Raffaela Vecchione

Liver iron excess in patients with hepatocellular carcinoma developed on non-alcoholic steato-hepatitis

BACKGROUND/AIMS Liver iron deposits are frequent in patients with non-alcoholic steato-hepatitis (NAFLD), but their role is not well defined. To investigate the effect of liver iron excess on the prevalence of hepatocellular carcinoma (HCC) in patients with NASH-related cirrhosis. METHODS Hepatic iron was measured retrospectively with a semiquantitative method in liver biopsies of 153 patients with NASH-related cirrhosis: 51 with HCC and 102 controls without HCC, matched for age, sex and stage of liver disease. The corrected total iron score (0-60) was the sum of three scores: the hepatocytic iron score (0-36), sinusoidal iron score (0-12), and portal iron score (0-12), multiplied by 3/3, 2/3, or 1/3 depending on the localisation of the iron in the nodules. RESULTS Conditional logistic regression analysis showed that iron deposits (corrected total iron score>0) were more frequent in HCC patients than in controls. The median corrected total iron score was significantly higher in HCC patients than in controls. The liver iron overload was sinusoidal. CONCLUSIONS Iron deposition in the liver was more frequent in patients with NASH-related cirrhosis with HCC than in HCC-free controls. Liver iron overload may be associated with development of HCC in patients with NASH-related cirrhosis.

Silybin combined with phosphatidylcholine and vitamin E in patients with nonalcoholic fatty liver disease: A randomized controlled trial

The only currently recommended treatment for nonalcoholic fatty liver disease (NAFLD) is lifestyle modification. Preliminary studies of silybin showed beneficial effects on liver function. Realsil (RA) comprises the silybin phytosome complex (silybin plus phosphatidylcholine) coformulated with vitamin E. We report on a multicenter, phase III, double-blind clinical trial to assess RA in patients with histologically documented NAFLD. Patients were randomized 1:1 to RA or placebo (P) orally twice daily for 12 months. Prespecified primary outcomes were improvement over time in clinical condition, normalization of liver enzyme plasma levels, and improvement of ultrasonographic liver steatosis, homeostatic model assessment (HOMA), and quality of life. Secondary outcomes were improvement in liver histologic score and/or decrease in NAFLD score without worsening of fibrosis and plasma changes in cytokines, ferritin, and liver fibrosis markers. We treated 179 patients with NAFLD; 36 were also HCV positive. Forty-one patients were prematurely withdrawn and 138 patients analyzed per protocol (69 per group). Baseline patient characteristics were generally well balanced between groups, except for steatosis, portal infiltration, and fibrosis. Adverse events (AEs) were generally transient and included diarrhea, dysgeusia, and pruritus; no serious AEs were recorded. Patients receiving RA but not P showed significant improvements in liver enzyme plasma levels, HOMA, and liver histology. Body mass index normalized in 15% of RA patients (2.1% with P). HCV-positive patients in the RA but not the P group showed improvements in fibrogenesis markers. This is the first study to systematically assess silybin in NAFLD patients. Treatment with RA but not P for 12 months was associated with improvement in liver enzymes, insulin resistance, and liver histology, without increases in body weight. These findings warrant further investigation.

Hepatoid adenocarcinoma with liver metastasis mimicking hepatocellular carcinoma: an immunohistochemical and molecular study of eight cases.

Hepatoid adenocarcinoma (HAC) is a special type of extrahepatic adenocarcinoma, which has a striking morphologic similarity to hepatocellular carcinoma. Seven HACs arising in the stomach and one in the lung, all with liver metastasis, were studied. They shared clinical features, such as old age, high serum alpha-fetoprotein level, aggressive behavior, and hepatic tumor in absence of risk factors for hepatocellular carcinoma (HCC). Morphologically, tumors were characterized by an admixture of tubulo-and/or papillary adenocarcinoma with hepatoid foci. In six cases, liver metastases showed an exclusive hepatoid differentiation, virtually indistinguishable from HCC with solid growth pattern. As HAC and HCC cannot be differentiated on the basis of morphology alone, differences in immunohistochemical reaction patterns would be of considerable diagnostic help. Immunostaining for CK7, CK8, CK18, CK19, CK20, alpha-fetoprotein, p-CEA, and HepPar1 revealed that hepatoid areas of both primary and metastatic HAC have a specific immunoprofile, distinctive of this entity. On the one hand, positivity of virtually all HACs for alpha-fetoprotein, CK8, CK18, and the membranous, canalicular staining for polyclonal carcinoembryonic antigen underline its hepatoid nature. On the other hand, positive staining for CK19 and CK20 and frequent negativity for HepPar1 in both primary tumors and their metastases were distinctive features of HAC. Furthermore, HAC differs from combined hepatocellular cholangiocarcinoma, being negative for CK7. In addition, for comparison of immunohistochemical results, we stained with the same antibody panel a tissue microarray of 121 HCCs. Comparative genomic hybridization study of three HAC supports their hepatoid differentiation as aberrations found in HAC are common in HCC (4q−, 8p−), and hepatoblastoma (Xq+), respectively.

