Lory Saveria Crocè

Genome-wide meta-analyses identify three loci associated with primary biliary cirrhosis

A genome-wide association screen for primary biliary cirrhosis risk alleles was performed in an Italian cohort. The results from the Italian cohort replicated IL12A and IL12RB associations, and a combined meta-analysis using a Canadian dataset identified newly associated loci at SPIB (P = 7.9 × 10−11, odds ratio (OR) = 1.46), IRF5-TNPO3 (P = 2.8 × 10−10, OR = 1.63) and 17q12-21 (P = 1.7 × 10−10, OR = 1.38).

Incidence and natural course of fatty liver in the general population: the Dionysos study.

Using the general population of the Dionysos Study, we followed up 144 subjects without fatty liver (FL−) and 336 with fatty liver (FL+) for a median time of 8.5 years. All subjects had suspected liver disease (SLD) defined as altered liver enzymes, high mean corpuscular volume, or low platelet count in the absence of HBV and HCV infection. Ethanol intake was assessed using a food frequency questionnaire, and FL was diagnosed using ultrasonography. The incidence and remission rates of FL were 18.5 and 55.0 per 1,000 person‐years. Progression to cirrhosis or HCC was rare in both cohorts (incidence rate: 1.7 versus 1.1 and 0.8 versus 0.4 per 1,000 person‐years for FL− versus FL+). Multivariable Poisson regression was performed to identify predictors of FL incidence and remission among sex, age, body mass index, ethanol, and liver enzymes. Every increase of 20 g/day of ethanol intake at baseline was associated with a 17% increase in the rate of incident FL (P = 0.019), a 10% decrease in the rate of remitting FL and SLD (P = 0.043), a 19% decrease in the rate of remitting FL with persistent SLD (P = 0.002), and a 10% increase in mortality rate (P = 0.005) in the FL+ cohort. Conclusion: In the general population of the Dionysos Study, FL regressed in nearly 1 of every 2 cases and had a substantially benign course. Ethanol intake was the most important risk factor for FL remission and incidence and a predictor of mortality in subjects with FL. (HEPATOLOGY 2007.)

Subcellular localization of APE1/Ref-1 in human hepatocellular carcinoma : Possible prognostic significance

APE1/Ref-1, normally localized in the nucleus, is a regulator of the cellular response to oxidative stress. Cytoplasmic localization has been observed in several tumors and correlates with a poor prognosis. Because no data are available on liver tumors, we investigated APE1/Ref-1 subcellular localization and its correlation with survival in 47 consecutive patients undergoing hepatocellular carcinoma (HCC) resection. APE1/Ref-1 expression was determined by immunohistochemistry in HCC and surrounding liver cirrhosis (SLC) and compared with normal liver tissue. Survival probability was evaluated using Kaplan-Meier curves (log-rank test) and Cox regression. Cytoplasmic expression of APE1/Ref-1 was significantly higher in HCC than in SLC (P = 0.00001); normal liver showed only nuclear reactivity. Patients with poorly differentiated HCC showed a cytoplasmic expression three times higher than those with well-differentiated HCC (P = 0.03). Cytoplasmic localization was associated with a median survival time shorter than those with negative cytoplasmic reactivity (0.44 compared with 1.64 years, P = 0.003), and multivariable analysis confirmed that cytoplasmic APE1/Ref-1 localization is a predictor of survival. Cytoplasmic expression of APE1/Ref-1 is increased in HCC and is associated with a lower degree of differentiation and a shorter survival time, pointing to the use of the cytoplasmic localization of APE1/Ref-1 as a prognostic marker for HCC.

