was no mortality. Conclusion CSPEB occurs in 6.0% and is more frequent with right colon lesions. Most bleeding settles spontaneously without re-intervention. In those who do undergo colonoscopy bleeding has already ceased in nearly 40%. Patients who respond to initial resuscitation without ongoing bleeding can be managed conservatively. Colonoscopy should be reserved for the minority (40 mm, with defect contamination or multiple vessels. – Transparent cap or gastroscope if required. Statistical analysis was performed using SPSS version 19 (SPSS Inc, Chicago IL). Results EMR was performed on 122 patients with rectal AMN: 64% males; mean age 66 yo; mean lesion size 47 mm; range 20–180 mm. 18 lesions involved the ARJ. The degree of dysplasia was significantly lower at the ARJ compared to proximal lesions (17.6% vs. 34.6% have at least high grade dysplasia, P = 0.048). Adenoma histology was significantly different, with all ARJ lesions being tubulovillous adenomas or sessile serrated adenomas (P = 0.004). Procedural success, and adenoma recurrence and admission rates were similar between the two groups. 3 patients with ARJ resections required admission (1 for pain and bleeding and 2 for observation). 20 adenocarcinomas were diagnosed, and all were located above the ARJ. On multivariate analysis, significant independent predictors of malignancy were location above the ARJ (OR 5.8, 95% CI 0.8 to infinity, P = 0.087) and Kudo pit pattern 5 (OR 23.2, 95% CI 2.1 to 1234.5, P = 0.006). 14 patients proceeded to surgery, with no residual adenoma or carcinoma seen in 46% of surgical specimens. Conclusion Simple modification of standard EMR technique allows safe and effective treatment of AMN at the ARJ. AMN of the ARJ has low risk of invasive disease compared to proximal rectal lesions.

Univariate analysis of rectal AMN involving the ARJ vs. above the ARJ

Polyp size Paris classification

Morphology

Intraprocedural bleeding Successful EMR Procedure duration Residual (mean 4th follow up) Histology

Dysplasia Admission

Is IIa IIa+Is IIb IIc Granular Nongranular Mixed Yes No Yes No

Involves ARJ

Above ARJ

5 4 7 0 2 17 1 0 15 2 17 0

23 33 45 1 2 80 14 10 87 17 95 9

(27.8%) (22.2%) (38.9%) (0%) (11.1%) (94.4%) (5.6%) (0%) (88.2%) (11.8%) (100%) (0%)

(22.1%) (31.7%) (43.3%) (1.0%) (1.9%) (76.9%) (13.5%) (9.6%) (83.7%) (16.3%) (91.3%) (8.7%)

P value 0.499 0.313

0.322

1.000 0.357 0.960

Yes No

2 (16.7%) 10 (83.3%)

22 (31%) 49 (69%)

0.679

Tubular TVA Villous SSA TVA + SSA Adenocarcinoma LGD HGD or cancer Yes No

0 11 0 5 1 0 14 3 3 14

18 55 1 1 9 20 68 36 12 92

0.0001

(0%) (64.7%) (0%) (29.4%) (5.9%) (0%) (82.4%) (17.6%) (17.6%) (82.4%)

(17.3%) (52.9%) (1%) (1%) (8.7%) (19.2%) (65.4%) (34.6%) (11.5%) (88.5%)

0.048 0.442

Endoscopic mucosal resection using mechanical submucosal dissection: a novel technique BRONTE A HOLT,1 JAMES T LIM,2 MILAN S BASSAN,1 DUNCAN MCLEOD,3 HEMA MAHAJAN,3 VISHNU SUBRAMANIAN,3 ERIC Y LEE,1 MICHAEL BOURKE1 1 Gastroenterology and Hepatology, Westmead Hospital, Sydney, NSW, Australia, 2Colorectal surgery, Westmead Hospital, Sydney, NSW, Australia, 3Tissue Pathology and Diagnostic Oncology, Westmead Hospital, Sydney, NSW, Australia Background En bloc wide-field Endoscopic Resection (ER) is technically challenging and currently requires endoscopic submucosal dissection. Conventional snare resection limits specimen size to 20 mm and less, and larger lesions require piecemeal resection. Alternative submucosal injectates may enable larger en bloc resections, which is particularly important for early malignant lesions Aims To assess the feasibility and outcome of en bloc ER using a viscous, biocompatible Gel with conventional snare or hybrid ER technique. Methods In an in vivo porcine model, two 5 mL submucosal injections with Gel (Cook™ Medical Gel) were made in each of 12 animals, followed by snare resection. This resulted in 24 resections in total. Four of these resections used a combination IT knife and snare resection technique. Four additional ERs were performed in each animal using conventional submucosal fluids (Normal Saline (NS) and Succinylated Gelatin (Gelofusine®, SG)) and snare resection. The submucosal cushion height and the extent

Journal of Gastroenterology and Hepatology 2012; 27 (Suppl. 4): 30–49 © 2012 Journal of Gastroenterology and Hepatology Foundation and Wiley Publishing Asia Pty Ltd

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of the elevated mucosa were estimated against snares of known size. Ease of injection and resection, histology specimen sizes and technical issues were recorded. Euthanasia and autopsy was performed day 4 post-procedure. Linear mixed effects models were used to analyse the within pig effect of solution type on continuous outcomes. McNemar’s test was used to test for within pig differences in dichotomous responses under different solution regimes. Results Significantly higher submucosal cushions were obtained with Gel compared to NS (3.0 mm higher, P = 0.007) and SG (1.8 mm higher, P = 0.057). In addition, Gel cushion volume was significantly larger than NS (3675 mm3 larger, P = 0.007) and SG (2308 mm3 larger, P = 0.020). Histology specimen sizes obtained with Gel was similar to NS (NS 46.9 mm2 smaller, P = 0.7329) and SG (SG 97.4 mm2 larger, P = 0.4214). Conventional snare resection was limited by both the size of the submucosal cushion and the Gel viscosity, leading to difficulty with snare closure. The hybrid resection technique was successful in all cases. Resections performed with Gel took a median of 7:14 minutes; 3.3 times longer on average than with NS (95% CI 2.5 to 4.2, P < 0.0001), and 2.9 times longer than with SG (95% CI 2.3 to 3.7, P < 0.0001). Conclusion This pilot study demonstrates submucosal Gel injection for ER is feasible, and results in reliable tissue separation. However, the precise technique to optimise resection requires refinement. The snarebased technique has limitations, and a hybrid technique may enable en bloc resection of adenomatous lesions that are currently removed by piecemeal ER or endoscopic submucosal dissection.

Endoscopic mucosal resection using a novel submucosal injection fluid: hydroxyethyl starch BRONTE A HOLT,1 JAMES T LIM,2 MILAN S BASSAN,1 DUNCAN MCLEOD,3 HEMA MAHAJAN,3 VISHNU SUBRAMANIAN,3 ERIC Y LEE,1 MICHAEL BOURKE1 1 Gastroenterology and Hepatology, Westmead Hospital, Sydney, NSW, Australia, 2Colorectal Surgery, Westmead Hospital, Sydney, NSW, Australia, 3Tissue Pathology and Diagnostic Oncology, Westmead Hospital, Sydney, NSW, Australia Background The submucosal injection is a crucial component of successful Endoscopic Mucosal Resection (EMR). The ideal submucosal injection fluid is readily available, safe, and inexpensive and results in a substantial, sustained and discrete elevation. This facilitates tissue capture with snare closure, increasing resection size and procedural efficacy. Normal saline (NS) and Succinylated Gelatin (Gelofusine®, SG) are both used as submucosal injection fluids (1), however they have some limitations (NS – diffuse and nonsustained lift; SG – derived from beef gelatine). Hydroxyethyl Starch (Voluven®, HES) is an intravenous plasma volume expander, with a higher mean molecular weight and more prolonged intravenous volume effect compared to NS and SG (2), and may be a superior submucosal injection fluid. Aims To assess whether HES as the submucosal injection fluid for EMR is more effective than NS and SG. Methods A blinded, randomised trial of HES, SG and NS was performed by two operators, using 12 live pigs. Two 5 mL submucosal injections using each fluid was made, followed by maximal resection with a 20 mm snare, resulting in 6 resections per animal, and 24 resections using each solution in total. The primary outcome measure was the size of the specimen. Secondary outcome measures were the ease of injection, elevation steepness, cushion size, ease of resection, resection time and complications. Euthanasia and autopsy was performed day 4 post-procedure. Linear mixed effects models were used to analyse the within pig effect of solution

36

type on continuous outcomes. McNemar’s test was used to test for within pig differences in dichotomous responses under different solution regimes. Results All resections were completed successfully. HES resulted in similar specimen sizes to NS (NS 4.9 mm2 larger, P = 0.9673) and SG (SG 149.2 mm2 larger, P = 0.4195). There were no significant differences in ease of injection and resection, and the height, volume, and steepness of the submucosal fluid cushion between HES and either NS or SG (P > 0.05). Resection time was similar between solutions. Similar intra-procedural complication rate and colonic histology was observed for resections performed with each of the three solutions. Conclusion In a porcine model, submucosal injection with HES does not result in significantly different procedural outcomes compared to NS or SG. Further studies are required to assess outcomes in humans. Outcome measures compared to Hydroxyethyl Starch Outcome measures

Comparisonsolution

Value

P value

Specimen size (mm2) – in room Specimen size (mm2) – in pathology Ease of injection (1–10) (1 = easy, 10 = hard) Ease of resection (1–10) (1 = easy, 10 = hard) Cushion height (mm) Cushion width (mm) Cushion length (mm) Cushion volume (mm3)

Normal Saline Succinylated Gelatin Normal Saline Succinylated Gelatin

140.417 more than HES 228.542 more than HES 4.917 more than HES 149.167 more than HES

0.2107 0.1937 0.9673 0.4195

Normal Saline Succinylated Gelatin

0.292 more than HES 0.042 more than HES

0.188 0.850

Normal Saline Succinylated Gelatin

0.125 easier than HES 0.375 harder than HES

0.7332 0.2929

Normal Saline Succinylated Gelatin Normal Saline Succinylated Gelatin Normal Saline Succinylated Gelatin Normal Saline Succinylated Gelatin

0.083 less than HES 1.083 more than HES 0.083 less than HES 1.042 more than HES 1.958 less than HES 0.417 less than HES 333.958 less than HES 1032.292 more than HES

0.9347 0.2323 0.9228 0.3220 0.1075 0.6842 0.6864 0.2167

References 1. Moss A, Bourke MJ, Metz A. Am J Gastroenterol 2010; 105: 2375. 2. http://www.fresenius-kabi.com.au/files/Voluven_PI.pdf

Safety study of haemostatic spray following endoscopic resection BRONTE A HOLT,1 JAMES T LIM,2 MILAN S BASSAN,1 DUNCAN MCLEOD,3 HEMA MAHAJAN,3 VISHNU SUBRAMANIAN,3 ERIC Y LEE,1 MICHAEL BOURKE1 1 Gastroenterology and Hepatology, Westmead Hospital, Sydney, NSW, Australia, 2Colorectal Surgery, Westmead Hospital, Sydney, NSW, Australia, 3Tissue Pathology and Diagnostic Oncology, Westmead Hospital, Sydney, NSW, Australia Background Following wide field endoscopic mucosal resection (EMR), significant delayed bleeding necessitating hospital admission, with associated patient morbidity and hospital costs, occurs in 7% and 30% of colonic and duodenal cases respectively (1,2). Most bleeding occurs in the first 36 hours. Haemostatic spray is highly effective for acute upper gastrointestinal bleeding in animal (3) and human studies (4). Prophylactic application of Haemostatic spray on the mucosal defect after EMR may alter the natural history of delayed bleeding, reduce delayed bleeding and significantly increase the safety of the procedure. Aims This study aimed to assess the safety of Haemostatic spray applied to the EMR defect.

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Methods Colonoscopy with 8 EMRs of normal distal colonic mucosa was performed in each of 12 live pigs, followed by haemostatic spray to the EMR defects. Repeat colonscopy was performed at 4–6 hours. Animals were monitored for adverse clinical effects, and euthanised day 4 postprocedure. The colon and end organs (brain, lung, kidney, spleen) were collected and assessed. Colon species were compared to historical porcine histology specimens. Primary outcome measure was abnormal colon histology. Secondary outcome measures were abnormal end-oral histology, mortality, and haemostatic spray location and adherence to the EMR defect at repeat colonoscopy. Results Autopsy results showed no significance difference in inflammatory or reparative changes compared to historical controls. No adverse end-organ effects such as emboli or ischaemia were identified on analysis. One animal died within 24 hours of the procedure, and was found to have evidence of aspiration on autopsy, and one animal was euthanised following a full thickness perforation that could not be closed endoscopically. Haemostatic spray effectively coated all defects, and subsequent colonoscopy showed haemostatic spray was retained on defeats in the dependent position. Conclusion Post wide-field EMR bleeding is an important clinical problem. In this animal study, haemostatic spray did not result in significant adverse local or distant effects, and may have a role in prevention as well as acute management of gastrointestinal bleeding. References 1. Metz A, Bourke MJ et al. Endoscopy 2011; 43: 506–11. 2. Hopper A, Bourke MJ, Williams SJ, Swan MP. Gastrointest Endosc 2010; 71: 967–75. 3. Giday SA, Kim Y, Krishnamurty DM et al. Endoscopy 2011; 43: 296–99. 4. Sung J, Luo D, Wu J et al. Endoscopy 2011; 43 (4); 291–95.

What is the increase of demand for endoscopic services? Results of a state-wide survey of the public sector in Queensland GERALD HOLTMANN,1 MARK APPLEYARD,2 LUKE HOURIGAN,1 JENNY SCOTT,1 MARY-ANNE STOCKWELL,1 JANE INGRAM3 1 Gastroenterology & Hepatology, Princess Alexandra Hospital, Woolloongabba, QLD, Australia, 2 Gastroenterology & Hepatology, Royal Brisbane & Women’s Hospital, Herston, QLD, Australia, 3 Workforce Design & Planning, Queensland Health, Brisbane, QLD, Australia Background Resource restrictions can increase waiting list times and affect patients who wait for prolonged periods for diagnostic procedures. Alternatively, providing excess resources may not result in a tangible improvement in health services. Aims In order to more accurately define the growth of demand, we preformed a state-wide survey of the endoscopic services provided and the waiting list dynamics. Whilst the population grows annually by 1–2%, it also needs to be noted that the high demand demographic group (e.g. subjects aged 65 and above) is growing in some areas substantially more rapidly. Data is needed to support a planning process that meets these future demands. Methods All Queensland Health facilities which provide endoscopic services were asked to participate. A total of 27 sites were asked to provide data on endoscopic activity and waiting lists for the 2009/2010 and 2010/11 financial years. Total numbers for the various types of procedures were calculated. The change in demand was calculated based on activity and then adjusted for the changes in waiting list numbers for specific procedures.

Results In total 46,119 endoscopic procedures were performed in 2009/2010 as compared to 52,299 in 2010/11 (+13.4%). At the same time the medical workforce involved in the provision of endoscopic services grew by 8.1% compared to a 25.2% increase in the nursing workforce. Combining the changes in endoscopic activity and the increase of the waiting list numbers for endoscopic procedures, there was a 22.3% increase in demand. Interestingly, there is considerable variability with regard to the increase of procedures (table). Conclusion From 2009/2010 to 2010/2011 the demand for endoscopic services grew by 25.4%. This is mainly driven by the demand for colonoscopy and EUS. The increase in the demand for colonoscopy can be at least partially explained by the colon cancer screening activities which may have considerable spin off effects and may plateau in the future. Interestingly, the demand for ERCP grew by 12%. Since ERCP has the most rigorous indication, an overall growth of demand for endoscopic services by 12% (+ 3%) can be seen as the most likely long-term scenario. While resources are required to provide more endoscopic services, it is obvious that there is also a need to grow consulting capacity to provide appropriate care for patients who have received these endoscopic services. Table

Growth of endoscopic procedures in 2009/10 vs 2010/11

Procedure Type

Endoscopy

Colonoscopy

ERCP

EUS

Sigmoidoscopy

Other

Growth %

12

22

12

18

26

7

The diagnostic yield of colonoscopy in patients with isolated abdominal pain ANTHONY (SHAW-HUA) KUEH, LIFENG ZHOU, RUSSELL WALMSLEY Waitemata District Health Board, Auckland, New Zealand Background Colonoscopy is an overstretched resource and there is no consensus on whether isolated abdominal pain is an appropriate indication for colonoscopy. Aims We evaluated the proportion of patients referred for colonoscopy with isolated abdominal pain and determined the diagnostic yield for this indication. Methods All patients registered as having a colonoscopy at Waitemata District Health Board on EndoscribeTM reporting database between March 2005 and February 2010 were included. Patients were recruited based on the indication for colonoscopy of: abdominal pain, anaemia or overt rectal bleeding. All investigations and electronic clinical documents for patients with abdominal pain were retrieved and patients with concurrent anaemia, rectal bleeding, weight loss, altered bowel habit, abdominal mass, previous abnormal investigations and history of inflammatory bowel disease or bowel malignancy were excluded. The diagnostic yield between the 3 study groups were compared using Chi-square test, Wilcoxon rank sum test and multiple logistic regression models. Results Total of 10,052 colonoscopies were performed. The abdominal pain group accounted for 1.2% of all colonoscopies performed and had the lowest diagnostic yield (48.8%, P < 0.001). Among those with abdominal pain, significant neoplasia was found in 3.3% and was significantly lower than those with anaemia or overt rectal bleeding groups (P < 0.001). Among those 50 and older, the abdominal pain group continued to have less significant neoplasia (3.8%, P = 0.001) but no difference was seen among the 3 groups in younger patients. Conclusion Colonoscopy is not indicated in patients with isolated abdominal pain.

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Diagnosis based on indication

Total Number Age Range Median Age 18 years, with dysplastic Barrett’s oesophagus referred to our quaternary referral centre were enrolled in this prospective study. Patients were excluded if they had an oesophageal stricture that did not respond adequately to dilatation or ≥T2 lesion. EUS was performed to evaluate for early oesophageal cancer or lymphadenopathy. CEM was performed to assess for dysplastic areas within Barrett’s mucosa. In the absence of nodules, the first RFA treatment was performed. If a nodule was identified, EMR was performed, followed by two RFA treatments at 8 week intervals. Patients were reassessed with CEM and Seattle protocol biopsies after two RFAs. Subjects underwent further treatment if there was residual dysplasia. Adverse events were defined as bleeding requiring blood transfusion, unplanned repeat endoscopic intervention, hospital admission, and surgery. Results A total of 148 procedures were performed in 38 included patients in the study between January 2010 and May 2012. 45 patients were enrolled and 7 met exclusion criteria. Mean age was 66 ( ± 12) years, 34 were men. 23 had high grade dysplasia (HGD), 14 had low grade dysplasia (LGD), 1 had intramucosal carcinoma (IMC). Six patients required EMR prior to RFA. Eleven patients have completed the protocol. At median follow-up of 18 months, 100% have complete remission of dysplasia (CR-D) and 73% have complete remission of metaplasia and dysplasia (CR-IM, CR-D) after 2 ± 0.63 RFAs and 0.36 ± 0.92 EMRs. Three patients were found to have early cancer whilst progressing through the protocol. For 772 patient-years of follow-up, there were 4 adverse events: 1 patient had post procedure chest pain, 1 patient had a minor post EMR bleed, 1 patient had minor bleeding post RFA and 1 had a mucosal tear post RFA. Therefore, the complication rate was 2.70% (95% CI 1.06–6.74%). The sensitivity, specificity, NPV and PPV of CEM were 92%, 72%, 93% and 71% respectively. Accuracy was 81%. Conclusion Multimodal endoscopic therapy for dysplastic Barrett’s in an Australian quaternary referral setting is associated with high remission rates and a low complication rate. Confocal endomicroscopy offers high sensitivity, negative predictive value and accuracy as a test for dysplasia in the setting of Barrett’s.

Narrow band imaging and white light endoscopy with optical magnification in the diagnosis of dysplasia in Barrett’s Oesophagus: results of the Asia Pacific Barrett’s Consortium RAJVINDER SINGH,1 MAHESH JAYANNA,1 JENNIE WONG,2 XIA ZHANG,3 JING LV,3 MING-LAN HAN,4 VIKNESWARAN NAMASIVAYAM,5 NORIYA UEDO,6 WAH KEONG CHAN,7 CHI-YANG CHANG,8 RUPA BANERJEE,9 DONG LIU,3 SHI-YAO CHEN,10 SHOBNA BHATIA,11 KOHEI FUNASAKA,13 WILLIAM TAM,1 KEE WOOK JUNG,12 COSMAS R LESMANA,14 WEN-LUN WANG,8 LEE GUAN LIM,15 HWOON-YONG JUNG,12 KENSHI YAO,16 SHIAW-HOOI HO,17 PRATEEK SHARMA,18 KHEK-YU HO2 1 Gastroenterology, Lyell McEwin Hospital, Elizabeth Vale, SA, Australia, 2Yong Loo Lin School of Medicine, Singapore, Singapore, 3Gastroenterology, The Second Affiliated Hospital, Xi’an Jiaotong University, Xi’an, China, 4National Taiwan University Hospital, Taipei, Taiwan, 5Singapore General Hospital, Singapore, Singapore, 6Osaka Medical Center for Cancer and Cardiovascular Diseases, Osaka, Japan, 7 Gastroenterology, University of Malaya, Kuala Lumpur, Malaysia, 8E-Da Hospital/I-Shou University,, Kaohshiung, Taiwan, 9Asian Institute of Gastroenterology, Hyderabad, India, 10Zhongshan Hospital, Shanghai, China, 11Seth GS Medical College and KEM Hospita, Mumbai, India, 12Asan Medical Cente, Seoul, Democratic People’s Republic of Korea, 13 Nagoya University Graduate School of Medicine, Nagoya City, Japan, 14Cipto Mangunkusumo Hospital, Jakarta, Indonesia, 15National University Health System, Singapore, Singapore, 16Fukuoka University Chikushi Hospital, Fukuoka, Japan, 17University of Malaya Medical Centre,, Kuala Lumpur, Malaysia, 18 University of Kansas Medical Centre, Kansas, KS Background The current recommended guideline of surveying patients with Barrett’s Oesophagus (BE) is time consuming and poorly adhered to. With the advent of new endoscopic imaging modalities, predicting histology in BE in real time with high accuracies appears promising and could potentially obviate the need to perform random biopsies. We evaluated the utility of white light endoscopy with magnification (WLE-z), Narrow Band Imaging with magnification (NBI-z) and a combination of both modalities (WLE-z and NBI-z), amongst a group of Endoscopists from the Asia Pacific region. Aims To assess the utility of WLE-z and NBI-z in patients with Barrett’s Oesophagus. Methods A total of 27 Endoscopists from 10 countries who have interest and expertise in BE participated in this online study. Fifty areas in BE imaged on WLE-z and NBI-z with corresponding histology were prospectively collected and randomly rearranged in 100 slides. All procedures were performed using the commercially available Olympus 160Z endoscope with optical magnification (115×) capability. After a short learning power point presentation was viewed (15 minutes), the assessors were invited to grade these images on an online website. A simplified classification based on the pit pattern and microvascular architecture was used to grade the images: (i) IR: irregular or absent pits, with irregular microvasculature (high-grade dysplasia), (ii) R: regular or absent pits, with regular

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microvasculature (no dysplasia), or (iii) Eq: equivocal/unsure [note: (i) & (ii) graded with confidence, (iii) – no confidence]. To simulate real-time endoscopy where both imaging modalities are often interchanged, each WLE-z and the corresponding NBI-z images were shown side by side on a single slide (50 slides in total). The assessors were then requested to grade these images with the same classification. The overall Accuracy (Acc), Sensitivity (Sn), Specificity (Sp), Positive Predictive Value (PPV), Negative Predictive Value (NPV) of WLE-z, NBI-z and the combination was then determined. Results A total of 4050 images were available for evaluation. The assessors were able to grade 3370 images with confidence (83.2%). The overall Acc for WLE-z and NBI-z was 87.1% and 88.7% respectively. When images from the two modalities were placed side by side, the Acc increased to 90.3%. The Sn, Sp, PPV, and NPV of WLE-z was 48%, 92%, 45%, 93% whilst with NBI-z, this improved to 89%, 89%, 56%, 98% respectively. When both imaging modalities were viewed together, this improved further to 93%, 90%, 61%, and 99%. Conclusion The high NPV (99%) when both WLE-z and NBI-z were used simultaneously indicates that regular appearing areas which are diagnosed with confidence can effectively be left alone and not biopsied. This approach could potentially lead to a paradigm shift of how patients with BE are surveyed and may enable random 4 quadrant biopsies to be abandoned altogether.

EUS for CBD and PD dilatation ALINA STOITA,1 IAN D PENMAN2 1 St Vincent’s Hospital Sydney, Darlinghurst, NSW, Australia, 2Centre for Liver and Digestive Disorders, Royal Infirmary, Edinburgh, United Kingdom Background One of the commonest endoscopic ultrasound (EUS) referrals is to clarify the cause of dilatation of common bile duct (CBD) and/ or pancreatic duct (PD) seen on a conventional imaging (CT/US/MRI). Aims The aim of this study was to determine the accuracy of EUS in these cases, to explore the cause of CBD/PD dilatation and analyse difficulties encountered. Methods Analysis of EUS cases performed for dilatation of CBD and/ or PD on imaging from a prospectively maintained database in an experienced EUS centre between Jan 2008–Jan 2010 was performed. The results of EUS+/− FNA were recorded. All patients were followed up for 1 year. Patient’s status (alive/dead), further interventions (ERCP) and imaging, blood results, operations, clinical follow up and alternative diagnoses were recorded. Results 102 patients had EUS for dilated CBD and/or PD during this period and 98 patients (100 procedures) were followed up for a minimum of 1 year. EUS had 95% sensitivity, 94% specificity for detecting any pathology and a 93% positive predictive value and a 96% negative predictive value. A cause of dilatation was found in 51% of cases. The commonest cause of CBD+/− PD dilatation was stones (14%) followed by benign biliary strictures (12%), malignancy (6%), ampullary adenomas (5%), chronic pancreatitis (6%), CBD papillomas (4%) and duodenal diverticulum (3%). Difficulties were encountered in interpreting CBD strictures post sphinctectomy with stent in situ and two of those cases were false positive. Another false positive case was due to a soft tissue lesion in the distal CBD (interpreted as papilloma) with a negative duct trawl at biliary sphincterotomy. There were two false negative cases: on a background of severe pancreatitis an ampullary carcinoma and a cholangiocarcinoma were interpreted as part of chronic pancreatitis rather than malignancy. Conclusion In patients with dilated CBD and/or PD on imaging, EUS has a high accuracy (95%) and high negative predictive value and should be considered as the first line modality. Difficulties arise in interpreting severe chronic pancreatitis and post sphincterectomy ± CBD stent images. We recommend having a low threshold to FNA abnormal areas in cases

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of severe chronic pancreatitis. Contrast enhanced EUS may help target the most appropriate area for FNA. Small soft tissue lesion in distal CBD (‘papilloma’) is a poorly defined entity of uncertain significance. In our cases it seems to run a benign course and ERCP was not helpful in diagnosing it.

EUS FNA for solid pancreatic masses lessons from practice and pitfalls ALINA STOITA,1 IAN D PENMAN2 1 Gastroenterology, St Vincent’s Hospital Sydney, Darlinghurst, NSW, Australia, 2Center for Liver and Digestive Disorders, Royal Infirmary Hospital, Edinburgh, United Kingdom Background Endoscopic ultrasound (EUS) has been shown to be the best modality to stage pancreatic cancer loco-regionally. EUS guided fine needle aspiration (FNA) is a safe and reliable modality of confirming malignancy with accuracy between 78–95%(1). Aims The aim of this study was to determine the causes which decrease the EUS FNA accuracy and to compare the accuracy of sonographic diagnosis with that of EUS FNA when no cyto-pathologist is on site. Methods Analysis was undertaken of all EUS cases performed for solid pancreatic masses from a prospectively maintained database at an experienced EUS centre between Jan 2008–Jan 2010. The sonographic diagnosis and FNA results were compared. All patients were followed up for one year. Patient’s status (alive/dead), further imaging, blood results, operations, clinical follow up and alternative diagnoses were recorded. Results 125 patients (128 procedures) had EUS for solid pancreatic masses and 124 patients (127 procedures) were followed up over a minimum of 1 year. EUS FNA was performed in 102 patients and was positive for malignancy in 76% of cases (62/81 true positive cases for pancreatic adenocarcinoma). In addition, based on the sonographic images alone the endoscopist unequivocally and accurately diagnosed another 19 malignancies in cases with FNA results of atypia (7), benign cells (8), insufficient samples (2) as well as two patients that went straight to surgery. Overall, EUS and EUS FNA had a sensitivity of 95%, specificity of 97% and positive predictive value of 98% and negative predictive value of 89%. However, four cases were false negative: chronic pancreatitis with a mass, mixed solid cystic malignant NET, a necrotic tumour with a secondary abscess and an ampullary cancer. One case was false positive as a pancreatic mass was called malignant but the patient is well at 2 years. There were 23 cases of chronic pancreatitis (CP) and EUS correctly diagnosed 13 cases of benign CP and 2 malignancies in CP but difficulties were encountered in differentiating between focal chronic pancreatitis and malignancy (8 cases). Half of these cases (4/8) were malignant and EUS FNA provided a diagnosis in 3/4 cases and showed atypia in 1 case. Conclusion Combined EUS appearances and EUS FNA have a high accuracy (96%) for detecting pancreatic malignancy even without an on site cyto-pathologist. In experienced hands EUS alone has a high positive predictive value for malignancy and in suspicious cases with negative cytology alternative means of confirming the diagnosis should be employed or the procedure should be repeated. Difficulties arise in chronic pancreatitis with a possible mass when the endoscopist has difficulties making an accurate assessment on the sonographic appearances alone. We recommend that all cases of chronic pancreatitis with a possible mass undergo FNA and should be repeated if cytology is negative or doubt persists. Reference (1) Yoshinaga S, Suzuki H, Oda I, Saito Y. Role of endoscopic ultrasound-guided fine needle aspiration (EUS-FNA) for diagnosis of solid pancreatic masses. Dig Endosc. 2011 May;23 Suppl 1:29–33.

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Dysplasia in normal appearing ampulla in familial adenomatous polyposis surveillance JONATHAN (YONG) C TAN, IAIN J SKINNER, ROBERT CHEN Western Hospital Footscray, PARKVILLE, VIC, Australia Background Familial Adenomatous Polyposis (FAP) syndrome is a hereditary autosomal dominant condition where the APC gene defect results in numerous adenomatous polyps through the colon. These can also develop in the upper gastrointestinal track in particular the ampullary region. Recommendations for surveillance are based on the presence of dysplasia. However, routine biopsies are currently not recommended with normal appearing ampulla due to the risk of pancreatitis. Aims To examine the incidence of dysplasia in normal appearing ampulla and complications related to ampullary biopsies in particular pancreatitis and duodenal polypectomies during surveillance in FAP patients Methods Western Hospital Footscray is one of the tertiary hospitals in Melbourne. We analysed retrospectively all surveillance gastroscopies performed on FAP patients in our centre. Various sub analyses were performed: 1) Endoscopic appearance of the ampulla were correlated with the degree of dysplasia found on biopsies. 2) Duodenal polypectomies and the associated complications. Results 15 FAP patients were identified having regular endoscopic surveillance in our centre. A total of 95 upper endoscopy procedures were performed where 87 ampullary biopsies were taken. Ampullary appearance on endoscopy with various histology findings were as follows (see table). No complications were noted in relation to ampullary biopsies in particular pancreatitis. Incidentally, gastro-oesophageal junction carcinomas were found in 2/15 (13.3%) patients during routine surveillance. 26 duodenal polypectomy procedures were performed with a total of 51 polyps removed. Bleeding (26.9%) was the only complication noted. Of the 7 episodes, 3 required utilising of coagulation graspers during the procedure, 1 settled spontaneously, 2 overnight hospital stay for observation and 1 needed a three day hospital admission. None of the procedures required a repeat endoscopy with therapeutic intervention for haemostasis. Conclusion In this retrospective study, we demonstrate that dysplasia is present in a significant proportion of ampullary biopsies despite normal ampullary appearance on endoscopy. Complications in particular pancreatitis was not observed in our cohort. This study lends support to routinely perform ampullary biopsies in the surveillance for dysplasia in FAP patients despite normal looking ampulla. Careful observation of the gastrooesophageal junction during surveillance gastroscopy is also essential by the findings of gastro-oesophageal carcinoma from our study cohort. This study also demonstrates that experienced endoscopists can perform duodenal polypectomies safely with minor bleeding as the main complication. Ampullary Appearnce

No Dysplasia

Low Grade Dysplasia

Moderate Dysplasia

Normal (27) Prominent (34) Appeared adenomatous (26)

13 (48.15%) 13 (38.24%) 7 (26.29%)

13 (48.15%) 16 (47.06%) 17 (65.38%)

1 (3.7%) 5 (14.71%) 2 (7.69%)

Infection related complications of percutaneous endoscopy gastrostomy tube insertion are high despite prophylactic antibiotic use ALVIN Y TING, NIK SHENG (JOHN) DING, SUMAYA ALUKAIDEY, DAMIAN DOWLING, CHRISTOPHER HAIR Gastroenterology, Barwon Health, Geelong, VIC, Australia Background Percutaneous endoscopic gastrostomy (PEG) insertion has become a common intervention since its introduction in 1980 to aid enteral nutrition in compromised patients. There is substantial evidence that prophylactic parental antibiotic use reduces infection rates. Despite advances in endoscopic techniques, localised infections still occur in one third of PEG insertions. Aims We aimed to assess the rate of infection related complications of PEG insertion within our practice, to evaluate the use of prophylactic antibiotics and to describe the rate of infection amongst those who received prophylactic antibiotic and those that did not. Methods We conducted a retrospective review of 100 cases of PEG catheter insertions performed between 2009 and 2012 at Geelong Hospital, Victoria. Exclusion criteria included incomplete documentation in medical records. Data collection included patient baseline demographics, PEG indications, type, duration, dosage and route of antibiotic use, and postoperative outcomes. Statistical analysis was performed using t-test and Chi squared test where appropriate. Results 2 of 100 patients were excluded from the study. 98 patients underwent PEG catheter insertion (64 males, 34 females), of age range 18–91 years (mean age 69 years). The main indication for PEG insertion was head and neck malignancy in 50% (49/98), followed by stroke in 22% (22/98). Prophylactic antibiotics were used in 76% (74/98) of patients; as a single dose at the time of procedure in 80% (59/74), or combination of pre and post procedure in 20% (15/74). Prophylactic antibiotics used include cephalosporins in 80% (73% cephazolin, 7% ceftrixone), ticarcillin in 20% and vancomycin in 1%. Post-operative complications were documented in 27% (26/98) of the cohort. Localised infection accounted for 73% (19/26) of all complications. Subgroup analysis of all patients who received prophylactic antibiotics revealed local infection rates of 24% (18/74). Of the 19 with localised infections, most (18/19) had received prophylactic parenteral antibiotics. Infectious complications were less frequently seen in patients who did not receive prophylactic antibiotics as compared to those that did (p = 0.02). There was no association of infection risk with indication for PEG (p > 0.05). Conclusion There is a high rate of local infectious complications within this cohort, and a suboptimal adherence to guidelines for prophylactic antibiotic use during PEG insertion. However, despite this finding the majority of complications were seen in patients receiving antibiotic prophylaxis. It is likely that other factors such as patient comorbidity and insertion technique are important in the development of local infections. Further study is warranted regarding risk factors for complications of this commonly performed procedure.

