Stefano Ginanni Corradini

Association between the PNPLA3 (rs738409 C>G) variant and hepatocellular carcinoma: Evidence from a meta-analysis of individual participant data.

The incidence of hepatocellular carcinoma (HCC) is increasing in Western countries. Although several clinical factors have been identified, many individuals never develop HCC, suggesting a genetic susceptibility. However, to date, only a few single‐nucleotide polymorphisms have been reproducibly shown to be linked to HCC onset. A variant (rs738409 C>G, encoding for p.I148M) in the PNPLA3 gene is associated with liver damage in chronic liver diseases. Interestingly, several studies have reported that the minor rs738409[G] allele is more represented in HCC cases in chronic hepatitis C (CHC) and alcoholic liver disease (ALD). However, a significant association with HCC related to CHC has not been consistently observed, and the strength of the association between rs738409 and HCC remains unclear. We performed a meta‐analysis of individual participant data including 2,503 European patients with cirrhosis to assess the association between rs738409 and HCC, particularly in ALD and CHC. We found that rs738409 was strongly associated with overall HCC (odds ratio [OR] per G allele, additive model = 1.77; 95% confidence interval [CI]: 1.42‐2.19; P = 2.78 × 10−7). This association was more pronounced in ALD (OR = 2.20; 95% CI: 1.80‐2.67; P = 4.71 × 10−15) than in CHC patients (OR = 1.55; 95% CI: 1.03‐2.34; P = 3.52 × 10−2). After adjustment for age, sex, and body mass index, the variant remained strongly associated with HCC. Conclusion: Overall, these results suggest that rs738409 exerts a marked influence on hepatocarcinogenesis in patients with cirrhosis of European descent and provide a strong argument for performing further mechanistic studies to better understand the role of PNPLA3 in HCC development. (Hepatology 2014;59:2170–2177)

Hepatocellular Carcinoma in Patients with Cirrhosis: Qualitative Comparison of Gadobenate Dimeglumine–enhanced MR Imaging and Multiphasic 64-Section CT

PURPOSE To prospectively investigate whether combined interpretation of dynamic and hepatobiliary phase magnetic resonance (MR) images can improve the accuracy of gadobenate dimeglumine-enhanced MR imaging in the detection of hepatocellular carcinoma (HCC) compared with either dynamic MR or multiphasic multidetector computed tomographic (CT) images alone. MATERIALS AND METHODS Institutional review board approval and informed patient consent were obtained. Fifty-two patients (39 men, 13 women; mean age, 68 years; range, 38-81 years) suspected of having HCC underwent gadobenate dimeglumine-enhanced MR imaging and multiphasic 64-section multidetector CT. Images were qualitatively analyzed independently by three observers in three separate reading sessions. The alternating free-response receiver operating characteristic (AFROC) method was used to analyze the results. Differences in sensitivity and positive predictive values were calculated at a statistical significance of P < .05. RESULTS A total of 67 HCCs were detected in 36 patients. The mean area under the AFROC curve (A(z)) was significantly higher for either the combined interpretation of dynamic and hepatobiliary phase MR images (A(z) = 0.95) or dynamic MR images alone (0.91) than for CT images (0.77) (P = .01 for both comparisons). The mean sensitivity of combined interpretation of MR images (0.72) was significantly higher than those of dynamic MR images alone (0.63) and multidetector CT images (0.61) (P = .008 and .001, respectively). The mean positive predictive value was not significantly different among the three imaging sets. CONCLUSION The combined interpretation of dynamic and hepatobiliary phase MR images improves diagnostic accuracy of gadobenate dimeglumine-enhanced MR imaging for the detection of HCC compared with either dynamic MR or multiphasic multidetector CT images alone.

