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Hepatocellular carcinoma (HCC) is a heterogeneous tumor associated with several risk factors, with non-alcoholic fatty liver disease (NAFLD) emerging as an important cause of liver tumorigenesis. Due to the obesity epidemics, the occurrence of NAFLD has significantly increased with nearly 30% prevalence worldwide. HCC often arises in the background of chronic liver disease (CLD), such as nonalcoholic steatohepatitis (NASH) and cirrhosis. Gut microbiome (GM) alterations have been linked to NAFLD progression and HCC development, with several investigations reporting a crucial role for the gut-liver axis and microbial metabolites in promoting CLD. Moreover, the GM affects liver homeostasis, energy status, and the immune microenvironment, influencing the response to immunotherapy with interesting therapeutic implications. In this review, we summarize the main changes in the GM and derived metabolites (e.g., short-chain fatty acids and bile acids) occurring in HCC patients and influencing NAFLD progression, emphasizing their potential as early diagnostic biomarkers and prognostic tools. We discuss the weight loss effects of diet-based interventions and healthy lifestyles for the treatment of NAFLD patients, highlighting their impact on the restoration of the intestinal barrier and GM structure. We also describe encouraging preclinical findings on the modulation of GM to improve liver functions in CLD, boost the antitumor immune response (e.g., probiotic supplementations or anti-hypercholesterolemic drug treatment), and ultimately delay NAFLD progression to HCC. The development of safe and effective strategies that target the gut-liver axis holds promise for liver cancer prevention and treatment, especially if personalized options will be considered.

Due to the lack of biomarkers predictive of response to atezolizumab-bevacizumab, the standard of care for advanced HCC, we analyzed baseline and early on-treatment variation of peripheral lymphocyte populations of 37 prospective patients treated by atezolizumab-bevacizumab and in 15 prospective patients treated by sorafenib or lenvatinib (TKIs). RNAseq analysis followed by RT-PCR validation on patients-derived PBMC was also performed. At first imaging, re-evaluation 13 patients receiving atezolizumab-bevacizumab, showed an objective response, 17 stable disease, while 7 were nonresponders. Baseline CD8+ and CD8+PD-L1+ peripheral lymphocytes were lower in responders versus nonresponders (T-test, p = 0.012 and 0.004, respectively). At 3 weeks, 28 of 30 responders displayed a rise of CD8+PD1+ lymphocytes with a positive mean fold change of 4.35 (±5.6 SD), whereas 6 of 7 nonresponders displayed a negative fold change of 0.89 (±0.84 SD). These changes were not observed in patients treated by TKIs. TRIM56, TRIM16, TRIM64, and Ki67 mRNAs were validated as upregulated in responders versus nonresponders after 3 weeks after treatment start, providing possible evidence of immune activation. Baseline CD8+ and CD8+PD-L1+ peripheral lymphocytes and early changes in CD8+PD1+ lymphocytes predict response to atezolizumab-bevacizumab providing noninvasive markers to complement clinical practice in the very early phases of treatment of HCC patients., (© 2023 The Authors. European Journal of Immunology published by Wiley-VCH GmbH.)

Background: Metabolic reprogramming is a well-known marker of cancer, and it represents an early event during hepatocellular carcinoma (HCC) development. The recent approval of several molecular targeted agents has revolutionized the management of advanced HCC patients. Nevertheless, the lack of circulating biomarkers still affects patient stratification to tailored treatments. In this context, there is an urgent need for biomarkers to aid treatment choice and for novel and more effective therapeutic combinations to avoid the development of drug-resistant phenotypes. This study aims to prove the involvement of miR-494 in metabolic reprogramming of HCC, to identify novel miRNA-based therapeutic combinations and to evaluate miR-494 potential as a circulating biomarker., Methods: Bioinformatics analysis identified miR-494 metabolic targets. QPCR analysis of glucose 6-phosphatase catalytic subunit (G6pc) was performed in HCC patients and preclinical models. Functional analysis and metabolic assays assessed G6pc targeting and miR-494 involvement in metabolic changes, mitochondrial dysfunction, and ROS production in HCC cells. Live-imaging analysis evaluated the effects of miR-494/G6pc axis in cell growth of HCC cells under stressful conditions. Circulating miR-494 levels were assayed in sorafenib-treated HCC patients and DEN-HCC rats., Results: MiR-494 induced the metabolic shift of HCC cells toward a glycolytic phenotype through G6pc targeting and HIF-1A pathway activation. MiR-494/G6pc axis played an active role in metabolic plasticity of cancer cells, leading to glycogen and lipid droplets accumulation that favored cell survival under harsh environmental conditions. High miR-494 serum levels associated with sorafenib resistance in preclinical models and in a preliminary cohort of HCC patients. An enhanced anticancer effect was observed for treatment combinations between antagomiR-494 and sorafenib or 2-deoxy-glucose in HCC cells., Conclusions: MiR-494/G6pc axis is critical for the metabolic rewiring of cancer cells and associates with poor prognosis. MiR-494 deserves attention as a candidate biomarker of likelihood of response to sorafenib to be tested in future validation studies. MiR-494 represents a promising therapeutic target for combination strategies with sorafenib or metabolic interference molecules for the treatment of HCC patients who are ineligible for immunotherapy., (© 2023. The Author(s).)

Treatments aimed to reverse the tumor-induced immune tolerance represent a promising approach for advanced hepatocellular carcinoma (HCC). Notwithstanding, primary nonresponse, early, and late disease reactivation still represent major clinical challenges. Here, we focused on microRNAs (miRNAs) acting both as modulators of cancer cell hallmarks and immune system response. We outlined the bidirectional function that some oncogenic miRNAs play in the differentiation and program activation of the immune system development and, at the same time, in the progression of HCC. Indeed, the multifaceted spectrum of miRNA targets allows the modulation of both immune-associated factors and oncogenic or tumor suppressor drivers at the same time. Understanding the molecular changes contributing to disease onset, progression, and resistance to treatments might help to identify possible novel biomarkers for selecting patient subgroups, and to design combined tailored treatments to potentiate antitumor approaches. Preliminary findings seem to argue in favor of a bidirectional function of some miRNAs, which enact an effective modulation of molecular pathways driving oncogenic and immune-skipping phenotypes associated with cancer aggressiveness. The identification of these miRNAs and the characterization of their 'dual' role might help to unravel novel biomarkers identifying those patients more likely to respond to immune checkpoint inhibitors and to identify possible therapeutic targets with both antitumor and immunomodulatory functions. In the present review, we will focus on the restricted panel of miRNAs playing a bidirectional role in HCC, influencing oncogenic and immune-related pathways at once. Even though this field is still poorly investigated in HCC, it might represent a source of candidate molecules acting as both biomarkers and therapeutic targets in the setting of immune-based treatments.