Acute necrotising pancreatitis complicating intragastric balloon placement ROSLYN VONGSUVANH, DAVID VAN DER POORTEN Department of Gastroenterology and Hepatology, Westmead Hospital, Westmead, NSW, Australia Background Endoscopic intragastric balloon (IGB) placement offers a less invasive alternative to weight loss than bariatric surgery. Nevertheless, IGBs are associated with complications, the most serious including bowel obstruction. The Spatz adjustable balloon system (ABS), with a

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non-collapsible catheter, was designed to decrease the risk of balloon migration and bowel obstruction. Aims We report the first case of severe acute necrotizing pancreatitis following migration of a Spatz balloon catheter into the duodenum. Methods Case History A 30-year-old female with a BMI of 29 and insulin resistance was referred for IGB insertion after failing to reduce weight by lifestyle measures. A Spatz ABS was inserted into the fundus and required two adjustments to a final volume of 700 mls at 8 months, which was well tolerated. At 11 months, the patient presented with a 24-hour history of severe abdominal pain. On abdominal examination, she was diffusely tender with guarding. Biochemistry was consistent with severe acute pancreatitis with amylase 2114 U/L, lipase 10,700 U/L, white cell count 11.2 × 10 9/L, CRP 21 mg/L, corrected calcium 2.00 mmol/L and albumin 25 g/L. Her liver enzymes were normal and she had no risk factors for pancreatitis. Abdominal CT showed extensive portal venous gas. The stomach was distended with possible intramural gas at the fundus but no perforation. The balloon catheter had migrated into D2 with adjacent mucosal inflammation and oedema. On gastroscopy, the balloon was in the fundus but the catheter was impacted at the D1/D2 junction. From mid-body to antrum, was an area of ischemia thought secondary to a pressure effect from the tightly angulated catheter. Given the possible intramural gas and the inability to retrieve the catheter endoscopically, the patient proceeded to laparotomy. At laparotomy, the gastric wall was ischemic but viable. The pancreas, however, was inflamed with fat saponification, necrosis and haemorrhage. The catheter was coiled at D1/D2 and removed with difficulty. Results The patient’s post-operative course in high dependency was complicated by pneumonia and wound dehiscence. She was discharged home on day 14. Conclusion This is the first case of severe, acute necrotizing pancreatitis secondary to IGB catheter impaction into the duodenum. The Spatz ABS is purported to have a non-collapsible catheter that lowers the risk of balloon migration and is thus approved for 12-month implantation. This case demonstrates that catheter migration with this device can occur even in the absence of deflation and with serious sequelae. A third generation Spatz ABS with an additional migration prevention function was designed to improve safety, but this was recently withdrawn from the market. Endoscopic weight loss devices are a necessary development in the face of the obesity epidemic, however the technology is far from perfect.

Endoscopic ultrasound (EUS)-guided endoscopic anterograde cholangioancreatography (EACP) with anterograde biliary interventions: single centre experience FRANK WEILERT, AMANDA F IRETON Peter Stokes Endoscopy Unit, Waikato Hospital, Hamilton, New Zealand Background EUS has been used to perform cholangiopancreatography and gain access for rendezvous ERCP. This technique can be a useful platform for anterograde interventions when retrograde access in not possible. We describe our single-centre experience with this technique that we refer to as EACP. Aims Assess technical and clinical success of EACP with anterograde biliary interventions when ERCP fails. Methods We retrospectively reviewed a database to identify all patients in which EUS-guided EACP with anterograde interventions was attempted from 6/11–2/12. The following data were collected: patient demographics, clinical indication, EUS puncture technique and procedural outcome. Results EUS-guided EACP with anterograde biliary intervention was attempted in 8 patients over the study period. Mean (median) patient age

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was 69 years (range 45–87) and most were male (75%). Indications included cholangiocarcinoma (n = 1), pancreatic cancer (n = 4), other malignant bile duct obstruction (n = 2) and one patient with gallstones post-Roux-Y-gastric bypass. Standard ERCP failed in 75% (n = 6) and/or previous percutaneous trans-hepatic cholangiogram (PTC) was performed in 25% (n = 2), while 25% (n = 2) had primary EUS-guided therapy. Standard ERCP was not possible due to: inability to pass wire anterograde through ductal strictures for ERCP rendezvous (62.5%), non-traversable duodenal stricture (25%) or surgically altered anatomy (12.5%). EUS puncture was transgastric-intrahepatic (100%) using a 19G EUS-FNA needle (100%). EACP interventions were technically successful in 7/8 (87.5%). Anterograde biliary interventions for malignant obstruction involved placement of anterograde metal stents in 7/8 patients: transpapillary stent (n = 4) or trans-gastric (hepato-gastrostomy) stent (n = 3). The patient with CBD stones post-RYGB had an anterograde sphincteroplasty and only partial clearance of stones and developed a post-operative bile leak treated definitively with surgery, which included CBD clearance. Conclusion EUS-guided EACP allows successful biliary drainage in most cases when ERCP fails or is not possible. EACP may be an option in surgically altered anatomy. Complications are comparable to PTC, however, specific tools for this procedure are needed to optimize efficacy and safety.

EUS-guided translumenal drainage of peri-pancreatic fluid collections using an exchange-free platform (NAVIX): 53 consecutive drainage procedures FRANK WEILERT, STEVE KANE, KENNETH F BINMOELLER Interventional Endoscopy Services, California Pacific Medical Center, San Francisco, CA Background Translumenal peri-pancreatic fluid collection (PFC) drainage with currently available tools remains technically challenging, which requires multiple device exchanges. The NAVIX access device was designed to enable secure exchange-free PFC access, tract dilation, and guidewire placement under EUS-guidance. Aims To evaluate the safety and efficacy of EUS-guided drainage of pancreatic fluid collections using the NAVIX access device. Methods Patients with pancreatic fluid collection treated by EUS-guided drainage between February 2010 and March 2012 were identified in a prospectively collected database. A curved linear array echoendoscope with colour Doppler was used to identify and access the fluid collection. Sequential steps: 1) trocar on the NAVIX used to gain access to the pseudocyst, 2) balloon catheter advanced directly over the trocar followed by inflation of the anchor balloon component to secure access, 3) aspiration of a diagnostic fluid sample and Isovue contrast injection to document the radiological outline of the pseudocyst cavity, 4) first 0.035 guidewire inserted 5) cystenterostomy tract dilated to 10 mm with the dilation balloon component, followed by a second 0.035 guidewire insertion, 6) device was removed leaving the two guidewires in place, 7) two double pigtail stents (7 Fr and 10 Fr × 3 cm) were inserted in sequence. For poorly adherent fluid collections (WOPN or abscesses), balloon dilation of the cystenterostomy was not performed and a fully covered SEMS (10 mm × 4 cm) was inserted. Results Fifty-one patients with peri-pancreatic fluid collections underwent 53 procedures (32M:19F; mean age 49.7 yrs, range 18–86 yrs). The mean size of the fluid collection was 101 mm (median 85 mm, range 41–175 mm). The procedure was technically successful in all patients. The mean procedure time was 49 min (median 47 min, range 29–81 min) and 38.5 min (median 34.5 min, range 22–69 min) for plastic (n = 24) and SEMS (n = 27) insertion, respectively. There were no acute procedure-

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related complications. CT was followed to cyst resolution or further intervention in all the patients. Conclusion 1. EUS-guided pseudocyst drainage using the NAVIX access device followed by one SEMS or two plastic stents is safe and effective. 2. A single drainage procedure accomplishes cyst resolution in most patients. 4. Cyst drainage from the distal duodenum may be riskier and warrants caution.

Closing the hole is the goal MMC’s experience of the novel OVESCO over the scope clip (OTSC) in the treatment of GI fistula PATRICK WILSON, DAVID DEVONSHIRE, MICHAEL SWAN, DANIEL CROAGH Gastroenterology, Monash Medical Center, Melbourne, VIC, Australia Background Perforations and post anastomotic leaks of the GI tract are a significant source of morbidity. Surgery has been the standard of care but advances in endoscopic treatment may change this. The OTSC system Incorporates a clip fixed over a cap fitted on the endoscope. We present our experiences with this novel endoscopic procedure. Aims To assess the effectiveness of the new OTSC in the closure of different GI defects Methods From October 2011 until May 2012 we collected data from all patients referred for consideration of endoscopic closure of GI fistula. All patients had failed conservative management and were being considered for surgical intervention. The outcome measures recorded were the success of the procedure (both early and late) and 30 day adverse event rate. Results 12 patients (8 male) with a mean age of 55 (range 36 to 82) underwent a total of 15 procedures over the specified time period. Indications for the OTCS included Gastro-cutaneous fistula post surgery (4), entero-cutaneous fistula post surgery (4), leak post bariatric surgery (3), Leak post HPB surgery (2), perforating GU (1), and perforating DU (1). Two patients had undergone previous endoscopic attempted closure using different technology and one (perforated DU) patient had failed surgical management. Initial success rate was 73% with a long term success rate (LTSR) of 63%. If we include initial technical failure to deploy the OTSC (intention to treat) the LTSR was 46%. LTSR was more likely in patients who had normal anatomy (4 patients w/100% LTSR) compared to post surgical anatomy and placement of the OTSC in the stomach (4/7 LTSR) vs the small bowel (2/5 LTSR). Reasons for failure included inability to deploy the OTSC due to technical difficulties (4 procedures), complex fistula site with multiple fistula (2 procedures) and suspected OTSC detachment (2 procedures) Failure to deploy the OTSC was due to inability to intubate the oesophagus with the OTSC clip (3 patients) and distal location of the fistula site (one patient). Two patients who were unsuccessful initially underwent retreatment, of these one achieved LTSR. There were no significant complications. Conclusion The new OVESCO OTSC is a significant advance in the treatment of gastrointestinal fistula. Larger studies are needed to confirm the durability of this new technique.

Spyglass® cholangioscopy system enhances diagnostic certainty in biliary and pancreatic disorders PATRICK WILSON, DAVID DEVONSHIRE, CHRISTOPHER HAIR Gastroenterology, Monash Medical Center, Melbourne, VIC, Australia Background Spylgass Spyscope® is a single operator endoscope that can aid in the diagnosis of various pancreatic and biliary diseases. The aim of the current study was to evaluate Spyglass® cholangiopancreatoscopy for its performance and safety. Aims To look at our practice over a three year period to examine our outcomes. Methods We performed a prospective clinical audit of consecutive patients undergoing cholangiopancreatoscopy at our institution commencing in December 2008. The major outcomes were adequacy of visualisation, diagnostic success rate, and 30 day adverse event rate. Results Spyglass® endoscopy was performed in 37 patients (15 male, 22 female, mean age 59). Diagnostic indications included indeterminate biliary strictures (27), pancreatic lesions (2), common bile duct filling defect (9). Visualisation was adequate in 100% of patients. Spyglass® assisted biopsy was performed in 8 patients and achieved a specimen that was adequate for histopathological assessment in 100% cases. The diagnosis of indeterminate biliary strictures was modified in 23/27 cases (85% of cases). 10 malignancies were diagnosed (9 cholangiocarcinoma, 1 intraductal papillary mucinous tumour (IPMT) of pancreas). Management outcomes were altered in 85% of cases of patients. The 30 day adverse event rate was 8% (3 cholangitis). One patient was readmitted after 3 weeks due to acute severe sepsis with multiorgan failure and died despite intensive support. Conclusion Spyglass® cholangioscopy system enhances diagnostic certainty in biliary and pancreatic disorders and should be considered in selected patients where there is diagnostic doubt.

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Journal of Gastroenterology and Hepatology (2012) 27 (Suppl. 4)

Hepatology Clinical Liver transplantation for erythropoietic protoporphyria (EPP) – case series ZAID S ARDALAN,1 SUJIEVVAN CHANDRAN,1 ADAM TESTRO,1 ILANA GORY,1 XINYI ZHOU,2 MARCIA RODOV,3 SIMONE STRASSER,3 GRAEME A MACDONALD,2 PAUL GOW,1 PETER ANGUS1 1 Liver Transplant, Austin Health, Melbourne, VIC, Australia, 2Liver Transplant, Princess Alexandra, Brisbane, QLD, Australia, 3Liver Transplant, Royal Prince Alfred, Sydney, NSW, Australia Background Erythropoietic protoporphyria (EPP) is a rare genetic disorder in which excessive production and biliary excretion of protoporphyrin can lead in a minority of patients to progressive liver disease ultimately requiring liver transplantation (OLT). Since first described in 1980 there has been limited experience with the natural history of liver disease and the unique peri and post-operative complications of OLT including disease recurrence in the allograft. Aims To review the Australian experience of EPP related liver disease across the transplant centres. Methods Utilizing the Australian transplant registry and the databases at each transplant centre we identified four patients referred to these

units over the last three years. Case notes and investigations were then reviewed. Results Four female patients with a background of EPP related liver disease were identified. The mean age at the time of referral for transplantation of 26.75 years (range = 20–41). All four patients were worked up for OLT and managed with various measures to reduce protoporphyrin levels. Despite falling porphyrin levels two patients died two months after being referred from complications related to end stage liver disease with severe sensory-motor neuropathy heralding deterioration in one of them. The remaining two were transplanted 3 and 8 months later. There were no remarkable intra or perioperative complications. The early post-operative course was remarkable in one patient for acute allograft rejection, 10 days post OLT, responding to 3 days of pulse methylprednisolone, and in both for anastamotic billiary stricture requiring endoscopic therapy. Early disease recurrence in the allograft occurred in both patients (20 days postOLT in one and 70 days in the other). Measures to reduce porphyrin levels were reinstituted and successfully lowered porphyrin levels. One patient has had two haematopoietic stem cell transplantation. At last review both patients had stable liver biochemistries and were on regular RBC exchange. Conclusion Management of end stage liver failure in EPP patients is difficult. Liver transplantation can be lifesaving, however it requires specific peri-operative interventions. Despite OLT, hepatic recurrence is common and the only definitive treatment of EPP is Haematopoietic stem cell transplantation.

Patient

Age

Pre treatment complications

Received liver transplant

Measures to reduce porphyrin levels pre transplant or death

Time of EPP recurrence post OLT

Post OLT complications

Current treatment

Bone marrow transplant

1

20

No

Haem arginate

N/A

N/A

N/A

N/A

2

21

ESLF HRS Variceal bleed ESLF

Yes

70 days

Anastomotic biliary stricture, EPP recurrence

RBC exchanges fortnightly

No

3

25

ESLF Recurrent bacterial infections

Yes

Cholestyramine, Ursodeoxycholic acid, Plasmaphoresis, haem arginate, blood transfusions Plasmaphoresis, haem arginate, iron supplementation

20 days

RBC exchange fortnightly

Yes

4

41

ESLF Sensory motor neuropathy

No

Haem arginate, Plasmaphoresis, blood transfusions

N/A

Acute allograft rejection, anastomotic biliary stricture, EPP recurrence N/A

N/A

N/A

Rifaximin for refractory hepatic encephalopathy: impact on hospital admissions and length of stay at a liver transplant centre ZAID S ARDALAN, SUJIEVVAN CHANDRAN, PAUL GOW, ADAM TESTRO, PETER ANGUS Liver Transplant Unit Victoria, Austin Health, Heidelberg, VIC, Australia Background Hepatic encephalopathy (HE) is a chronic and relapsing complication of end stage liver disease often resulting in frequent

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hospital admissions. Rifaximin, a minimally absorbed oral antibiotic, has recently been shown to maintain remission from HE and to reduce hospitalization. Aims Rifaximin has recently become available at our centre for the management of patients with recurrent HE. We therefore sought to audit our experience, particularly with regards to impact on hospitalization and length of stay, in order to assess both efficacy and cost-effectiveness. Methods Rifaximin has been available at Austin Health since October 2011. Due to the cost of the drug (approximately A$160/patient/month) commencement of rifaximin currently requires approval by the Clinical Pharmacology Unit. The Clinical Pharmacology database was therefore

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used to identify all patients who were prescribed rifaximin for the indication of HE. The medical record, correspondence and pathology results were reviewed to directly compare the 6-month periods pre- and postcommencement of rifaximin with regards primarily to number of hospital admissions, cause of admission, and length of stay. Results 12 patients have commenced rifaximin at a dose of 1000 mg daily in divided doses. Median age is 61.5 years with a male predominance (83.3%). Aetiology of liver disease included NASH (4 patients), HCV (4 patients), alcohol (3 patients) and combination of HCV/alcohol (1 patient). 2 patients died at 0.5 and 1-month post commencement of rifaximin, and were therefore excluded from further analysis due to inadequate follow-up. All other patients (10) were followed up for 6 months. Results demonstrated in the table below represent the median (range). Non-parametric statistical analysis using the Mann-Whitney U test was performed. Conclusion Within the limitations of this small audit, the introduction of rifaximin has led to a 50% reduction in both the number of admissions with HE and the length of stay, supporting the available literature and suggesting that when HE does occur, the severity of the encephalopathic episode is reduced. With a daily bed cost exceeding A$1000, rifaximin appears to be highly cost effective in this cohort of patients. Comparison of 6-month period pre- and post-rifaximin administration ChildPugh Score

PreRifaximin PostRifaximin P=

10 (8–12) 10 (7–12) 0.68

MELD Score

17.5 (9–32) 15 (8–30) 0.48

Total daily lactulose dose mL

90 (40–160) 85 (0–480) 0.40

Admissions with encephalopathy per patient

2 (1–5) 1 (0–7) 0.07

Length of stay (days) per admission

6 (3–46) 3 (2–37) 0.005

Total length of stay (days) over 6 month period 16 (5–65) 8 (2–37) 0.30

Acute liver failure in familial HLH: a case of 2 siblings highlighting the importance of early diagnosis to avoid futile liver transplantation and improve survival FARIHA BALOUCH,1 CHRIS FRASER,2 PETER LEWINDON1 1 Gastroenterology and hepatology, Royal Children hospital, Brisbane, QLD, Australia, 2Oncology, Royal children hospital, Brisbane, QLD, Australia Background HLH is a genetically determined, rapidly progressive, potentially fatal hyperinflammatory condition of a highly stimulated but ineffective immune response. Early diagnosis can be difficult but progressive liver dysfunction culminating in massive hepatic necrosis prompting consideration for what would be a futile Liver Transplantation can occur in days. Aims To emphasize that timely diagnosis and institution of therapy in HLH may reduce ultimate morbidity and mortality. Methods Review of medical records. Results Case 1: 11 months old boy presented with prolonged fever, lethargy and worsening LFTs (Bilirubin 67/53 μmol/L, Albumin 28 g/L, ALT 326 U/L, AST 61 U/L, GGT 480 U/L, ALP 94 U/L, and LDH 1330 U/L) weeks after a proven CMV infection. Synthetic function was normal; there was a mild cytopenia (Hb 85 g/L, WCC 15.5, platelets 122). Ferritin was 1000 μmol/L, Triglycerides 6.3 mmol/L and fibrinogen 2.8. Liver biopsy demonstrated non-specific portal inflammation with lymphocytes and bone marrow failed to demonstrate heamophagocytosis. 4 days after biopsy he died of fulminant, undiagnosed liver failure. Liver

Transplantation was considered. Autopsy revealed progression to massive hepatic necrosis but failed to confirm a diagnosis. Case 2: 4 years later his 2 year old sibling presented with 10 days of fever and moderate liver dysfunction (INR 1.5, albumin 16 g/L, ALT 468 U/L, AST 1080 U/L, ALP 632 U/L, GGT 448 U/L). After reviewing the notes of the deceased sibling, urgent Ferritin 5000 μmol/L and Triglycerides 2.9 mmol/L led to urgent BMA that evening which demonstrated haemophagocytic syndrome. Precipitating EBV infection was subsequently confirmed. Further review of liver biopsy from Case 1 confirmed that he also had EBV hepatitis. Chemotherapy commenced that evening but failed. Bone Marrow Transplantation was initially successful then complicated by graft rejection. Treatment with donor lymphocyte infusion reversed falling chimerism but lead to severe, resistant GVHD. Persistent EBV infection led to Post Transplantation Lymphoproliferative disorder and death 7 months later. Genotyping for common mutations associated with HLH was unsuccessful in these boys and only successful in 50% of cases. Conclusion This report confirms a typical catastrophic course of Familial HLH precipitated by EBV infection. It highlights the need to consider HLH in any child with rapidly progressive liver failure especially if there is a family history or identified precipitating event such as EBV, CMV, and preceding history of auto-immune condition or immunosuppression. Early diagnosis by checking Ferritin, Triglycerides, Fibrinogen and BMA or liver biopsy is crucial as patients deteriorate rapidly and liver transplantation is futile. Early intervention offers the best chance of survival and consideration of pre-emptive bone marrow transplantation for subsequent siblings can be considered. References Janika GE. Familial and acquired hemophagocytic lymphohistocytosis. Eur J Pediatr 2007; 16692:95–109. Janka G. Hemophagocytic lymphohistocytosis: when the immune system runs amoke. Klin Padiatr 2009; 221:278–285. Weitzman S. Approach to hemophagocytic syndromes. Haematology 2011; 2011:178–182.

Nurse assessment clinic: an efficient and effective delivery of hepatitis C virus (HCV) assessment and review MARIAN L BIDDLE,1 NIKKI R ADLER,1 MELISSA J HEATH,2 SUSAN E STREAT,2 MARGARET M WARDROP,2 JON P WATSON1 1 School of Medicine, Deakin University, Waurn Ponds, VIC, Australia, 2Department of Medicine, Barwon Health, Geelong, VIC, Australia Background As health care delivery moves towards a chronic disease model, a multidisciplinary approach is essential. One strategy for implementation is the establishment of a nurse assessment clinic. This approach facilitates timely medical review while maintaining high-quality, patientcentred care. Qualitative data indicates that patients are equally satisfied with being reviewed by a nurse or a doctor on initial screening visits [1]. Historically, there has been a conflict between the medical and nursing professions as the latter has struggled to achieve full implementation and acceptance [2]. While nurses and doctors have distinct roles in health care delivery, their contribution synergistically leads to enhanced patient care. In May 2010, a nurse assessment clinic was established in the Liver Clinic at Geelong Hospital to provide assessment, counselling, education and treatment of patients infected with HCV. It was also hoped that this clinic would drive efficiency within the service by facilitating patient flow and maximising attendance. Aims The aim of this study was to assess the first two years of operation of the nurse assessment clinic at Geelong Hospital.

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Methods Patients are seen at the clinic, firstly by a nurse and on a subsequent day by a physician. The purpose of the initial nurse appointment is to ensure standard tests are ordered and results are available before a medical review, as well as to educate the patient regarding HCV disease. Those requiring rapid assessment can be fast tracked to see a physician while non-attenders are identified, negating their need for further specialist appointments. Patients with specific psychiatric needs can also be identified early in the treatment process. Requests for tests are reviewed and ordered by the physicians, and the nurses are well supported within this team environment. Data was obtained retrospectively from clinical records at the Liver Clinic at Geelong Hospital. The number of patient appointments, the waiting times for appointments and lost to follow up rates were analysed before and after the institution of the nurse assessment clinic. Results The nurse assessment clinic has been successfully established and is functioning autonomously within the Hepatology Service at Geelong Hospital. We have already seen a reduction in the percentage of patients lost to follow up from 19.2% to 2.1%. Waiting times for an initial patient appointment at the clinic have reduced from nine months to 6 weeks. Conclusion The nurse assessment clinic at the Geelong Hospital improves efficiency and enhances outcomes for patients with HCV. We look forward to further developments within the clinic in an attempt to further improve patient outcomes. References 1. Munday P, Allan A, Hearne S, Gubbay A. The role of the nurse in screening asymptomatic male and female patients in a sexual health clinic. Int J STD AIDS. 2005;16:281–283. 2. Appel A, Malcolm P. The triumph and continuing struggle of nurse practitioners in New South Wales, Australia. Clin Nurse Spec. 2002;16(4):203–210.

Lessons learnt: implementation of transient elastography as a routine clinical service MARRIANNE BLACK, CAROLINE TALLIS, ANDREW ST JOHN, KATHERINE A STUART, GERALD HOLTMANN Department of Gastroenterology & Hepatology, Princess Alexandra Hospital, Brisbane, QLD, Australia Background Transient Elastography Service (FibroscanR) is a technology that is believed to facilitate the staging of liver disease. Thus this technology is high on the wish-list of many centres providing services for patients with chronic liver disease. However, there is limited data on the performance of this technology during implementation. Aims To assess the implementation of a routine Transient Elastography Service (FibroscanR) at the Princess Alexandra Hospital accompanied by systematic monitoring of performance data. Methods After continuous standardised training of staff, the inter-rater reliability (newly trained operator vs. experienced consultant) was calculated. In addition anonymous surveys among referring consultants and patients who had had a FibroScan were conducted to assess the ‘subjective’ value of the technology for patient care. Data analysis was performed 3 months after commencing the service. Results 146 patients had been studied, and there was a significant correlation in the median liver stiffness measurement (LSM) between the newly trained nurse and experienced operator (r > 0.83, p < 0.01). Additionally it is notable that the interquartile ranges for the novice operator and the expert decreased during the 3 month implementation period. 10 out of 10 consultants considered the Transient Elastography Service useful in assisting their clinical decision making. 8 out of 10 (80%) felt FibroScanR reduced the need for a liver biopsy. Notably the number of patients undertaking liver biopsies compared to the same period in 2011

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shows a reduction of 60%, which may be in part due to the availability of FibroScanR, however will require further assessment. Patients currently rate the availability of FibroScanR,, and the service provided very highly and are highly receptive to the new service with the failure to attend rate for the period reported being 4%. Conclusion The next steps of our evaluation will focus on the impact of the technology on the clinical management of patients (i.e. time interval between first appointment in the liver clinic and initiation of definitive therapy). The available data suggest that after standardised training the validity of readings is high. However, our project revealed that the newly trained as well as the experienced operator improved their performance over the reported time period. Thus it appears important that the implementation is done in a well controlled setting to ensure accuracy of findings. Continuous assessment of the high level of satisfaction through the availability of this technology for both clinicians and patients is paramount.

Conventional transarterial chemoembolization versus doxorubicin bead transarterial chemoembolization for the treatment of hepatocellular carcinoma, a retrospective cohort study STEPHEN BLOOM,1 RICHARD KAI YUAN CHENG,2 LUCY LIM,2 PAUL GOW,2 JOHN S LUBEL1 1 Department of Gastroenterology, Eastern Health, Melbourne, VIC, Australia, 2Gastroenterology and Liver Transplantation, Austin Health, Melbourne, VIC, Australia Background Hepatocellular carcinoma (HCC) represents the fifth most common cancer in men and the seventh in women(1). Despite increased detection, approximately 80% of patients die within one year of diagnosis and recurrence exceeds 70% following resection. For patients precluded from surgery or ablative therapy, transarterial chemoembolization (TACE) is recommended as first line non-curative treatment(2). The development of drug eluting bead (DEB)-TACE provides an alternative method of treatment to conventional (c)TACE. The Precision V study compared these two techniques and showed that the 6-month response was better in DEBTACE compared to cTACE (26.9% v 22.2%), although this failed to reach statistical significance(3). Aims Determine whether DEB-TACE in a non-trial setting is superior to cTACE and whether there was any difference in treatment response and time to progression. Methods A retrospective cohort study was performed at two metropolitan hospitals offering TACE. Subjects who had received either DEB TACE or cTACE between 2008 and 2011 with at least 6 months follow-up were included. Diagnosis of HCC required either typical radiological features or non-typical radiological features associated with either a significantly elevated AFP or histological confirmation. Patients were excluded on the basis of concomitant primary malignancy, age less than 18 years and a BCLC stage that precluded treatment. The DEB and cTACE patients were matched by ECOG status, BCLC stage, age, number of lesions and gender. The primary end-point was treatment response at 6 months or earlier disease progression as interpreted by mRECIST criteria for solid tumour assessment in HCC(4). Per treatment and per subject responses were analysed. Data are expressed as mean ± sem. Analysis was performed using Fisher’s exact test and Mantel-Cox logistic regression. Results Thirty patients (15 DEB-TACE, 15 cTACE) were included with a total of 62 procedures (30 DEB-TACE, 32 cTACE). The median age was 66.7 ( ± 12.9) with a male predominance (93.3%). ECOG status 0/1/2 were 28/2/0, Child Pugh Grade A/B/C = 23/7/0 and BCLC 0/A/B/C = 0/16/14/0. On average each subject received 2.0 procedures with no significant

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difference between the two groups. Median time to progression was 3 months for cTACE versus 5 months for DEB TACE (CI 0.02 to 1.18 p = 0.02). Cumulative measurements of treatment response demonstrated that object response was 17% more common in DEB-TACE versus cTACE (57% v 40% p = 0.2037). DEB-TACE objective response was 7% greater following TACE1 (53.3% v 46% p = 0.2234) and 32.5% greater following TACE2 (77.8% v 33.3% p = 0.0805). Conclusion In this retrospective study, DEB-TACE offered a statistically significant improvement in time to progression in comparison to cTACE. Objective response to treatment was improved in DEB-TACE versus cTACE but this did not reach significance probably reflecting sample size.

References 1. Ferlay J, Shin HR, Bray F, Forman D, Mathers C and Parkin DM. GLOBOCAN 2008 v1.2, Cancer Incidence and Mortality Worldwide: IARC CancerBase No. 10. Lyon, France: International Agency for Research on Cancer; 2010. http://globocan.iarc.fr, accessed on 15/3/2012. 2. Bruix J, Sherman M. Management of hepatocellular carcinoma: An update. Hepatology 2011; 53 (3): 1020–1022. 3. Lammer J and Malagari K. Prospective Randomized Study of Doxorubicin-Eluting-Bead Embolization in the Treatment of Hepatocellular Carcinpoma: Results of the PRECISION V study. Cardiovascular Interventional Radiology 2010; 33:41–52. 4. Lencioni R, Llovet JM. Modified RECIST (mRECIST) assessment for hepatocellular carcinoma. Semin Liver Dis. 2010; 30(1):52–60.

Timing of variceal haemorrhage in patients with cirrhosis – is there a circadian variation? MOHAMED A CHINNARATHA,1 GAWRI RAJAKARUNA,1 OYEKOYA T AYONRINDE2 1 Gastroenterology, Fremantle Hospital, Fremantle, WA, Australia, 2School of Medicine and Pharmacology, University of Western Australia, Perth, WA, Australia

Methods A single centre review of variceal haemorrhage presentations between January 2006 and December 2010 (5 year period). Patients were identified from the endoscopy procedure reporting database using terms for gastroscopy, haematemesis, melaena, bleeding and varices. Inclusion criteria were active oesophageal or gastric variceal bleeding or high risk stigmata for bleeding on varices. Patient demographics, time of hospital presentation, site, severity, aetiology of the varices and use of prophylactic propranolol prior to presentation were collected retrospectively. Time of hospital presentation was analysed in 6 groups of 4-hour blocks. Results During the study period there were 1943 presentations to our endoscopy unit with acute upper GI bleeding. Variceal haemorrhage associated with cirrhosis was diagnosed during gastroscopy in 49 (2.5%) patients. Mean ( ± SD) age was 55.7 ( ± 12.9) years, systolic BP: 113 ( ± 28.5) mmHg, pulse rate: 89 ( ± 14)/minute, haemoglobin: 90 ( ± 26) g/L, INR: 1.7 ( ± 0.5), bilirubin: 51.5 ( ± 71.2) μmol/L and albumin of 29.6 ( ± 5.8) g/L. Patients were predominantly male (79.6%). Common aetiologies for cirrhosis were alcohol (53.1%), and the combination of chronic hepatitis C infection and alcohol (18.4%). Child-Pugh class A/B/C accounted for 20.4%/42.9%/36.7% respectively. Twelve (24.5%) episodes occurred in patients who were already taking propranolol prophylaxis. Similar proportions of patients presented between 0800–2000 hours (49%) as between 2000–0800 hours (51%). The majority of presentations were between 22.00–02.00 hours and 14.00–18.00 hours (22.4%) or between 10.00–14.00 hours (20.4%), however there was no statistically significant difference between presentations during these and other hours (p = 0.2, figure 1). Propranolol-treated patients mostly presented between 22.00– 02.00 hours. There was no significant difference in presentations attributable to gender, age or child-Pugh class (all p > 0.05). Conclusion Acute variceal haemorrhage presentations in cirrhotics were highest around midnight and late afternoon however, there was no significant correlation between time of presentation and patient characteristics. Prophylactic propranolol did not appear to influence the timing of presentations with variceal bleeding. Larger sample sizes are required to discern any real differences in variceal bleeding rates by time of day.

References 1. Alvarez et al. Hepatology. 1997; 25(3):548-50. 2. Sugano et al. J Hepatol. 2001; 34(1):26–31.

Background Previous studies have reported a nocturnal increase in portal venous pressure and portal blood flow in patients with cirrhosis(1). Anecdotally, gastro-oesophageal variceal haemorrhage events appear to occur predominantly overnight. Propranolol reduces portal blood flow and abolishes the nocturnal peak in portal pressure(2). Aims We aimed to describe the occurrence of variceal haemorrhage by time of day and the association of prophylactic propranolol with this.

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Survival of patients with viral hepatitis related hepatocellular carcinoma is better than those with non viral hepatitis related disease BRITT CHRISTENSEN, MICHAEL BAILEY, WILLIAM KEMP, STUART ROBERTS Alfred Hospital, Prahran, VIC, Australia Background Hepatocellular carcinoma (HCC), the 5th most common cancer worldwide, is an increasing public health problem in Australia. Limited data suggests viral hepatitis B (HBV) and C (HCV) are major risk factors for HCC in Australia, with development of cirrhosis an important co-contributor to increased risk. However, it is unclear if the overall survival of patients with HBV- and HCV-related HCC differs from those with non-viral-related HCC. Aims To compare survival outcomes of patients with HBV- and HCVrelated HCC to those with non-viral disease managed at a single tertiary referral centre. Methods A retrospective analysis was conducted on HCC cases seen at The Alfred from 1999–2012. Patient demographics, severity of liver disease, tumour characteristics, and patient outcomes were retrieved from the HCC database and computer records. Patients with known co-infection were excluded. Overall survival was calculated and compared between the three cohorts of HBV, HCV and non-viral related HCC. Predictors of survival in HCC were determined by univariate and multivariate logistic regression analysis according to HCC aetiology, and baseline clinical, liver disease, and tumour characteristics. Results 196 HCC patients were identified: 83% male; 18% HBV, 33% HCV, 49% non-viral. 3% of HBV, 47% of HCV and 59% of non-viral cases were born in Australia. Non-viral cases were older at diagnosis compared to HCV and HBV cases (p = 0.008). In non-viral HCC cases extra-hepatic spread was more common (p = 0.025) and there was a trend for increased tumour burden at diagnosis (p = 0.054) compared to HBV and HCV cases. 5-year overall survival was worse in those with non-viral related HCC compared to those with HBV and HCV-related disease (5% vs 26% vs 16% respectively; p = 0.013). There was no difference in overall survival when comparing HBV versus HCV-related HCC (p = 0.49). On mutlivariate analysis independent factors associated with overall survival were initial treatment response (HR 7.57; CI 4.46–12.08), ALP (HR 1.002; CI 1.001–1.004), GGT (HR 1.001; CI 1.001–1.002), Child Pugh Score (HR 1.208; CI 1.208–1.058), age (HR 1.03; C 1.011–1.048) and WBC count (HR 1.053; CI 1.011–1.097). HCV patients with HCC had higher overall survival rates (HR 0.56; CI 0.335–0.853). Conclusion The overall survival of patients with HBV and HCV related HCC is better compared to those with non-viral HCC. Patients with viralrelated HCC were more likely to be younger and born overseas, and less likely to have extensive disease at diagnosis. On multivariate analysis a diagnosis of HCV was associated with improved survival outcomes. Further prospective studies are warranted to confirm these findings.