PNPLA3 I148M (rs738409) genetic variant is associated with hepatocellular carcinoma in obese individuals

BACKGROUND Obesity is a risk factor for cancer, including hepatocellular carcinoma. Patatin-like phospholipase domain-containing 3 (PNPLA3) I148M (rs738409) genetic variant has been associated with hepatocellular carcinoma (HCC) in individuals with chronic alcohol abuse or hepatic viral infection. In the present study we examined the association between the PNPLA3 I148M genetic variant and hepatocellular carcinoma in obese individuals from the Swedish Obese Subjects cohort (n = 4047). METHODS We performed a matched, prospective, controlled, interventional trial, investigating the effect of bariatric surgery (surgery group) compared to conventional treatment (control group) for obesity. RESULTS A total of 9 events were observed in the 15-year median follow up (5 in the control group and 4 in the surgery group). A significantly higher incidence of hepatocellular carcinoma in PNPLA3 148M allele carriers was found in obese individuals in the control group (log-rank P-value = 0.001), but not in the surgery group (log-rank P-value = 0.783). Consistently, an increased risk (for each PNPLA3 148M allele, hazard ratio: 5.9; 95% confidence interval 1.5-23.8; P-value = 0.013) of developing hepatocellular carcinoma was observed only in the control group. CONCLUSION The current study is the first prospective report showing the association of the PNPLA3 I148M genetic variant and hepatocellular carcinoma in severely obese individuals.

Serum and Biliary Insulin-like Growth Factor I and Vascular Endothelial Growth Factor in Determining the Cause of Obstructive Cholestasis