Aflatoxin B1 (AFB1) is a class 1 carcinogen with an ascertained role in the development of hepatocellular carcinoma (HCC) in high exposure areas. Instead, this study aimed to assay whether chronic/intermittent, low-dose AFB1 consumption might occur in low-exposure geographical areas, ultimately accumulating in the liver and possibly contributing to liver cancer. AFB1-DNA adducts were assayed by immunostaining in liver tissues from three Italian series of twenty cirrhosis without HCC, 131 HCC, and 45 cholangiocarcinoma, and in an AFB1-induced HCC rat model. CD68, TP53 immunostaining, and TP53 RFLP analysis of R249S transversion were used to characterize cell populations displaying AFB1-DNA adducts. Twenty-five HCCs displayed AFB1-adducts both in neoplastic hepatocytes and in cells infiltrating the tumor and non-tumor tissues. Nuclear immunostaining was observed in a few cases, while most cases showed cytoplasmic immunostaining, especially in CD68-positive tumor-infiltrating cells, suggestive for phagocytosis of dead hepatocytes. Similar patterns were observed in AFB1-induced rat HCC, though with higher intensity. Cholangiocarcinoma and cirrhosis without HCC did not displayAFB1-adducts, except for one case. Despite not providing a causal relationship with HCC, these findings still suggest paying attention to detection and control measures for aflatoxins to ensure food safety in low exposure areas.

The Notch family includes evolutionary conserved genes that encode for single-pass transmembrane receptors involved in stem cell maintenance, development and cell fate determination of many cell lineages. Upon activation by different ligands, and depending on the cell type, Notch signaling plays pleomorphic roles in hepatocellular carcinoma (HCC) affecting neoplastic growth, invasion capability and stem like properties. A specific knowledge of the deregulated expression of each Notch receptor and ligand, coupled with resultant phenotypic changes, is still lacking in HCC. Therefore, while interfering with Notch signaling might represent a promising therapeutic approach, the complexity of Notch/ligands interactions and the variable consequences of their modulations raises concerns when performed in undefined molecular background. The gamma-secretase inhibitors (GSIs), representing the most utilized approach for Notch inhibition in clinical trials, are characterized by important adverse effects due to the non-specific nature of GSIs themselves and to the lack of molecular criteria guiding patient selection. In this review, we briefly summarize the mechanisms involved in Notch pathway activation in HCC supporting the development of alternatives to the γ-secretase pan-inhibitor for HCC therapy.

Competing Interests: Declaration of Competing Interest None.

Purpose: After 10 years of clinical practice and research studies, there are still no validated prognostic or predictive factors of response to sorafenib for hepatocellular carcinoma (HCC). On the basis of the results of our two retrospective studies, we designed the multicenter INNOVATE study with the aim to validate the role of nitric oxide synthase 3 ( NOS3 ) and ANGPT2 polymorphisms in patients with HCC treated with sorafenib [NCT02786342]., Patients and Methods: This prospective multicenter study was conducted at 10 centers in Italy. All eligible patients received a continuous oral treatment with 400 mg of sorafenib twice daily. Genotyping analysis was performed for NOS3 (rs2070744) and ANGPT2 SNPs (rs55633437). The primary outcome was progression-free survival (PFS), whereas secondary outcomes included overall survival (OS) and disease-control rate., Results: A total of 165 patients were enrolled between March 2016 and June 2018. NOS3 rs2070744 CC/CT genotypes were significantly associated with a higher median PFS (5.9 months vs. 2.4 months; HR = 0.43; P = 0.0007) and OS (15.7 months vs. 8.6 months; HR = 0.38; P < 0.0001) compared with TT genotype. There was no statistically significant association between ANGPT2 rs55633437 TT/GT genotypes and PFS (2.4 months vs. 5.7 months; HR = 1.93; P = 0.0833) and OS (15.1 months vs. 13.0 months; HR = 2.68; P = 0.55) when compared with the other genotype. Following adjustment for clinical covariates, multivariate analysis confirmed NOS3 as an independent prognostic factor for PFS (HR = 0.50; P = 0.0128) and OS (HR = 0.29; P = 0.0041)., Conclusions: The INNOVATE study met the primary endpoint, confirming that patients with advanced HCC with NOS3 rs2070744 CC/CT genotypes had a better prognosis with respect to TT genotype patients., (©2020 American Association for Cancer Research.)

Background & Aims: Only limited therapeutic options are currently available for hepatocellular carcinoma (HCC), making the development of effective alternatives essential. Based on the recent finding that systemic or local hypothyroidism is associated with HCC development in humans and rodents, we investigated whether the thyroid hormone triiodothyronine (T3) could inhibit the progression of HCCs., Methods: Different rat and mouse models of hepatocarcinogenesis were investigated. The effect of T3 on tumorigenesis and metabolism/differentiation was evaluated by transcriptomic analysis, quantitative reverse transcription PCR, immunohistochemistry, and enzymatic assay., Results: A short treatment with T3 caused a shift in the global expression profile of the most aggressive preneoplastic nodules towards that of normal liver. This genomic reprogramming preceded the disappearance of nodules and involved reprogramming of metabolic genes, as well as pro-differentiating transcription factors, including Kruppel-like factor 9, a target of the thyroid hormone receptor β (TRβ). Treatment of HCC-bearing rats with T3 strongly reduced the number and burden of HCCs. Reactivation of a local T3/TRβ axis, a switch from Warburg to oxidative metabolism and loss of markers of poorly differentiated hepatocytes accompanied the reduced burden of HCC. This effect persisted 1 month after T3 withdrawal, suggesting a long-lasting effect of the hormone. The antitumorigenic effect of T3 was further supported by its inhibitory activity on cell growth and the tumorigenic ability of human HCC cell lines., Conclusions: Collectively, these findings suggest that reactivation of the T3/TRβ axis induces differentiation of neoplastic cells towards a more benign phenotype and that T3 or its analogs, particularly agonists of TRβ, could be useful tools in HCC therapy., Lay Summary: Hepatocellular carcinoma (HCC) represents an important challenge for global health. Recent findings showed that systemic or local hypothyroidism is associated with HCC development. In rat models, we showed that administration of the thyroid hormone T3 impaired HCC progression, even when given at late stages. This is relevant from a translational point of view as HCC is often diagnosed at an advanced stage when it is no longer amenable to curative treatments. Thyroid hormones and/or thyromimetics could be useful for the treatment of patients with HCC., Competing Interests: Conflict of interest The authors who have taken part in this study declared that they do not have anything to disclose regarding funding or conflict of interest concerning this manuscript. Please refer to the accompanying ICMJE disclosure forms for further details., (Copyright © 2020 European Association for the Study of the Liver. Published by Elsevier B.V. All rights reserved.)

The molecular background of hepatocellular carcinoma (HCC) is highly heterogeneous, and biomarkers predicting response to treatments are an unmet clinical need. We investigated miR-30e-3p contribution to HCC phenotype and response to sorafenib, as well as the mutual modulation of TP53/MDM2 pathway, in HCC tissues and preclinical models. MiR-30e-3p was downregulated in human and rat HCCs, and its downregulation associated with TP53 mutations. TP53 contributed to miR-30e-3p biogenesis, and MDM2 was identified among its target genes, establishing an miR-30e-3p/ TP53 / MDM2 feedforward loop and accounting for miR-30e-3p dual role based on TP53 status. EpCAM , PTEN , and p27 were demonstrated as miR-30e-3p additional targets mediating its contribution to stemness and malignant features. In a preliminary cohort of patients with HCC treated with sorafenib, increased miR-30e-3p circulating levels predicted the development of resistance. In conclusion, molecular background dictates miR-30e-3p dual behavior in HCC. Mdm2 targeting plays a predominant tumor suppressor function in wild-type TP53 contexts, whereas other targets such as PTEN , p27 , and EpCAM gain relevance and mediate miR-30e-3p oncogenic role in nonfunctional TP53 backgrounds. Increased circulating levels of miR-30e-3p predict the development of sorafenib resistance in a preliminary series of patients with HCC and deserve future investigations. SIGNIFICANCE: The dual role of miR-30e-3p in HCC clarifies how the molecular context dictates the tumor suppressor or oncogenic function played by miRNAs., (©2020 American Association for Cancer Research.)