Pretransplant obesity predicts long-term survival in adults undergoing orthotopic liver transplantation PAOLO DE SCISCIO,1 MICHELE DE SCISCIO,1 ALAN WIGG2 1 Hepatology and Liver transplant Medicine Unit, Flinders Medical Centre, Bedford Park, SA, Australia, 2 South Australian Liver Transplant Unit, Flinders Medical Centre, Bedford Park, SA, Australia Background Obesity is a recognised risk factor for morbidity in patients undergoing Orthotopic Liver Transplantation (OLT). Several studies have consistently demonstrated increased incidence of postoperative complications and longer hospital admission in obese patients. In contrast, there is

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conflicting evidence regarding the effect of obesity on posttransplantation survival. Aims The objective of this study was to determine whether body mass index (BMI), at the time of listing and/or organ offer, predicts long term patient and graft survival following OLT. Methods Two hundred and nine consecutive patients who underwent primary, single organ liver transplantation at Flinders Medical Centre between January 1992 and April 2011 were studied; Male: 135 (64%), Age: 49.3 ± 11.6 yrs, MELD: 18.8 ± 8.9, BMI: 27.0 ± 5.4 kg/m2; median follow-up: 5.1 yrs (minimum 1 yr). BMI was corrected for ascites. Cox regression models were fitted to assess the association between BMI (at time of listing and/or organ offer) and posttransplant patient and graft survival; adjusted for age, gender, underlying aetiology and MELD score. Results Of the 209 patients, 39% were within normal BMI (18.5–24.9), 38% were overweight (BMI 25–29.9), 16% were obese class I (BMI 30–34.9), 5% were obese class II (35–39.9) and 2% were obese class III (BMI ≥ 40). Patient and graft survival rates, stratified by BMI are presented in table 1. Obese patients (BMI ≥ 30) experienced a significantly higher covariate-adjusted risk of death compared to normal weight (hazard ratio [HR] = 2.27; confidence interval [CI]: 1.17–4.41; p = 0.02) and overweight (HR = 2.16; CI: 1.12–4.37; p = 0.02) patients. Increase in BMI was associated with a significant reduction in overall patient survival (HR = 1.06; CI: 1.01–1.11; p = 0.01) and graft survival (HR = 1.06; CI: 1.01–1.1; p = 0.01). Conclusion Pretransplant obesity and corrected BMI were independently associated with survival following OLT in this population. These findings suggest that obesity should be carefully considered, as part of the total risk assessment, when considering a patient for OLT. Table 1 BMI

Posttransplant survival, stratified by BMI n (%)

< 25

81 (39)

25–29.9

79 (38)

≥ 30

49 (23)

Total

209

Posttransplant Survival, Years

Patient Graft Patient Graft Patient Graft Patient Graft

1

5

10

94% 91% 96% 95% 88% 84% 93% 91%

86% 83% 85% 83% 76% 75% 83% 81%

74% 72% 73% 72% 43% 42% 67% 65%

Hazard Ratio [HR]

95 [CI] Exp(B) lower

p-value

upper

0.441 0.227 0.855 0.456 0.241 0.862 0.463 0.240 0.894 0.489 0.260 0.918 Reference Category

0.02 0.02 0.02 0.03

Frequency, costs and predictors of hospital readmission(s) following orthotopic liver transplantation PAOLO DE SCISCIO,1 MICHELE DE SCISCIO,1 ALAN WIGG2 1 Hepatology and Liver transplant Medicine Unit, Flinders Medical Centre, Bedford Park, SA, Australia, 2 South Australian Liver Transplant Unit, Flinders Medical Centre, Bedford Park, SA, Australia Background Liver Transplantation is widely accepted as the standardof-care treatment for end-stage liver disease. Although highly successful, liver recipients require ongoing follow-up which may include hospital readmission. Unfortunately, limited data exists on the incidence, costs and outcomes of readmission following transplantation. Aims The aims of this study were to identify the frequency, costs and predictors of hospital readmission following Orthotopic Liver Transplantation (OLT). Methods Hospital admissions (emergency or elective) up-to one year following the index hospitalization were analysed for 185 consecutive patients who underwent primary, single organ liver transplantation at

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Flinders Medical Centre between July 1996 and April 2011. Seven patients were excluded; 5 in-hospital deaths, 2 moved from health region. For the remaining 178 patients, cox regression models were fitted to assess predictors of time to first readmission and associations between readmission rate and mortality, adjusted for age, gender, underlying aetiology, Charlson comorbidity index and preoperative MELD. Negative binomial regression was used to assess predictors of readmission rate. Total cost for each readmission was obtained from hospital financial records. Results At 1 year post-OLT, 143 (83%) patients had at least one readmission (emergency or elective); median time to first readmission was 26 days (range: 1, 352). Within the first week post-OLT, 23 (13%) patients had been readmitted. The rate of readmission was 3.5 per person-year; 43% of readmissions were classified as emergency. Patients who were more frequently readmitted demonstrated a significantly higher covariate-adjusted risk of subsequent mortality (HR = 1.22; CI: 1.10–1.34; p < 0.001). HCV (HR = 1.61, CI: 1.13–2.30; p = 0.009) and MELD (HR = 1.02; CI: 1.01–1.04; p = 0.008) were identified as predictors of time to first readmission. Likewise, HCV (p = 0.001) and MELD (p = 0.003) were identified as predictors of readmission rate. On analysis of emergency readmissions only, HCV remained an independent predictor of time to first readmission (p = 0.048) and readmission rate (p = 0.002). The mean cost per readmission was $7,710 (emergency vs. elective: $11,458 vs. $4,181, p < 0.001). The mean cost per person-year was $29,785. Conclusion Hospital readmission during the first year following OLT is common and is associated with substantial cost and increased mortality. Patients with HCV and/or high preoperative MELD are at increased risk of readmission. These findings highlight the need for preventative approaches targeted towards patients at increased risk of readmission as a strategy for reducing subsequent hospitalization and costs associated with post-OLT patient care.

Involvement of a lifestyle intervention leader (LIL) improves compliance in diet and exercise programs prescribed to patients with NAFLD TEDDI DWYER,1 SHIVAKUMAR CHITTURI,1 JASMINE CRIBB,1 MARGARET EVERS,1 TONY HARBER,2 PETE BURNS,2 GEOFFREY FARRELL1 1 Liver Research Group, ANU Medical School at The Canberra Hospital, Garran, ACT, Australia, 2YMCA, Chifley, ACT, Australia Background Lifestyle intervention to improve diet and physical activity via an organised program has been shown effective in diabetes intervention studies that involve a lifestyle trainer. Similar intervention is the preferred strategy for overweight patients with non-alcoholic fatty liver disease (NAFLD) and has been shown effective in study participants followed for 3 to 6 months. In clinical practice, this involves a self or dietician-directed diet and exercise regimen. However, the success of such programs is eroded by high attrition rates. Aims In this study, we evaluate the impact of a lifestyle intervention leader (LIL) on compliance and success of a lifestyle intervention program (LIP) on NAFLD. Methods Between 2010–2012, we invited NAFLD patients of Canberra liver clinics to participate in a 3 month LIP focusing on diet and exercise. Consenting participants took part in 1 of 4 rounds of a 12 week LIP. Due to logistics the study was not randomized, with 2 including an LIL (one case TD, another JC) and 2 without (self-motivated). In all rounds, access to a fully equipped gym with experience in conduct of LIPs was subsidized. Training programs were comparable, adjusted based on age and ability. Dietary advice was given by professionally trained staff. The key role of the LIL was to modulate the program and encourage motivation in order to maintain an appropriate environment for individual success. In addition to program compliance and formal feedback, anthropometric

measurements, BP, and blood chemistry were evaluated before and after the LIP. Compliance was defined as >33% of scheduled gym visits (an avg of ≥1 visit/week). Results Of 67 participants, 34 received the version with LIL. In the LIL-assisted LIPs, compliance was 62%, with only 24% in those without LIL. A Chi square test revealed the difference in compliance between LIL and non-LIL was significant (p < 0.001). Patients who completed the LIP showed significant improvements in weight, systolic BP, waist, waist hip ratio, ferritin, albumin, cholesterol, triglycerides, and glycaemic control. No differences in blood chemistry or anthropometrics were observed between the 2 versions of LIP. Feedback from participants indicated that support from the LIL was crucial to their success. Specific examples include: ‘The [LIL] was always in contact with us’, ‘The [LIL] showed me a great deal of support’, ‘I liked having the [LIL] as a constant support/ motivator’. An assessment of 12-month follow-up will be completed before Oct 2012. Conclusion Traditionally implemented diet and exercise programs can see attrition rates up to 76%. This significantly reduces the benefit of implementing and funding an LIP for the treatment of NAFLD. With the inclusion of an LIL for groups of up to 25 participants, the overall value and likely cost-efficacy of such programs would be greatly augmented.

CDT as a biomarker of alcohol excess in patients with chronic liver disease: BMI affects diagnostic sensitivity Kevin Fagan,1 Brett McWhinney,2 Leigh Horsfall,1 Jacobus P Ungerer,2 Carel J Pretorius,2 Lambro A Johnson,2 Andrew Clouston,3 Linda M Fletcher,1 Julie Jonsson,3 Peter O’Rourke,4 Elizabeth Powell3 1 Gastroenterology and Hepatology, Princess Alexandra Hospital, Brisbane, QLD, Australia, 2Pathology Queensland, Royal Brisbane and Women’s Hospital, Brisbane, QLD, Australia, 3School of Medicine, The University of Queensland, Brisbane, QLD, Australia, 4 Biostatistics, Queensland Institute of Medical Research, Brisbane, QLD, Australia Background Alcohol remains a major global cause of liver injury and is a common co-factor in subjects with other chronic liver diseases (CLD). Failure of healthcare providers to recognise excessive alcohol consumption is common, and hinders the implementation of treatment strategies. Carbohydrate-deficient transferrin (CDT) is the most specific marker of chronic alcohol excess and its potential clinical application has increased with development of new high-performance liquid chromatography (HPLC) methods. However its utility in subjects with CLD remains unclear Aims The aim of this study was to evaluate the role of biomarkers of alcohol excess, in particular CDT, in 254 patients who underwent liver biopsy for assessment of liver disease. Methods Information regarding average alcohol intake (g/wk) during the preceding 2 weeks was obtained by nurse consultation at time of liver biopsy and corroborated by a detailed review of medical records. An HPLC method for CDT (Recipe®, Munich, Germany) was used to quantify individual transferrin glycoforms in serum collected at liver biopsy. Results 12 patients acknowledged heavy alcohol intake (male >420 g/ wk, female >350 g/wk) at time of liver biopsy. An additional 13 patients were drinking > recommended weekly allowance (RWA) (male > 210 g/ wk, female > 140 g/wk) and 229 reported drinking ≤RWA. CDT ≥ 1.7% (consistent with heavy alcohol intake) was found in 13 patients (5 with alcoholic liver disease, 5 with HCV and 3 with HBV). 7 of these 13 patients acknowledged heavy drinking at time of liver biopsy, 3 were

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drinking > RWA, 2 ≤RWA and 1 stated he was a ‘non-drinker’. Furthermore, 11 of the 12 ‘drinkers’ with CDT ≥ 1.7% had a history of prior alcohol abuse. Interestingly, only 7 of the 12 current heavy drinkers had a CDT ≥ 1.7% and these subjects were lean with a BMI < 25 kg/m2. All 5 heavy drinkers with CDT < 1.7% had a BMI > 28 kg/m2 (Figure 1) and had a γGT : ALP ratio >2.5. In the 34 subjects with alcohol intake > RWA at referral, only 38% of the referral letters cited alcohol. Conclusion There is a need for an objective means of identifying problem drinking. In subjects with CLD, CDT ≥ 1.7% indicates alcohol excess, and specificity was not affected by disease aetiology or presence of compensated cirrhosis. However, %CDT had low sensitivity for identifying heavy alcohol intake and was impaired by overweight/obesity. Further studies will investigate the combination of CDT with other markers, e.g. γGT : ALP ratio > 2.5, to develop a robust scoring system to identify heavy drinkers.

Figure 1. Log %CDT in subjects with heavy alcohol intake separated according to BMI.

Analysis of the discordance between the clinical assessment and the liver stiffness measurement (LSM) in patients with chronic hepatitis C (CHC) GRACE FLORESCU,2 STUART ROBERTS,1 WILLIAM KEMP1 1 Gastroenterology, Alfred Hospital, Melbourne, VIC, Australia, 2Medical School, Monash University, Melbourne, VIC, Australia Background Transient Elastography (TE) is frequently used to guide management decisions and establish treatment priorities in patients with CHC. TE has been shown to improve prediction of fibrosis versus the clinician assessment alone. Previous studies have indicated hepatic inflammation and cholestasis are among factors associated with discordance between LSM and fibrosis stage. However factors associated with discordances in TE evaluation of fibrosis and the clinician prediction, have not yet been investigated. This is important as understanding reasons for clinical-LSM discordance will assist in appropriate use and interpretation of this technology. Aims To evaluate the frequency of discordance between the clinician assessment and the LSM as assessed by TE in subjects with CHC. Furthermore to determine patient characteristics that results in clinicalLSM discordance. Methods A prospectively maintained database was interrogated to identify all subjects with CHC (PCR positive) who underwent TE assessment at The Alfred from 1st August 2010–31st December 2011 (n = 1011), and a valid TE result (≥10 shots were obtained, SR ≥ 60%, IQR: M ≤ 30%). Data included in the analysis was derived from the patient referral form, a patient completed questionnaire and the TE results. Complete demographic, virological, biochemical and haematological data was available

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on 530 patients and of these, 208 had a pre-TE clinician directed assessment of fibrosis stage. The clinician fibrosis assessment was divided into 3 groups; group 1 (F = 0/1), group 2 (F = 2/3) and group 3 (F = 4). To assess discordance, the clinical assessment was compared to the LSM results according to previously published cutoffs; 2 × ULN, Phase 3 (low replicative) = HBeAg-negative, DNA < 2000, Phase 4 = HBeAg-negative, DNA > 2000. ALT and HBV DNA levels were measured every 3–6 months. In a subset of patients with phase 3 CHB, we evaluated the association between HBsAg titre at first visit, and the risk of reactivation of CHB. Results There were 232 treatment naïve patients identified. Median age was 48 yrs, 41% were female, 72% were Asian. The distribution of disease phase was: phase 1 (9%), phase 2 (11%), phase 3 (36%) and phase 4 (44%). Median HBsAg level was strongly associated with the phase of

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CHB: phase 1 = 22,427 (IQR 3980–65,320), phase 2 = 16360 (4760– 62,120), phase 3 = 304 (72.5–3471), phase 4 (555–7266); P = 2631). HBsAg level was correlated with HBV DNA (R = 0.41, P < 0.001). Of patients with phase 3 disease, 51/83 (61%) had an HBsAg level < 1000 IU/mL. Conclusion HBsAg levels vary significantly through the course of CHB, with levels highest early in the natural history. HBAg levels correlate with HBV DNA levels. Of patients with phase 3 disease, the majority of patients had a low HBsAg level, and may be suitable for less intensive monitoring. Longitudinal follow-up studies are required.

The safety and efficacy of tenofovir disoproxil fumarate (TDF) substitution for HBIg in liver transplantation patients receiving long-term low dose IM HBIg/lamivudine (LAM) prophylaxis ILANA GORY,1 ED GANE,2 PAUL GOW,1 JULIE PAVLOVIC,1 PETER ANGUS1 1 Gastroenterology and Hepatology, Austin Hospital, Heidelberg, VIC, Australia, 2Gastroenterology and Hepatology, The Auckland City Hospital, Auckland, New Zealand Background Prevention of graft reinfection remains the most important issue in liver transplantation of hepatitis B virus (HBV) infected patients. When prophylaxis is not given, reinfection is almost inevitable. Standard protocol comprises combination use of lamivudine/HBIg, however HBIg is expensive and requires patients to receive lifelong painful intramuscular injections. Furthermore breakthrough HBV infections have been documented. The finding that tenofovir (TDF) is highly active against lamivudine resistant virus suggests that the combination of this drug with lamivudine may at least be as effective as HBIg/lamivudine. Aims The primary objective of this study is to assess the safety and efficacy of the substitution of TDF for HBIg whilst continuing LAM therapy in preventing the recurrence of HBV in patients post liver transplantation. Methods This open-label, multi-centre switch study evaluates the safety and efficacy of TDF plus LAM in HBsAg negative patients following liver transplantation who have been maintained for at least 12 months on combination HBIg and LAM. Patients were switched from combination therapy to TDF 300 mg daily plus LAM 100 mg daily. Patients were excluded if they have evidence of HBV viral recurrence or baseline creatinine >150 μmol/L. An initial analysis of outcomes was performed. The primary endpoint is at 96 weeks with continuation to 5 years post enrolment.

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Results A total of 18 patients received TDF plus LAM with a mean follow up time of 20 months (range 12–24). There was no case of treatment failure (defined as reappearance of HBsAg and HBV DNA) and no side effects reported. There was no significant loss of kidney function (defined as creatinine >50 μmol/L or >25% above baseline) or evidence of phosphate loss from commencement of TDF. In addition there was no difference in BMD from commencement of TDF. Mean creatinine at baseline was 109.1 ± 24 μmol/L. At latest follow-up the mean difference in renal function was −3.14 μmol/L (CI −10. 3 to 4.01; p = 0.36). The mean phosphate at baseline was 1.12 ± 24 mg/L with a mean difference of 0.07 (CI −0.04 to 0.19; p = 0.21). Four patients had dose reductions of TDF and one patient had the drug stopped due to minor declines in renal function. However, all 5 patients had significant co morbidities known to contribute to nephrotoxicity including use of calcineurin inhibitors and poorly controlled diabetes mellitus. Subsequent to dose reduction 3 of these patients’ creatinine returned to baseline with 2 patients showing progressive worsening of renal failure and 1 of these progressing to haemodialysis in the setting of diabetic nephropathy. Conclusion Replacement of HBIG with tenofovir appears to be an effective strategy in patients receiving HBIG/LAM prophylaxis post liver transplantation. Further follow up is required to confirm the long term safety and efficacy of this approach.

Radiofrequency ablation versus surgical resection for the treatment of early stage hepatocellular carcinoma – a retrospective multicentre study ILANA GORY,1 WILLIAM KEMP,1 MICHAEL A FINK,3 SALLY BELL,2 PAUL GOW,3 AMANDA NICOLL,4 VIRGINIA KNIGHT,5 ANOUK DEV,5 MATTHEW KITSON,1 ANTHONY RODE,4 STUART ROBERTS1 1 Gastroenterology and Hepatology, The Alfred Hospital, Melbourne, VIC, Australia, 2Gastroenterology and Hepatology, St Vincents Hospital, Melbourne, VIC, Australia, 3Gastroenterology and Hepatology, The Austin Hospital, Melbourne, VIC, Australia, 4 Gastroenterology and Hepatology, The Royal Melbourne Hospital, Melbourne, VIC, Australia, 5 Gastroenterology and Hepatology, Monash Medical Centre, Melbourne, VIC, Australia Background While radiofrequency ablation (RFA) and surgical resection (SR) are well established curative therapies for early stage (BCLC 0-A) HCC, it remains unclear as to which treatment provides optimal patient outcomes. We therefore compared survival outcomes of RFA to SR in patients with early stage HCC. Aims To compare the overall survival and other clinical outcomes of RFA versus surgical resection in patients with previously untreated early stage HCC. Methods A multicentre retrospective analysis was performed from 5 tertiary academic hospitals of patients with BCLC 0-A HCC having RFA or SR as primary therapy. Detailed information was collected from electronic databases, medical records and national death registries. Comparisons were made using Kaplan Meier method, log rank test and Cox proportional hazard model where appropriate. Results From 2000–2010, 146 patients (78% male, mean age 63 ± 16 years) with BCLC 0-A HCC who received primary treatment with RFA (n = 96) or SR (n = 52) were identified. Mean MELD score was 9 ± 3 and 83% had Child Pugh A disease. Mean tumour size was 25 ± 10 mm. Patients in the RFA group were older, with more advanced liver disease and smaller tumours. There was 1 death in the SR group with 14 (13%)

minor complications that occurred in the RFA group and 13 (25%) minor complications in the SR group. In patients with very early stage small HCCs (0–30 mm) there was no difference in terms of overall survival in those treated with RFA compared with SR (1, 3, and 5 year survival was 98%, 70% and 45% vs 95%, 72% and 60% respectively; p = 0.75). At multivariate analysis factors associated with overall survival were BCLC stage (HR 0.30, CI 0.11–0.82; p 0.02) and Child Pugh Score (HR 1.55, CI 1.09–2.11; p = 0.01). There was no increase in local recurrence in the RFA group (p = 0.1) and no difference in recurrence free survival (p = 0.11). However, in patients with larger tumours (30–50 mm) SR appears better in terms of overall survival compared with RFA (1, 3 and 5 year survival was 90%, 75% and 60% vs. 90%, 60% and 30% respectively; p = 0.02) In this group, at multivariate analysis RFA was associated with lower survival (HR 2.75, CI 1.09–6.7; p = 0.003) and there was a 3 fold increased risk of local recurrence with RFA (HR 3.31; CI 1.41–7.20; p = 0.003). Conclusion In this Multicentre study, in patients with very early stage small HCCs (0–30 mm) RFA and Resection are comparable treatments in terms of overall survival and recurrence free survival. BCLC and Child Pugh Score are both independent predictors of overall survival in this group. However in patients with larger tumours (30–50 mm) Surgical Resection appears better compared with RFA in terms of overall survival and local recurrence. RFA is an independent predictor of overall survival in this group. Further prospective data is warranted to compare these two modalities.

Do Tenofovir and Entecavir affect renal function in patients with chronic hepatitis B (CHB)? A two-year observational study from a single Australian centre PHIL HA,2 TONY HE,2 JULIAN LIM,2 LUKAS SAHHAR,2 SUONG LE,1 FERRY RUSLI,1 DARCY Q HOLT,1 DILIP RATNAM,1 STEPHEN PIANKO,1 GREGORY MOORE,1 ANOUK DEV,1 WILLIAM SIEVERT1 1 Gastroenterology and Hepatology, Monash Medical Centre, Clayton, VIC, Australia, 2Monash University, Clayton, VIC, Australia Background Tenofovir (TDF) and Entecavir (ETV) are nucleos(t)ide (NUC) analogues which are potent inhibitors of hepatitis B virus. However, the long-term effect of TDF on renal function in CHB patients remains unclear. Aims To compare the incidence, severity and clinical significance of changes in renal function over 2 years amongst CHB patients treated with TDF 300 mg daily or ETV 0.5 mg or 1.0 mg daily versus an untreated cohort. Methods We retrospectively identified 124 patients treated with either ETV (n = 74) or TDF+/− LMV (n = 50) for a minimum of 2 years. 73 untreated CHB patients were used as controls. Renal function was assessed by estimated glomerular filtration rate (eGFR) using the Modification of Diet in Renal Disease formula (MDRD) at baseline, 6, 12 and 24 months. Exclusion criteria included concomitant use of potential nephrotoxins, decompensated cirrhosis and previous NUC exposure apart from lamivudine if renal function was normal upon commencement of TDF. Baseline demographics, MELD score, presence of cirrhosis and risk factors for renal impairment were recorded. Outcomes assessed included kidney function (decline in eGFR ≥40% and change in eGFR over time) and dose adjustment or cessation of either therapy. Analysis was performed using a mixed linear model to incorporate repeated measures. Results Baseline characteristics were closely matched between the three cohorts (p = NS). Older age (p = 0.0043) and cirrhosis (p < 0.0001) were more prevalent in the ETV group. The proportion of patients with ≥40% reduction in eGFR from baseline was 3.92% in the TDF ± LMV group vs. 2.7% in the ETV group (p = NS). In the univariate mixed linear model,

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compared to control subjects at baseline, eGFR at 2 years declined in the ETV group by −7.6 ml/min (95% CI −15.8−+0.6, p = 0.07) and −8.7 ml/ min (CI −18.3−+1.0, p = 0.08) in the TDF ± LMV group while in the control it remained stable at +7.4 ml/min (CI 0.78 − 14.1, p = 0.03). After adjustment for age, gender, hypertension, diabetes and cirrhosis, eGFR decline at 2 years was attenuated in the ETV group (−1.8 ml/min, −10.2 −6.5, p = 0.67) but not in the TDF group (−6.5 (−16.1−+3.0, p = 0.18) while in the control it remained higher 7.2 ml/min (0.58−13.8, p = 0.03). There were no episodes of treatment cessation or dose adjustment. Conclusion In this single centre study of 124 treated CHB patients, there was a suggestion of decline in renal function in both ETV and TDF treated patients. However, we did not observe any significant changes in eGFR over 2 years of treatment. Observation in a real world cohort over a longer period may provide further insights into the potential renal complications of NUC therapy. Baseline characteristics

Age (years) Sex, Male Cirrhosis HTN DM eGFR ml/min (MDRD)

Untreated

Entecavir

Tenofovir

40.82 ± 11.12 55% 06.6 ± 0.9 10 (13.7%) 4 (5.8%) 100

46.69 ± 11.29 68% 32 (43%) 12 (16%) 7 (9.5%) 97

41.75 ± 11.44 65% 19 (37%) 4 (7.8%) 2 (3.9%) 98

p-value 0.0043 8000 μg/L. Patients who were admitted to either Calvary Mater or John Hunter Hospital between 01.01.2012–30.05/2012 and who had a ferritin level or complete iron studies were screened. Medical records of cases were then reviewed to define the demographic characteristic, diagnosis and outcomes for these patients. Results A search using the electronic pathology database yielded 21 episodes of hyperferritinaemia >8000 μg/L from 6 unique patients. This represented 0.4% of all requested ferritin levels at these hospitals during the audit period. Of the 6 patients 5 were male. The age, peak ferritin level and selected biochemical test are shown in Table 1. A diagnosis that explained the extreme hyperferritinaemia was established in all of the patients and is shown in Table 1. 3 of the patients had passed away at the end of the audit period. Conclusion Extreme hyperferritinaemia was found in a minority of the patients screened with iron studies in our inpatient population. The most common cause was not hereditary haemochromatosis, a disorder often considered in the setting of elevated ferritin. A malignant diagnosis was found in 4 of the patients, whilst 2 were due to direct hepatic causes. 3 of the patients died during the audit period of the illness directly linked to their hyperferritinaemia. A finding of extreme hyperferritinaemia should prompt a diagnostic search for haematological malignancy.

Results

Age Peak Ferritin μg/L T.Sat CRP ALT Diagnosis

Patient 1

Patient 2

Patient 3

Patient 4

Patient 5

Patient 6

18 24157 105% N/A 1855 Acute lymphoblastic leukaemia

38 76764 109% 170 410 Haemophagocytic syndrome

54 65060 60% 75 910 Haemophagocytic syndrome

57 75893 77% 83 7668 Ischaemic hepatitis

71 10388 94% N/A 96 Haemachromatosis C282Y Homocygous

87 9305 78% 4 161 NHL + Haemolytic anemia. Transfusion dependent

Reference 1. Finch CA, Bellotti V, Stray S, et al. Plasma ferritin determination as a diagnostic tool. West J Med 1986; 145:657.

Rifaximin prevents episodic hepatic encephalopathy: a real world experience in WA CLAIRE HARMA, YI HUANG, JASMINE BEAMAN, GARY JEFFREY, LEON ADAMS, GERRY MACQUILLAN Hepatology, Sir Charles Gairdner Hospital, Perth, WA, Australia Background Hepatic encephalopathy is a common complication of cirrhosis which is associated with a poor prognosis. Rifaximin is a semisynthetic, minimally absorbed antibiotic, which has been shown to maintain remission from encephalopathy and reduce rates of hospitalisation when compared to placebo, in the clinical trial setting (NEJM 2010, Bass et al).

Aims We sought to determine if the reported benefits of rifaximin translated to real world outcomes for patients with refractory hepatic encephalopathy. Methods A prospective cohort study of all patients prescribed Rifaximin for hepatic encephalopathy in our institution was performed (Sir Charles Gairdner Hospital, Nedlands, WA). Demographic data, Child Pugh and MELD scores, concomitant lactulose use, as well as episodes of and admissions with encephalopathy data while on rifaximin was collected. Each patient’s episodes of and admissions with hepatic encephalopathy in the 12 months prior to starting rifaximin was used as a historic control. Mortality and transplant data was also collated. Results Between November 2010 and January 2012, 23 patients were prescribed Rifaximin at a dose of 600 mg BD. They were followed for an average of 236 days. The cohort was predominantly male (82.6%) and had mean MELD of 17.34 ± 9.05. The mean number of episodes of encephalopathy in the cohort pre-rifaximin was 2.43 ± 1.70, compared to following the introduction of rifaximin with a mean of 1.33 ± 1.49 (p 0.028, 95% CI 0.12 to 2.08). The mean admission rate following the introduction of rifaximin with hepatic encephalopathy was 0.74 ± 1.39, compared to 1.13

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± 1.10 pre-rifaximin (p 0.295), however when the two patients with postTIPS encephalopathy were excluded from the analysis, there was a trend towards fewer admissions (p 0.072). There was no significant difference in the rates of SBP or infections following the introduction of rifaximin in our cohort. There was fewer episodes of variceal bleeding in the cohort after the introduction of rifaximin (6/23 vs 0/23, p 0.0216). Conclusion In a real world clinical setting, rifaximin reduces the frequency of episodes of hepatic encephalopathy and has a trend towards fewer hospital admissions, in our experience. In the setting of recalcitrant hepatic encephalopathy following TIPS in 2 patients, rifaximin was not of benefit.

Age (mean, yrs) Tumour size (mean, cm) Single tumour at presentation Curative treatment Survival (median, months)

Surveillance n = 138

Non-surveillance n = 90

Significance

61.32 3.46

65.83 5.67

p < 0.0001 p < 0.0001

63%

61%

p = 0.7814

41% 24.23

27% 7.99

p = 0.0340 p = 0.0030

Reference 1. Bass NM, Mullen KD, Sanyal A et al. Rifaximin Treatment in Hepatic Encephalopathy. NEJM 2010; 362(12): 1071.

Reference 1. Zhang BH, Yang BH, Tang ZY. Randomized controlled trial of screening for hepatocellular carcinoma. J Cancer Res Clin Oncol 2004;130:417–422.

Surveillance for hepatocellular carcinoma improves survival outcomes: the experience at St Vincent’s Hospital THAI HONG, NATASHA JANKO, MARNO C RYAN, BARBARA DEMEDIUK, TARA MCKENZIE, NIRANJAN J ARACHCHI, ALEX THOMPSON, PAUL DESMOND, SALLY BELL Gastroenterology, St Vincent’s Hospital, Melbourne, VIC, Australia

Different measures of HCV rapid fibrosis post liver transplant have different abilities to predict rapid graft cirrhosis JESSICA HOWELL, MICHAEL A FINK, ROHIT SAWHNEY, PETER ANGUS, PETER CROWLEY, KUMAR VISVANATHAN, ROBERT JONES, BAO-ZHONG WANG, PAUL GOW Gastroenterology, Austin Hospital, Ivanhoe, VIC, Australia

Background Hepatocellular carcinoma (HCC) is a common and deadly disease worldwide, for which survival is improved by earlier detection and treatment. Surveillance for HCC in patients at increased risk has been shown to reduce mortality in the clinical trial setting.1 Aims Our aims were to review the real-world experience of surveillance for HCC at St Vincent’s Hospital and compare outcomes of those patients who were on a surveillance program and those who were not. Methods A retrospective analysis of the St Vincent’s Hospital HCC database was performed for patients diagnosed with HCC between 1995 and 2012. Outcomes were compared between patients diagnosed with HCC as part of a defined surveillance program (6 monthly ultrasound and alpha-feto-protein follow-up) and those patients diagnosed with HCC outside of a surveillance program. The primary outcome was survival. We also considered disease aetiology, tumour size, tumour number, and treatment modality offered. Results There were 138 and 90 patients in the surveillance and nonsurveillance groups respectively. Patients in the surveillance group were younger (mean age 61.32 years vs 65.83 years, p < 0.0001). The severity of liver disease was similar between the two groups (median Child-Pugh score 6). Patients with cirrhosis due to viral hepatitis were more likely to be on surveillance than those patients with alcoholic liver disease. Patients on surveillance presented with smaller tumours compared to those not on surveillance (mean size 3.46 cm vs 5.67 cm respectively, p < 0.0001) but tumour number was not significantly different (single tumour: 87/138 (63%) vs 55/90 (61%), p = 0.7814). Curative treatment was offered significantly more frequently in the surveillance group (41% vs 27%, p = 0.0340). The median survival was significantly longer in the surveillance group compared to the non-surveillance group (24.23 vs 7.99 months, p = 0.0030). Conclusion Patients who had HCC diagnosed within a surveillance program were more likely to have early stage disease that was potentially amenable to curative therapy. Survival was longer for patients on surveillance than those diagnosed with HCC outside of a surveillance program. The data supports the efficacy of surveillance programs for patients at high risk of HCC.

Background Hepatitis C (HCV) is the most common indication for liver transplantation worldwide. Recurrence post transplant is universal, with a subgroup of patients developing rapid fibrosis progression. Various clinical definitions of rapid fibrosis have been used to identify risks for the subgroup of patients with rapid progression, but their ability to predict poor outcomes has not been directly compared. Aims To compare the ability of different measures of rapid fibrosis progression to predict the development of cirrhosis within five years of transplantation in patients with HCV. Methods Prospective data analysis was conducted on 131 adult patients with HCV who underwent liver transplantation at a single centre. We measured fibrosis rate (calculated using liver biopsies graded by Metavir scoring F0–4; FR = fibrosis stage/year post transplant), year one fibrosis progression (rapid fibrosis defined as Metavir F score ≥1 at 1 yr liver biopsy) and time to Metavir F2 stage fibrosis (years). We determined the correlation between these measures of fibrosis progression and time to cirrhosis and the ability of these measures to predict cirrhosis development within five years of transplantation (dichotomized about the median time to cirrhosis in our cohort) using ROC and Pearson correlation statistics. Results 131 consecutive adult patients with HCV were included in the study; 100 had liver biopsies allowing determination of fibrosis rate (minimum post-transplant follow-up 6 mths, mean 6.5 yrs). There was a significant correlation between fibrosis rate (p < 0.0001, r = −0.76), time to F2 stage fibrosis (p < 0.0001, r = 0.92) and fibrosis stage on year one biopsy (p = 0.012, r = −0.67) and time to cirrhosis development. Fibrosis rate calculated using the most recent biopsy was the strongest predictor of rapid graft cirrhosis development (p < 0.0001, AUC 0.979). Fibrosis rate calculated early on year 3 post transplant liver biopsy and time to F2 fibrosis was also significantly predictive of rapid graft cirrhosis (p = 0.004, AUC 0.848, p = 0.012, AUC 0.791 respectively). F stage on year one liver biopsy was not significantly predictive of rapid graft cirrhosis (p = 0.06). Conclusion Fibrosis rate measured on the most recent liver biopsy and at 3 years post transplant was the most accurate predictor of the rapid development of graft cirrhosis after transplantation for HCV.