Context Extrahepatic cholangiocarcinoma is difficult to diagnose. Contribution The researchers measured serum and biliary fluid levels of insulin-like growth factor I (IGF-I) and vascular endothelial growth factor (VEGF) in 73 patients referred for endoscopic retrograde cholangiopancreatography. Biliary IGF-I levels were 15- to 20-fold greater in patients with extrahepatic cholangiocarcinoma than in patients with pancreatic cancer or benign biliary diseases. Serum IGF-I levels and biliary VEGF levels did not distinguish accurately among extrahepatic cholangiocarcinoma, benign pancreatic disease, and pancreatic cancer. Caution Most patients did not have histologic confirmation of diagnoses. Implication Measurement of biliary IGF-I levels may help to differentiate patients with cholangiocarcinoma, pancreatic cancer, and benign biliary disease. The Editors Cholangiocarcinoma is a malignant tumor whose incidence and mortality are progressively increasing worldwide; its prognosis and response to current therapies are poor (18). Cholangiocarcinoma arises from a malignant transformation of the epithelial cells (cholangiocytes) lining the biliary tree and is usually diagnosed at an advanced stage, when fewer than 50% of patients are eligible for surgery (58). Markers for early diagnosis are lacking (58). The most frequently tested marker is serum Ca19-9, but its performance for early diagnosis of cholangiocarcinoma is considered unsatisfactory (59). The clinical challenge mostly concerns the extrahepatic form of cholangiocarcinoma, in which differential diagnosis compared with other causes of biliary obstruction is still based on several clinical, radiologic, and endoscopic approaches (58). Tissue-proven diagnosis of extrahepatic cholangiocarcinoma is daunting because a combination of brush cytologic examination and endoscopic biopsy yields definitive positive findings in only about one third of cases (58). Different approaches aimed at improving the diagnostic performance of current techniques are being investigated (1013). Along with others, we have shown in human and experimental studies that expression of insulin-like growth factor I (IGF-I) and vascular endothelial growth factor (VEGF) is marked in human cholangiocarcinoma (1416). All biopsy samples in patients with intrahepatic cholangiocarcinoma showed intense immunohistochemical positivity for IGF-I and its receptors, whereas cholangiocytes from normal livers were negative (14). In experimental studies, we showed that cholangiocarcinoma cell lines express and secrete IGF-I and VEGF, which play a major role in promoting growth and proliferation of neoplastic cells through autocrine mechanisms (14, 15). In other types of cancer, the neoplastic cells secrete IGF-I and VEGF (1721), and high serum levels of IGF-I are associated with an increased risk for prostate, breast, pancreatic, lung, and colorectal cancer, with prognostic significance in some cases (2225). We measured IGF-I and VEGF in bile and serum of patients undergoing endoscopic retrograde cholangiopancreatography (ERCP) for biliary obstruction and evaluated the performance of these substances as diagnostic markers in differentiating extrahepatic cholangiocarcinoma from pancreatic cancer or benign biliary abnormalities. Methods Patients We studied 73 patients (Table) with biliary obstruction, as demonstrated by abdominal ultrasonography (bile duct enlargement), who were consecutively referred to our division of gastroenterology (at a tertiary center for cholestasis) for ERCP from January 2004 to July 2005. All patients had abdominal magnetic resonance cholangiography and different imaging studies thereafter (Table), depending on the presumptive diagnosis. On the basis of this clinical work-up, ERCP was considered diagnostic in patients in whom the work-up did not provide a conclusive diagnosis or therapy (such as stenting, stricture balloon dilation, or gallstone removal) in the remaining patients. Endoscopic retrograde cholangiopancreatography was performed in all patients within 1 week after the ultrasonographic demonstration of biliary obstruction. We included patients who were admitted for biliary obstruction and referred for ERCP. Exclusion criteria were unsuccessful intubation of the common bile duct; combined laparoscopic cholecystectomy and ERCP; bile duct leak; biliary stenosis in patients who received a liver transplant; very rare diseases of the biliary tree (for example, Caroli disease); and lack of informed consent to participate in the study. The endoscopist who performed ERCP was aware of the definitive, presumptive, or undefined diagnosis. Table. Patient Characteristics Native bile fluid was aspirated immediately after endoscopic retrograde selective intubation of the common bile duct. Blood samples were obtained just before ERCP. Patients gave signed informed consent, and our institutional review board approved the study protocol. Follow-up Patients with benign biliary abnormalities were seen every 6 months or sooner if they developed disease symptoms or if indicated by their own physicians. The follow-up visit included upper abdominal ultrasonography and blood tests for liver enzymes; serum pancreatic enzymes; and, for patients with primary sclerosing cholangitis, serum carcinoembryonic antigen and Ca19-9. Patients with extrahepatic cholangiocarcinoma and pancreatic cancer were followed if they were inpatients in our or another division of the same hospital (admitted for surgery or complications related to disease progression), or follow-up was based on consulting medical records if they were dismissed from our division to other hospitals. Patients undergoing radical surgery were seen every 3 months or sooner if they developed disease symptoms or if indicated by their own physicians. The follow-up visit included upper abdominal ultrasonography; tests for liver enzymes, serum pancreatic enzymes, and different serum oncomarkers; and total-body computed tomography every 6 months. Further information was collected by telephone. Bile and Serum Analysis Bile and blood samples were collected in glass tubes. Bile samples were immediately stored in small aliquots at 20C. Blood samples were centrifuged, and the serum was immediately stored in small aliquots at 20C. Measurements were performed in groups of 30 samples by laboratory personnel who were blinded to the final diagnosis. Bile and serum samples were identified by a numeric code, which was deciphered only at the end of the study when all measurements were performed. Statistical analysis and comparisons were performed only at the end of the study. Insulin-like growth factor I in bile and serum was measured by using a commercial enzyme-linked immunosorbent assay kit consisting of extraction and separation of IGF-I from binding proteins followed by 1-step sandwich-type immunoassay (DSL-10-5600, Diagnostic System Laboratories, Webster, Texas). Vascular endothelial growth factor in bile and serum was measured by using a commercial VEGF enzyme-linked immunosorbent assay kit (VEGF165 and VEGF121, Pierce Biotechnology, Rockford, Illinois). Measurements of IGF-I and VEGF in all bile samples were repeated twice, at different times but within 3 months of one another and under the same conditions (coefficient of variation for repeated measures [26], 3.49% for IGF-I and 3.74% for VEGF). Total salts in bile were measured by using the 3-hydroxysteroid dehydrogenase method (27). Total and fractionated serum bilirubin and serum alkaline phosphatase were measured by using established routine methods. Statistical Analysis We used SPSS software, version 13.0.1 (SPSS, Chicago, Illinois), for all statistical analyses. Nonparametric statistical tests were used throughout. The KruskalWallis analyses of variance was used for between-group comparisons. A P value of 0.05 or less was considered statistically significant. Receiver-operator characteristic analysis of biliary IGF-I and serum VEGF levels was used to evaluate the performance (28) of these values in differentiating extrahepatic cholangiocarcinoma from benign abnormalities of the biliary tract or pancreatic cancer. The area under the receiver-operating characteristic curve was obtained by using a nonparametric method (28). Role of the Funding Source This study was funded exclusively by institutional grants (Italian Ministry of Education, University and Research). The funding source had no role in the design, conduct, and reporting of the study. Results The Table shows patient characteristics. Patients with benign biliary abnormalities were, on average, 12 to 13 years younger (Table) than the patients in the other 2 groups. Patients with extrahepatic cholangiocarcinoma had greater cholestasis than the other 2 groups. Total and direct (conjugated) serum bilirubin and serum alkaline phosphatase levels were statistically significantly higher (Table) in patients with cholangiocarcinoma than in patients with pancreatic cancer or benign biliary abnormalities. Accordingly, total biliary bile salt concentration was statistically significantly lower (Table) in the cholangiocarcinoma group than in the other 2 groups. In patients with cholangiocarcinoma (n= 29), according to the current classification (5), the tumor was located at the bifurcation (perihilar [Klatskin] tumor) in 18 cases, in the middle common bile duct in 3 cases, and at the distal common bile duct in 8 cases. The diagnosis of cholangiocarcinoma was based on histologic examination, cytologic examination, or both during ERCP in 5 of 8 cases of distal cholangiocarcinoma (histologic confirmation at surgery for the 3 remaining cases) and in 1 case of middle cholangiocarcinoma; for most patients, however, the results of radiologic documentation of ERCP and conventional radiology (abdominal spiral computed tomography, magnetic resonance imaging, and magnetic resonance cholangiopancreatography) were considered together. Four of 8 patients with distal cholangi