Several staging systems for hepatocellular carcinoma (HCC) have been developed. The Barcelona Clinic Liver Cancer staging system is considered the best in predicting survival, although limitations have emerged. Recently, the Italian Liver Cancer (ITA.LI.CA) prognostic system, integrating ITA.LI.CA tumor staging (stages 0, A, B1-3, C) with the Child-Turcotte-Pugh score, Eastern Cooperative Oncology Group performance status, and alpha-fetoprotein with a strong ability to predict survival, was proposed. The aim of our study was to provide an external validation of the ITA.LI.CA system in an independent real-life occidental cohort of HCCs. From September 2008 to April 2016, 1,508 patients with cirrhosis and incident HCC were consecutively enrolled in 27 Italian institutions. Clinical, tumor, and treatment-related variables were collected, and patients were stratified according to scores of the Barcelona Clinic Liver Cancer system, ITA.LI.CA prognostic system, Hong Kong Liver Cancer system, Cancer of the Liver Italian Program, Japanese Integrated System, and model to estimate survival in ambulatory patients with hepatocellular carcinoma. Harrell's C-index, Akaike information criterion, and likelihood-ratio test were used to compare the predictive ability of the different systems. A subgroup analysis for treatment category (curative versus palliative) was performed. Median follow-up was 44 months (interquartile range, 23-63 months), and median overall survival was 34 months (interquartile range, 13-82 months). Median age was 71 years, and patients were mainly male individuals and hepatitis C virus carriers. According to ITA.LI.CA tumor staging, 246 patients were in stage 0, 472 were in stage A, 657 were in stages B1/3, and 133 were in stage C. The ITA.LI.CA prognostic system showed the best discriminatory ability (C-index = 0.77) and monotonicity of gradients compared to other systems, and its superiority was also confirmed after stratification for treatment strategy., Conclusion: This is the first study that independently validated the ITA.LI.CA prognostic system in a large cohort of Western patients with incident HCCs. The ITA.LI.CA system performed better than other multidimensional prognostic systems, even after stratification by curative or palliative treatment. This new system appears to be particularly useful for predicting individual HCC prognosis in clinical practice. (Hepatology 2018;67:2215-2225)., (© 2017 by the American Association for the Study of Liver Diseases.)

Hepatocellular carcinoma (HCC) represents the second cause of cancer-related mortality worldwide and is associated with poor prognosis, due to a high recurrence rate after curative treatments and a drug resistance phenotype. In this scenario, the identification of innovative and effective therapeutic strategies is an unmet clinical need. The safety and efficacy of microRNA (miRNA) mediated approaches in preclinical models and clinical trials have been widely described in cancer. MicroRNA-199a downregulation is a common feature of HCC where its reduced expression contributes to mTOR and c-Met pathways activation. Notch1 activation is also a common event in HCC, influencing epithelial-to-mesenchymal transition, tumor invasion and recurrence at least in part through E-cadherin regulation. Here we identified a negative correlation between miR-199a-3p and Notch1 or E-cadherin protein levels in HCC patients and demonstrated that miR-199a-3p regulates E-cadherin expression through Notch1 direct targeting in in vitro models. Moreover, we showed that a strong correlation exists between miR-199a-5p and miR-199a-3p in HCC specimens and that miR-199a-5p contributes to E-cadherin regulation as well, underlying the complex network of interaction carried out by miR-199a and its influence on tumor aggressiveness. In conclusion, our findings suggest the restoration of miR-199a-3p physiologic levels as a possible therapeutic strategy for the treatment of HCC., (Copyright © 2017 Elsevier GmbH. All rights reserved.)

Hepatocellular carcinoma (HCC) represents the second cause of cancer-related mortality worldwide and is associated with poor prognosis, especially in patients not amenable for curative treatments. The multi-kinase inhibitor sorafenib represents the first-line treatment option for advanced HCC; nevertheless, its effectiveness is limited due to tumor heterogeneity as well as innate or acquired drug resistance, raising the need for new therapeutic strategies. MicroRNAs (miRNAs) involvement in treatment response as well as their safety and efficacy in preclinical models and clinical trials have been widely documented in the oncologic field, including HCC. Here, we identified miR-494 upregulation in a subgroup of human and rat HCCs with stem cell-like characteristics, as well as multiple epigenetic mechanisms involved in its aberrant expression in HCC cell lines and patients. Moreover, we identified p27, puma and pten among miR-494 targets, contributing to speed up cell cycle progression, enhance survival potential in stressful conditions and increase invasive and clonogenic capabilities. MiR-494 overexpression increased sorafenib resistance via mTOR pathway activation in HCC cell lines and, in line, high miR-494 levels associated with decreased sorafenib response in two HCC animal models. A sorafenib-combined anti-miR-494-based strategy revealed an enhanced anti-tumor potential with respect to sorafenib-only treatment in our HCC rat model. In conclusion, our findings suggested miR-494 as a possible therapeutic target as well as a candidate biomarker for patient stratification in advanced HCC.

Aims: This multicentre cohort study evaluated the role of ageing on clinical characteristics, treatment allocation and outcome of new hepatocellular carcinomas (HCCs), in clinical practice., Material & Methods: From September 2008, 541 patients >70 years old (elderly group), and 527 ≤70 years old (non-elderly group) with newly diagnosed HCC were consecutively enrolled in 30 Italian centres. Differences in clinical characteristics and treatment allocation between groups were described by a multivariable logistic regression model measuring the inverse probability weight to meet the elderly group. Survival differences were measured by unadjusted and adjusted (by inverse probability weight) survival analysis., Results: Elderly patients were mainly females, hepatitis C virus infected and with better conserved liver function (P<.001). At presentation, HCC median size was similar in both groups while, in youngers, HCC was more frequently multinodular (P=.001), and associated with neoplastic thrombosis (P=.009). Adjusted survival analysis showed that age did not predict short-mid-term survival (within 24 months), while it was a significant independent predictor of long-term survival. Moreover, age had a significant long-term survival impact mainly on early HCC stages (Barcelona Clinic for Liver Cancer [BCLC] 0-A), its impact on BCLC B stage was lower, while it was negligible for advanced-terminal stages., Conclusions: Age per se does not impact on short-mid-term prognosis (≤24 months) of HCC patients, and should not represent a limitation to its management., (© 2017 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.)