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Collagen proportional area using sirius red stained liver biopsies is more accurate than trichrome staining and reliably predicts the development of hepatocellular carcinoma YI HUANG,1 BASTIAAN DE BOER,2 LEON ADAMS,1 GERRY MACQUILLAN,1 ENRICO ROSSI,3 PAUL J RIGBY,4 SPIRO C RAFTOPOULOS,1 GARY JEFFREY1 1 Gastroenterology and Hepatology, Sir Charles Gairdner Hospital, Perth, WA, Australia, 2Anatomical Pathology, PathWest, QEII Medical Centre, Perth, WA, Australia, 3Biochemistry, PathWest, QEII Medical Centre, Perth, WA, Australia, 4Centre for Microscopy, Characterisation and Analysis, University of Western Australia, Perth, WA, Australia Background The risk of developing hepatocellular carcinoma (HCC) is increased in those chronic hepatitis C (CHC) patients with more severe liver fibrosis. Liver biopsy analysis using collagen proportional area (CPA) measured by quantitative image analysis was shown to better quantify the severity of liver fibrosis than histological stages. However, the different methods of collagen staining and analysis among studies has to date limited the clinical utility of CPA. Aims First to compare CPA obtained by two commonly used collagen staining methods, Sirius red and Masson’s trichrome. Secondly to assess the ability of CPA to predict the risk of developing HCC in CHC patients. Methods Patients with CHC who had a liver biopsy and a Hepascore performed from 1998 to 2012 were included. Liver biopsies were stained with Masson’s trichrome and Sirius red and were analysed using quantitative image analysis. Clinical information was obtained from hospital records. Paired sample t test was used to compare the CPA values. Cox regression analysis and ROC curve analysis were used to evaluate variables associated with HCC. Results 250 patients were included: 59 patients had advanced fibrosis (Metavir F3-F4), 62 females (24.8%), mean age was 42.3 years (19–69), median Hepascore value was 0.41 (0.03–1), median Sirius red stained CPA (CPA (s)) was 6.6% (1.8–44.53%), median trichrome stained CPA (CPA (t)) was 2.4% (0.03–68.87%). The correlation between CPA (t) and CPA(s) was 0.705 (p < 0.001). Mean CPA (s) was higher than mean CPA (t) by 3.63% (95% CI: 2.91–4.34%, P < 0.001). Using stratified analysis, significant differences between CPA (s) and CPA (t) were only found in Metavir F1 to F3. Both CPA(s) and CPA(t) were well correlated with Metavir (CPA(s): rs = 0.612, p < 0.001, CPA(t): rt = 0.520, p < 0.001) and Ishak (CPA(s): rs = 0.607, p < 0.001, CPA(t): rt = 0.531, p < 0.001). 36 patients with advanced fibrosis underwent 6 monthly ultrasound surveillance for HCC with a median follow up of 3 years (1–8). 6 patients (16.7%) developed HCC at a median of 2 years (0–5) follow up. Univariate analysis found CPA (t), CPA(s), platelet count and Hyaluronate were associated with HCC. Multivariate analysis found that only CPA(s) correlated with HCC (p = 0.018, HR = 1.251, 95% CI: 1.039–1.506). The area under ROC curve (AUROC) of CPA(s) was 0.95 (95% CI, 0.889–1.000). It was higher than that of CPA (t) (AUROC = 0.704, 95% CI, 0.364–1.000), Ishak (AUROC = 0.900, 95% CI, 0.797–1.000), Metavir (AUROC = 0.883, 95% CI, 0.774–0.992) and Hepascore (AUROC = 0.744, 95% CI, 0.576–0.912). A cut-off point of CPA(s) >20% achieved a sensitivity of 100% and specificity of 93.3%. Conclusion Sirius red staining was more sensitive and accurate than Masson’s trichrome staining when using CPA to quantify hepatic collagen especially in the early stages of liver fibrosis. CPA(s) was highly predictive of the appearance of HCC and better than Ishak, Metavir and Hepascore.

Admissions of patients with complications of chronic liver disease to a tertiary hospital in Western Australia: differences between indigenous and non-indigenous population TEE CHING HUN,1 NIROSHAN MUWANWELLA,1 NICHOLAS KONTORINIS,1 LORENZO TARQUINIO,1 WENDY CHENG,1 WENDY CHENG2 1 Gastroenterology Dept, Royal Perth Hospital, Perth, WA, Australia, 2Curtin Health Innovation Research Institute, Curtin University, Perth, WA, Australia Background Chronic liver disease is a large public health issue and accounts for significant morbidity and mortality worldwide. In Australia, mortality from cirrhosis and other forms of liver disease ranks 19th in year 2010. We review our experience in chronic liver disease in the absence of previously published data from Western Australia. Aims (1) To review the commonest admissions and complications of chronic liver disease admitted to Royal Perth Hospital (2) To compare the admissions and outcomes between the Indigenous and non-Indigenous population. Methods A descriptive retrospective analysis of admissions of patients with chronic liver disease to Royal Perth Hospital over the period of 2000–2011 was undertaken. Primary diagnosis and complications were recorded according to the ICD-10 criteria and collected from the Performance Unit of the South Metropolitan Area Health Service. Results A total of 7313 liver-related admissions were observed from a period of 2000 to 2011 generated by 3413 patients with a mean age of presentation of 53 years (range 17–90). Of all admissions, 81% were non-Indigenous and 19% were Indigenous. The commonest admission diagnosis for non-Indigenous patients was chronic viral hepatitis whilst for the Indigenous population, the commonest cause was alcoholic liver disease. The average length of stay was 6.3 days (range 1 and 222 days). 2530 admissions were related to complications of chronic liver disease, 55.8% of which were non-Indigenous and 44.2% Indigenous. Complications comprised of hepatorenal syndrome (50%), hepatic failure (36.8%) and portal hypertension (13%). Hepatorenal syndrome and hepatic failure were seen mainly in the non-Indigenous population (55.2% vs 43.4% and 41.5% vs 31.1%, respectively). This is in contrast to portal hypertension where 25.6% were Indigenous and only 3.3% were nonIndigenous. Indigenous patients had a younger age of presentation (48 vs 54 years p < 0.0001), higher mortality rates (44.5% vs 25.7% p < 0.001) and twice the length of stay compared to the non-Indigenous (p < 0.001). Conclusion There is a significant difference in the admission diagnoses and complications of chronic liver disease between the non-Indigenous and the Indigenous population. Accurate data and knowledge of the burden of disease are essential for health care policy formulation to prioritise health interventions. Study is ongoing to assess mortality from WA Data Linkage.

Autoimmune hepatitis – clinical presentations in the older population TEE CHING HUN,1 NICHOLAS KONTORINIS,1 MARCUS CHIN,1 LORENZO TARQUINIO,1 WENDY CHENG,1 WENDY CHENG2 1 Gastroenterology Dept, Royal Perth Hospital, Perth, WA, Australia, 2Curtin Health Innovation Research Institute, Curtin University, Perth, WA, Australia Background Autoimmune hepatitis, characterised by presence of circulating autoantibodies and high serum globulin concentrations has a

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heterogeneous and fluctuating presentation ranging from asymptomatic to florid acute liver failure. Autoimmune hepatitis can be an aggressive indolent liver disease associated with delay in diagnosis and treatment in the older patients. Aims We describe our experience of patients with autoimmune hepatitis, aged 50 and above, who presented in the last 12 months. Methods A retrospective review of all patients (n = 5) admitted with autoimmune hepatitis with positive ANA+/− anti-SMA and high IgG in the absence of an alternative aetiology (drug, viral) in patients >50 years. Demographics (age, sex), presentation, laboratory tests, treatment and outcomes were recorded from iSoft patient management database and outpatient notes.

Results 4/5 patients were females and 4/5 patients presented with nonspecific symptoms. 4 patients had painless jaundice. All patients had severe inflammatory changes (A4) and abundant plasma cells and only 1 patient had advance fibrosis on liver biopsy. Case 3 had overlap syndrome with primary biliary cirrhosis (PBC). Their management and outcomes were all recorded in table 1. Conclusion Clinical presentations of autoimmune hepatitis in patients over 50 years can be variable and associated with severe inflammation and variable fibrosis requiring prompt diagnosis and treatment. These patients respond to treatment with prednisolone and immunosuppressive agents. Management can be problematic in the older population with concurrent medical problems and drug intolerance.

Summary of case series Details

Case 1

Case 2

Case 3

Case 4

Case 5

Age (years) Symptoms (Sx)

67 Non specific symptoms, dark urine, weight loss 3 weeks

65 Non-specific symptoms, jaundice 5 days

52 RUQ pain, weight loss, jaundice 6 weeks

69 Lethargy, athralgia, jaundice 6 weeks

57 Jaundice 4 weeks

53 919 1.3 32 1:320 1:160 33.12

197 1990 1.1 39 15 0 20.8

35 475 1.1 36 0 3 44.5

132 605 1.7 23 30(ds 49) 0 40.8

53 2900 1.1 36 4 1 31.9

A4 F2 Past history of breast Ca and cervical Ca Hypothyroidism 3/12 Prednisolone Azathioprine

A4 F2 Chronic lymphocytic leukaemia (active) Hyperlipidaemia 2/12 Prednisolone Azathioprine

A4 F2 T2DM Hypertension Hyperlipidaemia 2/52 Prednislone (ceased) 6-MP Ursofalk

A4 F3 Old pulmonary TB

A4 F1 nil

Hypertension 5/12 Prednisolone Azathioprine

6/12 Prednisolone Azathioprine

24 not done

28 8.6

13 10.2

58 9.6

47 10.5

Duration of Sx LFT Bili μmol/L ALT μ/L INR Alb g/L ANA Anti SMA IgG Biopsy A score F score Medical History

Time to normalisation/lowest ALT Treatment Latest Ix ALT IgG

Nutritional evaluation and management of hospitalised patients with chronic liver disease – do we really care? DEP HUYNH, NAM NGUYEN Gastroenterology and Hepatology, Royal Adelaide Hospital, Adelaide, SA, Australia Background Hospital admission with chronic liver disease accounts for a significant proportion of patients under Gastroenterology Units. Malnutrition in these patients has been shown to be associated with increased morbidity and mortality. Despite this, little is known about the nutritional care of hospitalised patients with cirrhosis. Aims The aim of this study is to evaluate the practice of nutritional assessment and care for patients with liver cirrhosis who were admitted to a tertiary hospital. Methods Data on all patients with liver cirrhosis admitted to the Gastroenterology Unit at the Royal Adelaide Hospital over 12 months were reviewed. Details related to the patients’ demographics, disease severity, nutritional status and assessment, biochemistry and micronutrients, and clinical outcomes were collected. Nutritional status was assessed by a dietician and determined by subjective global assessment. Estimated energy and protein requirements were calculated by Simple Ratio Method. Intake was estimated from dietary history and/or food charts, and

82

represented as a percentage of estimated daily requirements. Median duration of follow up was 10 (4–18) months Results Of the 112 cirrhotic patients (87 males, age: 57.8 ± 2.0 yrs, 12% Childs A, 43% Childs B and 45% Childs C), only 50 (45%) patients had formal nutritional assessment by a dietician during their admission. Based on subjective global assessment, 60% these patients were judged to have malnutrition with only 9% consuming sufficient oral intake to meet their daily caloric requirements. Overall, 58% patients had intake that was less than 50% of their caloric requirement, and 38% patients had intake that was less than 50% of their protein requirements. Micro-nutrient evaluation indicated that zinc (77%) and vitamin D (67%) deficiencies were highly prevalent. Only 7 (6%) patients received enteral nutrition during hospitalisation and 5 (4%) patients received ongoing dietetic review and assessment following discharge from hospital. The overall mortality rate was 36%, with significantly higher mortality in patients who were deemed to have ‘malnutrition’ than those without (53% vs. 20%, P = 0.02). Conclusion A substantial portion (60%) of hospitalised patients with liver cirrhosis is malnourished, with dietary intake insufficient to meet recommended daily requirements for both calorie and protein. Despite the higher mortality in these patients, less than half of hospitalised cirrhotic patients had formal dietetic evaluation during their admission and even fewer had outpatient dietetic follow-up. The current study highlights the need for meticulous nutritional evaluation and management in hospitalized patients with chronic liver disease.

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Impact of malnutrition on clinical outcomes in hospitalised patients with chronic liver disease DEP HUYNH, NAM NGUYEN Department of Gastroenterology and Hepatology, Royal Adelaide Hospital, Adelaide, SA, Australia Background Malnutrition is common in patients with chronic liver disease. The prevalence is dependent on disease severity and the population examined (reported range 16–90%). Evidence for the impact of malnutrition on morbidity and mortality has mostly been derived from data in the liver transplant population. Aims The aim of this study is to examine the impact of malnutrition on outcome in hospitalised patients with cirrhosis, in a non-liver transplant tertiary hospital. Methods Data related to hospitalized patients with liver cirrhosis who had formal dietetic assessment, at the Royal Adelaide Hospital, over 12 months were reviewed. Patients’ demographics, disease severity, nutritional status and assessment, biochemistry, and clinical outcomes were collated. Nutritional status was assessed by a dietician and determined by subjective global assessment. Median duration of follow up was 8 (0.5–18) months. Results Of the 50 cirrhotic patients (39 males, age 57.4 ± 2 year, 4% Childs A, 42% Childs B, 54% Childs C) who had dietician review and subjective global assessment, 30 (60%) were deemed to have malnutrition. There was no difference in Childs Pugh score (10 vs 9.1) or MELD score (17.2 vs 16.5) between patients with malnutrition and normal nutritional status. Malnourished patients were more likely to have an underlying alcoholic liver disease (87% vs. 50%, P = 0.01), as compared to other aetiologies. Although there was no difference in in-patient mortality (4/30 vs. 3/20), cirrhotic patients with malnutrition had significantly higher mortality (53% vs. 20%, P = 0.02) than those without, with a 1.7 (1.1–2.7) relative risk of death over 8 months. Malnutrition, however, did not have a significant impact of the length of stay (median: 14.5 (6.7–23) vs. 12.5 (5–20) days, P = 0.50) or the rate of readmission (1.0 (0–2.5) vs. 1.0 (0–2); P = 0.78; malnutrition vs. without, respectively). Conclusion Malnutrition is highly prevalent (60%) amongst hospitalized patients with chronic liver disease, especially those with an underlying aetiology related to alcohol. Although nutritional status does not affect hospital length of stay, number of readmissions or in-hospital mortality, it has a significant adverse impact on long term survival. These findings suggest that aggressive nutritional management during hospitalization and on-going outpatient dietetic follow-up are essential in the care of patients with chronic liver disease.

The relationship between nutritional support, serum albumin level and meld score prioritisation for liver transplantation RIA KANAZAKI,1 ROBERT CHENG,1 HELEN VIDOT,1 ALISON POTTER,2 STUART HAGGIE,1 NICHOLAS SHACKEL1 1 Royal Prince Alfred Hospital, Sydney, NSW, Australia, 2 Centenary Institute of Cancer Medicine and Cell Biology, Sydney, NSW, Australia Background Nutritional support may improve MELD score. Prioritisation for liver transplant includes an assessment using the Model of Endstage Liver Disease (MELD) score. This score had been improved with the addition of serum sodium (Na+ – MELD) and now the inclusion of serum albumin to MELD is being investigated. Therefore, we performed a preliminary study using albumin as a surrogate marker of nutrition, to see if an improvement in albumin level is associated with an improvement in MELD score. A second analysis was performed to assess the effect of enteral feeding on MELD score.

Aims Our aim was to assess retrospectively the effect of nutritional support and serum albumin level on MELD score over time. Secondary end points were: prolonged time to liver transplantation and longer liver transplant free survival. Methods Clinical and biochemical data were collected retrospectively from 250 patients from the liver clinic over 6 years (2005–2010) who were assessed for liver transplantation. Serum albumin level, nutritional support and MELD score were recorded from the time of enrolment and at the end of nutritional support. Significance testing was performed comparing: 1) nutritional support v’s MELD over time; 2) serum albumin level v’s MELD over time. Individuals were classified as being successfully transplanted or having a poor outcome (delisted, died, allograft failure or re-transplanted). Results Of the 250 patients, the mean albumin at enrolment was 33 g/L (SD 6.2) and the mean MELD 16 (SD 16.9). Following nutritional support the mean albumin was 27 g/L (SD 27.8) and the mean MELD 9 (SD 21.8). Chi-squared testing was performed between nutritional support and MELD (p = 0.15) and serum albumin and MELD (p = 0.18). The change in MELD (p < 0.002) but not the change in serum albumin (p = 0.08) from the time of listing to transplant or delisting distinguished individuals with a successful transplant compared to those with a poor outcome. Overall the change in serum albumin and MELD score from listing to being transplanted or poor outcome correlated significantly (p = 0.023). Further, there was a significant correlation between the change in MELD and albumin seen across all groups (p = 0.005). Whether or not a patient received nutritional support did not significantly impact survival. Finally the absence of nutritional support did correlate significantly with individual’s with a poor outcome (p < 0.05). Conclusion Changes in serum albumin accompanied the MELD score. There is a trend towards significance with nutritional support and it is foreseeable that serum albumin level may further refine MELD scores. We expect a statistical significance result pending inclusion of more patient data (still being collected). Therefore, we conclude that serum albumin appears to refine the risk assessed by the MELD score as well as predicting outcomes in individuals given nutritional support prior to liver transplantation.

Low rates of hepatitis C virus (HCV) treatment and seroclearance associated with reduced graft survival in renal transplant recipients in a single centre Australian cohort SUONG LE,1 VANESSA BULL,2 STEPHANIE SPRING,2 AMALINA BAHAROM,2 JANANI PARAMANANTHAM,2 LAUREN BESWICK,1 WILLIAM MULLEY,3 KEVAN POLKINGHORNE,3 DILIP RATNAM,1 ANOUK DEV,1 WILLIAM SIEVERT,1 STEPHEN PIANKO1 1 Gastroenterology and Hepatology, Monash Medical Centre, Clayton, VIC, Australia, 2Monash University, Clayton, VIC, Australia, 3Nephrology, Monash Medical Centre, Clayton, VIC, Australia Background In comparison to the general population, Hepatitis C virus (HCV) infection is more prevalent in patients with chronic kidney disease (CKD). Furthermore, renal transplant candidates infected with HCV experience low rates of antiviral treatment and increased morbidity and mortality from accelerated liver disease post-transplantation. Reduced renal allograft survival in HCV positive recipients has been reported in observational studies with 5 years of follow-up. However, the rates of HCV treatment and the longer-term clinical outcomes beyond 5 years for renal transplant recipients in Australia remain poorly characterised. Aims To describe the baseline demographics, determinants of HCV seroclearance and graft survival in a single-centre Australian cohort of renal transplant recipients.

Journal of Gastroenterology and Hepatology 2012; 27 (Suppl. 4): 68–95 © 2012 Journal of Gastroenterology and Hepatology Foundation and Wiley Publishing Asia Pty Ltd

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Methods We conducted a retrospective analysis of patients with chronic HCV who underwent renal transplantation between 1974 and 2011 (n = 19) at Monash Medical Centre, Victoria. Patient histories were interrogated for staging and management of both chronic kidney disease (CKD) and HCV. The determinants of HCV seroclearance and graft loss were identified. Results For a summary of baseline patient characteristics please refer to Table 1. 19 patients received a total of 26 renal grafts over a mean observation period of 15.7 ± 13 years (range 1 to 38 years). 22% of these patients have returned to dialysis. Of those referred for consideration of HCV assessment pre renal transplant, treatment was deferred in 84% due to either a low ALT, low viral load, low fibrosis scores or concerns regarding potential side effects. This is compared to a 50% rate of treatment deferral in normal HCV patients reviewed through our centre. Despite low

rates (3/19) of pre transplant HCV treatment there were 2 patients who achieved a sustained virological response (SVR) with combination pegylated interferon and ribavirin. These patients had HCV Genotype 1 and Genotype 2 respectively with low fibrosis scores (Metavir F1 or F2) on liver biopsy. No spontaneous seroclearance was recorded. Compared to the Australian mean graft half-life of 12 years in a non HCV renal transplant recipient who has received a cadaveric repeat renal transplant, the mean graft half life in our cohort was markedly reduced at 5.2 years ± 5. Conclusion There were low rates of HCV seroclearance amongst renal transplant patients at Monash Medical Centre, Victoria which was associated with high rates of graft loss. The barriers to HCV seroclearance were multi-factorial. In order to optimise outcomes for patients with a dual diagnosis of HCV and CKD, a collaborative treatment driven protocol is required to manage this special population.

Baseline characteristics N = 19

Age, years

Male sex

Ethnicity

Weight (kg)

ETOH > 10 g/day (past or current)

Aetiology of CKD

Duration of dialysis pre renal transplant(years)

Duration of HCV infection pre transplant(years)

ALT (U/L, RR < 45) at time of assessment for HCV treatment

Mean ± Standard deviation

35.37 ± 13.3

14 (74%)

Asian: 3 Caucasian: 16

76.7 ± 19.48

11 (58%)

Glomerulonephritis 21% IgA Nephropathy 21% Reflux nephropathy 36.8% Polycystic Kidney10% Diabetes 10%

5 ± 3.54

9.7 ± 10.6

58 ± 72

Reference 1. Lie CH, Kao JH. Treatment of hepatitis C virus infection in patients with end-stage renal disease. Journal of Gastroenterology and Hepatology 2011;26:228–239. http://www.anzdata.org.au

Establishment of a liver stiffness normal range using transient elastography in non-liver disease control children and the utility of transient elastography in the detection of liver disease in children with Cystic Fibrosis PETER LEWINDON,2 TAMARA N PEREIRA,1 CHARLTON NOBLE,2 MORA PUERTOLAS-LOPEZ,1 LOUISE RAMM,1 GRANT RAMM1 1 Hepatic Fibrosis Group, Queensland instItute of Medical Research, Herston, QLD, Australia, 2 Gastroenterology, Royal Children’s Hospital, Brisbane, QLD, Australia Background Advanced hepatic fibrosis develops in 5–10% of patients with Cystic Fibrosis (CF) in early adolescence but it is difficult to detect by conventional diagnostic tools such as ultrasound and liver function tests which prove non-specific. Liver biopsy remains the benchmark for detecting fibrosis however it is invasive and only samples a small portion of the liver. Transient elastography (TE) is used as a non-invasive method for detecting liver stiffness (fibrosis) in adults and may be useful in the detection of liver disease and fibrosis in children with CF. However, prior to its potential utility in children with liver disease, a normal range of TE-derived liver stiffness in non-liver disease control children is required to be established.

84

Aims To establish a normal range of TE-derived liver stiffness measurement (LSM) in healthy control children and to determine the utility of TE in the detection of liver disease in patients with CF. Methods TE (FibroscanTM) was performed on 37 healthy non-liver disease Control children, 35 children with CF without liver disease (CFnoLD) and in 8 children with CF who had biopsy-proven evidence of liver fibrosis or macronodular cirrhosis with portal hypertension on Ultrasound (CFLD). Ten valid LSMs were performed and the median value, expressed in kilopascals (kPa), was recorded. Valid TE measurements were determined as having an interquartile range (IQR)/LSM ≤ 30% and a success rate ≥ 60%. Results This study identified a normal range for TE-derived LSM in non-liver disease Control children of 2.5–8.3 kPa. LSM was elevated in CFLD patients (11.2 ± 3.2 kPa; 95% CI = 3.6–18.8 kPa) versus both CFnoLD patients (4.77 ± 0.18 kPa; 95% CI = 4.4–5.1 kPa) and Control subjects (4.71 ± 0.2 kPa; (95% CI = 4.2–5.2 kPa) (KW ANOVA, P = 0.001). There was no significant difference in LSM between CFnoLD patients and Control subjects. There was a positive correlation between Scheuer fibrosis staging and LSM in CFLD patients (Spearman Rank r = 0.85, P = 0.02), albeit with small numbers of patients enrolled to date. ROC analysis of CFLD vs CFnoLD patients showed an AUROC = 0.90 (P = 0.0004), with a Sensitivity = 87.5% and Specificity = 82.85% for detection of liver disease in CF (cut-off = 5.75 kPa; likelihood ratio = 5.10). There was no effect of age on LSM in either Control subjects (r = 0.1, P = 0.56) or CFnoLD patients (r = 0.09, P = 0.60), and there was no difference in the median age between the three groups. Finally, there was no statistical difference in LSM between males and females in either Controls (5.0 ± 0.3 kPa vs 4.3 ± 0.37 kPa, resp.) or CFnoLD subjects (4.6 ± 0.25 kPa vs 4.9 ± 0.26 kPa, resp.). Conclusion In this preliminary study we have shown the potential utility of Transient Elastography in the detection of liver disease in patients with CF and have established a normal range of LSM in a non-liver disease cohort of Control children.

Journal of Gastroenterology and Hepatology 2012; 27 (Suppl. 4): 68–95 © 2012 Journal of Gastroenterology and Hepatology Foundation and Wiley Publishing Asia Pty Ltd

Hepatology Clinical

ASMA predicts for pre-treatment liver biopsy and delays normalisation of ALT during the first 12 weeks of therapy with pegylated interferon and ribavirin but has no impact on SVR EU JIN LIM,1 AMY E CLEMENTS,2 JOHN S LUBEL1 1 Department of Gastroenterology, Eastern Health, Melbourne, VIC, Australia, 2Eastern Health Clinical School, Monash University, Melbourne, VIC, Australia Background The detection of autoantibodies in hepatitis C (HCV) infection is well recognized, but their significance is not well understood. It is thought that the presence of antinuclear antibodies (ANA) or anti-smooth muscle antibodies (ASMA) has no impact on clinical course or response to pegylated interferon and ribavirin treatment. The prevalence of these antibodies in an Australian population treated for HCV has not been well described. Aims To determine the frequency of ANA and ASMA in patients receiving HCV treatment. To ascertain if their presence has any impact on any pre-treatment or on-treatment parameters and sustained virological response (SVR) rates. Methods Using the Eastern Health HCV database we identified patients who received HCV treatment between 2004 and 2011. Patients who received at least one dose of pegylated interferon and were tested for either ANA or ASMA were included. Demographic data, baseline and on-treatment biochemical and haematological indices, and SVR results were collected. Statistical analysis was performed using t-tests and Fisher exact test, with p-values less than 0.05 considered statistically significant. Results Of 314 patients that received HCV treatment, 273 (87%) had been tested for ANA and/or ASMA. Mean patient age was 43 (range 19–73), average weight was 76 kg, and 61% of the cohort was male. Genotype 1 infection was found in 46% of patients, while 45% and 7% had genotypes 3 and 2 respectively. ANA was tested in 246 patients with 16% testing positive, and of 242 patients tested for ASMA, 35% tested positive. ASMA was positive significantly more often than ANA (P < 0.001). There was no difference in pre-treatment viral load or baseline haematological indices between the ANA or ASMA positive and negative groups, but having ASMA significantly predicted for undergoing a pretreatment liver biopsy (p < 0.05). Although baseline ALT showed no significant difference between ASMA positive and negative patients, ALT was significantly greater in patients with detectable ASMA at weeks 4 (57.7 ± 6.1 vs 44.2 ± 2.8), 8 (52.7 ± 6.1 vs 38.5 ± 2.9) and 12 (51.2 ± 6.8 vs 36.2 ± 2.3) into treatment (p < 0.05). After 24 weeks of therapy these differences became non-significant. ANA had no influence upon the ALT at baseline or during treatment. On-treatment effects (nadirs) upon haematological indices were unaffected by ANA and ASMA. In 72% of ANA negative patients SVR was achieved compared to 77% of ANA positive patients (p = 0.81), while 79% of patients who were ASMA negative attained SVR compared to 70% of ASMA positive patients (p = 0.51). Conclusion The presence of ASMA predicted for a pre-treatment liver biopsy and was associated with higher ALT during the first 12 weeks of therapy, suggesting the presence of an associated autoimmune component in the ASMA positive group. However, neither ANA nor ASMA had a significant impact on SVR, even when antibody titre was taken into account.

Tenofovir rescue therapy achieves long-term suppression of HBV replication in multi-drug resistant HBV patients: 4 year follow-up of the TDF109 cohort LUCY LIM,1 SCOTT J PATTERSON,1 JAMES M TRAUER,1 JACOB GEORGE,3 SIMONE STRASSER,4 ALICE LEE,5 WILLIAM SIEVERT,6 AMANDA NICOLL,7 STUART ROBERTS,8 PAUL DESMOND,9 D SCOTT BOWDEN,2 ALEX THOMPSON,9 STEPHEN LOCARNINI,2 PETER ANGUS1 1 Gastroenterology and Liver Transplant, Austin Hospital, COLLINGWOOD, VIC, Australia, 2Molecular Research and Development, VIDRL, Melbourne, VIC, Australia, 3Gastroenterology and Hepatology, Westmead Hospital, Sydney, NSW, Australia, 4AW Morrow Gastroenterology and Liver Centre, Royal Prince Alfred Hospital, Sydney, NSW, Australia, 5 Gastroenterology, Concord, Sydney, NSW, Australia, 6 Gastroenterology, Monash Medical Centre, Melbourne, VIC, Australia, 7Gastroenterology, Royal Melbourne Hospital, Melbourne, VIC, Australia, 8 Gastroenterology, The Alfred, Melbourne, VIC, Australia, 9Gastroenterology, St Vincent’s Hospital, Melbourne, VIC, Australia Background Multi-drug resistant (MDR) hepatitis B virus (HBV) presents a serious management challenge, particularly for patients with advanced liver disease. We present the long-term follow-up data from the TDF109 study that investigated the use of tenofovir (TDF) ± lamivudine (LAM) for the treatment of well-characterized chronic hepatitis B (CHB) patients who had previously failed LAM & adefovir dipivoxil (ADV) therapy. Aims To determine the efficacy of TDF in highly experienced CHB patients. Methods TDF109 was an investigator-initiated, prospective, multi-centre, open-label study of 60 patients treated with TDF. All patients had previously failed LAM and subsequent ADV add-on or switch therapy. The year 1 & 2 data have been previously reported (Patterson, S.J. et al GUT 2011;60). At study entry, 38/60 patients (63%) were switched from ADV monotherapy to TDF, and 22/60 (37%) were switched from ADV/ LAM to TDF/LAM combination. Those patients treated with TDF monotherapy with persistent viral replication at or after 24 weeks were switched to TDF/LAM. The primary end point for the current analysis was the number of patients with HBV DNA below the lower limit of quantification (LLOQ, DNA ≤ 20 IU/ml) after 4 years of follow-up. We also analysed the rate of viral decline based on the type HBV resistance associated substitutions at baseline. Results At year 2, 64% (38/59) had serum HBV DNA levels below the LLOQ. At 4 years, 63% (38/60) had achieved complete viral suppression by intent-to-treat analysis. 4 patients had discontinued TDF & 4 patients were lost to follow-up. 38/51 (75%) had achieved viral suppression per protocol. In patients with quantifiable serum HBVDNA levels at 4 years, the median viral load was 1.72 log10 IU/ml (serum HBV DNA level was 7 years with multiple NA, including at least 3 years of tenofovir (TDF). Methods TDF-109 was an investigator-initiated, prospective, multicentre, open-label study of 60 patients treated with TDF salvage/rescue therapy. All patients had previously failed LAM and subsequent ADVadd-on or switch therapy. The median age was 48.5 years (range 21–80), 77% were males, 67% were HBeAg positive, 67% were of Asian ethnicity and 52% were infected with HBV genotype C. Median follow-up is now 4 years; all patients have achieved an adequate virological response, with 75% (38/51) having undetectable HBV DNA levels. All patients have been maintained on TDF in long-term follow-up, now out to 4 years. QHBsAg titres were retrospectively quantified at baseline and every 6 months and expressed as IU/ml (Roche Elecsys). Results At baseline, median QHBsAg titre was 5226 IU/ml. Baseline QHBsAg titre were positively correlated with HBV DNA levels at baseline, R = 0.33, p = 0.0168). There was a trend for higher baseline QHBsAg levels in HBeAg positive patients (p = 0.1257). The median HBsAg titres 86

were 2772 IU/ml at year 1, 2284 IU/ml at year 2 and 1819 IU/ml at year 3. There was a trend for patients with detectable rtA181T/V variants to have a lower QHBsAg at baseline. There was a trend for patients with no detectable resistance changes or lamivudine (LAM) resistance HBV Pol to have higher QHBsAg at baseline, but surprisingly more significant decline in QHBsAg on TDF (table). Two patients achieved HBsAg loss at weeks 120 and 156 respectively. Conclusion More than 3 years of TDF therapy progressively declined the QHBsAg titres in the majority of patients over time. Even though the presence of rtA181T/V at baseline resulted in lower QHBsAg, the ontreatment rate of decline was significantly less. Median QHBsAg Titers IU/ml Variables

Number % (Total n = 59)

Median QHBsAg at Baseline

Median QHBsAg at Year 1

Median QHBsAg at Year 2

Median QHBsAg at Year 3

P-value (baseline – yr 3)

HBeAg Postitive

66% (39/59)

HBeAg Negative

34% 20/59

8759 Note n = 35 3893 Note n = 17

3032

3450

3138

0.0027

1900

1730

1289

0.0008

15855

7768

5632

4400

35% (21)

7462

2225

3450

2433

0.0027 (n = 12) 0.031 (n = 14)

8% (5)

3912

1530

862

1140

0.5468 (n = 2)

30% (18)

2992

2344

1664

1537

0.0020 (n = 16)

Resistance Changes None detected 25% (15) L-Nucleoside (LAM/LdT) M204I/V Acyclic phosphonate (ADV) N236T 181 Containing (LAM/LdT/ ADV/TFV) A181T/V +/− N236T

Trends and outcomes of hepatocellular carcinoma in a Western Australia tertiary hospital from 2000 to 2011 WAI SUN LOO, JEE Y KONG, WENDY CHENG, LORENZO TARQUINIO, NICHOLAS KONTORINIS Gastroenterology, Royal Perth Hospital, Perth, WA, Australia Background The incidence of hepatocellular carcinoma (HCC) in Australia has been rising over the past two decades and has been associated with high mortality rates due to the overall poor prognosis. Aims Our aim was to evaluate the changing trends in clinical presentation, treatment and survival of HCC over a 12-year period between 2000–2011. Methods A retrospective analysis of all patients who presented to Royal Perth Hospital with HCC between 2000–2011. Subjects were identified based ICD-10 codes and data was obtained from clinical notes and electronic databases. We recorded patient’s demographics, underlying and stage of liver disease, tumour characteristics, mode of presentation, treatment and survival outcomes. The cohort was divided into two groups 2000–2005 and 2006–2011, and comparison was made between groups. T-test was used to compare means and Fischer’s exact test was used to compare between groups with significant value used as p < 0.05. Results 204 patients with HCC presented to Royal Perth Hospital over a 12-year period. Mean age was 63.4 years and 84.3% of patients had liver cirrhosis with hepatitis C being the commonest cause. Child-Pugh and

Journal of Gastroenterology and Hepatology 2012; 27 (Suppl. 4): 68–95 © 2012 Journal of Gastroenterology and Hepatology Foundation and Wiley Publishing Asia Pty Ltd

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MELD score were similar in both cohorts; Child-Pugh A (63% of patients from 2000–2005; 59% from 2006–2011); B (22.1% and 21.6%); C (15.1% and 13.1%), respectively; mean MELD score (11.7). The percentage of patients diagnosed with HCC out of surveillance programme was 72% from 2000–2005 and 81% from 2006–2011 (p = NS). 16 patients received treatment with curative from 2000–2005 compared to only 9 patients from 2006–2011. There was no improvement in median survival between both cohorts (7 months in patients from 2000–2005 compared to 6 months from 2006–2011). Conclusion There has been an increase in number of HCC related to Hepatitis C and alcoholic liver disease. There did not appear to be an improvement in survival in patients with poor mean survival of 6–7 months in our cohort. The proportion of HCC detected by surveillance had not increased with time. A comprehensive and vigilant HCC surveillance program may provide patients with more treatment options and improve survival.