Impaired human gallbladder lipid absorption in cholesterol gallstone disease and its effect on cholesterol solubility in bile

BACKGROUND & AIMS The role of the gallbladder in gallstone pathogenesis is still unclear. We examined the effects of gallbladder mucosal lipid absorption on lipid composition and cholesterol crystallization in bile. METHODS The in vitro-isolated, intra-arterially perfused gallbladder model was used (1) to compare the absorption rates of lipids from standard bile by gallbladders obtained from 7 patients with cholesterol gallstones and 6 controls; and (2) to measure the microscopic cholesterol crystal detection time in cholesterol-enriched pig bile before and after lipid absorption by the pig gallbladder. RESULTS Control gallbladders, but not cholesterol gallstone gallbladders, significantly reduced cholesterol (P < 0.02) and phospholipid (P < 0.01) and increased bile salt (P < 0.01) molar percentages in bile over a 5-hour period by efficient and selective cholesterol and phospholipid absorption. A histomorphometric study of the epithelial cells showed significantly higher values for nuclear density (P < 0.01) and nuclear (P < 0.05) and cytoplasmic (P < 0.05) areas in the cholesterol gallstone than the control group. Sequential microscopy of cholesterol-enriched pig bile showed significantly shorter cholesterol filament (P < 0.01) and typical cholesterol plate (P < 0. 02) detection times before than after exposure of bile to the gallbladder lipid absorption. CONCLUSIONS In cholesterol gallstone disease, the human gallbladder epithelium loses its capacity to selectively and efficiently absorb cholesterol and phospholipids from bile, even if it is hyperplastic and hypertrophic. This epithelial dysfunction eliminates the positive effect that the normal gallbladder exerts on cholesterol solubility in bile and might be a pathogenetic cofactor for cholesterol gallstone formation.

Comparison of Changes in Lipid Profile after Bilio-intestinal Bypass and Gastric Banding in Patients with Morbid Obesity