Purpose: The aberrant expression of miR-221 is a hallmark of human cancers, including hepatocellular carcinoma (HCC), and its involvement in drug resistance, together with a proved in vivo efficacy of anti-miR-221 molecules, strengthen its role as an attractive target candidate in the oncologic field. The discovery of biomarkers predicting the response to treatments represents a clinical challenge in the personalized treatment era. This study aimed to investigate the possible role of miR-221 as a circulating biomarker in HCC patients undergoing sorafenib treatment as well as to evaluate its contribution to sorafenib resistance in advanced HCC. Experimental Design: A chemically induced HCC rat model and a xenograft mouse model, together with HCC-derived cell lines were employed to analyze miR-221 modulation by Sorafenib treatment. Data from the functional analysis were validated in tissue samples from surgically resected HCCs. The variation of circulating miR-221 levels in relation to Sorafenib treatment were assayed in the animal models and in two independent cohorts of patients with advanced HCC. Results: MiR-221 over-expression was associated with Sorafenib resistance in two HCC animal models and caspase-3 was identified as its target gene, driving miR-221 anti-apoptotic activity following Sorafenib administration. Lower pre-treatment miR-221 serum levels were found in patients subsequently experiencing response to Sorafenib and an increase of circulating miR-221 at the two months assessment was observed in responder patients. Conclusions: MiR-221 might represent a candidate biomarker of likelihood of response to Sorafenib in HCC patients to be tested in future studies. Caspase-3 modulation by miR-221 participates to Sorafenib resistance. Clin Cancer Res; 23(14); 3953-65. ©2017 AACR ., (©2017 American Association for Cancer Research.)

The role of microRNAs as oncogenes and tumor suppressor genes has emerged in several cancers, including hepatocellular carcinoma (HCC). The pivotal tumor suppressive role of p53-axis is indicated by the presence of inactivating mutations in TP53 gene in nearly all cancers. A close interaction between these two players, as well as the establishment of complex p53/miRNAs loops demonstrated the strong contribution of p53-effector miRNAs in enhancing the p53-mediated tumor suppression program. On the other hand, the direct and indirect targeting of p53, as well as the regulation of its stability and activity by specific microRNAs, underlie the importance of the fine-tuning of p53 pathway, affecting the cell fate of damaged/transformed cells. The promising results of miRNAs-based therapeutic approaches in preclinical studies and their entrance in clinical trials demonstrate the feasibility of this strategy in several diseases, including cancer. Molecularly targeted drugs approved so far for HCC treatment show intrinsic or acquired resistances with disease progression in many cases, therefore the identification of effective and non-toxic agents for the treatment of HCC is actually an unmet clinical need. The knowledge of p53/miRNA inter-relations in HCC may provide useful elements for the identification of novel combined approaches in the context of the "personalized-medicine" era., Competing Interests: The authors declare no conflict of interest.

Metabolic changes are associated with cancer, but whether they are just bystander effects of deregulated oncogenic signaling pathways or characterize early phases of tumorigenesis remains unclear. Here we show in a rat model of hepatocarcinogenesis that early preneoplastic foci and nodules that progress towards hepatocellular carcinoma (HCC) are characterized both by inhibition of oxidative phosphorylation (OXPHOS) and by enhanced glucose utilization to fuel the pentose phosphate pathway (PPP). These changes respectively require increased expression of the mitochondrial chaperone TRAP1 and of the transcription factor NRF2 that induces the expression of the rate-limiting PPP enzyme glucose-6-phosphate dehydrogenase (G6PD), following miR-1 inhibition. Such metabolic rewiring exclusively identifies a subset of aggressive cytokeratin-19 positive preneoplastic hepatocytes and not slowly growing lesions. No such metabolic changes were observed during non-neoplastic liver regeneration occurring after two/third partial hepatectomy. TRAP1 silencing inhibited the colony forming ability of HCC cells while NRF2 silencing decreased G6PD expression and concomitantly increased miR-1; conversely, transfection with miR-1 mimic abolished G6PD expression. Finally, in human HCC patients increased G6PD expression levels correlates with grading, metastasis and poor prognosis. Our results demonstrate that the metabolic deregulation orchestrated by TRAP1 and NRF2 is an early event restricted to the more aggressive preneoplastic lesions., Competing Interests: No conflict of interest to declare.

The miR-145-5p, which induces TP53-dependent apoptosis, is down-regulated in several tumors, including hepatocellular carcinomas (HCCs), but some HCCs show physiological expression of this miR. Here we demonstrate that in HCC cells carrying wild-type TP53 the steady activation of the miR-145 signaling selects clones resistant to apoptosis via up-regulation of the oncogenic miR-483-3p. Expression of the miR-145-5p and of the miR-483-3p correlated negatively in non-neoplastic liver (n=41; ρ=-0.342, P=0.028), but positively in HCCs (n=21; ρ=0.791, P<0.0001), which we hypothesized to be due to impaired glucose metabolism in HCCs versus normal liver. In fact, when liver cancer cells were grown in low glucose, miR-145-5p lowered miR-483-3p expression, allowing apoptosis, whereas when cells were grown in high glucose the levels of miR-483-3p increased, reducing the apoptotic rate. This indicates that depending on glucose availability the miR-145-5p has double effects on the miR-483-3p, either inhibitory or stimulatory. Moreover, resistance to apoptosis in clones overexpressing both miR-145-5p and miR-483-3p was abrogated by silencing the miR-483-3p. Our data highlight a novel mechanism of resistance to apoptosis in liver cancer cells harbouring wild type TP53 and suggest a potential role of miR-145-5p and miR-483-3p as druggable targets in a subset of HCCs., Competing Interests: The authors declare no conflicts of interest.

Background & Aims: l-2-Hydroxy acid oxidases are flavin mononucleotide-dependent peroxisomal enzymes, responsible for the oxidation of l-2-hydroxy acids to ketoacids, resulting in the formation of hydrogen peroxide. We investigated the role of HAO2, a member of this family, in rat, mouse and human hepatocarcinogenesis., Methods: We evaluated Hao2 expression by qRT-PCR in the following rodent models of hepatocarcinogenesis: the Resistant-Hepatocyte, the CMD and the chronic DENA rat models, and the TCPOBOP/DENA and TCPOBOP only mouse models. Microarray and qRT-PCR analyses were performed on two cohorts of human hepatocellular carcinoma (HCC) patients. Rat HCC cells were transduced by a Hao2 encoding lentiviral vector and grafted in mice., Results: Downregulation of Hao2 was observed in all investigated rodent models of hepatocarcinogenesis. Interestingly, Hao2 mRNA levels were also profoundly downregulated in early preneoplastic lesions. Moreover, HAO2 mRNA levels were strongly downregulated in two distinct series of human HCCs, when compared to both normal and cirrhotic peri-tumoral liver. HAO2 levels were inversely correlated with grading, overall survival and metastatic ability. Finally, exogenous expression of Hao2 in rat cells impaired their tumorigenic ability., Conclusion: Our work identifies for the first time the oncosuppressive role of the metabolic gene Hao2. Indeed, its expression is severely decreased in HCC of different species and etiology, and its reintroduction in HCC cells profoundly impairs tumorigenesis. We also demonstrate that dysregulation of HAO2 is a very early event in the development of HCC and it may represent a useful diagnostic and prognostic marker for human HCC., (Copyright © 2015 European Association for the Study of the Liver. Published by Elsevier B.V. All rights reserved.)