Marked genetic similarities between hepatitis B virus-positive and hepatitis C virus-positive hepatocellular carcinomas.

Hepatocellular carcinoma (HCC) is one of the most common neoplasms worldwide. Well‐established risk factors include infections with two very different viruses: the DNA virus causing hepatitis B (HBV) and the RNA virus inducing hepatitis C (HCV). In order to determine whether genetic differences exist between HBV‐ and HCV‐induced HCC, 41 HCC samples of known vival status were examined by comparative genomic hybridization (CGH). The analysis revealed frequent deletions of 1p (24%), 4q (39%), 6q (41%), 8p (44%), 9p (24%), 11q (24%), 12q (22%), and 13q (39%), as well as common gains of 1q (46%), 6p+ (20%), 8q+ (41%), 11q (27%), and 17q+ (37%). There was no significant difference in the number and type of chromosomal imbalances between 25 HCV‐ and 16 HBV‐infected tumours. This is consistent with models suggesting that HBV and HCV cause cancer through non‐specific inflammatory and regenerative processes, rather than through virus‐specific interactions with defined target genes. Chromosomal imbalances were also unrelated to the grade and stage of HCC. This may suggest that most gross genomic alterations occur early during HCC development and that further progression of these tumours may be associated with other types of genetic changes, not detectable by CGH. In summary, these data show that characteristic gross genomic changes occur in HCC, but these alterations at present do not appear to have diagnostic or prognostic applications. Copyright

Chromosomal alterations in hepatocellular nodules by comparative genomic hybridization: high-grade dysplastic nodules represent early stages of hepatocellular carcinoma.

Data from experimental hepatocarcinogenesis and recent studies in humans have suggested that the emergence of hepatocellular carcinoma (HCC) is a stepwise process. However, despite abundant experimental data, the precise molecular mechanisms and genetic alterations involved in human liver carcinogenesis are still unclear. Comparative genomic hybridization was used to analyze 26 hepatocellular nodules obtained from patients undergoing liver transplantation or surgical resection for HCC. According to the criteria proposed by the International Working Party, 16 nodules were classified as multiacinar regenerative nodules (MRN), 4 as low-grade dysplastic nodules (LG-DN), and 6 as high-grade dysplastic nodules (HG-DN). Our aim was to investigate the possible genetic differences between MRN, LG-DN, and HG-DN. The whole group of nodules showed only a few aberrations (mean 1.1/case), without any significant pattern. This finding is comparable to what happens in non-neoplastic tissue. On the contrary, in three of six HG-DN, we found deletions of 8p and gains of 1q. LG-DN and MRN did not show these chromosomal imbalances. These results confirm the important role of allelic losses on 8p as well as of gains of 1q in HCC. We conclude that the genes that are important in early stages of hepatocarcinogenesis are probably located on these chromosomal arms.

Diagnosis of chronic liver disease: reproducibility and validation of liver biopsy

at nucleotide 677) in the coding region of the gene for methylenetetrahydrofolate reductase (MTHFR), which is involved in the remethylation pathway of homocysteine. Recently, we performed a study in a population of 64 IBD patients in whom vitamin B12 and folic acid levels were determined together with the prevalence of the MTHFR genotypes. Among the 11 IBD patients carrying the TT MTHFR genotype, six had hyperhomocysteinemia, of whom five had concurrent folate and/or vitamin B12 deficiency. The relative risk of developing hyperhomocysteinemia was 5.3-fold (95% CI 2.9–9.6) in IBD patients with the TT MTHFR genotype associated with folate and/or vitamin B12 deficiency, in comparison to individuals with CC or CT MTHFR genotypes and adequate levels of folate and/or vitamin B12. Furthermore, in patients’ homozygotic for the C677T MTHFR gene mutation there is an increased folate requirement to mantain plasma homocysteine within normal levels (5). Thus, the IBD patients with this genetic background are at increased risk to develop hyperhomocysteinemia and may need vitamin B12 and folate supplementation. In conclusion, we suggest that determination of both the MTHFR genotype and vitamin status may predict the risk of developing hyperhomocysteinemia in IBD patients.