Background: The presence of hypercholesterolemia is currently not considered a selection criteria for performing gastric restrictive or diversionary bariatric surgery. Methods: We prospectively investigated the effects of the bilio-intestinal bypass (BI-bypass) with a wide cholecysto-jejunal anastomosis and of adjustable gastric banding (AGB) on blood lipid concentrations in obese patients. To clarify the mechanism of the hypocholesterolemic effect of the BI-bypass, daily fecal sterol excretion was measured by gas-liquid chromatography (GLC). Results: At 1 year after BI-bypass compared to baseline, the hypercholesterolemic (n=18) and the normocholesterolemic (n=19) patients significantly reduced total (−38% and −27%, respectively), LDL (−47% and −24%, respectively) and HDL (−11% and −13%, respectively) cholesterol and total / HDL cholesterol ratio (−25% and −13%, respectively). At 1 year after AGB, the total / HDL cholesterol ratio was significantly decreased (−11%) compared to baseline in hypercholesterolemic (n=12) but not in normocholesterolemic (n=6) patients, while total and LDL cholesterol were not affected in both groups. At 3 years after BI-bypass compared to baseline, the hypercholesterolemic (n=9) and the normocholesterolemic (n=11) patients significantly reduced total (−43% and −28%, respectively) and LDL (−53% and −29%, respectively) cholesterol and total / HDL cholesterol ratio (−38% and −21%, respectively). The BI-bypass induced a significant (P <0.005; n=7) 6-fold increase in mean fecal cholesterol output. Conclusions: The BI-bypass but not the AGB leads to a persistent and marked beneficial effect on blood LDL cholesterol associated with an increased cholesterol fecal output. BI-bypass but not AGB is indicated in morbidly obese patients with hypercholesterolemia.

Glial fibrillary acidic protein as an early marker of hepatic stellate cell activation in chronic and posttransplant recurrent hepatitis C

Activated alpha‐smooth muscle actin (α‐SMA)–positive hepatic stellate cells (HSCs) are pericytes responsible for fibrosis in chronic liver injury. The glial fibrillary acidic protein (GFAP), commonly expressed by astrocytes in the central nervous system, is expressed in vivo in the liver in a subpopulation of quiescent stellate cells. In the rat, increased GFAP expression in the acute response to injury and down‐regulation in the chronic response have been observed, whereas reports concerning GFAP expression in human liver are still conflicting. We investigated the utility of GFAP compared to α‐SMA as an immunohistochemical marker of early activated HSCs in chronic and posttransplant recurrent hepatitis C and correlated GFAP expression with vascular remodeling and fibrosis progression. With immunohistochemistry and a semiquantitative scoring system, the expression of GFAP and α‐SMA in HSCs and the microvessel density were analyzed in biopsies from normal livers obtained from cadaveric donors [donor liver (DL); n = 21] and from livers from posttransplant hepatitis C virus recurrent hepatitis (HCV‐PTR) patients (n = 19), hepatitis C virus chronic hepatitis (HCV‐CH) patients, (n = 12), and hepatitis C virus cirrhosis (HCV‐C) patients (n = 16). The percentage of α‐SMA–positive HSCs was significantly higher in the HCV‐PTR, HCV‐CH, and HCV‐C groups compared to the DL group (P < 0.01). The percentage of GFAP‐positive HSCs was significantly higher in the HCV‐PTR group compared to the DL, HCV‐C (P < 0.01), and HCV‐CH (P < 0.05) groups and in the HCV‐CH group compared to the DL group (P < 0.01), inversely correlating with the extent of fibrosis and microvessel density (P < 0.01). In the HCV‐PTR group, the percentage of GFAP‐positive HSCs correlated with fibrosis progression (P < 0.01). In conclusion, GFAP could represent a useful marker of early activation of HSCs in HCV‐CH and seems to predict fibrosis progression in HCV‐PTR. Liver Transpl 14:806–814, 2008.

PNPLA3 Gene Polymorphism Is Associated With Predisposition to and Severity of Alcoholic Liver Disease