Blood Flow Changes in Hepatocellular Carcinoma After the Administration of Thalidomide Assessed by Reperfusion Kinetics During Microbubble Infusion: Preliminary Results

Objectives:We sought to investigate whether thalidomide is able to produce tumor vascular changes in patients with untreatable hepatocellular carcinoma (HCC) that can be detected using microbubble contrast agents. Materials and Methods:Eleven consecutive patients with untreatable HCC underwent contrast-enhanced ultrasound before and during thalidomide administration. Real-time destruction reperfusion kinetics was obtained from a representative HCC nodule and from the surrounding liver parenchyma during SonoVue infusion (Bracco, Milan, Italy) at a constant rate of 0.10 mL/s by using a syringe pump and modelized according to the mathematical function SI = A(1 − exp(−βt)) where the plateau signal intensity A reflects the percent blood volume, the time constant β reflects the average speed of blood, and their product A*β reflects the nutrient blood flow. Results:Size of the representative nodule reduced significantly 3 to 6 months after the start of thalidomide treatment. Before thalidomide administration A, β, and A*β of the index lesion were 44 ± 60 LIU, 0.31 ± 0.40 seconds−1 and 8.1 ± 11.8 LIU/s, respectively). A and A*β reduced significantly after 15 days (26 ± 50 LIU and 2.9 ± 4.8 LIU/s, P < 0.01), 3 months (12 ± 18 LIU, and 4.3 ± 7.7 LIU/s, P < 0.01), and 6 months (13 ± 23 LIU and 2.4 ± 3.7 LIU/s, P < 0.05) of treatment. No statistically significant changes of the exponential time constant β were observed, nor changes of A, β and A*β in the liver parenchyma. Conclusions:Contrast-enhanced ultrasound can be used effectively to evaluate changes in perfusion parameters of HCC nodules during thalidomide administration.

Treatment options in Western hepatocellular carcinoma: a prospective study of 224 patients

BACKGROUND/AIMS Though hepatocellular carcinoma (HCC) is one of the most frequent malignant tumors in the world, the optimal therapeutic strategy is still poorly defined. This is mainly due to geographic differences in HCC which may affect the validity of treatment regimens in differents areas of the world. The aim of the present study was to analyze the natural course of the disease as well as to assess the efficacy of different therapeutical schemes in HCC observed in Ljubljana (Slovenia) and Trieste (Italy), two cities in Western Europe situated close to each other. METHODS During the period from January 1988 to December 1993, 224 consecutive patients (132 in Trieste and 92 in Ljubljana) with HCC were enrolled in the study. Patients were treated with the following 3 schemes: surgery 39 (17.4%), transcatheter chemoembolization (TACE) 116 (51.8%), and no treatment 69 (30.8%). The tumor was classified by Okuda staging and the liver disease by Child-Pugh score. Patients were followed up for 12-60 months, with an average of 40 months. The response rate to TACE and recurrence following surgery were evaluated. Comparative analysis of survival between different treatment groups was performed. RESULTS The natural course of the disease, and other characteristics of the HCC, showed a typical Western type of tumor. Liver disease was scored as Child A in 58%, Child B in 30% and Child C in 12%, and the tumor was staged as Okuda I in 52%, Okuda II in 37% and Okuda III in 11%, respectively. Treatment with TACE was followed by an objective response in 27%, with a median survival of 31 months. Surgery was followed by a recurrence rate of 77% within 19.5 months and median survival of 49 months. The overall median survival of nontreated patients was 8 months. Survival in each group of patients differed significantly between all three consecutive stages of Okuda (p<0.001). In contrast, the differences in survival were significant only between Child A and B (p<0.02). The differences between Child B and C were not significant. CONCLUSIONS This study emphasizes the importance of staging in the choice of treatment modality and diffusion of HCC in affecting an overall response to treatment and survival. Surgery is highly effective in monofocal HCC of Okuda I and II without cirrhosis. TACE is effective in Okuda I and II and Child A cirrhosis only. The treatment of HCC in Child B cirrhosis needs further studies. In Child C and/or Okuda stage III of HCC, any treatment except pure symptomatic relief is detrimental and should not be used.