The performance of circulating biomarkers for the diagnosis of hepatocellular carcinoma (HCC) is sub-optimal. In this study we tested circulating microRNAs as biomarkers for HCC in cirrhotic patients by performing a two stage study: a discovery phase conducted by microarray and a validation phase performed by qRT-PCR in an independent series of 118 patients. Beside miRNAs emerged from the discovery phase, miR-21, miR-221, miR-519d were also tested in the validation setting on the basis of literary and tissue findings. Deregulated microRNAs were assayed in HCC-derived cells in the intracellular compartment, cell culture supernatant and exosomal fraction. Serum and tissue microRNA levels were compared in 14 patients surgically treated for HCC. From the discovery study, it emerged that seven circulating microRNAs were differentially expressed in cirrhotic patients with and without HCC. In the validation set, miR-939, miR-595 and miR-519d were shown to differentiate cirrhotic patients with and without HCC. MiR-939 and miR-595 are independent factors for HCC. ROC curves of miR-939, miR-595 and miR-519d displayed that AUC was higher than AFP. An exosomal secretion of miR-519d, miR-21, miR-221 and miR-1228 and a correlation between circulating and tissue levels of miR-519d, miR-494 and miR-21 were found in HCC patients. Therefore, we show that circulating microRNAs deserve attention as non-invasive biomarkers in the diagnostic setting of HCC and that exosomal secretion contributes to discharging a subset of microRNAs into the extracellular compartment.

Background: Recent data suggest that outcome of hepatocarcinoma is improving., Aims: In order to explore whether survival is also increasing in clinical practice, we compared two multicenter independent in-field cohorts of cirrhotics with newly diagnosed HCCs., Methods: Cohort 1 (C1) consisted of 327 patients enrolled between January and December 1998, and cohort 2 (C2) included 826 patients enrolled between September 2008 and November 2012. Patients were stratified according to Child-Pugh score, MELD score, and HCC staged according to TNM, BCLC systems., Results: At baseline, C2 patients were significantly older, with more frequent comorbidities and better liver function. In C2, HCC was more frequently detected under regular ultrasound surveillance (P < 0.001), BCLC early stages were more frequent, and rates of smaller and uni/paucinodular tumors were significantly higher. Treatment of any type was more frequently offered to C2 patients (P < 0.001). Proportion of patients treated by TACE increased, and radiofrequency ablation was the most used ablative treatment. Survival rate was significantly higher in C2 being C1 and C2 survival at 1-3 years 72-25 and 75-44 %, respectively. Child-Pugh score A, BCLC stage A, single nodule, size ≤ 3 cm, belonging to cohort C2 and treatment per se independently predicted survival., Conclusions: This in-field study showed a trend on improved HCC outcomes over time, which seems to be mainly due to a better presentation thanks to the wider application of surveillance and increased propensity to treat patients. These encouraging data should support further efforts to implement such approach to HCC in everyday clinical practice.

Background/aim: The impact of radiofrequency ablation (RFA) and percutaneous ethanol injection (PEI) on survival in patients with small hepatocellular carcinoma (HCC) is unclear. We compared their efficacy in cirrhotics with single HCC ≤2 cm., Patients and Methods: Two hundred forty-four cirrhotics with single HCC ≤2 cm treated with PEI (108 cases) or RFA (136 cases) were enrolled in the study. Eighty-one patients in each group were selected for propensity score matching analysis., Results: The five-year survival was not significantly different (64.7% in PEI and 72.9% in RFA group) but the 5-year recurrence (73.3% in PEI and 49% in RFA group, p=0.023) and local tumor progression (49% in PEI and 30.1% in RFA group, p=0.018) were higher in the PEI group., Conclusion: PEI and RFA are equally effective in treating HCCs smaller than 2 cm in terms of 5-year survival, despite higher cumulative and local recurrence rates, in patients treated with PEI., (Copyright© 2015 International Institute of Anticancer Research (Dr. John G. Delinassios), All rights reserved.)

To successfully target Notch receptors as part of a multidrug anticancer strategy, it will be essential to fully characterize the factors that are modulated by Notch signaling. We recently reported that Notch3 silencing in HCC results in p53 up-regulation in vitro and, therefore, we focused on the mechanisms that associate Notch3 to p53 protein expression. We explored the regulation of p53 by Notch3 signalling in three HCC cell lines HepG2, SNU398 and Hep3B.We found that Notch3 regulates p53 at post-transcriptional level controlling both Cyclin G1 expression and the feed-forward circuit involving p53, miR-221 and MDM2. Moreover, our results were validated in human HCCs and in a rat model of HCC treated with Notch3 siRNAs. Our findings are becoming an exciting area for further in-depth research toward targeted inactivation of Notch3 receptor as a novel therapeutic approach for increasing the drug-sensitivity, and thereby improving the treatment outcome of patients affected by HCC. Indeed, we proved that Notch3 silencing strongly increases the effects of Nutilin-3.With regard to therapeutic implications, Notch3-specific drugs could represent a valuable strategy to limit Notch signaling in the context of hepatocellular carcinoma over-expressing this receptor.

Background & Aims: Although contrast-enhanced computed tomography (CT), dynamic magnetic resonance (MRI) and fine needle biopsy (FNB) are the standard of care to diagnose hepatocellular carcinoma (HCC), the clinical and economic benefits of the updated AASLD diagnostic algorithm, including the drop of contrast enhanced ultrasound (CEUS), have not been previously evaluated., Methods: 119 de novo liver nodules detected during ultrasound (US) surveillance in 98 cirrhotics, 7 <1cm, 67 1-2cm, 45 >2cm in size, were sequentially examined by CEUS and CT, using MRI as a rescue approach in patients lacking a typical vascular pattern for HCC by one or both contrast techniques in the 1-2cm nodules and by CT in the >2cm nodules. A FNB was performed when required to meet both 2005 and 2010 AASLD criteria., Results: Eighty-four (70%) nodules were HCC: the radiological diagnosis was done in 38 (88%) of those 1-2cm and in 38 (95%) for those >2cm HCCs according to 2010 AASLD criteria. CT or MRI detected 13 HCC nodules that were missed by unenhanced US. Despite an absolute specificity, CEUS failed to identify any HCC uncharacterized by CT or MRI. By updated AASLD criteria, 6 (17%) FNB procedures were spared in patients with 1-2cm nodules (p=0.025), as compared to 2005 criteria. The 2010 vs. 2005 AASLD per patient cost was similar in 1-2cm nodules, 432 € vs. 451 € (p=0.46), but lower in >2cm nodules, 248 € vs. 321 € (p<0.001)., Conclusions: A sequential study with either CT or MRI enhances the radiological diagnosis of HCC and reduces costs and liver biopsy need., (Copyright © 2014 European Association for the Study of the Liver. Published by Elsevier B.V. All rights reserved.)

Unlabelled: The overexpression of microRNA-221 (miR-221) is reported in several human cancers including hepatocellular carcinoma, and its targeting by tailored treatments has been proposed. The evidence supporting the role of miR-221 in cancer is growing and has been mainly focused on the discovery of miR-221 targets as well as on its possible therapeutic exploitations. However, the mechanism sustaining miR-221 aberrant expression remains to be elucidated. In this study, MDM2 (E3 ubiquitin-protein ligase homolog), a known p53 (TP53) modulator, is identified as a direct target of miR-221, and a feed-forward loop is described that sustains miR-221 aberrant expression. Interestingly, miR-221 can activate the p53/mdm2 axis by inhibiting MDM2 and, in turn, p53 activation contributes to miR-221 enhanced expression. Moreover, by modulating the p53 axis, miR-221 impacts cell-cycle progression and apoptotic response to doxorubicin in hepatocellular carcinoma-derived cell lines. Finally, CpG island methylation status was assessed as a causative event associated with miR-221 upregulation in hepatocellular carcinoma cells and primary tumor specimens. In hepatocellular carcinoma-derived cell lines, pharmacologically induced DNA hypomethylation potentiated a significant increase in miR-221 expression. These data were confirmed in clinical specimens of hepatocellular carcinoma in which elevated miR-221 expression was associated with the simultaneous presence of wild-type p53 and DNA hypomethylation., Implications: These findings reveal a novel miR-221-sustained regulatory loop that determines a p53-context-specific response to doxorubicin treatment in hepatocellular carcinoma.