The HOX gene network in hepatocellular carcinoma

Liver organogenesis and cancerogenesis share common mechanisms. HOX genes control normal development, primary cellular processes and are characterized by a unique genomic network organization. Less is known about the involvement of HOX genes with liver cancerogenesis. The comparison of the HOX gene network expression between nontumorous livers and hepatocellular carcinomas (HCCs) highlights significant differences in the locus A HOX genes, located on chromosome 7, with a consistent overexpression of HOXA13 mRNA thus validating this gene deregulation as a feature of HCC. HOXA13 is a determinant of gut primordia and posterior body structures. Transcriptome analysis of HCC/nontumorous liver mRNAs, selected on the basis of HOXA13 overexpression, recognizes a set of deregulated genes. The matching of these genes with previously reported HCC transcriptome analysis identifies cell‐cycle and nuclear pore‐related HCC phenotype displaying poor prognosis. HOXA13 and HOXA7 homeoproteins share a consensus sequence that physically links eIF4E nuclear bodies acting on the export of specific mRNAs (c‐myc, FGF‐2, vascular endothelial growth factor (VEGF), ornithine decarboxylase (ODC) and cyclin D1). We report the protein–protein interaction between HOXA13 and eIF4E in liver cancer cells and the deregulation of eIF4E mRNA and protein in cell cycle/nuclear pore HCC group phenotype and in T4 stage HCCs, respectively. Thus, transcriptional and post‐transcriptional HOXA13 deregulation is involved in HCC possibly through the mRNA nuclear export of eIF4E‐dependent transcripts.

Predicting Fibrosis Worsening in Obese Patients With NASH Through Parenchymal Fibronectin, HOMA-IR, and Hypertension

OBJECTIVES:Few published studies have examined the results obtained from repeat liver biopsies in obese patients with nonalcoholic fatty liver disease (NAFLD). The progressive form of this disease may be largely limited to a subgroup of NAFLD patients with nonalcoholic steatohepatitis (NASH). The presence of intralobular fibronectin (Fn) and other variables was investigated in relation to subsequent fibrosis progression.METHODS:In this prospective study, 271 obese patients admitted to the hospital with NAFLD and abnormal liver enzymes were scheduled to undergo a repeat liver biopsy at least 5 years after the initial biopsy. After excluding cirrhotic patients, basal biopsy specimens obtained from patients who underwent a second liver biopsy were stained with antibodies against Fn. The progression of fibrosis in the follow-up sample was correlated with the amount of Fn and other clinicopathological variables.RESULTS:We obtained a second liver biopsy from 149 patients after a median time of 6.4 years. Of these, 132 showed suitable Fn staining for semi-quantitative assessments. In all, 44 out of 83 patients (53%) with basal NASH showed fibrosis progression by at least one stage in the second liver biopsy. The amount of Fn (odds ratio=14.1; P<0.001), a diagnosis of hypertension (odds ratio=4.8; P=0.028), and homeostasis model assessment parameter of insulin resistance (HOMA-IR) scores (>8, odds ratio=1.9; P=0.004) were independent predictive factors of worsening fibrosis.CONCLUSIONS:A semi-quantitative assessment of the amount of parenchymal Fn present at an early stage in obese patients with NASH is valuable for predicting the progression of fibrosis. Similarly, lobular Fn deposition may be a sensitive and early indicator of active fibrogenetic processes in the liver. Hypertension and higher HOMA-IR scores are other clinical independent risk factors that predict the progression of fibrosis.

Preservation of nutritional-status in patients with refractory ascites due to hepatic cirrhosis who are undergoing repeated paracentesis.

Background and Aim:  Refractory ascites in liver‐cirrhosis is associated with a poor prognosis. We performed a prospective study to investigate whether aggressive nutritional‐support could improve outcomes in cirrhotic patients.

Validation of an extension of the international non-invasive criteria for the diagnosis of hepatocellular carcinoma to the characterization of macroscopic portal vein thrombosis

Background and Aim:  We aimed to validate the non‐invasive criteria for the characterization of portal vein thrombosis (PVT) in patients with cirrhosis and hepatocellular carcinoma (HCC). In a prospective study, we examined the impact of arterial hypervascularity, as established by the European Association for the Study of the Liver and the American Association for the Study of Liver Diseases recommendations for the non‐invasive diagnosis of HCC, as a criterion for characterizing macroscopic PVT (EASL/AASLD extension criteria).