The Expression of CD90/Thy-1 in Hepatocellular Carcinoma: An In Vivo and In Vitro Study

Although the CD90 (Thy-1) was proposed as biomarker of several tumors and cancer stem cells, the involvement of this molecule in the progression of hepatocellular carcinoma (HCC) and other less frequent hepatic neoplasms is still undefined. The distribution of CD90 was investigated both in in vivo (human tissues samples) and in vitro (human HCC cell line JHH-6). A total of 67 liver tumors were analyzed: 51 HCC, 6 cholangiocarcinoma and 10 hepatoblastoma. In all cases, paired tissue sample of both the tumor and cirrhotic liver was available. Hepatic tissue obtained in 12 healthy livers was used as control. CD90 gene expression was studied by RT-qPCR, protein expression was assessed by quantitative Western Blot, immunofluorescence and flow cytometry. The CD90 expression analysis showed a significant increment in tumor compared to both its paired cirrhotic tissue and normal liver (p<0.05 and p<0.001, respectively). This increase was accompanied by the up-regulation of stromal component in the cancer, as demonstrated by alpha smooth muscle actin staining. In vitro analysis of JHH-6 cell line showed a higher proliferation capacity of CD90+ compared to CD90- cells (p<0.001), also noticed in 3D clonogenic assay (p<0.05), associated by a significant higher expression of the promoting factors (hepatocyte growth factor, fibroblast associated protein and alpha smooth muscle actin 2). A higher expression of the breast cancer resistance protein was found in CD90+ subpopulation while the multidrug resistance protein 1 showed an opposite behavior. Collectively, these results point to the importance of CD90 in the HCC.

Gene and functional up-regulation of the BCRP/ABCG2 transporter in hepatocellular carcinoma.

BackgroundThe Breast Cancer Resistance Protein (BCRP/ABCG2) is one member of ABC transporters proteins super family responsible of drug resistance. Since data on ABCG2 expression in liver malignances are scanty, here we report the expression of ABCG2 in adult human hepatocellular carcinoma (HCC) in both in vivo and in vitro models with different degree of malignancy.MethodsIn cell lines derived from human hepatocellular carcinoma, ABCG2 gene expression was assessed by reverse transcription quantitative real time PCR and function by Hoechst 33342 efflux assay; protein content was assessed by SDS-PAGE Western blot.ResultsABCG2 expression was found to be highest in the most undifferentiated cell lines, and this was related with a higher functional activity. ABCG2 expression was sensitive to antineoplastic drugs since exposure to 5 μM doxorubicin for 24 hours resulted in significant up-regulations of ABCG2 in all cell lines, particularly in those lines with low basal ABCG2 expression (p<0.01). The gene expression was also investigated in 51 adult liver tissues with HCC and related cirrhosis; normal liver tissue was used as control. ABCG2 gene expression was higher in HCC than both cirrhotic paired tissue and normal tissue. This up-regulation was greater (p<0.05) in pathological poorly differentiated grade G3/G4 than in well-differentiated G1/G2 HCC.ConclusionsOur results suggest a correlation of ABCG2 gene expression and differentiation stage both in human and HCC derived cell lines. The rapid up-regulation of ABCG2 to exposure to doxorubicin emphasizes the importance of this transporter in accounting for drug resistance in liver tumors.

Systematic review and meta-analysis on the adverse events of rimonabant treatment: Considerations for its potential use in hepatology

