Eduardo Mullen

The Role of Serum Biomarkers in Predicting Fibrosis Progression in Pediatric and Adult Hepatitis C Virus Chronic Infection

Background/Aims Liver biopsy represents the gold standard for damage evaluation, but noninvasive serum markers that mirror liver fibrosis progression are actual goals both in adults and especially in children. The aim was to determine specific serum markers that correlate with liver fibrosis progression during chronic HCV infection. Methods Liver biopsies and concomitant serum samples from 22 pediatric and 22 adult HCV patients were analyzed. Histological parameters were evaluated. On serum TGF-ß1, tissue inhibitor of matrix metalloprotein inhibitor-1 (TIMP-1), hyaluronic acid (HA) and aminoterminal peptide of procollagen type III (PIIINP) were tested. Results Significant fibrosis (F≥2) and advanced fibrosis (F≥3) represented 64% and 20%, respectively in children; while 54% F≥2 and 23% F≥3 in adults. Hyaluronic acid (p = 0.011) and PIIINP (p = 0.016) were related to worse fibrosis stages only in adults, along with TIMP-1 (p = 0.039) just in children; but TGF-ß1 was associated with mild fibrosis (p = 0.022) in adults. The AUROC of TIMP-1 in children to discriminate advanced fibrosis was 0.800 (95%IC 0.598–0.932). In adults, the best AUROCs were that of HA, PIIINP and TGF-ß1 [0.929 (IC95% 0.736–0.994), 0.894 (IC95% 0.689–0.984) and 0.835 (IC95% 0.617–0.957)], respectively. In children, according to the cut off (165.7 ng/mL) value for TIMP-1, biopsies could have been avoided in 72% (18/25). Considering the cut off for HA (109.7 ng/mL), PIIINP (9.1 µg/L), and TGF-ß1 (10,848.3 pg/mL), biopsies could have been avoided in 87% (19/22) of adult patients by using HA and 73% (16/22) using PIIINP or TGF-ß1. Conclusions In adults given the diagnostic accuracy of HA, PIIINP, TGF-ß1, their combination may provide a potential useful tool to assess liver fibrosis. This first pediatric study suggests that TIMP-1 is clinically useful for predicting liver fibrosis in HCV patients.

Colorectal Cancer Staging: Reappraisal of N/PN Classification

PURPOSE: Current American Joint Committee on Cancer and the Union Internationale Contre le Cancer TNM classification disregards location of positive nodes, discontinuing N3 category, which constitutes a major modification to 1987 version. This study was designed to assess the impact of the recategorization of former N3 cases and the reliability of the current N1-N2 subcategorization of Stage III patients. METHODS: Prospectively collected data from 1,391 patients (55.8 percent males; median age, 64 (range, 21–97) years), operated on with curative intent between 1980 and 1999, were analyzed. The median follow-up was 60 (interquartile range, 27–97) months with 129 cases lost to follow-up. RESULTS: Of positive node cases, 25.3 percent were former N3. Among them, 30.5 percent migrated to the N1 group and 69.5 percent to the N2 group. The proportions of former N3 cases in N1 and N2 groups were 12.5 percent and 46.1 percent, respectively (P < 0.001). Node-positive patients had an actuarial five-year survival rate of 56.7 percent (95 percent confidence interval, 53–59), with a significant difference between N1/N2 categories (63.6 vs. 44.1 percent, respectively; P < 0.001). Although apical node involvement and more than three positive nodes were associated with poorer outcomes in univariate analysis, only the number of positive nodes had independent association (hazard ratio, 1.6 (range, 1.2–2.2); P < 0.001). Integration of former N3 cases did not modify outcomes. CONCLUSIONS: The recategorization of former N3 involved a high proportion of positive node cases. Current N1/N2 categories clearly defined different outcomes and were not modified by the integration of former N3.

Serum Apoptosis Markers Related to Liver Damage in Chronic Hepatitis C: sFas as a Marker of Advanced Fibrosis in Children and Adults While M30 of Severe Steatosis Only in Children

Background Liver biopsy represents the gold standard for evaluating damage and progression in patients with chronic hepatitis C (CHC); however, developing noninvasive tests that can predict liver injury represents a growing medical need. Considering that hepatocyte apoptosis plays a role in CHC pathogenesis; the aim of our study was to evaluate the presence of different apoptosis markers that correlate with liver injury in a cohort of pediatric and adult patients with CHC. Methods Liver biopsies and concomitant serum samples from 22 pediatric and 22 adult patients with CHC were analyzed. Histological parameters were evaluated. In serum samples soluble Fas (sFas), caspase activity and caspase-generated neoepitope of the CK-18 proteolytic fragment (M30) were measured. Results sFas was associated with fibrosis severity in pediatric (significant fibrosis p = 0.03, advanced fibrosis p = 0.01) and adult patients (advanced fibrosis p = 0.02). M30 levels were elevated in pediatric patients with severe steatosis (p = 0.01) while in adults no relation with any histological variable was observed. Caspase activity levels were higher in pediatric samples with significant fibrosis (p = 0.03) and they were associated with hepatitis severity (p = 0.04) in adult patients. The diagnostic accuracy evaluation demonstrated only a good performance for sFas to evaluate advanced fibrosis both in children (AUROC: 0.812) and adults (AUROC: 0.800) as well as for M30 to determine steatosis severity in children (AUROC: 0.833). Conclusions Serum sFas could be considered a possible marker of advanced fibrosis both in pediatric and adult patient with CHC as well as M30 might be a good predictor of steatosis severity in children.