Considerable variation in approach to chemoembolisation by interventional radiologists treating hepatocellular carcinoma – is standardised treatment feasible? JOHN S LUBEL,1 MARK D GOODWIN,9 MANFRED SPANGER,10 NIRANJAN J ARACHCHI,5 SALLY BELL,3 ANOUK DEV,2 MICHAEL A FINK,7 PAUL GOW,4 WILLIAM KEMP,8 VIRGINIA KNIGHT,2 IAN KRONBORG,5 AMANDA NICOLL,6 STUART ROBERTS,8 MARNO C RYAN3 1 Department of Gastroenterology and Hepatology, Eastern Health, Melbourne, VIC, Australia, 2Department of Gastroenterology, Monash Medical Centre, Melbourne, VIC, Australia, 3Department of Gastroenterology, St Vincent’s Hospital, Melbourne, VIC, Australia, 4Department of Gastroenterology, Austin Hospital, Melbourne, VIC, Australia, 5 Department of Gastroenterology, Western Hospital, Melbourne, VIC, Australia, 6Gastroenterology and Hepatology, Royal Melbourne Hospital, Melbourne, VIC, Australia, 7University of Melbourne Department of Surgery, Austin Hospital, Melbourne, VIC, Australia, 8 Department of Gastroenterology, Alfred Hospital, Melbourne, VIC, Australia, 9Department of Radiology, Austin Hospital, Melbourne, VIC, Australia, 10 Department of Radiology, Eastern Health, Melbourne, VIC, Australia Background Transarterial chemoembolisation (TACE) is frequently employed for the treatment of intermediate and advanced hepatocellular carcinoma (HCC). This procedure is usually performed by interventional radiologists (IRs), often with guidance from a multidisciplinary team (MDT) including hepatologists and hepatobiliary surgeons. The practices and preferences of IRs for the various techniques employed have not previously been examined in Australasia. Aims Primary aims were to characterise the clinical approach to TACE in Australasia and to record IR preferences for techniques such as drug eluting bead TACE (DEB-TACE) or conventional (c)TACE. Methods IRs with membership of The Royal Australian & New Zealand College of Radiologists were invited to complete an online questionnaire (‘RADIATE’ survey) designed by the Melbourne collaboration for the Study of Hepatocellular carcinoma (MeSH). This survey assessed; i) personal demographic data; ii) involvement in MDT; iii) details on procedural technique for TACE; iv) preference for TACE technique; v) clinical

threshold to perform procedure. Analysis was performed using Fisher exact test with a p value 790 moderate-severe inflammation. CDAI and CRP were measured at baseline, 3, 6, 9 and 12 months or, clinical relapse. Faecal calprotectin and S100A12 levels were obtained at baseline and study endpoint. Baseline CESI findings were correlated with CDAI, CRP, calprotectin and S100A12. Referring physicians were blinded to CE and faecal biomarker findings and therapy was only changed with a clinical relapse. Results 39 pts (19 M) median age 41 (20–67) yrs were studied. At enrolment, median CDAI was 73 (14–141); CRP 5.2 (0.5–31; NR < 5); calprotectin 120 (2–6390; NR < 100 mg/kg) and; S100A12 4 (0–2000; NR < 10 mg/kg). As assessed by CESI, 62% of pts in clinical remission had inflammatory changes. All pts without inflammation had normal calprotectin levels and all with mod-severe inflammation had an elevated calprotectin. CESI and baseline faecal biomarkers were significantly correlated, calprotectin R = 0.820 (p < 0.0001) and S100A12, R = 0.415 (p < 0.01). There was no correlation between CESI and CDAI or CRP. 31 pts reached study end point, 5 of whom had a clinical flare. Of these 5, all had mucosal inflammation at CE and 4 had elevated baseline calprotectin levels. None of the pts with normal CESI and calprotectin flared. Of interest, 25/31 pts remained in clinical remission, 16 (63%) of whom had CE evidence of mucosal inflammation. Conclusion In small bowel CD pts assessed in clinical remission: (1) 62% had mucosal inflammation on CE which significantly correlated with faecal calprotectin and S100A12 levels; (2) During 12 months follow-up only a minority of these pts developed clinical relapse; (3) Baseline CESI and faecal calprotectin levels may predict medium-term disease outcome. Sensitivity and specificity of baseline faecal calprotectin levels in predicting mucosal inflammation

Elevated calprotectin Normal calprotectin

Inflammation present

Inflammation absent

19 4 Sensitivity 83%

0 16 Specificity 100%

PPV 100% NPV 80%

Pregnancy-related inflammatory bowel disease knowledge amongst obstetricians is significantly lower than gastroenterologists SOLEIMAN BAHMANI KASHKOOLI,1 CHRISTIAN SELINGER,1 MELISSA J WESTON,1 MATTHEW B ROBERTS,2 JANE ANDREWS,2 RUPERT W LEONG,1 SHOALEH AKBARZADEH1 1 Gastroenterology, Concord Hospital, Concord west, NSW, Australia, 2Royal Adelaide Hospital, Adelaide, SA, Australia Background Inflammatory bowel diseases (IBD) are complex conditions that commonly affect women of a reproductive age. Management involves a number of medical professionals, but especially obstetricians. Poor practice or limited education provided may ultimately lead to voluntary childlessness or unfortunate pregnancy outcomes. CCPKnow is a recently validated tool that assesses knowledge of pregnancy-related

issues in IBD. It has not been used to evaluate IBD-related pregnancy knowledge in obstetrician-gynaecologists previously. Aims To assess the knowledge of IBD issues associated with pregnancy in obstetricians in comparison with gastroenterologists and determine predictors of good pregnancy-related knowledge. Methods The CCPKnow (range 0–17) was distributed to invited participants by post or online. Data were collected on demographics, experience and frequency of IBD consultations. The comparative knowledge of gastroenterologists and obstetricians about pregnancy-related issues in IBD was assessed. Analyses were for the composite score and individually on the domains of inheritance, fertility, disease activity, safe use of drugs, mode of delivery, pregnancy outcomes and breastfeeding using the nonparametric Mann Whitney U test and logistic regression to identify predictors of high knowledge. Results The response rates were 33/150 gastroenterologists (22%, median age 42 years) and 36/120 obstetricians (30%, median age 49.5 years). Years of experience favoured obstetricians (median 12 years versus 8, P = 0.131). The median CCPKnow scores of gastroenterologists was 16.0 and obstetricians 14.0 (P < 0.001). Gastroenterologists’ knowledge was higher regarding inheritance (P < 0.001), fertility (P = 0.019), effects of disease activity (P = 0.010), safe use of drugs (P < 0.001), but not mode of delivery (P = 0.460), pregnancy outcomes (P = 0.066) and breast feeding (P = 0.092). Assessing all respondents, the predictor of high CCPKnow was gastroenterology profession (odds ratio: 7.294, 95% confidence interval 2.100–25.335, P = 0.002). CCPKnow was not influenced by age, sex, years of experience, number of non-pregnant IBD patients, or number of pregnant IBD patients (all P > 0.05). Conclusion The overall obstetrician knowledge of pregnancy-related issues in IBD seems to be adequate but significantly poorer compared with gastroenterologists. This supports the concept of a combined multi-disciplinary management team involving gastroenterologists with IBD patients that are pregnant or planning pregnancy.

Predictive factors for colectomy during an admission of severe ulcerative colitis CHAMARA BASNAYAKE,1 ELDHO PAUL,2 MILES SPARROW1 1 Department of Gastroenterology, The Alfred Hospital, Melbourne, VIC, Australia, 2Department of Epidemiology and Preventive Medicine, Monash University, Melbourne, VIC, Australia Background Inpatient severe ulcerative colitis (UC) can be life-threatening without appropriate surgical or medical intervention. During an admission for severe UC that is failing medical therapy clinicians must decide when a ‘curative’ colectomy is required. Aims Our aim was to identify which clinical and biochemical variables predicted the need for a colectomy during that admission. Methods We retrospectively reviewed all admissions at The Alfred Hospital, between 2000–2009, that met Truelove and Witt’s criteria for a severe episode of UC. Our study compared two groups: admissions that resulted in a colectomy and admissions that responded to medical therapy. Standard demographic variables were compared. In addition daily clinical factors (HR, Temp, BP, number of bloody bowel motions [BM]), and investigations (Hb, WCC, PLT, Albumin, K+, CRP, ESR, endoscopic Mayo severity score, histology findings) were compared between groups. Medication use prior to, and during admissions, was also compared. Comparison between groups was performed using a Student t test or Mann-Whitney U test as appropriate. Logistic regression was used to identify the predictors of colectomy. The Travis Score (Day 3 bloody BM’s >8 or 3–8 bloody BM’s and CRP > 45 mg/L) was calculated for each patient determine its validity in this patient cohort.

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Results 57 admissions in 48 patients fulfilled the criteria of a severe flare of UC. When comparing the variables between admissions that resulted in colectomy and those that did not: The mean number of bloody BMs at Day 3 for colectomy patients was 6.8 and for non-colectomy patients was 3.3 (p = 0.01), the mean number of bloody BMs on day 5 for colectomy admissions was 4.7 and non-colectomy admissions was 1.9 (p = 0.01). Mean Mayo scores (0–3, assessed at any time during the admission) for colectomy patients were 2.9 and for non-colectomy 2.4 (p = 0.02). The overall mean Albumin levels were 24.7 amongst colectomy admissions and 29.1 amongst non-colectomy admissions (p = 0.01). The mean prednisolone dose at admission for admissions requiring a colectomy was 18.1 mg compared to 7.8 mg for non-colectomy admissions (p = 0.04). At Day 3, 80% of colectomy patients fulfilled the Travis criteria, compared to 43% of non-colectomy patients (p = 0.02), OR 5.2 (95% CI 1.3–21.8). Conclusion Amongst this cohort of patients with severe UC the following factors were predictive of colectomy during that hospital admission: prednisolone dose at admission, number of bloody BMs at day 3 and 5, Mayo endoscopic severity score, and the mean albumin level. Although patients fulfilling the Travis Score were five times more likely to require colectomy, 43% of responders to medical therapy also fulfilled these criteria for colectomy at Day 3. These data suggest that decisions regarding the need for colectomy need to be individualised in each patient, but should be made early during each hospital admission.

Use and outcomes of anti-TNF-α therapy in pregnant women with inflammatory bowel disease (IBD) SALLY BELL,1 METTE JULSGAARD2 1 Gastroenterology, St. Vincent’s Hospital, Melbourne, VIC, Australia, 2Gastroenterology, Aarhus University Hospital, Aarhus, Denmark Background IBD is common in the fertile years. Anti TNF-α antibodies (biologics) are used for serious IBD. Current reports suggest there is no increase in adverse outcomes or congenital anomalies in offspring of women on biologics (category B) but many patients express concern about their use. Data on outcomes in exposed children is limited. The drugs cross the placenta in increasing amounts from week 18–20 resulting in European guideline recommendations to suspend therapy in the late second and third trimester. The approach to biologic therapy in pregnancy and the outcomes of current practice are unknown in Australia and New Zealand (ANZ). Aims 1. To describe the use and outcomes of biologics for IBD in pregnant women in ANZ. 2. To identify patient concerns regarding biologics in pregnancy Methods Gastroenterologists were emailed an online questionnaire regarding their use of biologics in pregnancy and breastfeeding via the IBDA interest group, GESA e-newsletter and NZ GE society. Women with IBD who had received biologics at any time from 6 months prior to conception through to breastfeeding will be recruited through IBD A and the ANZ IBD consortium, and will complete 2 online questionnaires on pregnancy, lactation, and infant development and outcomes at 12 months. Demographic details, diagnosis, medical treatment for IBD, pregnancy complications and outcomes, breastfeeding practices, patient concerns, child development and vaccination compliance are collected. Results To date 82 gastroenterologists have replied (65% Australian). 95% prescribed anti-TNF-α therapy (53% in pregnancy). Only 12% did not counsel patients prior to conception regarding risks and benefits of biologics. 82–86% were happy to start or continue biologics in patients planning a pregnancy. During pregnancy, 48% recommended stopping by week 30 and 48% recommended continuing. 18% of women were advised not to breastfeed. Almost half the gastroenterologists indicated their practices had changed since they started prescribing. 13 women exposed

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during pregnancy have been enrolled to date. In half the pregnancy was unplanned. Two patients ceased anti-TNF-α therapy. During pregnancy 36% took immunomodulators. 12 had normal babies with 1 spontaneous abortion. 7 delivered by caesarean, 5 because of IBD. One third had concerns about breastfeeding. The majority were happy with their counselling and 75% would consider biologics in the next pregnancy. Conclusion Anti-TNF-α is used in pregnancy by the majority of ANZ gastroenterologists with an IBD interest. The prescribing practices in pregnancy varied with only half following European guidelines. Half the pregnancies were unplanned suggesting risk benefit counselling should be offered to all fertile women at initiation. There was a high caesarean rate due to IBD. Birth outcomes to date were equivalent to the normal population.

Does macrocytosis reflect therapeutic 6-thioguanine levels in patients with inflammatory bowel disease treated with azathioprine or 6-mercaptopurine? LAUREN BESWICK, ALVIN Y TING, CHRISTOPHER HAIR, DAMIAN DOWLING Department of Gastroenterology, Barwon Health, Geelong, VIC, Australia Background Thiopurines have a major therapeutic role in the management of inflammatory bowel disease (IBD). Optimal management depends on achieving therapeutic levels of 6-thioguanine (6-TGN) but measuring this metabolite is associated with significant cost. Thiopurines are known to cause an increase in mean corpuscular value (MCV). It is unknown whether any correlation exists between 6-TGN levels and MCV. Aims To investigate whether a correlation is present between MCV and 6-TGN levels in patients on azathioprine or 6-mercaptopurine for the treatment of IBD. Methods We conducted a retrospective case note review of 37 IBD patients treated with azathioprine and 6-mercaptopurine. 6-TGN levels were measured on 48 occasions. We analysed the data looking for any relationship between 6-TGN levels and MCV both within the cohort as a whole and in individual patients who had 6-TGN levels measured on multiple occasions. We excluded patients with iron and vitamin B12 deficiency. B12 deficiency was defined as a value less than 5 mg/L) was present in 9 of 20 patients with CD, and 8 of 15 with UC. Concentrations of all vitamin D-related indices were similar across 3 groups. Four patients with CD, 9 with UC and 5 HC were taking vitamin D3 supplementation. Total, free and bioavailable 25(OH) D significantly correlated with FC on univariate linear regression in the IBD group as a whole or with CD and UC alone (see Table), whereas DBP did not. These vitamin D indices correlated with CRP in patients with UC (Spearman r = −0.52 for 25(OH)D, r = −0.53 for free 25(OH)D, r = −0.58 for bioavailable 25(OH)D, p < 0.05 for all), but not in patients with CD. On multivariate analysis for all IBD patients in which each vitamin D index, BMI, albumin, calcium and phosphate were predictor variables and FC the dependent variable, only total 25(OH)D levels were associated with FC (p = 0.04, R2 = 42%). Conclusion Despite total, free and bioavailable 25(OH)D concentrations being similar to those in a healthy control population, they do inversely correlate with the degree of intestinal inflammation, and systemic inflammation in patients with UC. If such an association is causal, it provides further evidence that the supply of vitamin D to the intestinal mucosa may indeed be relevant in the control of local inflammation. Studies are required at the intestinal level. No.

IBD (all) Crohn’s disease Ulcerative colitis

35 20 15

Spearman correlation coefficient (r) – FC as dependent variable 25(OH)D

Free 25(OH)D

Bioavail 25(OH)D

−0.53a −0.47c −0.56b

−0.49a −0.49c −0.42d

−0.55b −0.55c −0.52e

a P < 0.005 b P < 0.001 c P < 0.05 d P = 0.151 e P = 0.07.

Total, free and bioavailable vitamin D inversely correlate with faecal calprotectin in patients with inflammatory bowel disease (IBD) MAYUR GARG,1 OURANIA ROSELLA,3 ROSEMARY ROSE,2 JOHN S LUBEL,1 PETER GIBSON3 1 Gastroenterology & Hepatology, Eastern Health, Box Hill, VIC, Australia, 2Eastern Health Clinical School, Monash University, Box Hill, VIC, Australia, 3 Gastroenterology & Hepatology, The Alfred Hospital, Prahran, VIC, Australia Background There is accumulating evidence that vitamin D may mediate immunomodulatory effects in patients with IBD. The relationships of objective markers of disease activity with circulating levels of total, free and bioavailable 25(OH) vitamin D, and of vitamin D binding protein (DBP) are poorly defined. Aims To measure circulating concentrations of the key components of the vitamin D axis in patients with IBD and in healthy controls, and to correlate these with markers of disease activity. Methods Healthy controls, and patients with Crohn’s disease (CD) and ulcerative colitis (UC) attending Eastern Health IBD Clinics were recruited, and demographic and clinical data recorded. Blood samples were analysed for 25(OH) vitamin D (25(OH)D) by chemiluminescence and DBP by ELISA. Systemic inflammation was assessed via the serum C-reactive protein (CRP) and intestinal inflammation via faecal calprotectin (FC). Results 20 healthy controls (11 female; mean age 38, range 23–68 y), 20 patients with CD (11 female; aged 45, 23–76 y) and 15 with UC (6 female; aged 42, 26–64 y) were studied. No significant demographic differences were noted between the groups. Active disease (defined by FC >

Circulating components of the alternative reninangiotensin system are upregulated in patients with inflammatory bowel disease MAYUR GARG,1 KAREN GRIGGS,4 ELENA VELKOSKA,4 OURANIA ROSELLA,3 PETER ANGUS,2 LOUISE BURRELL,4 PETER GIBSON,3 JOHN S LUBEL1 1 Gastroenterology & Hepatology, Eastern Health, Box Hill, VIC, Australia, 2Gastroenterology & Liver Transplant Unit, Austin Health, Heidelberg, VIC, Australia, 3Gastroenterology & Hepatology, The Alfred Hospital, Prahran, VIC, Australia, 4Medicine, Austin Health, Heidelberg, VIC, Australia Background There is accumulating evidence that the renin-angiotensin system (RAS) is active within the gastrointestinal tract, but its influence in intestinal inflammation, especially inflammatory bowel disease (IBD), is poorly understood. Aims To measure the circulating concentration of RAS enzymes and peptide products in patients with IBD and healthy controls, and to correlate these with markers of disease activity. Methods Healthy controls, and patients with Crohn’s disease (CD) and ulcerative colitis (UC) attending Eastern Health IBD Clinics were recruited, and demographic and clinical data recorded. Blood samples were analysed for components of the classical RAS – angiotensin converting enzyme (ACE) and angiotensin II (Ang II), and alternative RAS – ACE2 and Ang (1–7) – by radioimmuno- and enzymatic assays. Systemic inflammation was assessed using serum C-reactive protein

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(CRP) and intestinal inflammation using faecal calprotectin (FC). Participants taking ACE inhibitors and angiotensin receptor blockers were excluded. Results 19 healthy controls (11 female; mean age 38, range 23–68 y), 19 patients with CD (11 female; aged 45, 23–76 y) and 15 with UC (6 female; aged 42, 26–64 y) were studied. No significant demographic differences were noted between the three groups. Active disease (defined by FC > 100 μg/g or CRP > 5 mg/L) was present in 9 of 19 patients with CD, and 8 of 15 with UC. Although the classical RAS components were not significantly different between the three groups, both ACE2 and Ang (1–7) concentrations were significantly higher in IBD patients compared to

No.

Healthy controls IBD (all) Crohn’s disease Ulcerative colitis

19 34 19 15

controls, whether considered overall, as UC or CD alone, or after exclusion of 7 patients treated with corticosteroids. While no indices differed between active and inactive disease, regression analysis showed Ang (1–7) levels correlated with FC in all participants (Spearman r = 0.33; p = 0.028), and in patients with UC (r = 0.67; p = 0.018), but not in patients with CD (r = 0.20; p = 0.416). Conclusion This is the first description of alternative RAS axis upregulation in patients with IBD. This was independent of disease activity, except for Ang (1–7) in patients with UC. Further research into this novel pathway may pave the way for novel diagnostic and therapeutic approaches in IBD.

Classical RAS

Alternative RAS

ACE (U/L)

Ang II (pg/ml)

ACE2 (pmol/ml/min)

Ang (1–7) (pg/ml)

22.1 22.9 23.6 21.9

17.2 15.4 13.7 17.4

13.3 21.5 21.8 21.1

14.1 22.8 25.0 19.9

(17.1–27.2)# (19.3–26.5) (18.3–28.9) (16.8–27.1)

(10.6–23.9) (10.9–19.8) (10.8–16.7) (7.4–27.5)

(9.9–16.7) (17.2–25.9)** (16.5–27.2)* (13.1–29.2)**

(11.1–17.0) (20.0–25.5)*** (21.2–28.8)*** (16.2–23.7)***

# mean (95% confidence intervals) * P < 0.01 compared with healthy controls (t test) ** P < 0.005 *** P < 0.001.

Rescue therapy in ulcerative colitis – regional Queensland experience ENOKA GONSALKORALA, ENRICO ROCHE Gastroenterology, The Townsville Hospital, Townsville, QLD, Australia Background Infliximab is established therapy for steroid refractory Crohn’s disease. Under the Pharmaceutical Benefits Scheme (PBS) it is not indicated for treatment of refractory Ulcerative Colitis. Cyclosporine can be used to treat severe colitis under the PBS. Data suggest infliximab and cyclosporine may be equally efficacious at achieving short term remission and avoidance of urgent colectomy. Studies have however shown that more complications are associated with cyclosporine compared with infliximab. Experience in regional Australian centres with these two medications is unknown. Review the demographics, disease severity, other therapies, type of rescue therapy, complications and outcomes of patients with severe Ulcerative Colitis refractory to steroid therapy Aims Review the demographics, disease severity, other therapies, type of rescue therapy, complications and outcomes of patients with severe Ulcerative Colitis refractory to steroid therapy. Methods Retrospective chart review of patients with Ulcerative Colitis treated with rescue therapy The Townsville Hospital, Queensland, Australia was performed. A list of patients presenting to day therapy for infliximab with a diagnosis of Ulcerative colitis was obtained. Pharmacy records were used to obtain a list of patients who had received cyclosporine for Ulcerative Colitis. Information was obtained from medical records, imaging records and hospital pathology records. Results 7 patients were treated with rescue therapy from 2007 to 2012. Infliximab was given at week zero, two and six at 5 mg/kg. Cyclosporine was administered at 2 mg/kg infusion as an inpatient and changed to oral therapy at 5 mg/kg upon discharge. This was subsequently weaned as an outpatient. Three patients received infliximab alone, one patient received cyclosporine alone and three patients received both. To date none of the patients treated with infliximab alone have required colectomy. The patient treated with cyclosporine alone has not required a colectomy. All three patients treated with both therapies received cyclosporine followed by infliximab. Two subsequently proceeded to surgery at 0 and 23 months after rescue therapy with infliximab. Since 2010 patients have not been rescued using cyclosporine. The one major adverse event in the group consisted of an anaphylactoid reaction to cyclosporine leading to immediate cessation of treatment and switch to infliximab.

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Conclusion Since early 2010 cyclosporine has not been used to treat refractory Ulcerative Colitis at The Townsville Hospital. In this small sample group those who had rescue treatment with cyclosporine were more likely to need rescue with infliximab. Both patients who proceeded to colectomy were initially treated with cyclosporine. The one major adverse event was also associated with cyclosporine use. References 1. Dean et al. Infliximab or cyclosporine for acute severe ulcerative colitis. J Gastroenterol Hepatol 2012 27:487–92 2. Croft et al. Outcomes of salvage therapy for acute severe ulcerative colitis: cyclosporine versus infliximab (abstract) 2009 24:S112.

What is the unmet need in patients with inflammatory bowel disease and is this linked to inflammatory activity? CHRISTIAN GREIS, NORA MAYER, PETER HENDY, DANIEL BURGER, NEAL MARTIN, LOUISE YOUNG, GERALD HOLTMANN Gastroenterology and Hepatology, University of Queensland, Princess Alexandra Hospital, Brisbane, QLD, Australia Background Controlling inflammatory activity and healing of mucosal lesions as well as Quality of Life (QoL) are now considered to be major targets for treatment of patients with inflammatory bowel disease (IBD). However, in the clinical setting the patient’s subjective unmet need is rarely assessed. Aims Thus we aimed a) to develop and validate an instrument that assesses the unmet need of IBD patients and b) to identify the role of inflammatory activity on the unmet need. Methods In focus group interviews 14 items were identified. These items were tested in a cohort of 100 consecutive IBD patients (Age: 40.26 ± 1.52; years of disease: 9.4 ± 1.18), 67 patients with Crohn’s disease (CD; Montreal Classification [%] A1:5.3 A2:78.9 A3:15.8; L1:32.5 L2:22.5 L3:45.0; B1:61.1 B2:30.6 B3:8.3; p+:43.2) and 33 ulcerative colitis (UC; Montreal Classification [%] A1:0 A2:93.3 A3:6.7; E1:23.1 E2:46.2 E3:30.7).

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Results Factor analysis revealed 5 factors that explained more than 70% of variance. These factors were labelled: 1) Disease Control, 2) Treatment Simplicity, 3) Diarrhoea & Prevention, 4) Cure, 5) Abdominal Pain. The highest unmet need scores were reported for the factors Diarrhoea & Prevention (6.54 ± 0.11), Abdominal Pain (6.49 ± 0.13) and Disease Control (6.23 ± 0.11), followed by the factors Cure (5.78 ± 0.14) and Simplicity of Treatment (5.52 ± 0.13). Interestingly, the various unmet need scores of IBD-Patients were statistically not significant correlated with CRP (Table 1). Conclusion The newly developed ‘unmet need questionnaire’ is a tool to better characterise and target patients’ needs. Unmet need to treat ‘Diarrhoea’ and ‘Abdominal Pain’ were ranked highest by the patients. However, the ‘Unmet Need’ was not linked to disease activity as measured by CRP or other inflammatory markers. While the medical management primarily now targets control of inflammation and long term prevention of complications, there is a substantial unmet need in relation to symptoms and in a broader term ‘concerns’, that are not linked to inflammatory activity. Table 1 Mean values and SEM for the unmet need factors for patients with IBD in various CRP groups

CRP < 2 CRP > 2 < 10 CRP > 10 total

Disease Control

Treatment Simplicity

Diarrhoea & Prevention

Cure

Abdominal Pain

6.18 ± 0.26 6.19 ± 0.15

5.29 ± 0.30 5.56 ± 0.18

6.70 ± 0.17 6.34 ± 0.22

6.08 ± 0.20 5.50 ± 0.23

6.89 ± 0.15 6.54 ± 0.14

6.38 ± 0.22

5.44 ± 0.24

6.64 ± 0.19

6.00 ± 0.43

5.56 ± 0.60

6.23 ± 0.11

5.52 ± 0.13

6.54 ± 0.11

5.78 ± 0.14

6.49 ± 0.13

Symptom fluctuation during maintenance infliximab therapy for Crohn’s disease PETER J HENDY,1 CHRISTIAN GREIS,1 DANIEL BURGER,1 NEAL MARTIN,1 NICHOLAS TALLEY,2 GERALD HOLTMANN1 1 Gastroenterology, Princess Alexandra Hospital, Brisbane, QLD, Australia, 2Gastroenterology, Newcastle University, Newcastle, NSW, Australia Background The current prescribing recommendation for infliximab maintenance therapy for Crohn’s disease is 5 mg/kg every 8 weeks. Although this schedule is effective in preventing relapse, a number of patients suffer with breakthrough symptoms despite demonstrating mucosal healing. In addition to this, Infliximab pharmacokinetics studies demonstrate as few as 40% of patients have detectable trough serum levels prior to their next scheduled dose [1]. Aims To characterise loss of symptom control at infliximab dose nadir, by examining intestinal and extra-intestinal symptoms during a standard maintenance infliximab cycle. Methods Consecutive patients with Crohn’s disease on maintenance infliximab therapy at Princess Alexandra Hospital were included. As part of the routine clinical management, symptoms were assessed at day 7 and day 56 of their maintenance treatment cycle with a modified Psychosomatic Symptom Checklist (PSC) and Hospital Anxiety and Depression Scale (HADS). Symptoms within the PSC were defined as either ‘intestinal’ or ‘extra-intestinal’. CRP and CDAI were measured at day 56 to correlate symptom scores with disease activity. Results 6 patients were included to date. In 5 out of 6 patients, there was an increase in gastrointestinal (3.3 ± 1.5, p = 0.08) and extra-intestinal (6.8 ± 5.8, p = 0.297) symptom scores from day 7 to day 56, as well as HADS scores (5.0 ± 2.4, p = 0.106). There was a significant correlation between HADS scores and PSC scores (r = 0.89, p = 0.043), as well as between

PSC scores and CDAI (r = 0.895, p = 0.016) and PSC scores and CRP (r = 0.881, p = 0.021). Conclusion This is the first systematic assessment of gastrointestinal and extraintestinal symptoms during the course of the anti-TNFα treatment cycle. There is a trend for symptom breakthrough at day 56. Since it is well established that the 8 week interval is sufficient to control mucosal lesions, further research is needed to assess whether the symptom development precedes clinical inflammation and tissue damage. Reference 1. Seow CH, Newman A, Irwin SP, et al. Trough serum infliximab: a predictive factor of clinical outcome for infliximab therapy in acute ulcerative colitis. Gut 2010; 59: 49–54.

Pilot validation study of the international classification of functioning, disability and health score: a novel measure of disability in Crohn’s disease TONY HUANG,1 RUPERT W LEONG,2 YANNA KO,1 VIRAJ C KARIYAWASAM2 1 The Faculty of Medicine, The University of New South Wales, Sydney, NSW, Australia, 2Gastroenterology and Liver Services, Concord Hospital, Sydney, NSW, Australia Background Patients with Crohn’s Disease (CD) often suffer debilitating symptoms that impair their ability to participate in everyday activities. Disability is an objective measure that differs to subjective quality-of-life. Currently there is no standardised tool for assessing disability in inflammatory bowel diseases (IBD). The International Classification of Functioning, Disability and Health (ICF) questionnaire covering the range of limitations seen in IBD was recently published. This questionnaire is not yet validated for clinical use and a scoring system has not been developed. Aims This study aims to develop a new composite scoring system based on the ICF questionnaire and evaluate through construct validity its correlation with quality of life (convergent validity) and inverse correlation with disease activity (divergent validity) in CD. Methods Patients with CD were prospectively recruited at hospital IBD clinics. Subjects completed the ICF disability and Crohn’s Disease Activity Index (CDAI) questionnaires face-to-face, and the self-administered IBDQ quality-of-life survey. Their corresponding clinical data were recorded. In scoring the ICF questionnaire, negative scores were proportioned to self-reported severity of difficulties in the past week in areas such as sleeping, mood, abdominal pain, stool frequency, adverse effects of medications, and so on. Positive scores were proportioned to the alleviating effects of medication, food, family and health professionals. Disability was compared across phenotypes according to the Montreal Classification. Correlations of ICF total scores with CDAI and IBDQ total scores were analysed via Spearman rho bi-variate non-parametric statistics. Results This pilot study recruited 19 consecutive patients prospectively (64% males, aged 20–71 years). Divergent validity was demonstrated with lower ICF scores significantly correlating with higher CDAI score (rho = −0.599, P < 0.007) and convergent validity was demonstrated on the IBDQ scores (rho = 0.895, P < 0.001). Location phenotypes of CD were 26% ileal, 16% colonic and 58% ileocolonic. Respectively, median ICF scores were −5.0, −7.0, 2.0, (P = 0.288). Behavioural phenotypes of CD were 42% inflammatory, 26% stricturing and 32% penetrating. Respectively, median ICF scores were −5.5, −6.0, −5.5 (P = 0.992). Age at diagnosis, sex, perianal disease (32% of CD, P = 0.429), previous hospitalisation (58%, P = 0.400), previous surgery (53%, P = 0.840), previous or current steroid-used (58%, P = 0.840) all did not significantly influence the ICF.

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Conclusion The ICF disability score significantly correlates with greater disease activity and poorer quality of life. However, ICF only reflects functioning in the past week and does not measure the overall fluctuating disease course, previous treatments and complications, and may better suit illnesses that remain constant rather the relapsing-remitting nature of IBD.

Severe cardiac complications of aminosalicylic acid (ASA) drug therapy – 2 case reports and literature highlights TEE CHING HUN,1 SURESHKUMAR KALLIPPATTI PONNUSWAMY,1 NICHOLAS KONTORINIS,1 WENDY CHENG,1 WENDY CHENG,2 KANNAN VENUGOPAL1 1 Gastroenterology Dept, Royal Perth Hospital, Perth, WA, Australia, 2Curtin Health Innovation Research Institute, Curtin University, Perth, WA, Australia Background Aminosalicylic acid (ASA) compounds are common firstline agents used for treatment of mild to moderate ulcerative colitis. Severe adverse reactions to these drugs are uncommon and cardiac complications are rarely reported. Aims We report two cases of cardiac complications secondary to Sulfasalazine and Mesalazine at our hospital. Methods Retrospective description of cases based on patient case notes and investigations. Results Case 1: A 20-year-old gentleman with recent diagnosis of ulcerative colitis was commenced on mesalazine 1.5 g bd. Three weeks later, he presented with sharp retro-sternal pain associated with cough and fever. No pericardial rub or signs of heart failure were elicited. Investigations revealed neutrophilia, a raised CRP of 150 mg/l and a Troponin-I rise of 9 μg/l. ECG displayed inferolateral concave ST-segment elevation. Echocardiography demonstrated mild-moderate global systolic dysfunction (LVEF = 45%) and a small pericardial effusion. Viral serology and autoimmune markers were negative. Coronary angiogram was normal. A diagnosis of mesalazine-induced myo-pericarditis was made. Mesalazine was ceased and the patient was commenced on metoprolol, ramipril and hydrocortisone. By day 3, his pain had improved. ECG changes resolved, his troponin normalised and a repeat echocardiogram demonstrated normalisation in cardiac function (LVEF = 54%). A cardiac MRI confirmed appearances consistent with myo-pericarditis. Case 2: A 22-year-old gentleman with recently diagnosed ulcerative colitis was commenced on oral sulfasalazine and mesalazine enemas with no concurrent medications. He presented with a 2-day history of flu-like symptoms and rapidly deteriorated into cardiogenic shock secondary to massive pericardial effusion. This subsequently led to multi-organ failure with ischemic hepatitis and acute tubular necrosis of the kidneys. He required pericardiocentesis and inotropic support. Blood tests revealed negative viral serology, positive antinuclear antibody (ANA-30), low levels of complement (C3 & C4) and positive anti-histone antibodies consistent with drug-induced lupus. Diagnosis of mesalazine/sulfasalazine-induced lupus with manifestation of serositis and secondary pericardial effusion and cardiac temponade was established. Sulfasalazine and enema were ceased and he was commenced on corticosteroids with clinical improvement. Conclusion 1. Cardiac complications to sulfasalazine and 5-ASA are rare, with a limited number of case reports described in the literature including: – Pericarditis – 14 cases – Myocarditis – 6 cases with 1 fatality – Myopericarditis – 3 cases, excluding the current case

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2. These reactions typically occur within the first few weeks of commencement and improve with cessation of the offending drug. 3. Clinicians should be mindful of these potentially fatal adverse reactions.