BackgroundThe cannabinoid-1 receptor blockers have been proposed in the management of obesity and obesity-related liver diseases (fatty liver as NAFLD or NASH). Due to increasing number of patients to be potentially treated and the need to assess the advantage of this treatment in terms of risk/benefit, we analyze the side events reported during the treatment with rimonabant by a systematic review and meta-analysis of all randomized controlled studies.MethodsAll published randomized controlled trials using rimonabant versus placebo in adult subjects were retrieved. Relative risks (RR) with 95% confidence interval for relevant adverse events and number needed to harm was calculated.ResultsNine trials (n = 9635) were considered. Rimonabant 20 mg was associated with an increased risk of adverse event (RR 1.35; 95%CI 1.17-1.56), increased discontinuation rate (RR 1.79; 95%CI 1.35-2.38), psychiatric (RR 2.35; 95%CI 1.66-3.34), and nervous system adverse events (RR 2.35; 95%CI 1.49-3.70). The number needed to harm for psychiatric adverse events is 30.ConclusionRimonabant is associated with an increased risk of adverse events. Despite of an increasing interest for its use on fatty liver, the security profile and efficacy it is needs to be carefully assessed before its recommendation. At present the use of rimonabant on fatty liver cannot be recommended.

The role of multipotent cancer associated fibroblasts in hepatocarcinogenesis

BackgroundThe presence of tumor supporting cells in various cancer, including in hepatocellular carcinoma (HCC), has become an important target in the study of carcinogenesis. The cancer-associated fibroblast (CAF), one of the most important cellular components in the cancer stroma, might contribute to the progression of the disease due to its plasticity, a behavior of the stem cells. In this study, we investigate the significance of the CAF and its role in the HCC progression and metastasis.MethodsPrimary CAF and non-tumoral fibroblast (NTF) from nine paired HCC and distant non-tumoral liver tissues were isolated and cultured. The cells were characterized by flow cytometry, RT-PCR, anchorage-independent assay and in vitro cells directed trans-differentiation. Co-culture study was performed in Transwell system and xenograft assay was performed in immunodeficient mice.ResultsCAF and NTF were positive for CD90, CD44, αSMA, and vimentin and negative for CD34, CD45, CD117, and CD133. When stimulated, they showed the potential to differentiate into adipocytes, osteoblasts, and pancreatic cells. When co-cultured with human HCC cell lines, CAF up-regulated gene expressions of TGFB1 and FAP of HuH-7 and JHH-6 while NTF did not induced either of the genes. Xenograft assay showed that the CAF had the capacity to enter into circulation as confirmed by RT-PCR and DNA sequencing.ConclusionOur data provides evidence of the plasticity of the CAF and the NTF as stem cells in the process of hepatocarcinogenesis and metastasis. These cells mutually interacts with HCC cells. Their trans-differentiation flexibility may induce a switch from normal to cancerous microenvironment.

Abnormal apolipoprotein B pre-mRNA splicing in patients with familial hypobetalipoproteinaemia

Background: Familial hypobetalipoproteinaemia (FHBL) is a codominant disorder characterised by fatty liver and reduced plasma levels of low-density lipoprotein (LDL) and its protein constituent apolipoprotein B (apoB). FHBL is linked to the APOB gene in some but not all known cases. In a group of 59 patients with FHBL genotyped for APOB gene mutations, we found three novel splice-site mutations: c.904+4A→G in intron 8, c.3843−2A→G in intron 24 and c.4217−1G→T in intron 25. Objective: To assess the effects of these mutations on apoB pre-mRNA splicing. Methods: ApoB mRNA was analysed in the liver of one proband and in cells expressing APOB minigenes harbouring the mutations found in the other probands. Results: In the liver of the c.3843−2A→G carrier, an apoB mRNA devoid of exon 25 was identified, predicted to encode a truncated peptide of 1260 amino acids. The analysis of minigene transcripts in COS-1 cells showed that the c.904+4A→G mutation caused the formation of an mRNA devoid of exon 8, predicted to encode a short apoB of 247 amino acids. The minigene harbouring the c.4217−1G→T mutation in intron 25 generated an mRNA in which exon 25 joined to a partially deleted exon 26, resulting from the activation of an acceptor site in exon 26; this mRNA is predicted to encode a truncated protein of 1380 amino acids. All these truncated apoBs were not secreted as constituents of plasma lipoproteins. Conclusion: These findings demonstrate the pathogenic effect of rare splice-site mutations of the APOB gene found in FHBL.