Distinctive intrahepatic characteristics of paediatric and adult pathogenesis of chronic hepatitis C infection

Mechanisms leading to liver damage in chronic hepatitis C (CHC) are being discussed, but both the immune system and the virus are involved. The aim of this study was to evaluate intrahepatic viral infection, apoptosis and portal and periportal/interface infiltrate in paediatric and adult patients to elucidate the pathogenesis of chronic hepatitis C. HCV-infected, activated caspase-3(+) and TUNEL(+) hepatocytes, as well as total, CD4(+), CD8(+), Foxp3(+) and CD20(+) lymphocytes infiltrating portal and periportal/interface tracts were evaluated in 27 paediatric and 32 adult liver samples by immunohistochemistry or immunofluorescence. The number of infected hepatocytes was higher in paediatric than in adult samples (p 0.0078). In children, they correlated with apoptotic hepatocytes (activated caspase-3(+) r = 0.74, p < 0.0001; TUNEL(+) r = 0.606, p 0.0017). Also, infected (p = 0.026) and apoptotic hepatocytes (p = 0.03) were associated with the severity of fibrosis. In adults, activated caspase-3(+) cell count was increased in severe hepatitis (p = 0.009). Total, CD4(+), CD8(+) and Foxp3(+) lymphocyte count was higher in adult samples (p < 0.05). Paediatric CD8(+) cells correlated with infected (r = 0.495, p 0.04) and TUNEL(+) hepatocytes (r = 0.474, p = 0.047), while adult ones correlated with activated caspase-3(+) hepatocytes (r = 0.387, p 0.04). In adults, CD8(+) was associated with hepatitis severity (p < 0.0001) and correlated with inflammatory activity (CD8(+) r = 0.639, p 0.0003). HCV, apoptosis and immune response proved to be involved in CHC pathogenesis of both paediatric and adult patients. However, liver injury in paediatric CHC would be largely associated with a viral cytopathic effect mediated by apoptosis, while in adults it would be mainly associated with an exacerbated immune response.

Chronic hepatitis C liver microenvironment: role of the Th17/Treg interplay related to fibrogenesis

The role of the different lymphocyte populations in liver microenvironment of chronic hepatitis C (CHC) patients is still matter of debate. Since Th17 and Treg have opposite functions, their balance could affect disease progression. The aim was to explore liver microenvironment and its peripheral blood counterpart in adult CHC patients. CD4+ lymphocytes were predominant in the liver, with high Foxp3+ but low IL-17A+ frequency. IL-17A+ lymphocytes and IL-17A+/Foxp3+ ratio displayed association with advanced fibrosis (p = 0.0130; p = 0.0236, respectively), while Foxp3+ lymphocytes and IL-10 expression level inversely correlated with fibrosis severity (p = 0.0381, p = 0.0398, respectively). TGF-β/IL-6 ratio correlated with IL-17A+/Foxp3+ ratio (p = 0.0036, r = 0.5944) and with IL-17A+ lymphocytes (p = 0.0093; r = 0.5203). TNF-α and TGF-β were associated with hepatitis severity (p = 0.0409, p = 0.0321). Peripheral blood lymphocyte frequency was not associated with liver damage. There are functionally different immune cell populations actively involved in liver damage, but the liver cytokine milieu actually drives the pathogenesis. The intrahepatic Foxp3+ lymphocytes predominance beside the low IL-17A+ lymphocytes frequency, delineate a skewed IL-17A+/Foxp3+ balance towards Foxp3+ lymphocytes. However, the IL-17A+ lymphocytes association with advanced fibrosis denotes their role in the pathogenesis. Therefore, the interplay between Th17 and Treg conditions liver fibrogenesis.