Failure to improve inpatient thromboprophylaxis rates in inflammatory bowel disease after a re-education intervention AIESHA JAVED, JEREMY P DWYER, CHRISTOPHER HAIR, GREGORY MOORE Gastroenterology & Hepatology, Monash Medical Centre, Clayton, VIC, Australia Background Inflammatory bowel disease (IBD) is associated with an increased risk of thromboembolism (TE). Hospitalised patients with IBD have a higher morbidity and mortality associated with TE. Many IBD patients do not receive adequate thromboprophylaxis, despite the existence of hospital protocols. Aims The aim of this study was to assess whether education and reinforcement of hospital thromboprophylaxis protocols was effective in increasing the rates of thromboprophylaxis given to hospitalised IBD patients. Methods All patients with a diagnosis of IBD, coded according to the ICD tenth revision, were identified from a coding search of separations from Southern Health (Monash Medical Centre and Dandenong Hospital). An initial audit for patients admitted between December 2009 to May 2010, assessed rates of thromboprophylaxis. This was followed by reinforcement of pre-existing hospital thromboprophylaxis protocols via an email with the protocol and direct verbal education from the IBD physician to the registrars, highlighting the importance of thromboprophylaxis in IBD patients. Post-reinforcement, a further audit of all IBD admission from July 2010 to January 2011 assessed compliance with the thromboprophylaxis guidelines. Analysis of rates was performed using a two tailed Fisher’s exact test. Results There were 36 multi-day admissions for IBD prior to the intervention and 91 in the 6 months post intervention. Thirty out of 36 (83.3%) pre-intervention and 69 out of 91 (75.8%) post-intervention patients received appropriate thromboprophylaxis, a difference that was not statistically significant (p = 0.517). In the 30 pre-intervention patients who received thromboprophylaxis, 26 (86.7%) received 40 mg enoxaparin s/c daily and 4 received alternative thromboprophylaxis (heparin 5000 units s/c BD, 100 mg or 20 mg subcutaneous enoxaparin). 58 patients out of 91 receiving thromboprophylaixs post-intervention (63.7%) received 40 mg enoxaparin, with no statistical difference between the groups (p = 1). Rates of thromboprophylaxis for medical admissions were 67.6% pre-intervention and 67.7% post-intervention (p = 1.0) compared with 50% pre-intervention and 68.9% post intervention for surgical admissions (p = 0.539). Conclusion Hospitalised patients with IBD receive inadequate thromboprophylaxis with no difference in observed after reinforcement of protocols via e-mail and registrar education. Constant education and clinical auditing may be required to improve compliance with the guidelines.

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Natural history of Crohn’s disease and changes in the use of immunomodulators over time: the Sydney Inflammatory Bowel Disease Cohort 1942–2012 VIRAJ C KARIYAWASAM,1 PAUL C LUNNEY,1 ROSY R WANG,1 KATE L MIDDLETON,1 CHRISTIAN SELINGER,1 PETER KATELARIS,1 JANE ANDREWS,2 RUPERT W LEONG1 1 Gastroenterology and Hepatology, Concord Repatriation General Hospital, Concord, NSW, Australia, 2IBD Service, Royal Adelaide Hospital, Adelaide, SA, Australia Background Immunomodulators (IM) are proven to be effective in the induction and maintenance of remission in Crohn’s disease (CD). However, few studies have evaluated changes in its use over longitudinal follow-up and impact this has made on the natural history of CD. Aims The study aims to determine the changes in use of immunomodulators during longitudinal follow-up in a large metropolitan cohort of ambulatory inflammatory bowel disease (IBD) patients. Methods The Sydney IBD Cohort is a cohort study based around the Sydney Local Health District with recruitment by specialists in private rooms and hospitals. The medical records were reviewed for patient demographics, disease characteristics, drug treatments and surgical management. Three time periods were assessed: pre-1990, 1990–2000, and post-2000. Kaplan-Meier analysis was used to estimate the cumulative probability of initiation of IM and the log rank test to determine significance. Predictive factors affecting the likelihood of IM initiation over time were investigated using the Cox proportional hazard regression model. Results A total of 406 CD patients (53% females) with complete followup data were included in the analysis. The median length of follow-up was 9 years (IQR 4–17) with an overall 4,800 patient-years of follow-up. Age of diagnoses was over 16 years old in 92.6%. Location phenotypes were ileo-colonic 39.8%, ileal 24.6%, and colonic 35.6%. The stricturing phenotype was found in 20.4%, the penetrating phenotype in 5.3% of patients and perianal disease in 17.9%. Ever smokers comprised of 36% of the cohort. There were total of 241 (60.2%) patients who were exposed to IM with 146 (36.1%) being current users (31.8% thiopurines and 4.7% methotrexate at the time of follow-up). Percentage of patients on IM in the 3 time periods considered did not differ (P = 0.6). However, the time of introduction of an IM significantly reduced over the three time periods [P < 0001], Figure 1. On multivariate Cox regression analysis, earlier time periods of diagnosis (hazard ratio (HR): 7.81, 95% CI: 3.7–16.48, P < 0001 and 21.23, 95% CI: 9.51–47.40, P < 0.001), age at onset (HR: 0.977, 95% CI: 0.96–0.99, P < 0.0001), perianal involvement at diagnosis (HR: 1.69, 95% CI: 1.14– 2.52, P = 0.009), need for systemic steroids (HR: 2.83, 95% CI: 1.69–3.35, P < 0.001) and requirement for surgical resection (HR: 1.493, 95% CI: 1.00–2.22, P = 0.05) were all significantly associated with the time to initiation of IM therapy. In total, 138 first surgical resections were noted in the cohort, and 41.3% of these had surgical recurrence. IM significantly decreased the need for subsequent surgical recurrence (P = 0.039). Conclusion Earlier introduction of IM over time is associated with reduced surgical recurrence rate.

Figure 1. Kaplan Meier curve showing time from diagnosis to introduction of immunomodulators over three time periods.

The natural history of ulcerative colitis and predictors of the use of immunomodulators and colectomy: the Sydney Inflammatory Bowel Disease Cohort 1946–2012 VIRAJ C KARIYAWASAM,1 KATE L MIDDLETON,2 PAUL C LUNNEY,2 ROSY R WANG,2 JANE ANDREWS,3 CHRISTIAN SELINGER,2 PETER KATELARIS,1 RUPERT W LEONG1 1 Department of Gastroenterology, Concord Repatriation General Hospital, Concord, NSW, Australia, 2Faculty of Medicine, University of Sydney, Sydney, NSW, Australia, 3IBD Service, Royal Adelaide Hospital, Adelaide, SA, Australia Background Colectomy in ulcerative colitis (UC) may be indicated at initial diagnosis or during follow-up. The colectomy rate and whether colectomy rate has reduced with modern treatments is currently not well known. Predictors of colectomy may include disease phenotype but may also be influenced by medical treatment. Aims This study aims to describe the UC cohort of Sydney Local Health District, an extension of the Sydney IBD Cohort. The study describes the cumulative colectomy rates over two time periods: pre- and post-2000. Potential predictive factors of colectomy include its phenotype according to the Montreal Classification and medical treatment. Methods UC patients were recruited. Their medical records were reviewed for demographics, disease characteristics, treatments and surgery. Kaplan Meier analysis illustrates the cumulative probability of colectomy and statistical significance was determined by the log rank test. Predictive factors for colectomy and immunomodulator (IM) use were investigated using multivariate binary logistic regression. Results In total, 890 patients (52% males) were recruited, with a mean age at diagnosis of 37.4 years (IQR: 24–49). The median follow-up was 14 years (IQR: 5–24) with an overall 14,597 patient-years of follow-up. Ever smokers comprised of 22%. Immunomodulators use was 26%, 5-ASA 90% and long-term steroid use (defined as steroid use >6 months in a year) was 36%. Steroid-use significantly decreased in patients diagnosed post-2000 compared to pre-2000 (P = 0.013).

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A total of 137 (15.4%) colectomies were performed, most commonly total colectomy (TC) with ileostomy (61%) and TC with pouch anastomosis (23%). The early colectomy rate within the first year of diagnosis was 3.2%. Thereafter, the mean time to colectomy was 11.9 years (IQR: 3–18; Figure 1). The colectomy rate has significantly decreased in the post-2000 UC cohort compared to the pre-2000 UC cohort, whether analysed for all-time colectomies (P = 0.017) or only colectomies after the first year of diagnosis (P = 0.042). Extensive colitis (OR: 1.85, 95% CI: 1.34–2.56, P < 0.0001), steroid dependency (OR: 10.54, 95% CI: 5.36–20.73, P < 0.0001), smoking (OR: 3.03, 95% CI: 1.38–6.67, P < 0.0001) and younger age at diagnosis (OR: 0.96, 95% CI: 0.94–0.98, P < 0.0001) all significantly predicted the need for IM therapy. Extensive colitis (OR: 3.90, 95% CI: 1.61–9.47, P = 0.003) and steroid dependency (OR: 5.285, 95% CI: 2.03–13.78, P = 0.001) were significant predictors for colectomy, but IM did not reduce the colectomy rate. Conclusion This large cohort study showed a significant reduction in the cumulative colectomy rate in ulcerative colitis patients in the recent decade. However, immunomodulator use was not associated with this reduction. Significant predictors of colectomy included extensive colitis and corticosteroid dependency.

Aims The study aims to determine the changes in intestinal resection rates over longitudinal follow-up in a large specialist-based metropolitan cohort of CD patients. Methods The Sydney Inflammatory Bowel Disease Cohort is an ongoing epidemiological study recruiting metropolitan patients of Sydney Local Health District. CD patients were recruited and that demographics, disease characteristics, drug treatments and surgical procedures recorded. KaplanMeier analysis was used to estimate the cumulative probability of undergoing intestinal resection over 3 time periods and statistical analysis according to the log rank test. Factors that may affect the likelihood of intestinal surgery over time were investigated using the Cox proportional hazard multivariate regression model. Results In total, 751 CD patients (56.6% females) with complete followup data were recruited. The median length of follow-up was 12 years (IQR: 5–21) with an overall 10,383 patient years of follow-up. Inflammatory behaviour was noted in 74.3% patients at diagnosis and ileal, colonic and ileo-colonic locations of disease in 24.6%, 35.6% and 39.8% respectively. Perianal involvement was found in 18.0% at diagnosis. There were total of 305 (40.6%) patients who had intestinal surgery. Ileocaecal resections accounted for 54.8% of surgeries. A total of 32.1% of resections occurred in the first year of diagnosis (IQR: 0–7; Figure 1). There was a significant increase in time to first surgery between the three time periods (P = 0.0001). The proportion of CD patients having surgery in each subsequent time period significantly decreased with 68.1% having surgery in pre-1980 group compared to only 22.0% in the post-2000 group (P = 0.0001). Early introduction of immunomodulators prior to surgical resection was significantly associated with the reduced surgical rate in CD patients (Hazard Ratio [HR]: 0.02; 95% confidence interval [CI]: 0.001–0.438). Decade of diagnosis, disease location, behaviour, presence of perianal involvement at diagnosis and smoking status were all associated with time to intestinal resection (all P < 0.05). Conclusion There has been a significant decrease in the surgical rate of Crohn’s disease over the last 7 decade. This reduction is most likely associated with earlier introduction and greater use of immunomodulators.

Figure 1. Time to colectomy in ulcerative colitis according to year of diagnosis.

Surgical resection rate of Crohn’s disease over time: the Sydney Inflammatory Bowel Disease Cohort (1942–2012) VIRAJ C KARIYAWASAM,1 ROSY R WANG,1 KATE L MIDDLETON,1 PAUL C LUNNEY,1 CHRISTIAN SELINGER,1 GLEN D COLLINS,1 PETER KATELARIS,1 JANE ANDREWS,2 RUPERT W LEONG1 1 Gastroenterology and Hepatology, Concord Repatriation General Hospital, Concord, NSW, Australia, 2IBD Service, Royal Adelaide Hospital, Adelaide, SA, Australia

Figure 1. KaplaneMeier curve showing time from diagnosis to first intestinal surgery in the four hexiles.

Background Medical therapy for Crohn’s disease (CD) has changed significantly over the last two decades. Studies from Europe and Northern America have shown a trend towards reduced surgical rates in the recent times. However data from the Southern Hemisphere is lacking.

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care. Given this support and earlier treatment of flares it would be predicted that long-term outcomes would also be improved. Whilst the GP Plus Service Funding did not continue beyond 2011/12, the demonstration of effectiveness required by the funding agreement facilitated the acceptance of the role by Flinders Medical Centre enabling ongoing funding to be secured.

Figure 2. Number of Crohn’s patients undergoing resections according to decade of diagnosis.

The effect of an IBD nurse position on service delivery PETA LEACH, PETER BAMPTON, MAHINDA DE SILVA, REME MOUNTIFIELD, SAM EDWARDS, LAURIE D CHITTI Gastroenterology, Flinders Medical Centre, Bedford Park, SA, Australia Background IBD nurse positions are now regarded as standard of care, yet convincing funders of their importance remains difficult. Despite a moderate IBD service catering for 1200 patients, the IBD nurse position at FMC has remained unfunded from the hospital budget. In 2010–2011 an agreement was made between our local health network to fund the IBD nurse as part of the GP Plus Service Fund, in line with a strategic aim of minimising acute hospital presentations. Aims To report the effect of the presence of an IBD nurse position on an IBD service. This position is separate to the IBD Biologics nurse, which is funded through the endoscopy unit at Flinders Medical Centre. Methods As part of a funding agreement between all occasions of service were prospectively recorded by the IBD Nurse to gauge the effectiveness of the IBD Nurse position at reducing acute hospital presentations. Results Over the calendar year 2011, the IBD nurse position prevented 21 presentations to ED, facilitating direct admission to the ward and avoided 11 admissions. Given our average IBD admission is 6 days this represented 66 occupied bed days (OBD). The IBD nurse, through consultation with medical staff, changed/arranged treatment in 93 cases and initiated investigations in 48 cases. Urgent clinic review was arranged in 28 cases, although avoidance of clinic was achieved in 100 cases. Inpatient counselling and review occurred in 108 cases, using an inpatient admission to review issues such as bone density measurement, immunisation status and education. All patients over this time had their immunisation reminders sent and, if relevant, blood test reviewed for immunomodulator toxicity. Over 8000 occasions of service were provided by the IBD nurse over this period. Conclusion An IBD Nurse enables the avoidance of admission to hospital, saving significant OBD as well as enabling the prompt initiation of

Intestinal mucosal leakage is detected using in vivo confocal endomicroscopy in macroscopically-normal Crohn’s disease and ulcerative colitis RUPERT W LEONG,1 BRENDON WONG,2 JIM CHEN,2 EDMUND CHUNG,2 JUSTINE MILL,1 VIRAJ C KARIYAWASAM,1 CHRISTIAN SELINGER,1 DARREN PAVEY,1 NEIL MERRETT3 1 Gastroenterology and Liver Services, Bankstown Hospital, Bankstown, NSW, Australia, 2Faculty of Medicine, The University of New South Wales, Sydney, NSW, Australia, 3University of Western Sydney, Campbelltown, NSW, Australia Background Increased permeability of the intestinal tract allowing excessive intestinal luminal antigens to cross the mucosal barrier may underlie the pathogenesis of inflammatory bowel diseases. Confocal Endomicroscopy (CEM) may diagnose in vivo and quantitate mucosal barrier dysfunction of the intestinal tract and identify dynamic contrast leakage under real time. Aims To validate the features of ‘leaky gut’ in Crohn’s disease (CD), ulcerative colitis (UC) and controls, quantitate this against clinical disease activity and its reversibility after escalation of treatment. Methods Consecutive patients with CD, UC and controls were prospectively recruited for CEM using intravenous fluorescein as contrast agent. Blinded assessment of images of five macroscopically non-ulcerated terminal ileum sites was performed, using ‘fluorescein leak (FL)’, ‘cell junction enhancement (CJE)’ and ‘cell drop out (CDO)’ as features of intestinal mucosal barrier dysfunction. Scores for each individual feature as well as the composite confocal leakage score (CLS) were assessed and expressed as percentages. Correlation with clinical disease activity was performed, and paired procedures were performed to determine reversibility of leakiness following treatment. Kruskal-Wallis, Wilcoxon signed ranks and chi square non-parametric analyses were performed. Results A total of 36 subjects were recruited and yielded 6,565 images for the study (64% females; CD n = 17, UC n = 6 and controls n = 13; 2,362, 784 and 3,419 CEM images respectively; ages not significantly different). Median CLS for CD, UC and controls were 27.2, 17.5 and 5.4 (P = 0.007) respectively. The scores for individual features of mucosal leakiness are shown in Table 1 and were all significantly different (all P < 0.05). In UC CJE was significantly increased in comparison to controls (11.0 versus 3.2 respectively; P = 0.035). Reversibility of CLS was demonstrated after treatment in one UC and two CD patients (median CLS pre-treatment 36.4, post-treatment was 13.9; P = 0.028. CLS was independent to clinical disease activity (P = ns). Conclusion Confocal endomicroscopy can identify and measure intestinal mucosal leakage in the terminal ileum of macroscopically normal CD. There is also impaired terminal ileum barrier integrity in UC. Leaky gut does not correlate with disease activity and may represent an underlying intrinsic barrier dysfunction. Reversal of leaky gut occurs following medical therapy escalation. This technique also has the potential to measure functional healing of the mucosal barrier.

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Table 1

Intestinal mucosal leakage (%)

Crohn’s disease Ulcerative colitis Controls P value

Confocal leakage score

Fluorescein leakage

Cell junction enhancement

Cell drop out

27.2

12.4

13.3

1.4

17.5

3.9

13.8

1.2

2.7 P = 0.009

0.0 P = 0.037

5.4 P = 0.007

2.7 P = 0.044

Smoking prevalence in inflammatory bowel diseases and its effects on disease course and surgery PAUL C LUNNEY,1 KATE L MIDDLETON,1 ROSY R WANG,1 JANE ANDREWS,3 VIRAJ C KARIYAWASAM,2 JENNIFER PEAT,2 CHRISTIAN SELINGER,2 RUPERT W LEONG2 1 Faculty of Medicine, The University of Sydney, Sydney, NSW, Australia, 2Gastroenterology and Liver Services, Concord Repatriation General Hospital, Sydney, NSW, Australia, 3IBD Service, Royal Adelaide Hospital, Adelaide, NSW, Australia Background Smoking is an influential factor in the development and progression of inflammatory bowel diseases (IBD). It has a heightened prevalence in Crohn’s disease (CD) and detrimentally influences its natural history. Paradoxically, ulcerative colitis (UC) is predominantly a disease of non-smokers and ex-smokers, with varying reports of amelioration of disease in active smoking. Confirmation of the harmful effects of smoking in CD using local data may encourage patients to quit smoking. Aims This study aims to determine the prevalence of smoking and its effects on disease progression and outcomes. Disease progression was defined by the need for surgical intervention and requirement to commence a thiopurine or biological agent. Methods Ambulatory patients with IBD were recruited in Sydney from a metropolitan cohort diagnosed between 1942 and 2012. Case notes for each patient were reviewed to identify data on current smoking status, age at diagnosis, disease phenotype at diagnosis, thiopurine and biological agent use, need for surgery and number of hospital admissions. Chi square and the log-rank statistical tests were performed with cumulative surgery rate illustrated using the Kaplan-Meier method. Results In total 803 IBD patients were recruited (374 CD, 393 UC and 36 IBD-undefined). Clear smoking status was available in 82.3%. The mean years of follow-up were 11.4 for CD patients and 12.2 for UC patients. Of the CD patients 12.6% were current smokers, 15.5% ex-smokers and 51.6% never smokers. There was no statistically significant difference in age or phenotype of disease at diagnosis between these three groups. Frequency of thiopurine and biological agent use and hospital admission between the groups was equivocal. Surgical management of disease was required more frequently in current and ex-smokers than in never smokers (P = 0.041) and ever smoking was associated with an increased cumulative probability of intestinal resection (Figure 1, P = 0.045). In UC 5.9% were current smokers, 13.2% ex-smokers and 65.4% never smokers. Ex-smokers had a later age of diagnosis than current smokers (P = 0.039) and never smokers (P < 0.001) with UC (45.7 vs. 37.0 vs. 34.9 years, respectively). Extent of disease at diagnosis was not influenced by smoking habit. Ever smoking was associated with a greater likelihood of thiopurine therapy induction requirement (P = 0.014). In UC there was no significant difference between groups in the need for biological agent use, colectomy or hospital admission.

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Conclusion Smoking was associated with an increased need for surgery in CD. Smoking poorly correlated to other markers of disease severity. This study found UC as predominantly a disease of non-smokers and exsmokers but it did not protect against colectomy or hospitalisation. These data should strongly encourage smokers with IBD to quit smoking.

Figure 1. Kaplan-Meier curve showing the time from diagnosis to first intestinal resection in CD comparing ever smokers (current or ex-smokers) and never smokers.

A comparison of inflammatory bowel disease patients aged with disease and those diagnosed late in life KATE L MIDDLETON,1 VIRAJ C KARIYAWASAM,1 PAUL C LUNNEY,1 ROSY R WANG,1 CHRISTIAN SELINGER,1 PETER KATELARIS,1 JANE ANDREWS,2 RUPERT W LEONG1 1 Gastroenterology and Hepatology, Concord Repatriation General Hospital, Concord, NSW, Australia, 2IBD Service, Royal Adelaide Hospital, Adelaide, SA, Australia Background Australia has one of the highest incidences of inflammatory bowel diseases (IBD). Previous findings from our cohort have shown no reduction in life expectancy in patients with IBD compared to the general population. In an aging population this will lead to an increase prevalence of IBD in the elderly. Currently, there is limited data addressing disease progression and therapeutic interventions in the older IBD population. Aims The aim of this study was to describe IBD in the elderly and compare those aged with disease and those diagnosed late in life. Methods The Sydney IBD Cohort database (1942–2012) was interrogated. All patients older than 60 years at the time of last follow-up were included in the analysis. Patient demographics, disease characteristics, drug treatments and information on any surgical procedures were reviewed. Continuous variables are presented as medians and ranges. Categorical variables are presented as percentages and comparison of frequencies was made by the chi square test. Results A total of 205 elderly patients (51.2% females) were recruited with mean age of 71.2 years (IQR: 64–79). Of these, 121 (61.5%) were diagnosed before the age of 60 years. The mean length of follow-up was 16.5 years (IQR: 6–22) and there were 3,380 patient-years of follow-up. Crohn’s disease (CD) was found in 38% at diagnosis, ulcerative colitis (UC) in 54% and indeterminate colitis (IC) in 8%.

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In the diagnosis before 60 years-old group, CD-related surgery (P = 0.017) and immunomodulator use (P = 0.016) were significantly higher than the late-in life diagnosis group. No differences were found in the sex, disease location or disease behaviour at diagnosis, presence of perianal involvement at diagnosis, smoking, use of 5-ASA or use of biological agents in the two groups. In the UC group, the use of 5-ASA was significantly higher in patients diagnosed before 60 years old (P = 0.02). Immunomodulator use was higher but did not reach statistical significance (P = 0.06). No significant differences were observed in the sex, disease location at diagnosis, smoking and the use of biological agents. Conclusion In elderly IBD patients, younger age of onset was associated with increased prevalence of surgery and immunomodulator use in CD, as well as increased use of 5-ASA drugs and likely immunomodulators in UC. Disease severity in patients diagnosed after 60 is observed to be milder and less complicated compared to patients with an early age of diagnosis.

What are the implications of changing treatment delivery models for inflammatory bowel disease (IBD) sufferers? ANTONINA A MIKOCKA-WALUS,1 JANE ANDREWS,2 ROLAND VON KÄNEL,3 GABRIELE MOSER4 1 Nursing and Midwifery, University of South Australia, Adelaide, SA, Australia, 2Gastroenterology and Hepatology, Royal Adelaide Hospital, Adelaide, SA, Australia, 3Department of General Internal Medicine, Bern University Hospital, Bern, Switzerland, 4Division of Gastroenterology and Hepatology, Medical University of Vienna, Vienna, Austria Background An integrated model of care has been used effectively to manage chronic diseases; however, there is limited yet encouraging evidence on its introduction to manage inflammatory bowel disease (IBD), a chronic gastrointestinal condition. Aims To discuss the rationale for and implications of introducing an integrated model of care for IBD sufferers, with a particular focus on: psychology input, patient-centred care, efficiency as perceived by patients and doctors, financial implications and the possible means of model introduction. Methods A discussion policy paper of the integrated model of care for IBD against a background of what has been learned from an integrated model of care established in other chronic conditions. Results Although limited, the emerging data on an integrated model of care in IBD are encouraging with respect to patient outcomes and savings in healthcare costs. In other conditions, the model has been well received by both patients and practitioners, although the loss of autonomy by doctors is listed among its drawbacks. The cost-effectiveness data are now sufficiently convincing to recommend the model’s acceptance in principle. Conclusion The model should be promoted on the policy level rather than by individual practitioners to facilitate equal access for IBD suffers on a larger scale than currently.

Experience of biologic therapy during pregnancy for inflammatory bowel disease in a tertiary centre in Western Australia NIROSHAN MUWANWELLA, JEREMY S NAYAGAM, NICHOLAS M KELLY, JANINA PAVLIK, GEOFF FORBES Gastroenterology & Hepatology, Royal Perth Hospital, Perth, WA, Australia Background Infliximab (IFX) is known to cross the placenta during late second and third trimesters and is detectable in infants’ sera. There is currently limited data on the safety and efficacy of IFX therapy during pregnancy. Aims To evaluate the outcome of the use of biologic therapy in IBD during pregnancy at Royal Perth Hospital (2008–2011). Methods Retrospective analysis of hospital records was performed for details of disease activity, treatment profile, maternal and fetal complications. Results 7 patients, all with Crohn’s disease (mean age at delivery 28.4 years). Prior to pregnancy, 6 were in remission (5 on IFX, 1 on prednisolone and mesalazine). 1 new diagnosis during pregnancy. During pregnancy, of the 5 already on IFX, 2 had flares which were managed medically (reinduction and dose adjustment of IFX, and prednisolone respectively). Other 3 patients remained in remission. Two patients who were not on IFX prior to pregnancy required IFX rescue therapy during pregnancy both achieving remission. Patients received 3–6 doses of IFX during pregnancy, with 5 receiving the last dose in third trimester (32–36 weeks). Mean gestation was 37.6 weeks (32–41 weeks, national average 38.8 weeks). Delivery was uncomplicated in all but 2 patients (1 induced delivery due to poor disease control at 32 weeks, 1 emergency caesarean section due to pre-eclampsia at 37 weeks). Birthweights were 1.2–4.1 kg (mean 3.1 kg, national average 3.37 kg). The preterm infant was admitted to ICU for 2 months. There were no reported birth abnormalities. To date, all of the offspring are developing well. 2 of the 3 who had flares during pregnancy went onto have emergency colectomy for postpartum flares (7 weeks and 5 months). Conclusion In this small series no long-term adverse outcomes on maternal or fetal health were detected following IFX therapy during pregnancy. These patients present challenging clinical problems which may explain why many of our patients received IFX during the mid third trimester. References 1. Infliximab to treat Crohn’s disease: an update. Cottone M, Criscuoli V. Clin Exp Gastroenterol. 2011; 4:227–38. 2. Outcome of pregnancy in women with inflammatory bowel disease treated with antitumor necrosis factor therapy. Schnitzler F, Fidder H, Ferrante M, Ballet V, Noman M, Van Assche G, Spitz B, Hoffman I, Van Steen K, Vermeire S, Rutgeerts P. Inflamm Bowel Dis. 2011 Sep;17(9):1846–54. 3. Anti-TNFα therapy and its implication in gynecology and obstetrics. Ducarme G, Amate P, Seirafi M, Ceccaldi PF, Bouhnik Y, Luton D. J Gynecol Obstet Biol Reprod (Paris). 2011 Oct;40(6):492–7. 4. Safety of infliximab use during pregnancy. Djokanovic N, Klieger-Grossmann C, Pupco A, Koren G. Reprod Toxicol. 2011 Jul;32(1):93–7.

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Case study: secondary hamoephagocytic lymphohistiocytosis in ulcerative colitis: a rare complication in inflammatory bowel disease KATE NAPTHALI Gastroenterology, John Hunter Hospital, Newcastle East, NSW, Australia Background Secondary Haemophagocytic Lymphohistiocytosis (HLH) is a rare complication of systemic illness. We present a case of HLH in a 52 year old gentleman with poorly controlled ulcerative colitis who underwent colectomy after being treated intermittently with Azathioprine. Aims The aim of this case study is to describe a rare complication of thipurine use in inflammatory bowel disease. Methods Case notes and endoscopy images were reviewed and a literature review was performed. Results This is a case of a 57 year old gentleman who presented to a rural hospital with fulminant colitis on a background of poorly compliant thipuring use for suspected ulcerative colitis. The patient had also been recently diagnosed with adenocarcinoma of the large bowel and proceeded to total colectiomy. The post operative course was remarkable for progressive profound pancytopenina, deranged liver function tests and ultimately multiorgan failure. Upon admission to a tertiary centre intensive care unit, upper gastrointestinal bleeding was suspected and gastroscopy revealed necrotic and extensive fungal plaques adherent to the gastric mucosa. This was subsequently identified as aspecgillus fumigatus. Further investigation found this to be disseminated fungal sepsis and bone marrow biopsy confirmed the diagnosis of secondary haemophagocytic syndrome. Salvage therapy was commenced with intravenous immunoglobulin without success. Conclusion Haemophagocytic syndrome is a rare but important complication of thiopurine use and of ulcerative colitis. Given the frequency with which Ulcerative colitis is treated with azathioprine as a steroid sparing agent it is important to be aware of this complication. Treatment of HLH is difficult and chemotherapeutic options are limited to salvage chemotherapy using dexamethasone, etoposide and methotrexate. Case reports of immunomodulating treatment have been identified, including the use of intravenous immunoglobulin or plasma exchange. HLH is an important condition to be aware of when treating patients with ulcerative colitis with azathioprine, particularly in those presenting with bone marrow suppression.

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Bacterial 16S ribosomal RNA high-throughput sequencing analysis of lymph nodes and mucosa from Crohn’s disease resections CLAIRE L O’BRIEN,1 GWEN E ALLISON,2 JANE E DAHLSTROM,3 PAUL PAVLI4 1 Medical School, The Australian National University, Canberra, ACT, Australia, 2Research School of Biology, The Australian National University, Canberra, ACT, Australia, 3Anatomical Pathology, ACT Pathology, The Canberra Hospital, Canberra, ACT, Australia, 4 Gastroenterology & Hepatology Unit, The Canberra Hospital, Canberra, ACT, Australia Background Putative pathogens, shifts in microbiota composition (the dysbiosis hypothesis), excessive bacterial translocation, and reduced mucosal microbial diversity have all been implicated in Crohn’s disease (CD). Aims The aim of this study was to characterise and compare lymph node and mucosa microbial communities of CD and non-inflammatory bowel disease (IBD) patients. Methods Lymph nodes, involved and uninvolved mucosa were obtained from bowel resections of 58 patients (29 CD, 8 other-IBD, 21 non-IBD). All patients were screened for six mutations conferring CD susceptibility (the three NOD2 mutations: Arg702Trp [rs2066844], Gly908Arg [rs2066845], and Leu1007insC [rs2066847]; ATG16L1: Thr300Ala [rs2241880]; IRGM: 11386314T > C [rs13361189]; and IL23R: Arg381Gin). Universal bacterial primers targeting the V1-V3 region of the 16S rRNA gene were used to amplify all samples for high-throughput sequencing. Twenty patients (8 CD, 2 other-IBD, 10 non-IBD) had PCR-positive lymph nodes. Samples within and among patients were compared. Results Each sample was covered by an average of 8, 942 16S rRNA reads. Lymph node and mucosa samples from the same patient were highly similar. Overall, microbial diversity of CD samples was lower than non-IBD (p = 0.0062). Dysbiosis was common in all patient groups, however shifts in the microbiota were host-specific. No putative pathogen was present in 100% of CD samples, or single disease location. Campylobacter was detected in 3 CD (all with colonic disease and perianal involvement) and 1 non-IBD patient; Helicobacter in 1 CD and 1 non-IBD patient; and Yersinia in 1 other-IBD patient. Mycobacterium and Listeria were not detected in lymph nodes of any patient. Escherichia coli/Shigella were common in all patient groups. Patients with ileal CD had a significantly greater proportion of E. coli reads in their lymph nodes than other CD patients (p = 0.0475).

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Conclusion We conclude that it is unlikely a single bacterium is responsible for the onset or perpetuation of CD.

Addition of thiopurine can recapture lost response to anti-TNF monotherapy in Crohn’s disease DAVID E ONG, MARK LUST, MICHAEL A KAMM Gastroenterology, St Vincent’s hospital, Melbourne, Melbourne, VIC, Australia Background Anti-tumour necrosis factor (TNF) antibodies are effective in maintaining remission in Crohn’s disease. However a significant proportion of patients lose response to these agents with time. Established approaches to regaining response comprise anti-TNF dose increase or changing to alternative anti-TNF therapy. Given that combination thiopurine with anti-TNF therapy results in higher rates of clinical remission and mucosal healing than with anti-TNF monotherapy, we wished to examine whether the introduction of a thiopurine in patients who have lost response to anti-TNF monotherapy may result in regaining response. Aims To assess the clinical outcomes of adding thiopurine in patients who have lost response to anti-TNF monotherapy. Methods Five patients (four males, 1 female; age range 22–38 years) with active Crohn’s disease, who had an initial response to anti-TNF therapy but who had lost response, were commenced on azathioprine or mercaptopurine at standard doses while continuing anti-TNF therapy. All had previously failed thiopurine therapy. Details regarding diagnosis, treatment, symptoms, CRP, colonoscopy findings, and adverse effects were identified retrospectively via chart review. Response was defined as an improvement in clinical symptoms, reduction or normalisation of CRP, or improvement in endoscopic appearance. Results All five patients experienced improved clinical symptoms within two to six months, with benefit sustained over a mean follow-up of 18.2 months. Two patients with an elevated CRP at the time of thiopurine addition demonstrated a fall in CRP. Colonoscopy before and after thiopurine addition in four patients showed improvement in all, with mucosal healing achieved in two. No adverse effects of treatment were noted during the follow-up period. Conclusion To our knowledge this is the first demonstration that addition of a thiopurine in patients who have lost response to anti-TNF monotherapy is an effective strategy to recapture response, even if the patient has previously failed thiopurine therapy. Thiopurines may reduce immunogenicity or act synergistically with anti-TNF therapy.