OBJECTIVES:The genetic polymorphism with an isoleucine-to-methionine substitution at position 148 (rs738409 C>G) in the patatin-like phospholipase domain protein 3 (PNPLA3) gene confers risk of steatosis. PNPLA3 polymorphism is shown to be associated with alcoholic liver disease (ALD). We performed a systematic review and meta-analysis to examine association of this genetic polymorphism with ALD spectrum and its severity.METHODS:Medline, Embase, and Cochrane Library were searched for studies on association of PNPLA3 polymorphism and ALD spectrum: alcoholic fatty liver (AFL), alcoholic liver injury (ALI), alcoholic cirrhosis (AC), and hepatocellular carcinoma (HCC). Pooled data are reported as odds ratio (OR) with 95% confidence interval. Heterogeneity was assessed using the I2 statistics and publication bias using Egger’s test and Begg and Mazumdar’s test. Individual participant data obtained from five studies were used for subgroup analyses.RESULTS:Among 10 studies included in this pooled analysis, compared with controls, OR for rs738409 CG and GG among ALI patients was 1.45 (1.24–1.69) and 2.22 (1.50–3.28), respectively, compared with CC. Respective OR among AC patients was 2.09 (1.79–2.44) and 3.37 (2.49–4.58) and among AC patients with HCC was 2.87 (1.61–5.10) and 12.41 (6.99–22.03). Data for AFL were inconsistent. Among ALD patients, OR of CG and GG genotypes was 2.62 (1.73–3.97) and 8.45 (2.52–28.37), respectively, for AC compared with fatty liver (FL) patients. Similar OR for AC compared with ALI was 1.98 (1.24–3.17) and 3.86 (1.18–12.60). The OR for CG and GG genotypes among AC patients for HCC occurrence was 1.43 (0.76–2.72) and 2.81 (1.57–5.01), respectively. Individual participant data analysis showed age to predispose to AC among ALI patients.CONCLUSIONS:PNPLA3 genetic polymorphism (rs738409 C>G) is associated with increased risk for the entire spectrum of ALD among drinkers including ALI, AC, and HCC. Studies are needed to clarify association of PNPLA3 polymorphism and steatosis in alcoholics. PNPLA3 gene may potentially be a therapeutic target in ALD.

PNPLA3 I148M variant and hepatocellular carcinoma: A common genetic variant for a rare disease

Hepatocellular carcinoma (HCC) is highly associated with chronic liver disease. The rs738409 genetic variant in the patatin-like phospholipase domain-containing 3 (PNPLA3, adiponutrin) gene has been implicated as a genetic determinant of the entire spectrum of liver diseases, ranging from steatosis, chronic hepatitis, cirrhosis and ultimately to HCC. In this review, first we will examine the current genetic theories of disease susceptibility. Next, we will analyze the evidences for the association between PNPLA3 I148M variant and HCC. Moreover, we will exploit this association to propose a new paradigm in human genetics: a common genetic variant contributing to a rare disease. Finally, we will examine the molecular genetics of PNPLA3 and, specifically, the theories that have been proposed to explain the function of PNPLA3 in health and disease.

Bone Disorders in Patients With Chronic Liver Disease Awaiting Liver Transplantation

BACKGROUND An important complication of chronic liver disease is osteodystrophy, which includes osteoporosis and the much rarer osteomalacia. Both conditions are associated with significant morbidity through fractures resulting in pain, deformity, and immobility. Liver transplantation may further deteriorate bone metabolism. The aim of the present study was to investigate the frequency and severity of hepatic osteodystrophy among patients with liver cirrhosis who were referred for liver transplantation. We also evaluated modifications in bone metabolism after liver transplantation. MATERIALS AND METHODS We recruited 35 consecutive patients with chronic liver disease who were undergoing assessment for transplantation over a 1-year period. Bone mass in the total skeleton and proximal hip was evaluated using a dual-energy X-ray absorptiometry device (Lunar Prodigy Advance, GE Healthcare, USA). According to World Health Organization recommendations, osteoporosis was defined as a T score < -2.5 and osteopenia as T score between -1 and -2.5. RESULTS We enrolled in the study 35 patients, including 8 females and 27 males of overall mean age of 57 +/- 7, who showed a viral etiology (57%) or alcohol etiology (28%), Child-Pugh 8.7 +/- 2.3. The overall prevalence of osteodystrophy was 40% (26% osteopenia and 14% osteoporosis). No difference was evident according to gender, severity of liver disease (Child-Pugh, Model for End-stage Liver Disease), or origin of liver disease. A subgroup of 10 transplanted patients reached 3-month follow-up, showing total body T score with a significant decrease after 3 months while femoral T scores tended to decrease insignificantly. CONCLUSIONS This study revealed a high prevalence of low bone mineral density among cirrhotic patients before liver transplantation. We suggest that both bone mineral density and biochemical examinations should be considered to be routine tests to identify the status of bone mass and bone metabolism among recipients prior to liver transplantation.