α-lipoic acid reduces postreperfusion syndrome in human liver transplantation - a pilot study

A double‐blind randomized controlled trial was performed to compare the safety and efficacy of α‐lipoic acid (ALA) in liver transplantation (LT). The grafts were randomized to receive ALA or placebo before the cold ischemia time. Furthermore, patients transplanted with the ALA‐perfused graft received 600 mg of intravenous ALA, while patients with the nonperfused graft received the placebo just before graft reperfusion. Hepatic biopsy was performed 2 h postreperfusion. Blood samples were collected before, during and 1 and 2 days after reperfusion. Quantitative polymerase chain reaction (qPCR) analysis was performed on biopsies to assess genes involved in the response to hypoxia, apoptosis, cell growth, survival and proliferation, cytokine production and tissue damage protection. Nine of 40 patients developed postreperfusion syndrome (PRS), but seven of them belonged to the control group. There was a decrease in PHD2 and an increase in alpha subunit of hypoxia‐inducible factor‐1 (HIF‐1α) and baculoviral IAP repeat containing 2 (Birc2) transcript levels in the biopsies from the ALA‐treated versus the control group of patients. Additionally, plasma levels of alarmins were lower in ALA‐treated patients than control patients, which suggests that ALA‐treated grafts are less inflammatory than untreated grafts. These results showed that ALA is safe for use in LT, induces gene changes that protect against hypoxia and oxidative stress and reduces the appearance of PRS.

Compartmentalization of hepatitis C virus variants in patients with hepatocellular carcinoma.

Chronic Hepatitis C Virus (HCV) infection is a major risk for hepatocellular carcinoma (HCC) development. HCV Core protein has been associated with the modulation of potentially oncogenic cellular processes and E2 protein has been useful in evolutive studies to analyze the diversity of HCV. Thus, the aim of this study was to evaluate HCV compartmentalization in tumoral, non‐tumoral liver tissue and serum and to identify viral mutations potentially involved in carcinogenesis. Samples were obtained from four patients with HCC who underwent liver transplantation. Core and E2 were amplified, cloned and sequenced. Phylogenies and BaTS Test were performed to analyze viral compartmentalization and a signature sequence analysis was conducted by VESPA. The likelihood and Bayesian phylogenies showed a wide degree of compartmentalization in the different patients, ranging from total clustering to a more scattered pattern with small groups. Nevertheless, the association test showed compartmentalization for the three compartments and both viral regions tested in all the patients. Signature amino acid pattern supported the compartmentalization in three of the cases for E2 protein and in two of them for Core. Changes observed in Core included polymorphism R70Q/H previously associated with HCC. In conclusion, evidence of HCV compartmentalization in the liver of HCC patients was provided and further biological characterization of these variants may contribute to the understanding of carcinogenesis mediated by HCV infection.

S1022 Prevalence of Colorectal Premalignant Lesions Detected By Colonoscopy in Different Risk Groups for Colorectar Cancer

BACKGROUND: Colorectal cancer (CRC) is the second most frequent cancer and the second cause of mortality related to cancer in Argentina. The detection of premalignant lesions (PML) through colonoscopy has been demonstrated to be effective in the prevention of CRC. It is important to be aware of the prevalence of these lesions to estimate their impact on the prevention of CRC in our population. The aim of this study was to assess the prevalence of PML in asymptomatic subjects with different levels of risk of CRC. METHODS: A cross-sectional analysis was performed based on the colonoscopy records of asymptomatic subjects screened for CRC in a General Hospital of Buenos Aires City, Argentina, between July 2004 and March 2007. The prevalence of PML was assessed in 2 groups: 1) average risk and 2) first-degree relatives with CRC. Patients with incomplete colonoscopy, poor preparation or second-degree relatives with CRCwere excluded. PMLwas defined as polyps or flat adenomaswith low or high-grade dysplasia.Multivariate logistic analysis was performed to establish the association between individual characteristics and PML. RESULTS: Data from 1233 subjects was collected (female= 733, mean age= 67.6 years SD= ±11.2). We found a global prevalence of 24.1% for PML and 1.1% for CRC. Average risk group (n=476; female= 246; mean age=61.8 years SD= ±9,06) showed a prevalence of 22.1% for PML (CI 95%= 18.3-25.8), in which 19.2% had low-grade and 2.5% high-grade dysplasia. The prevalence of CRC was 1.05%. First-degree relatives group (n=757; female =487; mean age= 54.9 years SD= ±11.53) showed a prevalence of 25.2% (CI 95%=22.1-28.3), in which 20% had lowgrade and 4.8% high-grade dysplasia. The prevalence of CRC in this group was 1.2%. In the validation set a logistic regressionmodel showed that family history of CRCwas independently associated with increased risk of PML (odds ratio=1.54; CI 95%= 1.15-2.05; P=0.004). This difference was greater in younger subjects (table 1). Male sex was also independently associated with an increased risk of PML (odds ratio=1.50; IC 95%= 1.15-1.97; P=0.003). CONCLUSION: Approximately a quarter of this population presented at least one PML and a significantly increased risk in first-degree relatives with CRC and male sex was observed. These results may contribute to issue local guidelines for CRC prevention. Table 1. Prevalence in different age groups