The ASSURANCE program – surveillance for neoplasia in inflammatory bowel disease DAVID E ONG,2 MARK LUST,1 PAUL DESMOND,1 SIEW C NG,3 MICHAEL A KAMM1 1 Gastroenterology, St Vincent’s Hospital, Melbourne, VIC, Australia, 2Gastroenterology, National University Hospital, Singapore, Singapore, 3Gastroenterology, Chinese University of Hong Kong, Hong Kong, China Background Patients with inflammatory bowel disease (IBD) are at increased risk of developing colorectal cancer. The major Gastroenterological Societies advocate surveillance for colorectal cancer but despite the existence of accepted guidelines, adherence amongst clinicians remains poor. Aims To establish a database, algorithm and system to prospectively collect data and enable long-term management of patients with IBD enrolled in a colorectal cancer surveillance program. The system should incorporate a user friendly screening tool to allow clinicians to stratify patients according to risk of developing colorectal cancer, and to tailor surveillance accordingly.

Methods An online, cloud based data entry software system (EpiSoft), was used to create a database for clinicians to enter various demographic, clinical and endoscopic data. These data are used to estimate an individual’s risk of developing colorectal cancer. Based on published UK, US and Australian guidelines for colorectal cancer screening in IBD, a recommendation for colonoscopic surveillance interval or surgical management was developed. Results The database and algorithm has been established and a pilot project is currently underway to enter patients into the ASSURANCE program. Patients entered into the algorithm will be stratified into 6 risk categories with recommendations made for each risk category. For example, a 50 year old man with newly diagnosed extensive ulcerative colitis on a background of primary sclerosing cholangitis would be stratified as high risk and yearly colonoscopic surveillance would be recommended. How the algorithm functions will be illustrated in greater detail in the final presentation. Conclusion The ASSURANCE Program has been developed to enable clinicians to stratify patients with IBD according to their risk of developing colorectal cancer and to standardise colonoscopic surveillance. The colorectal cancer surveillance database will be maintained over time and outcomes will be measured prospectively. This is a collaborative project between St Vincent’s Hospital Melbourne, the National University Hospital of Singapore and the Chinese University of Hong Kong. We intend applying this throughout the Asia-Pacific as a collaborative project.

How good are we at screening for infections prior to anti-TNF-alpha therapy? SIM Y ONG, MICHAEL SWAN, DARCY Q HOLT, GREGORY MOORE Monash Medical Centre, Melbourne, VIC, Australia Background Anti-tumour-necrosis-factor-alpha (anti-TNF-α) is associated with increased susceptibility to infections, and reactivation of latent tuberculosis (TB) and hepatitis B. Screening in IBD patients is recommended for both vaccine preventable (Hepatitis B, varicella (VZV), measles, mumps and rubella (MMR)) and non-preventable (hepatitis C, HIV, cytomegalovirus (CMV), Epstein Barr virus (EBV)) infections however the cost effectiveness of this remains controversial, and the window for live vaccines when not immunosuppressed is small. Aims To assess the adherence to screening guidelines and clinical outcomes in patients starting anti-TNF-α therapy for IBD in a tertiary hospital. Methods A retrospective audit of all patients treated with anti-TNF therapy (adalimumab and infliximab) from 2008 to April 2012 in our centre identified 72 patients (55 with available data). Screening rates for TB (Chest X-Ray, Quantiferon gold), serology (hepatitis B surface antigen, surface antibody, core antibody, hepatitis C antibody, HIV, VZV, CMV, EBV, MMR) were analysed. Chart review was undertaken to identify any infectious complications. Results Quantiferon gold testing was performed in 94.5% of patients and 67.3% had a CXR. Quantiferon Gold was tested in 52 patients and was negative in 43 (82.7%), and indeterminate in 9 (19.1%, 7 on corticosteroids when tested, 2 data unavailable) but with low TB risks on history all were commenced on anti-TNF therapy with no TB reactivation. Hepatitis B surface antigen was assessed in 70.9% of patients (all negative), while 29.1% had surface antibody testing (10 out of 16 had no immunity) and 25.5% had core antibody testing which were all negative. Only 47.3% were tested for hepatitis C antibody. The proportion of patients tested for Hepatitis A, HIV, VZV, EBV and CMV were 9.1%, 30.9%, 32.7%, 21.8%, 23.6% and 0% respectively. One of 17 patients tested for HIV-1 was positive with known clinical risk factors and is receiving antiTNF and anti-retroviral therapy. There were no recorded infective complications.

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Conclusion Adherence to TB screening was high but poor for other infections. Most HBV screening was only for surface antigen. Screening for vaccine non-preventable infections was particularly low but without overt clinical sequelae. Larger studies are required to determine the cost effectiveness of the screening guidelines. References 1. E. Bélard et al. Prednisolone Treatment Affects the Performance of the Quantiferon Gold In-tube Test and the Tuberculin Skin Test in Patients with Autoimmune Disorders Screened for Latent Tuberculosis Infection. Inflamm Bowel Dis, Nov 2011, 17(11):2340–49. 2. W. Connell et al. Practical guidelines for treating inflammatory bowel disease safely with anti-tumour necrosis factor therapy in Australia. Int Med J, 2010, 40:139–49. 3. M. Naganuma et al. Poor Recall of Prior Exposure to Varicella Zoster, Rubella, Measles, or Mumps in Patients with IBD. Inflamm Bowel Dis, May 2012 Online publication.

Utility of thiopurine metabolite monitoring in the management of inflammatory bowel disease in an adult population: a local experience SOONG-YUAN OOI,1 MELITA ANDELKOVIC,2 JOHN EDWARDS2 1 Royal Brisbane and Women’s Hospital, Department of Gastroenterology and Hepatology, Brisbane, QLD, Australia, 2Department of Gastroenterology and Hepatology, Gold Coast Hospital, Gold Coast, QLD, Australia Background Thiopurine drugs azathioprine and 6-mercaptopurine (6-MP) are effective in treating inflammatory bowel disease (IBD). The metabolites 6-thioguanine nucleotide (6-TGN) and 6-methyl mercaptopurine (6-MMP) were shown in some studies to be associated with improved clinical response and prediction of toxicity. The role of thiopurine metabolite testing in managing patients with IBD is uncertain. Aims This study aimed to examine use of metabolite levels in asymptomatic and symptomatic patients with IBD and describe the impact upon management in a local Queensland hospital. Methods Retrospective chart review. Patients categorised into 4 groups: Routine (metabolite levels taken 1–4 months after starting thiopurines); Inadequate control (clinically or endoscopically active disease); Nonadherence (suspected failure of patient to consistently take prescribed thiopurine dose); and Adverse drug effect (symptoms suggestive of thiopurine adverse reaction or laboratory evidence of marrow toxicity or hepatotoxicity). Audit of 6-TGN and 6-MMP levels performed at a Queensland Hospital between January 2010 and September 2011 identified 82 consecutive IBD adult patients. 54 were treated with azathioprine, 28 with 6-MP. Results Routine group (n = 25): 3 patients had 6TGN levels (>800) with subsequent dose reduction; 3 shunters were identified and switched to thiopurine/allopurinol; 3 patients had subtherapeutic levels (all underdosed by weight based dosing). Inadequate control group (n = 34): 2 patients had subtherapeutic levels with both marginally under-dosed; 14 patients had supratherapeutic levels indicating thiopurine refractory disease; 5 patients were shunters with 3 changed to thiopurine/allopurinol (two attaining clinical remission). Shunters were not identifiable by TPMT, full blood count or liver enzymes. 11 patients had 6TGN levels 50%. Median time between test-retest was 4.5 (range 3.4–5.9) weeks. No predictors of a large difference between AUC on the two occasions were identified, including time between testing and patient characteristics. Conclusion Repeated breath hydrogen testing after lactulose showed a consistent reduction in total breath hydrogen produced, despite similar methodologies and no change to diet or therapy. The application beyond semi-quantitatively estimating the degree of malabsorption for subsequent sugar breath tests is limited. Reasons for the fall in hydrogen production require elucidation. Table 1. Initial Test Adequate hydrogen response, n (%) Median [IQR] H2 AUC (ppm.2 h)

Re-Test

50/50 (100%)

43/50 (86%)

2500 [1820–3610]

1222 [615–2040]

p-value 1 ULN GGT at start of HPN. 28% had ALT > 1 ULN increase and 25% had GGT > 1 ULN increase. The frequency of central access vascular device (CVAD) complications included sepsis (3% per patient year), thrombosis (1.5% per patient-year) and CVAD change (4% per patient-year). 7 (22%) died whilst on TPN, however only 1 patient (3%) died as a result of TPN itself (sepis). Conclusion HPN can be delivered effectively and safely in a tertiary referral centre. The indications are varied and dictate the patients’ outcomes. The major complications encountered include CVAD related sepsis and thrombosis, as well as liver dysfunction. Complication rates are similar to other centres around the world. The mortality of HPN is related to the underlying disease state, rather than HPN itself.

Why don’t you use our guideline? LYN GILLANDERS,1 KATERINA ANGSTMANN,2 THEODORIC WONG,3 PATRICK BALL,4 MARGIE OCALLAGHAN,6 ANDREW THOMSON5 1 Nutrition Services, Auckland City Hospital, Auckland, New Zealand, 2Royal North Shore Hospital, Sydney, NSW, Australia, 3The Royal Brisbane Children’s Hospital, Brisbane, QLD, Australia, 4Charles Sturt University, Wagga Wagga, NSW, Australia, 5The Canberra Hospital, Canberra, ACT, Australia, 6Flinders Medical Centre, Adeleide, SA, Australia Background Research based guidelines provide the best evidence and should be the cornerstone of achieving quality outcomes including improved quality of life and reduced health care costs for home parenteral nutrition (HPN) patients and their families. However evidence about the implementation of HPN guideline recommendations is rarely reported. Aims The aim of this study was to determine factors associated with adherence to the 2008 Australasian Society for Parenteral and Enteral Nutrition (AuSPEN) Guideline (1) and to inform a review of the Guideline which is due by 2013. Methods We conducted a survey of all known clinicians (doctors, nurses, dietitians, and pharmacists, n = 102) who care for HPN patients in Australia and New Zealand using an on-line survey tool. Clinicians were identified through the AuSPEN HPN Register and network of reporters. They were asked to grade familiarity, scientific basis, accessibility, applicability of guideline recommendations and barriers to usage using a five point Likert scale. Results The response rate was 62%. Two thirds of respondents agreed or somewhat agreed that scientific validity and accessibility were of high value but most identified multidisciplinary staff shortages as barriers to implementation. The ten key recommendations in the Guideline were graded useful or very useful by 88% of respondents. No relationship in responses was identified between discipline, geographical setting or experience level. Greater than 66% of respondents identified that they had 80% of their nutritional requirements whilst on PN and 20% would have benefited from an earlier transition to enteral nutrition or earlier commencement of PN. The median cost of PN was $581 per patient. Conclusion This study emphasises inconsistencies in the prescription and monitoring of PN and has demonstrated the potential impact on complication rates, nutritional adequacy and cost implications for the organisation. Recommendations include piloting a specialist multidisciplinary PN team to evaluate the efficacy, clinical and organisational outcomes of such a model to oversee best practice in PN. Funding has since been secured and a pilot is currently nearing completion. References 1. Saalwachter R, Evans H, Willcutts K, O’Donnell K. A nutrition support team led by general surgeons decreases inappropriate use of total parenteral nutrition on a surgical service. The American Surgeon 2004; 70(12); 1107–1111. 2. Kohli-Seth R, Sinha R, Wilson S, Bassily-Marcus. Adult parenteral nutrition utilization at a tertiary care hospital. Nutrition in Clinical Practice 2009; 24(6): 728–732. 3. Maurer J et al. Reducing the inappropriate use of parenteral nutrition in an acute care teaching hospital. JPEN 1996; 20(4): 272–274. 4. Piquet M, Bertrand P, Roulet M. Role of a nutrition support team in reducing the inappropriate use of parenteral nutrition. Clinical Nutrition. 2004; 23(3): 437–438. 5. Trujillo E, Young L, Chertow G, Randall S, Clemons T, Jacobs D, Robinson M. Metabolic and monetary costs of avoidable parenteral nutrition use. JPEN 1999; 23(2): 109–113. 6. Kennedy J, Nightingale J. Cost savings of an adult hospital nutrition support team. Nutrition 2005; 21(11–12): 1127–1133. 7. Naylor C, Griffiths R, Fernandez, R. Does a multidisciplinary total parenteral team improve patient outcome? A systematic review. JPEN 2004; 28(4): 251–258.

An evaluation of the safety and success of placing postpyloric feeding tubes by a dietitian in critically ill patients with gastroparesis ROSALIE YANDELL,1 STEPHANIE O’CONNOR,2 ALISON SHANKS,1 MARIANNE CHAPMAN,2 ADAM DEANE2 1 Clinical Dietetics, Royal Adelaide Hospital, Adelaide, SA, Australia, 2Critical Care Services, Royal Adelaide Hospital, Adelaide, SA, Australia Background Placement of a postpyloric feeding tube to facilitate nutrient delivery in critically ill patients with gastroparesis may be delayed due to a lack of availability of trained staff. A bedside technique for postpyloric feeding tube placement that can be taught to, and administered by, a range of care providers would potentially benefit enteral feeding outcomes for critically ill patients. Aims To determine whether placement of a postpyloric feeding tube can be safely and effectively taught to a critical care dietitian. Methods This was a prospective observational study conducted in a tertiary mixed medical-surgical, adult intensive care unit (ICU), between

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December 2008 and December 2010. Twenty attempts at postpyloric intubation by the dietitian in 18 patients with gastric stasis (defined by at least 2 gastric residual volumes > 250 ml in 24 hs; resistant to pro-motility agents) or who required a postpyloric feeding tube for research purposes. Attempts were classified as learning and consolidation stages of training. The initial 11 ‘learning’ attempts were performed by the dietitian under the direction of a physician using the technique previously described (1). To develop independence the following 9 ‘consolidation’ attempts were performed by the dietitian under the responsibility of the intensive care consultant on duty, who had varying amounts of experience with the technique. Data were collected on success of feeding tube placement (as defined by the tip of the feeding tube being visible in or distal to the duodenum on x-ray) and time (min) from commencement to completion of successful feeding tube placement. Results The dietitian was naive to this technique prior to training. In 18 patients [52 (23–70) yrs, ICU admission diagnoses included: trauma n = 4, respiratory failure n = 3, burns, pancreatitis, renal failure n = 2 each] 20 postpyloric tube placements were attempted. Most (75%) patients were sedated and mechanically ventilated. Prokinetics were used to assist the tube placement in 10% of attempts. Postpyloric catheter placement was successful in 10/20 (50%) of attempts. Whilst training the success rate was 27% (3/11) compared with 78% (7/9) once training was consolidated (P = 0.063). In the successful attempts the median time to placement was 9.63 min (3.9–27.1 min). No adverse events occurred. Conclusion A dietitian can be trained to safely place postpyloric feeding tubes in critically ill patients. Increasing the number of care providers able to place postpyloric tubes has the potential to lead to improvements in nutritional therapy in critically ill patients. Reference 1. Deane AM et al, Crit Care Resus:2009;11(3),180–3.

Effects of a D-xylose preload, with or without sitagliptin, on gastric emptying and postprandial glycaemia in patients with type 2 diabetes REBECCA B ZHAO, TONGZHI WU, KAREN L JONES, MICHAEL HOROWITZ, CHRISTOPHER RAYNER Discipline of Medicine, University of Adelaide, Hillcrest, SA, Australia Background Macronutient ‘preloads’ taken before a meal can trigger gastrointestinal hormonal feedback to slow gastric emptying (GE) and can reduce postprandial glycaemic excursions in health and type 2 diabetes, but entail additional energy intake (1,2). The combination of a preload with a dipeptidyl peptidase-4 (DPP-4) inhibitor could potentially increase its efficacy (3). Aims Therefore, we sought to determine the effects of a low energy preload, D-xylose, with or without sitagliptin, on gastric emptying and postprandial glycaemia in patients with type 2 diabetes. Methods 10 type 2 patients were studied on 4 occasions in randomised, double-blinded, order. Subjects took either 100 mg sitagliptin or placebo on the night preceding each of the studies (~2200), and drank a 200 mL ‘preload’, containing either 50 g D-xylose, or 80 mg sucralose (control of equivalent sweetness) after fasting overnight [i.e. treated by either sitagliptin + D-xlyose (SX), sitagliptin + control (SC), placebo + D-xylose (PX), or placebo + control (PC)]. 40 min after the drink, subjects ate a potato meal labelled with 100 μg 13C octanoic acid. Blood glucose and GE (breath test) were evaluated at frequent intervals. Data are mean ± SEM. Results There was a significant treatment effect on half-emptying time (T50, P < 0.0001), such that GE was slower after PX (T50 223 ± 28 min) and SX (T50 253 ± 25 min) than PC (T50 153 ± 7 min) or SC (T50 167

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± 13 min) (P < 0.01 for each), without significant difference between either PX and SX, or PC and SC. The incremental areas under the blood glucose curves were comparable between PC and PX, and between SC and SX, but less after SC and SX than PC and PX (P < 0.05 for each). Both the postprandial glycaemic peak and range (i.e. maximum minus minimum value on each day) were greatest after PC (peak 14.3 ± 0.7 mmol/L, range 8.6 ± 0.6 mmol/L), and least after SX (peak 11.9 ± 0.5 mmol/L, range 5.4

± 0.5 mmol/L) (P < 0.001 for each), without significant difference between SC (peak 12.8 ± 0.6 mmol/L, range 7.1 ± 0.6 mmol/L) and PX (12.8 ± 0.7 mmol/L, range 6.3 ± 0.6 mmol/L). Postprandial glycaemic range was associated with the rate of GE (r = −0.50, P = 0.001). Conclusion In type 2 diabetes, a preload of D-xylose generates feedback to slow GE and reduce postprandial glycaemic excursions, and the effects on glycaemia can be enhanced by addition of a DPP-4 inhibitor.

References 1. Ma J, Stevens JE, Cukier K, et al. Effects of a protein preload on gastric emptying, glycemia, and gut hormones after a carbohydrate meal in diet-controlled type 2 diabetes. Diabetes Care 2009;32(9):1600–2. 2. Gentilcore D, Chaikomin R, Jones KL, et al. Effects of fat on gastric emptying of and the glycemic, insulin, and incretin responses to

a carbohydrate meal in type 2 diabetes. J Clin Endocrinol Metab 2006;91(6):2062–7. 3. Wu T, Rayner CK, Jones K, Horowitz M. Dietary effects on incretin hormone secretion. Vitam Horm 2010;84:81–110.

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Motility/Neurogastroenterology Assessment of rectal afferent neuronal function and brain activity in patients with constipation and rectal hyposensitivity REBECCA BURGELL,1 DINA LELIC,2 EMMA V CARRINGTON,1 PETER J LUNNISS,1 SOREN S OLESEN,2 SUSAN M SURGUY,1 ASBJORN DREWES,2 MARK SCOTT1 1 GI Physiology Unit, Wingate institute, Blizard Institute, Queen Mary, University of London, London, United Kingdom, 2Mech-Sense, Department of Gastroenterology and Hepatology, Aalborg University hospital, Aalborg, Denmark Background Blunted rectal sensation (rectal hyposensitivity: RH) is present in almost one quarter of patients with chronic idiopathic constipation (Gladman et al. 2003). The mechanisms contributing to its develonutripment are not fully understood, but in a significant proportion, afferent neuronal dysfunction appears to be likely. Aims Rectal evoked potentials (EPs) and inverse modelling of cortical dipoles produced in response to electrical stimulation in the rectum were examined in patients with constipation and RH in comparison to healthy controls to determine if, in patients, alteration of the sensory pathways to the rectum exists. Methods Rectal EPs (using a 64 channel cap) were recorded in 13 patients with constipation and RH to balloon distension, and 11 healthy controls in response to electrical stimulation (50 stimuli, 0.2 Hz frequency, 0.2 ms pulse width) of the rectum at 10 cm from the anal verge using a specialized bipolar stimulating electrode. Stimuli were delivered at reported pain threshold. EP peak latencies and amplitudes were analysed, and inverse modelling was performed on the traces obtained to determine the location of cortical generators in response to rectal stimulation. Results Pain threshold was higher in patients than controls (median 59 [range 23–80] mA vs. 24 [10–55]; P = 0.007 mA). Median latency to the first negative peak was 142 (±24) ms in subjects with RH compared to 116 (±15) ms in control subjects (P = 0.004). There was no significant difference in topographic analysis of EPs between groups, and no difference in the location of cortical activity seen on inverse modelling. Conclusion This study is the first to show objective measures indicating alteration in the afferent pathways of individuals with RH and constipation. Prolonged latencies suggest a primary defect in sensory neuronal function, while cerebral processing of visceral sensory information appears normal. Reference 1. Gladman MA, Scott SM, Chan CL, Williams NS, Lunniss PJ. Rectal hyposensitivity: prevalence and clinical impact in patients with intractable constipation and faecal incontinence. Dis Colon Rectum 2003;46:238–46.

Quality of perception of the desire to defaecate and viscerosomatic referral patterns differ between patients with chronic constipation and healthy subjects REBECCA BURGELL, EMMA V CARRINGTON, SAHAR MOHAMMED, PETER J LUNNISS, MARK SCOTT GI Physiology Unit, Wingate institute, Blizard Institute, Queen Mary, University of London, London, United Kingdom Background The ability to sense stool in the anorectum is essential to normal defaecation. Constipation is known to be associated with impaired rectal sensation (predominantly hyposensitivity), and some available evidence indicates that patients with constipation report either altered, or indeed an absent call to stool. However, whether an alteration in viscerosomatic referral area or quality of sensation exists has not been robustly tested. Aims This study aims to objectively assess pre-defaecatory sensations in patients with chronic constipation (CC) in comparison to healthy, asymptomatic volunteers (HV). Methods Twenty-two patients presenting for investigation of CC and 20 HV were sent a standardized questionnaire, completed over a three day period, which assessed the quality and perception of the desire to defaecate and associated areas of viscerosomatic referral. The questionnaire included a stylized drawing, where the area of sensation experienced immediately prior to defaecation was shaded, a list of descriptive terms/word triggers for the subject to select, and space for free text to ensure all potential sensory descriptors were captured. Results All subjects completed the questionnaire. Three patients (14%) did not report any urge sensation to defaecate, and evacuated ‘out of routine’, usually with the assistance of laxatives/enemas. By contrast, all HV evacuated following the urge to defaecate. Ninety-five percent of healthy individuals and 50% of CC (p = 0.002) located the urge to defaecate as either a perianal or suprapubic sensation, most frequently described as pressure/fullness (73% vs. 80% respectively). Patients were also more likely to identify greater than one location in which sensations arose (i.e. right iliac fossa, periumbilical, left iliac fossa, left flank, epigastrium etc.) than HV (82% vs. 55%; P = 0.09). Patients also used multiple, more varied descriptive terms (e.g. heaviness, butterflies, cramping, sickness/nausea, aching, bloating) than healthy individuals (86% vs. 50%; P = 0.01). Conclusion This study confirms altered perception of the call to stool in constipated patients, indicating that aberrant afferent signalling or attenuated sensory processing may contribute to the development of their symptoms. A larger study is currently underway, examining subgroups of constipated patients stratified by rectal sensory status, which may shed light on the clinical significance of this finding.

Solid boluses increase the diagnostic yield in esophageal motility studies CARLY M BURGSTAD, ALISON K THOMPSON, RICHARD HEDDLE, CHARLES COCK, ROBERT J FRASER Investigation & Procedures Unit, Repatriation General Hospital, Adelaide, SA, Australia Background High resolution manometry (HRM) is important in the assessment of dysphagia, reflux symptoms and non-cardiac chest pain. HRM is generally performed with water boluses, although patients may comment on dysphagia with solids only.

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Aims To evaluate the diagnostic yield of including solid boluses in manometric assessment. Methods Consecutive manometric reports from the Motility Laboratory; Repatriation General Hospital were reviewed. Patients with diabetes mellitus, achalasia, cardiomyotomy or incomplete data were excluded. A detailed swallowing history was taken prior to procedure. Esophageal manometry performed using a 16-channel water-perfused catheter. Ten 5 ml water boluses in the RL and UR postures and 5 solid boluses (2 × 2 cm piece bread) in UR posture only. Swallows classified according to standard criteria for amplitude and organisation. Studies categorised ‘normal’ if ≥70% liquid and ≥60% solid swallows were normal1. Ineffective esophageal motility (IEM) and spastic esophageal motility (SEM) diagnosed if amplitude and migratory abnormalities were present in ≥50% liquid and/or ≥60% solid swallows2. Studies classified as mixed if both IEM and SEM characteristics were present. Data compared using Student’s t test. Results Data obtained for 100 patients who met the inclusion criteria (39M; 53.4 ± 1.6 yrs). Indication for assessment: dysphagia (13%), reflux symptoms (78%) and chest pain (9%). Forty-seven percent of all patients reported dysphagia during swallowing history. During manometric assessment, 47 patients reported dysphagia, 36 of whom reported difficulty with solids only (77%). Of these, approximately 1/3 had not reported a history of dysphagia. Classification of IEM, analysing liquid boluses alone, was 34%. With the addition of solids, this increased to 45% (p = 0.11). Analysis of both solid and liquid boluses altered SEM classification from 2 to 12% (p < 0.01). Furthermore, with the addition of solids, 5 IEM and 4 normal patients were subsequently changed to mixed (9%). Conversely, normal diagnosis decreased from 64 to 34% (p < 0.01). Overall, in this study, the inclusion of solids altered classification in 35% of patients. Analysis of both liquid and solid boluses altered classification of IEM (36%), SEM (83%) and mixed (100%) motility disorders. Patients with history of dysphagia were more likely to be classified with SEM than those with no history. Conclusion These data suggest inclusion of solids in esophageal manometric assessment alters diagnosis of motility disorders in 35% of patients. This was independent of a prior history of dysphagia, suggesting that the addition of solids may increase the sensitivity of diagnosis in motility disorders. Diagnosis of spastic motility disorders was particularly enhanced by the addition of solids. Further studies are needed to clarify if solid boluses should be routinely used during esophageal motility studies. References 1. Bernhard A et al. Influence of bolus consistency and position on esophageal high resolution manometry findings. Dig Dis Sci 2008; 53;1198–1205. 2. Blonski W et al. Revised criterion for diagnosis of ineffective esophageal motility is associated with more frequent dysphagia and greater bolus transit abnormalities. Am J Gastroenterol 2008; 103:699–704.

(AIM). Correlation of SRI with video fluoroscopic swallowing studies with or without laryngeal aspiration, suggest a score of more than 15 indicates aspiration. Aims We investigated the effect of ageing and bolus volume on swallowing risk index (SRI). Methods Data were collected on 15 subjects aged 20–39 (mean 27 years), 14 subjects aged 40–59 (mean 48 years), 17 subjects aged 60 to 79 (mean 67 years) and 17 subjects aged over 80 (mean 84 years). No subjects reported swallowing difficulties. Swallowing of liquid boluses (5 × 5 ml and 5 × 10 ml) was recorded by solid state manometry-impedance catheter (OD 3.2 mm, 25 pressure at 1 cm, 12 impedance at 2 cm). AIMplot software was used to derive swallow functional variables. The SRI was derived by the formula; SRI = (FI * PNadImp)/(PeakP * (TNadImp-PeakP + 1))*100. Age group differences in variables and the SRI were assessed allowing for the effects of differences in liquid bolus volume using two way repeated measures ANOVA. The effect of bolus volume was assessed using paired t-test. Results Increasing age was associated with an increase in SRI. The mean SRI remained below the cut-off value of 15 for all age groups (see graph). Increasing age was likewise associated with lengthening the pharyngeal flow interval variable, indicative of residual bolus. Other pharyngeal swallowing variables were not affected by increasing age. Larger bolus volume increased pharyngeal bolus propulsion (TNadImp-PeakP). Other swallowing function variables were not affected by increased bolus volume and there was no net effect of increasing bolus volume. Conclusion SRI increases with increasing age, without reaching the cut-off value indicative of aspiration set previously at 15. Pharyngeal AIMplot can reliably be used to determine safe swallowing in the aged group. Subtle deterioration of swallowing can be non-radiologically assessed, allowing for timely intervention before major symptoms develop.

References 1. Omari et al. Gastroenterology 2011; 140: 1454–1463. 2. Omari et al. Clinical Gastroenterology and Hepatology 2001; 9: 862–867.

Effect of ageing on measurement of swallowing risk index CHARLES COCK,1 STAMATIKI KRITAS,2 CARLY M BURGSTAD,1 ALISON K THOMPSON,1 LAURA K BESANKO,1 NATHALIE ROMMEL,3 RICHARD HEDDLE,1 ROBERT FRASER,1 TAHER OMARI2 1 Motility Laboratory, Repatriation General Hospital, Daw Park, SA, Australia, 2Gastroenterology, Women and Children’s Hospital, Adelaide, SA, Australia, 3 ExpORL, Neurosciences, Catholic University Leuven, Leuven, Belgium Background Swallowing risk index (SRI) is derived from pharyngeal swallowing variables acquired with Automated Impedance Manometry

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Colonic manometry demonstrates the mode of action of sacral nerve stimulation in patients with faecal incontinence PHIL DINNING,4 VICKI E PATTON,1 JOHN W ARKWRIGHT,2 DAVID Z LUBOWSKI3 1 Anorectal Physiology, St George Hospital, Sydney, NSW, Australia, 2Material Science & Engineering, CSIRO, Sydney, NSW, Australia, 3Colorectal Surgery, St George Hospital, Sydney, NSW, Australia, 4Human Physiology, Flinders University, Adelaide, SA, Australia Background Sacral nerve stimulation (SNS) is an effective treatment for patients with neurogenic faecal incontinence. However symptomatic improvement is not correlated with changes in sphincter function, and the mode of action by which SNS improves continence remains unknown. Aims To examine the effect of SNS on colonic propagating sequences (PS) in patients with neurogenic faecal incontinence. Methods Under general anaesthesia, in 10 patients with neurogenic faecal incontinence, a high-resolution fibre-optic manometry catheter (90 recording sites at 1 cm intervals) was positioned colonoscopically, with the tip clipped to the caecum to prevent displacement. A unipolar or quadpolar tined electrode (Medtronic, Minneapolis, USA) was implanted in 10 patients into the S3 or S4 sacral nerve foramen. In the fasted state, 2 sets of parameters were evaluated in a double-blind randomized fashion (1. 300us, 14 hz, supra-sensory; 2. Sham;). Each stimulation period lasted 2 hrs and was preceded by a 2 hr basal period. All data are expressed as mean deltas (i.e. stimulation period – basal period). Results Compared to sham stimulation, SNS had no effect upon the frequency of antegrade PSs (sham: −5.0 + 10.48 vs SNS: 1.3 + 15.97PS/2 hr; NS). The frequency of retrograde PSs was significantly increased during SNS in comparison to sham (sham 1.0 ± 9.75 vs SNS; 19.6 + 18.52 PS/2 hr; P = 0.03). Subsequent analysis of specific colonic regions revealed that this increase was due to a significant increase in retrograde PSs in the left colon (descending colon and sigmoid) (sham 0.0 ± 6.83 vs SNS; 12.6 + 9.52 PS/2 hr; p = 0.02). SNS had no effect upon the amplitude of PSs. Conclusion SNS modulates colonic motility in patients with neurogenic faecal incontinence, and increases retrograde PS in the left colon. These data suggest that a potential mode of action for SNS improving continence is through alteration of colonic motility, particularly by increasing retrograde propagating sequences.

Manometric aliasing. Have we been labelling colonic motility patterns incorrectly? PHIL DINNING,1 VICKI E PATTON,2 LUKASZ WIKLENDT,3 PETER BAMPTON,4 DAVID Z LUBOWSKI,2 JOHN W ARKWRIGHT5 1 Human Physiology, Flinders University, Adelaide, SA, Australia, 2Department of Anorectal Physiology, St. George Hospital, Sydney, NSW, Australia, 3Medicine, University of New South Wales, Sydney, NSW, Australia, 4Department of Gastroenterology and Hepatology, Flinders Medical Centre, Adelaide, SA, Australia, 5Materials Science and Engineering, CSIRO, Sydney, NSW, Australia Background Colonic manometry has defined dysmotility in patients with constipation, yet correlation with patient symptoms remains elusive; as does the identification of a specific biomarker to define constipation subtypes. Typically colonic manometry is recorded with sidehole spacing ≥7 cm, however propagated sequences (PS) in the colon can extend over 152

segments as short as 3 cm. Thus in practice, we have been measuring manometric patterns at spacing up to twice that of many of the propagated events which can lead to mislabelling of many of the events of interest. Aims To determine if accurate identification of PSs is dependent upon the spatial-resolution of recording sites. Methods A fibre-optic catheter with 90 sensor spaced at 1 cm intervals was placed colonoscopically to the caecum in 8 patients with scintigraphically confirmed slow transit constipation, 9 patients with neurogenic faecal incontinence, and 4 healthy controls. From each of these subjects a 2 hr section of colonic manometry was selected. These consisted of either the colonic response to a 1000 kCal meal (healthy controls) or the colonic response to sacral nerve stimulation (patient population). Each data set was analysed in 5 different ways by sub-sampling different sets of the full array to simulate different sensor spacing. This allowed us to analyse the data at sensor spacing of 1 cm, 3 cm, 5 cm, 7 cm, 10 cm intervals. For each trace, using our well established criteria, antegrade and retrograde PSs (APS & RPS) were identified. Data set were analysed in a blinded fashion. Results For ease of presentation averages across all groups have been presented. The findings for the complete data set also apply to each individual group. At 1 cm intervals an average of 34 ± 25 APSs and 114 ± 140 RPS were identified. The average extent of propagation was 11 ± 4 cm (APS) and 9 ± 3 cm (RPS). The number of identified APS and RPS decreased significantly (P < 0.0001) with increasing sensor spacing (Table 1). The ratio of APS to RPSs also differed significantly (P < 0.0001) as the sensor spacing increased; from pre-dominantly retrograde activity at 1 cm spacing to predominantly antegrade at 10 cm spacing. Conclusion This study indicates the potential to miss colonic PSs if inadequate sensor spacing is used. High resolution colonic manometry is helping us to correctly identify both the normal physiology and pathophysiology of colonic motility. This is turn may lead to the identification of specific manometric markers of dismotility in patient populations. Supported by NHMRC and CSIRO Australia. APS & RPS frequency at each sensor spacing

APS RPS Ratio APS:RPS

1 cm

3 cm

5 cm

7 cm

10 cm

34 (26) 114 (140) 0.5 : 1

20 (14) 68 (99) 0.9 : 1

19 (14) 23 (26) 1.6 : 1

16 (13) 15 (15) 1.4 : 1

10 (7) 4 (5) 3:1

Assessment of risk of bronchiolitis obliterans syndrome in the lung transplant population using combined oesophageal manometry and 24 h pH-impedance monitoring ANDREW FINCH, RUTH S HODGSON Gastroenterology, The Prince Charles Hospital, Brisbane, QLD, Australia Background Lung allograft has one of the shortest graft survival times compared with other solid organ transplants, mainly due to the development of bronchiolitis obliterans syndrome (BOS)1 which develops in almost half of lung transplant recipients within 5 years.2,3 No causal link has been demonstrated, but GORD has been proposed as a risk factor in the development of BOS.4 Proximal reflux in particular is closely correlated to aspiration and respiratory complications after lung transplantation2,4 and oesophageal pH-impedance has confirmed that treatment with PPIs alter the pH of gastric refluxate, but do not significantly change the number of reflux events.2 Aims We are interested in the advantage of using 24 h impedance in addition to pH measurements in the post lung transplant population. Methods We reviewed 24 lung transplant recipients for investigation of reflux disease. High resolution oesophageal manometry and 24 h

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Oesophageal pH-impedance monitoring was performed in each patient. Measurements included the total number of oesophageal refluxes, total acid reflux time (significant > 4.2%), DeMeester score and percentage of total proximal refluxes at 17 cm corrected for 24 h. Reflux events were further grouped into acid, weakly acid or non acid and patients were off PPI or motility agents for at least 3 days prior to examination where possible. Results 24 lung transplant recipients had the study at a median postoperative day of 241 ± 1331. 20 of the patients were investigated whilst off PPI and only one had had prior anti-reflux surgery. The mean LOS pressure was 15 mmHg ± 13 mmHg (normal = 15–30 mmHg). Total acid reflux time > 4.2% and significant DeMeester score was seen in 12 of 20 (60%) patients while off PPI. The mean percentage of total refluxes to the maximum proximal extent was 23% (range 0% to 91%), with only one patient experiencing no refluxes to the maxiumum proximal extent. More than half (52%) of all reflux events were weakly acid or non acid. Conclusion Most of the lung transplant recipients reviewed here demonstrated evidence of proximal reflux events that potentially put them at risk of developing BOS from silent aspiration of gastric contents. The mean LOS was at lower end of normal which may be attributed to vagal nerve injury, ischaemia, and local scarring related to surgery.2 More than 50% had weakly acid or non acid reflux which would not be expected to respond to PPI therapy and would not be detected by conventional 24 h pH investigations. This places the individual at a higher risk of silent aspiration and subsequent lung complications. Investigation in this group using combined oesophageal manometry and pH-impedance contributes significantly to the ongoing management of patients post lung transplant.