Background & Aims: The aim of this study was to compare liver resection and radiofrequency ablation in patients with single hepatocellular carcinoma ≤3 cm and compensated cirrhosis., Methods: The study involved 544 Child-Pugh A cirrhotic patients (246 in the resection group and 298 in the radiofrequency group) observed in 15 Italian centers. Overall survival and tumor recurrence rates were analyzed using the Kaplan Meier method before and after propensity score matching. Cox regression models were used to identify factors associated with overall survival and tumor recurrence., Results: Two cases of perioperative mortality were observed in the resection group and the rate of major complications was 4.5% in the resection group and 2.0% in the radiofrequency group (p=0.101). Four-year overall survival rates were 74.4% in the resection group and 66.2% in the radiofrequency group (p=0.353). Four-year cumulative HCC recurrence rates were 56% in the resection group and 57.1% in the radiofrequency group (p=0.765). Local tumor progression was detected in 20.5% of ablated patients and in one resected patient (p<0.001). After propensity score matching, both survival and tumor recurrence were still not significantly different although a trend towards lower recurrence was observed in resected patients. Older age and higher alpha-fetoprotein levels were independent predictors of poor overall survival while older age and higher alanine-aminotransferase levels resulted to be independent factors associated with higher recurrence rate., Conclusions: In spite of a higher rate of local tumor progression, radiofrequency ablation can provide results comparable to liver resection in the treatment of single hepatocellular carcinoma ≤3 cm occurring in compensated cirrhosis., (Copyright © 2013 European Association for the Study of the Liver. Published by Elsevier B.V. All rights reserved.)

Background: Radiofrequency Ablation is the most widely performed percutaneous treatment for Hepatocellular Carcinoma. This multicentre study was aimed at assessing the complication, overall survival and disease-free survival rates in cirrhotic patients with single Hepatocellular Carcinoma nodule ≤3 cm undergoing Radiofrequency Ablation., Methods: Data of 365 patients (59% males; mean age 67 ± 8 years), Child-Pugh A/B, with single Hepatocellular Carcinoma nodule ≤3 cm (tumours >2-3 cm = 127/236), showing complete necrosis after Radiofrequency Ablation between 1998 and 2010 in 7 Italian Centers were retrospectively reviewed. Complication, overall survival and disease-free survival rates were analyzed as main clinical end-points., Results: Major complications were observed in 8 patients (2.2%) and minor complications in 23 patients (6.3%). The 3-, and 5-year overall survival rates were 80% and 64%. One hundred and seven patients (29.5%) died, being 41 deaths (38.3%) Hepatocellular Carcinoma-related. At multivariate analysis only age (p = 0.04; OR 2.29), ascites (p < 0.001; OR 3.74) and Child-Pugh class ≥B8 (p = 0.003; OR 2.42) were confirmed as independent predictors for overall survival. The disease-free survival rates at 3- and 5-year were 50%, and 41.8%., Conclusions: Radiofrequency Ablation is an effective and safe tool for the treatment of single Hepatocellular Carcinoma ≤3 cm providing excellent 5-year overall survival and disease-free survival rates. Patient's age and liver status appeared as main determinants of outcome., (Copyright © 2012 Editrice Gastroenterologica Italiana S.r.l. Published by Elsevier Ltd. All rights reserved.)

Aim: Adherence to and the applicability of practice guidelines for the management of hepatocellular carcinoma (HCC) in field practice have not been fully addressed. We designed a multicenter field practice prospective study to evaluate the adherence to the 2005 American Association for the Study of Liver Diseases guidelines in Italy., Materials & Methods: The study began in September 2008 and consecutively enrolled cirrhotic patients with newly diagnosed HCC from 30 local, nonreference centers in Italy. Patients were stratified according to Child-Pugh, the model for end-stage liver disease, tumor-node metastasis, performance status and the Barcelona Clinic Liver Cancer (BCLC) classifications. The diagnostic and therapeutic strategies adopted in each individual patient were recorded. Statistical analysis was carried out on 536 patients using all of the valuable data., Results: A total of 286 (54.5%) patients were ≥70 years old. Comorbidities, recorded in 397 (74%) patients, were classified as moderate to severe in 170 patients (43%). Overall, 174 (59%) patients with early-stage BCLC were ≥70 years; 104 (35%) of these had moderate-to-severe comorbidities and 54% were under a regular US surveillance program. Diagnosis was performed by computed tomography in 93% of patients, contrast-enhanced ultrasound in 62% and MRI in 17%. In patients with nodules of ≤2 cm, adherence to noninvasive diagnostic criteria was 56%. Adherence to the BCLC classification was shown to be suboptimal overall, particularly regarding allocation to surgical procedures, and a total of 119 patients (40%) with BCLC stage A did not receive curative therapies., Conclusions: This multicenter survey showed that, in the 'real world', adherence to the both the diagnostic and therapeutic American Association for the Study of Liver Diseases 2005 algorithms was low, particularly in patients with early-stage HCC. Difficulties in applying the algorithms in routine clinical practice and the high prevalence of older patients with relevant comorbidities may account for our findings. Strategies to help improve adherence to international guidelines for HCC in field practice are required.

Unlabelled: MicroRNA-221 (miR-221) is one of the most frequently and consistently up-regulated microRNAs (miRNAs) in human cancer. It has been hypothesized that miR-221 may act as a tumor promoter. To demonstrate this, we developed a transgenic (TG) mouse model that exhibits an inappropriate overexpression of miR-221 in the liver. Immunoblotting and immunostaining confirmed a concomitant down-regulation of miR-221 target proteins. This TG model is characterized by the emergence of spontaneous nodular liver lesions in approximately 50% of male mice and by a strong acceleration of tumor development in 100% of mice treated with diethylnitrosamine. Similarly to human hepatocellular carcinoma, tumors are characterized by a further increase in miR-221 expression and a concomitant inhibition of its target protein-coding genes (i.e., cyclin-dependent kinase inhibitor [Cdkn]1b/p27, Cdkn1c/p57, and B-cell lymphoma 2-modifying factor). To validate the tumor-promoting effect of miR-221, we showed that in vivo delivery of anti-miR-221 oligonucleotides leads to a significant reduction of the number and size of tumor nodules., Conclusions: This study not only establishes that miR-221 can promote liver tumorigenicity, but it also establishes a valuable animal model to perform preclinical investigations for the use of anti-miRNA approaches aimed at liver cancer therapy., (Copyright © 2012 American Association for the Study of Liver Diseases.)

MiR-519d belongs to the chromosome 19 miRNA cluster (C19MC), the largest human miRNA cluster. One of its members, miR-519d, is over-expressed in hepatocellular carcinoma (HCC) and we characterized its contribution to hepatocarcinogenesis. In HCC cells, the over-expression of miR-519d promotes cell proliferation, invasion and impairs apoptosis following anticancer treatments. These functions are, at least in part, exerted through the direct targeting of CDKN1A/p21, PTEN, AKT3 and TIMP2. The mechanisms underlying miR-519d aberrant expression in HCC were assayed by genomic DNA amplification, methylation analysis and ChIP assay. The aberrant hypomethylation of C19MC and TP53 were respectively identified as an epigenetic change allowing the aberrant expression of miR-519d and one of the factors able to activate its transcription. In conclusion, we assessed the oncogenic role of miR-519d in HCC by characterizing its biological functions, including the modulation of response to anticancer treatments and by identifying CDKN1A/p21, PTEN, AKT3 and TIMP2 among its targets., (Copyright © 2012 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.)

We described the optimization, by molecular modelling, of small pyrazole derivatives, as kinase inhibitors, obtained through a 1,3-dipolar cycloaddition between nitrile imines and functionalized acetylenes. The two compounds, selected as potential agents active against hepatocellular carcinoma (HCC) were then evaluated in vitro for their biological activity on HCC-derived cell lines. The compounds show a promising inhibitory growth efficacy (IC(50) 50-100 μM) in SNU449 cell line, as well as block of cell cycle progression and induction of apoptosis, and can be considered as lead compounds for further SAR developments., (Copyright © 2011 Elsevier Masson SAS. All rights reserved.)

Hepatocellular carcinoma (HCC) is the most common primary liver cancer and the third most common cause of cancer-related death worldwide. In 90% of cases, HCC arises on a background of cirrhosis which, in turns, results from hepatitis (HBV and HCV) infections, alcohol abuse, metabolic disorders including NASH, and genetic metabolic diseases, autoimmune hepatitis, primary biliary cirrhosis and exposure to environmental carcinogens. The molecular mechanisms underlying HCC development are still only partially known. Despite a high molecular variability, the deregulation of definite oncogenic pathways has been confirmed as a common finding in HCC. Among these, the molecular ways controlling proliferation, apoptosis and migration play a major role. In recent years, a new class of regulatory RNAs, the microRNAs, has been discovered and their deregulated expression has been linked to the molecular pathogenesis of many cancers because of their ability to strongly impact on the expression of crucial messenger RNAs. This review focuses on some of the most relevant evidence concerning the contribution of microRNA aberrant expression to HCC development.

Unlabelled: Deregulated cell proliferation and apoptosis play a major role in hepatocellular carcinoma (HCC). MicroRNAs participate in the modulation of key molecules linked to hepatocarcinogenesis., Purpose: This study aims to investigate the role of miR-221 in the modulation of Bmf, a proapoptotic BH3-only protein, and to characterize miR-221 contribution to hepatocarcinogenesis through modulation of apoptosis., Experimental Design: Transfection of miR-221 and anti-miR-221 in HCC-derived cell lines and luciferase reporter assay were used to assess Bmf as a target of miR-221. Modulation of miR-221 and Bmf expression contributed to characterize their role in anoikis. Primary HCC tissues were analyzed to assess the clinical relevance of in vitro findings., Results: Enforced miR-221 expression caused Bmf down-regulation, whereas anti-miR-221 induced its up-regulation. A luciferase reporter assay confirmed Bmf as a target of miR-221. Following matrix detachment, miR-221 silencing led to increased apoptotic cell death. The analysis of HCC tissues revealed an inverse correlation between miR-221 and Bmf expression and a direct correlation between Bmf and activated caspase-3, as a marker of apoptosis. High miR-221 levels were associated with tumor multifocality and reduced time to recurrence after surgery., Conclusions: Our results indicate that miR-221, by targeting Bmf, inhibits apoptosis. Moreover, in HCC, miR-221 overexpression is associated with a more aggressive phenotype. These findings, together with the previously reported modulation of CDKN1B/p27 and CDKN1C/p57, show that miR-221 simultaneously affects multiple pro-oncogenic pathways and suggest miR-221 as a potential target for nonconventional treatment against HCC.

Hepatocellular carcinoma (HCC) is the third cause of cancer-related death worldwide. Curative options for HCC are limited and exclusively available for patients carrying an early stage HCC. In advanced stages, traditional chemotherapy proved to be only marginally effective or even toxic. Thus, the identification of new treatment options is needed. New targets for non-conventional treatment will necessarily take advantage of progresses on the molecular pathogenesis of HCC. MicroRNAs (miRNAs) are a group of tiny RNAs with a fundamental role in the regulation of gene expression. Aberrant expression of several miRNAs was found to be involved in human hepatocarcinogenesis. miRNA expression signatures were correlated with bio-pathological and clinical features of HCC. In some cases, aberrantly expressed miRNAs could be linked to cancer-associated pathways, indicating a direct role in liver tumourigenesis. For example, up-regulation of mir-221 and mir-21 could promote cell cycle progression, reduce cell death and favour angiogenesis and invasion. These findings suggest that miRNAs could become novel molecular targets for HCC treatment. The demonstration of in vivo efficacy and safety of anti-miRNA compounds has opened the way to their use in clinical trials.

The identification of target mRNAs is a key step for assessing the role of aberrantly expressed microRNAs in human cancer. MiR-221 is upregulated in human hepatocellular carcinoma (HCC) as well as in other malignancies. One proven target of miR-221 is CDKN1B/p27, whose downregulation affects HCC prognosis. Here, we proved that the cyclin-dependent kinase inhibitor (CDKI) CDKN1C/p57 is also a direct target of miR-221. Indeed, downregulation of both CDKN1B/p27 and CDKN1C/p57 occurs in response to miR-221 transfection into HCC-derived cells and a significant upregulation of both CDKN1B/p27 and CDKN1C/p57 occurs in response to antimiR-221 transfection. A direct interaction of miR-221 with a target site on the 3' UTR of CDKN1C/p57 mRNA was also demonstrated. By controlling these two CDKIs, upregulation of miR-221 can promote growth of HCC cells by increasing the number of cells in S-phase. To assess the relevance of these studies in primary tumors, matched HCC and cirrhosis samples were assayed for miR-221, for CDKN1B/p27 and CDKN1C/p57 expression. MiR-221 was upregulated in 71% of HCCs, whereas CDKN1B/p27 and CDKN1C/p57 proteins were downregulated in 77% of cases. A significant inverse correlation between miR-221 and both CDKN1B/p27 and CDKN1C/p57 was found in HCCs. In conclusion, we suggest that miR-221 has an oncogenic function in hepatocarcinogenesis by targeting CDKN1B/p27 and CDKN1C/p57, hence promoting proliferation by controlling cell-cycle inhibitors. These findings establish a basis toward the development of therapeutic strategies aimed at blocking miR-221 in HCC.

Background: Contrast enhanced ultrasound (CEUS) is an imaging technique which appeared on the market around the year 2000 and proposed for the detection of liver metastases in gastrointestinal cancer patients, a setting in which accurate staging plays a significant role in the choice of treatment., Methods: A total of 109 patients with colorectal (n = 92) or gastric cancer prospectively underwent computed tomography (CT) scan and conventional US evaluation followed by real time CEUS. A diagnosis of metastases was made by CT or, for lesions not visible at CT, the diagnosis was achieved by histopathology or by a malignant behavior during follow-up., Results: Of 109 patients, 65 were found to have metastases at presentation. CEUS improved sensitivity in metastatic livers from 76.9% of patients (US) to 95.4% (p <0.01), while CT scan reached 90.8% (p = n.s. vs CEUS, p < 0.01 vs US). CEUS and CT were more sensitive than US also for detection of single lesions (87 with US, 122 with CEUS, 113 with CT). In 15 patients (13.8%), CEUS revealed more metastases than CT, while CT revealed more metastases than CEUS in 9 patients (8.2%) (p = n.s.)., Conclusion: CEUS is more sensitive than conventional US in the detection of liver metastases and could be usefully employed in the staging of patients with gastrointestinal cancer. Findings at CEUS and CT appear to be complementary in achieving maximum sensitivity.