Methods Daily bowel charts are maintained by nursing staff at the facility. Residents with ≥5 days without faecal output were identified and 10 residents (75–92 years) with faecal impaction were randomly selected from the ward list. Residents unable to perform independent toileting were excluded. Subjects were given Movicol (PEG + electrolytes, Norgine) 8 sachets/day plus 15–25 Dulcolax Drops (sodium picosulphate, BohringerMannheim), for 2 consecutive days. Defecation frequency, volume & consistency, food and water intake were monitored in bowel charts. Maintenance medication (1 satchet Movicol, 5–10 drops Dulcolax) was given daily to continue producing 1 defecation/day (Bristol Stool Type 4). Patients were followed up for 5 weeks to monitor stool frequency, consistency and output volume. Results Subjects began to defecate within 10–12 hours (as charted) and emptied their impacted stool over 2 days without soiling. Patients then defecated daily without soiling on the maintenance dose of Movicol and Dulcolax. Time commitment from nursing staff was ten minutes/per day/ per subject. Patients reported feeling much better and there was enthusiastic compliance reported by all patients and staff. There was no reported re-impaction of patients on low maintenance medication. Conclusion High doses of Movicol and Dulcolax Drops are effective to disimpact elderly people with chronic constipation. Subjects avoided suppositories and soiling. Success and compliance was high with minimal staff time. This could be a useful method to manage bowel habits in agedcare facilities with improved quality of life outcomes in context of continence/medical care.

References 1. Hartwig MG, Anderson DJ, Onaitis MW, et al. Fundoplication after lung transplantation prevents the allograft dysfunction associated with reflux. Annals of Thoracic Surgery 2011;92:462–9. 2. Castor JM, Wood RK, Muir AJ, Palmer SM, Shimpi RA. Gastroesophageal reflux and altered motility in lung transplant rejection. Neurogastroenterology and Motility 2010;22:841–50. 3. Davis CS, Shankaran V, Kovacs EJ, et al. Gastroesophageal reflux disease after lung transplantation: Pathophysiology and implications for treatment. Surgery 2010;148:737–45. 4. Fisichella PM, Davis CS, Gagermeier J, et al. Laparoscopic antireflux surgery for gastroesophageal reflux disease after lung transplantation. Journal of Surgical Research 2011;170:e279-e86.

Diagnostic utility of high resolution manometry and pH study in a regional setting SAMANTHA KOSCHEL, NEIL BRIGHT UNSW Albury Rural Clinical School, Albury, NSW, Australia

High dose polyethelene glycol plus electrolytes and sodium picosulphate for disimpaction of patients in aged-care setting JULIE A JORDAN-ELY,3 BRIDGET SOUTHWELL,1 JOHN M HUTSON2 1 Surgical Research Group, Murdoch Children’s Research Institute, Parkville, VIC, Australia, 2 Paediatrics, Royal Children’s Hospital, Parkville, VIC, Australia, 3Bowel Health Clinic, Lilydale, VIC, Australia Background Chronic constipation (CC) is a common feature in aged care. Daily suppositories and high dose Lactulose are common treatments but relief is poor. Colon preps for colonoscopy use high dose polyethelene glycol (PEG) with a stimulant laxative. Current management for nurses within aged-care facilities means for the patients treatment is invasive and often compounding patients’ already dehydrated state together with higher incidence vaso-vagal episodes. Aims The aim of this study was to determine the success of colonic disimpaction using high levels of PEG+E in combination with stimulant laxatives followed by low levels for maintenance in patients with CC at a suburban aged-care facility.

Background High resolution manometry (HRM) and 24-hour pH monitoring provide a high level of diagnostic accuracy when investigating oesophageal motility. It has been available in Albury-Wodonga since mid 2009. Aims This study aimed to compare the diagnostic utility of HRM in a regional setting with that of international centres. Methods 108 referred patients underwent 111 manometries and 94 pH studies. The studies were interpreted using previously defined parameters to diagnose motility disorders, reflux and/or hiatus hernia (HH). Statistical analysis of studies with each type of oesophageal dysfunction was performed, and statistical tests were used to determine relationships between variables. Results Diagnoses of the 111 studies included 57 (60.6%) with reflux, 54 (48.6%) with HH, 16 (14.4%) with achalasia, 39 (35.1%) with spasm, and 61 (55%) with peristaltic dysfunction. Statistically significant relationships between reflux and HH, and diffuse oesophageal spasm (DOS) and peristaltic dysfunction were found. Clinically significant changes in diagnosis were reported in 34 patients (31.2%). Conclusion The results of our study were comparable to international centres. The clinically significant changes in diagnosis emphasise the clinical utility of HRM and its utility in a regional centre.

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HRM allowed identification of achalasia by its two definitive criteria: inadequate lower oesophageal sphincter relaxation (LOS), and aperistalsis. References 1. Kahrilas, P.J. and D. Sifrim. High-resolution manometry and impedance pH/manometry: valuable tools in clinical and investigational esophagology. Gastroenterology, 2008. 135: p. 756–769. 2. Ayazi, S. and P.F. Crookes. High-resolution esophageal manometry: using technical advances for clinical advantages. J Gastrointest Surg, 2010. 14(Suppl 1): p. S24–S32.

Radiation exposure during modified barium swallow studies measuring oesophageal clearance DAPHNE MOK,1 JULIA GREIG,1 ALLEGRA STOCKS,1 CHARLES COCK2 1 Speech Pathology, Repatriation General Hospital, Daw Park, SA, Australia, 2Gastroenterology, Repatriation General Hospital, Daw Park, SA, Australia

year 2010 from radiological records. These data included all studies done with (n = 207) or without (n = 22) assessment of oesophageal clearance. A comprehensive literature search was undertaken of studies assessing radiation exposure time and/or dose during modified barium swallow studies. Mean radiation exposure time and calculated radiation dose was determined for studies with or without oesophageal clearance. Mean values for radiation time and calculated exposure with and without oesophageal clearance were compared using Student’s t-test. Results Mean radiation exposure time for MBS at our institution was 202.9 sec (114 to 450 sec) for studies without assessment of oesophageal clearance vs. 201.7 sec (66 to 510 sec) for those studies including oesophageal clearance (p = 0.27). The included graphic shows a comparison of mean radiation exposure time in our institution compared to the published literature. Mean calculated dose of radiation for patients increased from 218.6 (174.7 to 262.5) mrem/cm2 for studies without oesophageal clearance to 262.4 (236.1 to 288.8) mrem/cm2 for studies including oesophageal clearance (p = 0.63). Conclusion Adding oesophageal clearance to modified barium swallow studies does not significantly increase radiation exposure time or calculated radiation dose. Actual measurement of radiation exposure during this procedure would add valuable data.

Background Modified barium swallow studies (MBS) uses video fluoroscopy, which has a low level of radiation exposure. We have added oesophageal screening studies to MBS conducted at our institution in order to assess oesophageal clearance of various consistencies. Due to concerns regarding the oncogenic potential of X-rays, we conducted an audit to compare radiation exposure for studies done at our institution to those in the published literature. Aims To determine the radiation exposure time and calculated radiation dose for modified barium swallow studies with or without oesophageal clearance. To compare radiation exposure times at our institution with those in the published literature. Methods After obtaining ethics approval, data were collected for all modified barium swallow studies done at our institution for the calendar

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Automated impedance manometry detects esophageal motor dysfunction in patients who have non-obstructive dysphagia with normal manometry NAM NGUYEN,1 RICHARD HOLLOWAY,1 ANDRE J SMOUT,3 TAHER OMARI2 1 Gastroenterology & Hepatology, Royal Adelaide Hospital, Adelaide, SA, Australia, 2Gastroenterology, Women’s & Children’s Hospital, Adelaide, SA, Australia, 3Gastroenterology, Amsterdam Medical Centre, Amsterdam, Netherlands Background Automated impedance manometry (AIM) analysis is a novel method for objective assessment of esophageal motor function by measuring time from bolus movement to peak, intrabolus and intrabolus ramp pressure. This method has previously been reported to be useful in identifying subtle pre-operative esophageal dysfunction in patients who developed post-fundoplication dysphagia (Myers et al DDW 2011). A significant proportion of patients who complained of non-obstructive dysphagia (NOD) have no abnormality found on manometric assessment. Aims To investigate the role of AIM analysis in the evaluation of patients who have NOD with normal manometry. Methods Combined manometry-impedance was prospectively performed in 42 patients (27F:15M; 56.2 ± 5.1 yrs) referred to our Unit for assessment of dysphagia without evidence of obstruction on gastroscopy (i.e. NOD). Data were compared to those from 24 healthy subjects (8F:16M; 48.2 ± 2.9 yrs). A 9 channel perfusion manometric catheter incorporating 4 impedance segments (5 cm spacing) was used. Ten × 5 ml viscous boluses were administered. Exported text files of swallows (30 sec at 30 Hz) were analysed objectively using MATLAB-based algorithms which defined the median intra-bolus pressure (IBP), the IBP slope, timing of bolus flow relative to peak pressure (PNadImp) and peak pressure (PP). Results Diagnoses based on conventional manometric assessment were: achalasia (n = 7), DOS (n = 5), NSMD (n = 19) and normal (n = 11). There was no difference in age between healthy controls and patients with NOD and normal manometry (NOD/NM: 49.8 ± 4.6 yrs). Only 2/11 (18%) patients with NOD/NM had evidence of flow abnormality on conventional impedance assessment. Several esophageal variables derived by AIM analysis were significantly different in patients as compared to healthy subjects, including higher PNadImp (7.0 ± 1.1 vs. 2.8 + 0.7 sec; P = 0.002), IBP (11.2 ± 1.8 vs. 6.1 ± 1.1 mmHg, P = 0.01) and IBP slope (10.1 ± 1.8 vs. 6.4 ± 0.9 mmHg/sec, P = 0.05) and shorter TNadImp_PP (2.8 ± 0.2 vs. 3.6 ± 0.1 sec, p = 0.01). There was no difference in PP between the NOD/ NM (70.7 ± 10.3 mmHg) and healthy (69.4 ± 8.4 mmHg) subjects. An index of abnormal esophageal function was derived from these variables ((whole IBP*distal_IBPslope)/distal TNadImp-PeakP), which was significantly higher in the NOD/NM patients compared to healthy. (48.6 ± 16.1 vs 9.5 ± 1.9; P = 0.001). Based on AIM analysis, 9/11 NOD/NM patients had abnormal esophageal function index > 10.4 (75th percentile). Conclusion Compared to conventional manometry-impedance assessment, AIM analysis is more sensitive in detecting subtle abnormalities in esophageal functional variables related to peristaltic pressure and bolus movement in patients with NOD/NM. Clinically, AIM may be useful in providing an explanation for these patients’ dysphagia and warrants further evaluation using high resolution manometry-impedance in a larger cohort.

Involvement of 5-HT3 neural pathways in oesophageal sensitivity and acid-induced viscerosomatic sensitisation MICHAL SZCZESNIAK, SERGIO E FUENTEALBA, IAN COOK Department of Gastroenterology and Hepatology, St George Hospital and University of NSW, Kogarah, NSW, Australia Background We have limited understanding of oesophageal afferent pathways and their pathophysiological role in heartburn symptoms. A high density of 5-HT3 receptors exist in the dorsal medulla, dorsal horn neurones, vagal afferent terminations and in the enteric nervous system, implying that 5-HT3 may be implicated in pain transmission. Aims The study aim was to investigate whether neural pathways involving 5-HT3 mediate oesophageal mucosal sensitivity to acid and electrical stimuli, and viserosomatic sensitization following esophageal acid exposure. Methods Effect of 5-HT3 blockade with ondansetron on sensory perception was investigated in nine healthy participants. Oesophageal sensory and pain thresholds to electrical stimulation were measured in the oesophagus, on the chest wall and on the foot, before subjects were randomized to receive either Ondansetron (8 mg i.v.) or NaCl (0.9% w/v). HCl (0.15 M, 30 min) was then infused into distal esophagus and electrical thresholds were reassessed. Following electrical sensory threshold testing, subjects received a second oesophageal infusion of HCl to evaluate specifically oesophageal sensitivity to acid. Results Ondansetron had no effect on oesophageal sensitivity to HCl or acid-induced sensitisation. However, blockade of 5-HT3 receptors did reduce somatic pain thresholds in the viscerosomatic referral area (anterior chest wall). The mean baseline somatic pain thresholds mid-sternum were 4.21 ± 1.8 mA and 5.36 ± 1.78 mA in the NaCl and Ondansetron arms respectively. Sixty minutes after esophageal acid exposure pain thresholds were significantly lower in the ondansetron arm (mean Δ−1.36 ± 0.4 mA) when compared to NaCl (mean Δ−0.14 ± 0.58 mA) (p < 0.05). Conclusion This study does not support the hypothesis that in health, 5-HT3 receptors play a significant role in oesophageal sensitivity to acid or electrical stimulation. It does provide new evidence for involvement of 5-HT3 receptors in viscerosomatic sensitisation.

Sensitivity to electrical stimulation in the viscerosomatic referral area. Reduction in chest wall pain thresholds to electrical stimulation follwing esophageal HCl exposure (* p < 0.01, # p = 0.09) (left). No effect on the pain and sensory thresholds on the dorsum of the foot (right).

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Inflammatory changes in human small intestine following ischaemia-reperfusion: potential mechanisms for dysmotility post intestinal transplantation ADAM TESTRO,1 TONY FRUGIER,2 MEHRDAD NIKFARJAM,3 GENE VENABLES,2 LOUISE PONTELL,2 ROBERT JONES,1 JOHN FURNESS2 1 Intestinal Transplantation Unit, Austin Health, Melbourne, VIC, Australia, 2Department of Anatomy and Neuroscience, The University of Melbourne, Melbourne, VIC, Australia, 3Department of Surgery, Austin Health, The University of Melbourne, Melbourne, VIC, Australia

Home-based transcutaneous electrical stimulation (TES) improved symptoms and reduced laxative use in children with slow-transit constipation (STC) YEE IAN YIK,4 KHAIRUL A ISMAIL,3 JOHN M HUTSON,2 BRIDGET SOUTHWELL1 1 Surgical Research Group, Murdoch Children’s Research Institute, Parkville, VIC, Australia, 2 Department of Urology, Royal Children’s Hospital, Parkville, VIC, Australia, 3Department of Paediatrics, University of Melbourne, Parkville, VIC, Australia, 4 Department of General Surgery, Faculty of Medicine, University of Malaya, Parkville, Malaysia

Background Intestinal transplantation is offered to patients with irreversible intestinal failure and life-threatening complications of parenteral nutrition. Ischaemia-reperfusion (I/R) injury to the intestine is universal following transplantation. Whilst I/R can occasionally result in graft failure, more commonly it results in intestinal dysmotility, often resulting in significant morbidity. The mechanism by which I/R results in dysmotility remains unclear. Aims The aim was to describe the histologic and molecular changes occurring in the human intestine following I/R injury in an attempt to elucidate mechanisms to explain post transplant dysmotility. Methods I/R was applied to a segment of jejunum (5–8 cm) in patients undergoing pancreaticoduodenectomy (Whipple’s procedure). Adjacent segments of intestine were isolated by a cutting stapler. One segment was subjected to vascular occlusion for 30 minutes, followed by a subsequent 30 minutes or 2 hours of reperfusion prior to the segment being removed. The adjacent region (control) had no vascular occlusion. Fresh tissue was processed for histology, immunohistochemistry and molecular analyses. Results 15 patients were included. 7 patient samples were exposed to 30 minutes reperfusion whilst 8 were exposed to 2 hours. With 30 minutes reperfusion histological examination revealed structural damage centred at the tips of the villi, with the surface of the epithelium separated from the underlying tissue or occasionally sloughed off. There was significant villus neutrophil and macrophage infiltration (P < 0.05) and acute inflammation present in and around the venules of the longitudinal muscle and serosal areas, but not around the submucosal vessels. At 2 hours the same histological trend was seen. The major difference was further infiltration of neutrophils and macrophages into the serosal layer and longitudinal muscle, with conspicuous neutrophil aggregates around myenteric ganglia (ganglionitis). Concomitant with histological features, mRNA levels of major inflammatory cytokines show significant increase in external muscle layers of the segments subjected to I/R including IL-1 (P < 0.0001), IL-6 (P < 0.0001) and IL-8 (P < 0.001). Similar changes were not seen in the mucosa. Conclusion This novel study clearly shows in human intestine that following I/R there is a rapid and targeted immune cell infiltration of the serosal and external muscle layers associated with significant local upregulation of inflammatory cytokines. Within 2 hours ganglionitis is present. These findings highlight the exquisite sensitivity of the intestine to relatively brief periods of I/R and the need to further characterise the extremely complex association between I/R injury, inflammation and damage to intestinal ganglia.

Background Chronic constipation (CC) occurs in 3% of children. CC can be due to retention in the anorectum, slow motility in the upper colon (slow transit) or both. Children with slow-transit constipation (STC) are often resistant to medical treatments and are sent for surgery. Rather than removing the bowel, current management is to place a stoma through the appendix and then perform antegrade enemas to wash stool out through the anus. Recently, a physiotherapy method of non-invasive electrical stimulation applied across the abdomen has been shown to speed up colonic transit (1), reduce soiling (2) and increase defecation (3). Transcutaneous electrical stimulation (TES) can be given at home using battery-operated machines. Aims This study aimed to assess symptoms (defecation, soiling and abdominal pain) and laxative use in STC children before and after homebased TES. We hypothesized TES would improve symptoms and reduce laxative use. Methods STC was diagnosed by radio-nuclear colonic transit study (4). Parents were trained to administer TES. Four electrodes (4 cm × 4 cm) were placed, 2 on the belly and 2 on the back and currents crossed. Interferential stimulation (3) (4000 Hz carrier frequency, 80–160 Hz beat frequency) was self-administered for 1 hr/day for 3–6 months. Daily continence diary and laxative use were recorded before and throughout treatment. (Ethics30116A, 30059A) Statistical analyses performed with STATA 12: paired t-test & chi-square, with p < 0.05 significant. Appendicostomy for antegrade enemas was offered if TES failed to improve symptoms. Results Sixty-two STC children (34 female; 2–16 yrs, mean 7 yrs) selfadministered home-based TES successfully. Symptoms improved significantly in 56/62 (90%): defecation frequency increased (mean ± SD, pre 1.6 ± 1.6 vs post 3.5 ± 1.9 bowel actions/wk, soiling reduced (pre 4.6 ± 2.4 vs post 0.7 ± 1.1 days soiling/wk, p < 0.001, t-test), p < 0.05) and abdominal pain reduced (pre 1.7 ± 1.9 vs post 0.2 ± 0.5 days abdominal pain/wk, p < 0.001). Laxative use significantly reduced (p < 0.001, Pearson chi-square) : 15 (24%) children stopped laxative, 30 (48%) reduced laxative, 15 (24%) remained on same laxative dose and 2 children who stopped laxative prior to TES had symptom improvement without further laxative use. Six children (10%) had no improvement and required appendicostomy for antegrade enemas. Conclusion Home-based TES is a promising non-invasive treatment for children with treatment-resistant STC. TES treatment improved symptoms and stopped laxative use in ¼ and reduced laxative use in half of these children. Long-term follow-up is required to determine the duration of symptom improvement and whether further intervention is necessary. References 1. Clarke MC, et al. J Pediatr Surg 2009;44:408–12. 2. Leong LC, et al. J Pediatr Surg 2011;46:2309–12. 3. Ismail KA, et al. J Pediatr Surg 2009;44:2388–92. 4. Sutcliffe JR, et al. Pediatr Surg Int 2009;25:465–72.

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Motility/Neurogastroenterology

Transcutaneous electrical stimulation (TES) therapy improves gastric emptying in children with slow-transit constipation YEE IAN YIK,3 DAVID J COOK,2 DUNCAN M VEYSEY,2 BS KING,2 KYLIE A MORRIS,2 CORAL F TUDBALL,2 TIMOTHY M CAIN,2 ANTHONY CATTO-SMITH,5 JOHN M HUTSON,4 BRIDGET SOUTHWELL1 1 Surgical Research Group, Murdoch Children’s Research Institute, Parkville, VIC, Australia, 2Medical Imaging, Royal Children’s Hospital, Parkville, VIC, Australia, 3Department of General Surgery, Faculty of Medicine, University of Malaya, Kualar Lumpur, Malaysia, 4Department of Paediatrics, University of Melbourne, Parkville, VIC, Australia, 5Department of Gastroenterology, Royal Children’s Hospital, Parkville, VIC, Australia Background Chronic constipation (CC) can be due to retention in the anorectum, slow motility in the upper colon (slow transit) or both. At our hospital, STC is diagnosed by radio-nuclear colonic transit study. Gastric emptying (GE) is also measured and we observed that some children with STC had delayed GE. In previous studies, we found that transcutaneous electrical stimulation (TES) speeds up colonic transit in children with slow-transit constipation (STC). Aims To assess (1) proportion of children with STC and delayed GE, (2) effect of medical therapy on GE and (3) effect of TES on GE in STC children using NTS. We hypothesized that TES may speed up gastric emptying in children with STC. Methods TES was applied at home using interferential current (4000Hz carrier frequency, 80–160 Hz beat frequency), at the level of the umbilicus using quadripolar stimulation for 1 hr/day for 3–6 mths. Our NTS database of 344 studies on STC children was reviewed. The 48-hour NTS

protocol involved Gallium-67 citrate milk drink with acquisition of images (anterior and posterior) at 0–2 hrs for gastric emptying study and at 6, 24, 30 and 48 hours for small bowel and colonic transit. Colonic transit was measured by geometric centre (% of radioactivity in each region of interest). Six regions of interest were employed (1 = pre-colonic, 2 = ascending colon, 3 = transverse colon, 4 = descending colon, 5 = recto-sigmoid colon and 6 = toilet). GE was reported as t (rate of tracer emptied to 50% of its original activity from stomach, normal 100 IU/mL for treatment-experienced) and timepoints (Weeks 4, 12 for treatment-naïve and Weeks 4, 6, 8, and 12 for treatment-experienced) that were applied in the studies to identify patients unlikely to achieve SVR. A viral dynamic model of T/PR, developed based on data from Phase 3 studies, was used to simulate achievement of SVR with different futility rules. Results At Week 4, 1.7% (14/844) treatment-naive patients, 0.7% (1/138) prior relapsers, no (0%, 0/46) prior partial responders, and 14% (10/70) prior null responders had HCV RNA levels > 1000 IU/mL; none of these patients achieved an SVR with continued PR treatment (telaprevir was discontinued per protocol). 23/25 patients reached HCV RNA nadir prior to Week 4, typically at Week 2, with a subsequent increase in HCV RNA levels by Week 4. Among 16/844 treatment naïve patients and 7/254 treatment-experienced patients with HCV RNA levels between 100– 1000 IU/mL at Week 4, 22% (5/23) achieved SVR with continued treatment. Modelling data confirmed patients with 100–1000 IU/mL at Week 4 would benefit from continued telaprevir and PR treatment but patients with greater than 1000 IU/mL would not.

Conclusion A futility rule of greater than 1000 IU/mL at Week 4 identified and predicted treatment-naïve or -experienced patients unlikely to achieve an SVR, and prevented unnecessary exposure to telaprevir and peginterferon/ribavirin in patients unlikely to benefit from further treatment. Additionally, 23/25 of these patients had reached HCV RNA nadir by Week 4 and were experiencing viral breakthrough. References 1. Jacobson IM et al. N Engl J Med. 2011;364:2405–16. 2. Sherman KE, et al. N Engl J Med. 2011;365:1014–24. [Erratum: N Engl J Med. 2011;365:1551]. 3. Zeuzem S, et al. N Engl J Med. 2011;364:2417–28.

Peginterferon Lambda-1a (Lambda) compared to peginterferon alfa-2a (alfa) in treatment-naïve patients with HCV genotypes (G) 2 or 3: first SVR24 results from emerge phase IIb JACOB GEORGE,1 JAN L HILLSON,2 BARBARA LEGGETT,3 STEFAN ZEUZEM,4 ANDREW MUIR,5 EMERGE STUDY GROUP FOR THE,1 LAURA ISHAK,2 DAVID FONTANA,2 DONG XU,6 JENNIFER LESTER,2 TODD E GRAY,2 ARANTXA HORGA,6 JUAN CARLOS LOPEZ-TALAVERA6 1 Westmead Hospital, Westmead Millennium Institute and University of Sydney, Westmead, NSW, Australia, 2 Zymogenetics, Bristol-Myers Squibb, Seattle, WA, 3 Royal Brisbane and Woman’s Hospital, Herston, QLD, Australia, 4Klinikum der Johann-Wolfgang GoetheUniversität, Frankfurt/Main, Germany, 5Duke University Medical Center, Durham, NC, 6Bristol-Myers Squibb, Wallingford, CT Background Lambda exerts antiviral effects through a unique receptor with limited distribution outside the liver and is expected to have an improved tolerability profile vs alfa. Methods 118 non-cirrhotic, treatment-naïve patients infected with HCV G2 (n = 60) or G3 (n = 58) received ribavirin (RBV) and weekly Lambda (120, 180 or 240 μg) or alfa for up to 24 weeks. HCV RNA and safety were assessed through Week 48 (SVR24). Results Baseline characteristics were similar across arms. Adverse events were mostly low grade, self-limited and unrelated to dose. SVR24 was similar in Lambda-treated patients infected with HCV G2 vs G3. Conclusion Lambda/RBV was associated with a comparable SVR24 rate in patients with HCV G2,3 with fewer musculoskeletal and flu-like symptoms, less hematologic toxicity and fewer peginterferon or RBV dose modifications vs alfa/RBV. The primary dose-limiting toxicities associated with Lambda were > 5×ULN ALT elevations, which were less common with Lambda 180 μg than alfa, and bilirubin elevations; all resolved with dose modification or discontinuation.

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Viral Hepatitis

Efficacy and safety results, n (%)

Lambda 120 μg N = 29

Lambda 180 μg N = 29

Lambda 240 μg N = 30

Lambda ALL N = 88

Alfa N = 30

HCV RNA < LODa

13 (44.8) 24 (82.8) 19 (65.5) 7/12 (58.3) 12/17 (70.6) 0 2 (6.9) 7 (24.1) 5 (17.2) 8 (27.6) 3 (10.3)

22 (75.9) 27 (93.1) 22 (75.9) 12/17 (70.6) 10/2 (83.3) 1 (3.4) 2 (6.9) 2 (6.9) 6 (20.7) 6 (20.7) 2 (6.9)

20 (66.7) 21 (70.0) 18 (60.0) 10/16 (62.5) 8/14 (57.1) 4 (13.3) 4 (13.3) 7 (23.3) 7 (23.3) 5 (16.7) 3 (10.0)

55 (62.5) 72 (81.8) 59 (67.0) 29/45 (64.4) 30/43 (69.8) 5 (5.7) 8 (9.1) 16 (18.2) 18 (20.5) 19 (21.6) 8 (9.1)

9 (30.0) 22 (73.3) 16 (53.3) 10/15 (66.7) 6/15 (40.0) 1 (3.3) 8 (26.7) 13 (43.3) 12 (40.0) 19 (63.3) 13 (44.8)d

SVR24 (W5ek 48)

{

RVR (Week 4) ETVR (Week 12) Overallb G2, n/N (%)b G3, n/N (%)b

Serious adverse events Interferon dose reductions RBV dose held/reduced Flu-like symptomsc Musculoskeletal symptomsc Hemoglobin 3.4 g/dL decrease from BL (≥Grade 2) Neutrophils < 1000/mm3 (≥Grade 2) Platelets 5×ULN (≥Grade 3) AST > 5×ULN (≥Grade 3) Direct bill rubin >1.2 mg/dL (≥Grade 3)

0 0 1 (3.4) 0 3 (10.3)

0 0 2 (6.9) 1 (3.4) 1 (3.4)

0 0 5 (16.6) 3 (10.0) 3 (10.0)

0 0 8 (9.1) 4 (4.5) 7 (8.0)

15 (51.7)d 7 (24.1)d 4 (13.8)d 1 (3.4)d 0d

a HCV RNA < limit of detection (LOD) by Roche COBAS HPS Taqmanv2.0. bP > 0.05 for all comparisons (Lambda vs. alfa). cCategories of interest: composite terms composed of pre-specified sets of MedDRA preferred terms. dN = 29 (patients with at least one post baseline laboratory evaluation).

The hepatitis B bear: novel patient orientated information enhances patient understanding of the phases of hepatitis B ASTRID-JANE GREENUP,1 USHMI CHATTERJEE,2 LOUISE E SMITH,1 MIRIAM LEVY1 1 Gastroenterology, Liverpool Hospital, Liverpool, NSW, Australia, 2Clinical School, University of NSW, Sydney, NSW, Australia Background Chronic Hepatitis B (CHB) compromises a prominent focus of outpatient hepatology, especially in the South Western Sydney Local Health District where it has a population prevalence of 1.6%. An ongoing challenge is the explanation to patients of CHB phases so that patients are engaged correctly for a life-long journey of monitoring and possible treatment. There are currently few educational tools available to assist with patient understanding of CHB and the phases. Aims 1. To develop illustrations and terminology to more effectively describe CHB phases in a way patients can understand. 2. To evaluate the effectiveness for patients and general practitioners. Methods 1. Replace immune-tolerant, immune-clearance, immune-control and immune-escape terminology with Silent, Damage, Control, Escape and Clear (for HBsAg loss) 2. Develop illustrations using a bear to demonstrate each phase: Hibernating, Attacking, In a cage, Breaking out of cage and Dead (depicted as bear head mounted on wall) 3. Survey a random selection of 70 English-speaking CHB patients, using self-response questionnaires to describe their understanding of the phases of CHB before and after viewing the bear illustrations 4. Survey 50 general practitioners, known to have patients with viral hepatitis, using self-response questionnaires to review usage of traditional CHB phases and diagram and their opinion of the bear illustrations. Results 30 patients (43%) responded. Few patients understood the terms immune tolerance (30%), immune clearance (27%), immune control (40%) or immune escape (20%). Correct identification of damaging phases and indication for treatment was poor, but increased significantly by viewing the bear illustrations. Overall, 83% of patients reported that the bear illustrations were more effective in educating them about the phases of CHB compared to the traditional diagram. Nine general practitioners responded. Few (22%) responders used the traditional phase terminology and diagram when explaining to patients. There were no responders who believed such terminology could be easily

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understood by the patient. Contrastingly, 78% favoured use of the bear brochure and suggested that the proposed terminology and images could be easily explained to patients. The bear illustrations were generally not thought to be upsetting or confusing for patients. Conclusion 1. Most patients do not understand the traditional CHB phase diagram or terminology, similarly reflected by few responding general practitioners using such material 2. Knowledge and understanding was significantly improved when patients examined the bear illustrations; a similar outcome was anticipated by the majority of the responding general practitioners 3. Most patients and general practitioners preferred the bear illustrations and terminology to the traditional phase diagram and as such these should be considered as more favourable terms and images for patient education material

IL28B genotype is not useful for predicting pegylated-interferon-α treatment outcome in Asian chronic hepatitis B cohorts JACINTA A HOLMES,1 TIN NGUYEN,1 DILIP RATNAM,2 NEEL M HEERASING,2 JANE V TEHAN,1 ROBERT CHEN,1 ANOUK DEV,2 SALLY BELL,1 STEPHEN PIANKO,2 SARA BONANZINGA,3 KUMAR VISVANATHAN,4 FERRY RUSLI,2 STEPHEN LOCARNINI,3 WILLIAM SIEVERT,2 D SCOTT BOWDEN,3 PAUL DESMOND,1 ALEX THOMPSON1 1 Gastroenterology, St Vincent’s Hospital, Melbourne, Fitzroy, VIC, Australia, 2Gastroenterology, Monash Medical Centre, Monash University, Clayton, VIC, Australia, 3Molecular Microbiology, Victorian Infectious Diseases Reference Laboratory, North Melbourne, VIC, Australia, 4Infectious Diseases, Innate Immunity, Monash Medical Centre, Monash University, Clayton, VIC, Australia Background Host IL28B genotype predicts response to peg-IFN in patients with chronic hepatitis C and was associated with HBeAg and HBsAg clearance in a European study of HBeAg-positive chronic hepatitis

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Viral Hepatitis

B (CHB) patients treated with peg-IFN. However, the utility of IL28B genotyping in Asian CHB cohorts who are treated with peg-IFN is not clear. Aims We investigated whether IL28B genotype is associated with treatment outcomes in a predominantly Asian CHB cohort treated with peg-IFN. Methods This was a retrospective analysis of consecutive CHB patients treated with 48 weeks of peg-IFN monotherapy from two large Australian centres. IL28B genotype (rs12979860) was determined retrospectively on stored sera using the TaqMan allelic discrimination kit. Baseline HBV DNA, ALT, and liver histology were available. The primary treatment outcome for HBeAg-positive patients was HBeAg seroconversion with HBV DNA

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