We investigated the role of microRNAs (miRNAs) in the pathogenesis of human hepatocellular carcinoma (HCC). A genome-wide miRNA microarray was used to identify differentially expressed miRNAs in HCCs arisen on cirrhotic livers. Thirty-five miRNAs were identified. Several of these miRNAs were previously found deregulated in other human cancers, such as members of the let-7 family, mir-221, and mir-145. In addition, the hepato-specific miR-122a was found down-regulated in approximately 70% of HCCs and in all HCC-derived cell lines. Microarray data for let-7a, mir-221, and mir-122a were validated by Northern blot and real-time PCR analysis. Understanding the contribution of deregulated miRNAs to cancer requires the identification of gene targets. Here, we show that miR-122a can modulate cyclin G1 expression in HCC-derived cell lines and an inverse correlation between miR-122a and cyclin G1 expression exists in primary liver carcinomas. These results indicate that cyclin G1 is a target of miR-122a and expand our knowledge of the molecular alterations involved in HCC pathogenesis and of the role of miRNAs in human cancer.

Background: Because hepatic cirrhosis is a major risk factor for hepatocellular carcinoma, recent guidelines by the European Association for the Study of the Liver (EASL) on clinical management of hepatocellular carcinoma recommend periodic ultrasound surveillance of cirrhotic patients with immediate workup for nodules >1 cm; an increase in the frequency of screening is considered sufficient for smaller lesions., Aims: To determine the actual risk of hepatocellular carcinoma associated with the latter lesions and to assess the role of ultrasound guided-fine needle biopsy in their diagnosis., Patients and Methods: Data were analysed for 294 new nodular lesions <20 mm, including 48 that were <10 mm, detected during a prospective multicentre study involving ultrasound surveillance of 4375 patients with hepatic cirrhosis. In the absence of alpha fetoprotein (AFP) levels diagnostic of hepatocellular carcinoma, ultrasound guided-fine needle biopsy was performed (n = 274). AFP and fine needle biopsy diagnoses of malignancies (hepatocellular carcinoma and lymphoma) were considered definitive. Non-malignant fine needle biopsy diagnoses (dysplastic or regenerative nodule) were verified by a second imaging study. Diagnoses of hepatocellular carcinoma based on this study were considered definitive; non-malignant imaging diagnoses were considered definitive after at least one year of clinical and ultrasound follow up., Results: Overall, 258/294 (87.6%) nodules proved to be hepatocellular carcinoma, including 33/48 (68.7%) of those < or =10 mm. Overall typing accuracy of ultrasound guided-fine needle biopsy was 89.4%, and 88.6% for lesions < or =10 mm., Conclusions: In a screening population, well over half of very small nodules arising in cirrhotic livers may prove to be hepatocellular carcinoma, and approximately 90% of these malignancies can be reliably identified with ultrasound guided-fine needle biopsy.

Background/aims: Diagnosis of hepatocellular carcinoma (HCC) relies strongly on the detection of hypervascularity in the arterial phase and, in this setting, spiral computed tomography (CT) is the most widely used method. This prospective study aimed to investigate the usefulness of low mechanical index harmonic ultrasound (US), using a second generation contrast-enhanced technique, in the assessment of vascular pattern of HCC shown to be hypervascular at spiral CT., Methods: A total of 79 cirrhotic patients with 103 nodules (mean+/-SD 28+/-13 mm) with arterial hypervascularity at spiral CT were studied. US examination was performed by perfusional sonography, using a new dedicated technology (CnTI-Esaote trade mark ), operating at low mechanical index, after injection of a second generation contrast agent (SonoVue trade mark -Bracco), allowing detection of tumoral flow during arterial phase., Results: Selective arterial enhancement on perfusional sonography was observed in 94 /103 nodules (91.3%), with a sensitivity of 66.6, 87.5, 91.7, and 97.3% in nodules 123 cm respectively., Conclusions: Perfusional sonography shows good diagnostic agreement with spiral CT in hypervascular HCC and may be proposed for the immediate vascular characterization of nodules detected at US and used as second imaging technique to confirm hypervascularity in cirrhotic nodules.

Purpose: To evaluate the usefulness of percutaneous radio-frequency (RF) thermal ablation of nonresectable hepatocellular carcinoma (HCC) after occlusion of the tumor arterial supply., Materials and Methods: Sixty-two patients with cirrhosis and biopsy-proved HCC underwent RF ablation after interruption of the tumor arterial supply by means of occlusion of either the hepatic artery with a balloon catheter (40 patients) or the feeding arteries with gelatin sponge particles (22 patients)., Results: After a single RF procedure in 56 patients and after two procedures in six patients, spiral computed tomography (CT) demonstrated a nonenhancing area corresponding in shape to the previously identified HCC, which was suggestive of complete necrosis. No major complications occurred. Two patients subsequently underwent surgical resection; the remaining 60 patients were followed up with spiral CT. During a mean follow-up of 12.1 months, 11 HCC nodules showed areas of local progression; 49 were identified as nonenhancing areas with a 40%-75% reduction in maximum diameter. The 1-year estimate of failure risk was 19% for local recurrence and 45% for overall intrahepatic recurrence. The estimated 1-year survival was 87%. Histopathologic analysis of one autopsy and two surgical specimens revealed more than 90% necrosis in one specimen and 100% necrosis in two., Conclusion: HCC nodules 3.5-8.5 cm in diameter can be ablated in one or two RF sessions after occlusion of the tumor arterial supply.

In two consecutive series of patients with hepatocellular carcinoma (HCC), we compared clinico-laboratory and ultrasonographic characteristics, diagnostic work-up, survival of untreated patients and, finally, therapeutic choices. In addition of the clinical examination, we tested for blood serum alpha-fetoprotein levels, HBsAg and anti-HCV antibodies. Ultrasonography was performed in all the patients. In most cases, a pathologic diagnosis was obtained by ultrasound-guided fine-needle biopsy. As curative treatment we considered open surgery, percutaneous alcohol injection and radio frequency thermal ablation. In the second series, we observed an increased number of patients with compensated cirrhosis and with small HCCs, therefore the number of patients undergoing a potentially curative treatment was higher. The percentage of multiple tumours was comparable in two series implying the presence of two kinds of HCC, different ¿ab initio'. The survival rate of untreated patients was better in the second series.

In a consecutive series of 317 patients with hepatocellular carcinoma (HCC), 32 (10.1%) had 35 extrahepatic primary malignant neoplasms (PMNs) (3 patients had triple cancers). Twenty-five PMNs occurred before the diagnosis of HCC, 7 were synchronous and 3 metachronous. These 35 PMNs were: 6 cancers of the colon, 3 of the stomach, 1 of the rectum, 4 of the breast, 2 of the lung, 1 of the larynx, 3 of the prostate, 1 of the penis, 1 of the urinary bladder, 1 of the uterus, 2 of the skin, and the remaining 10 were immunoproliferative cancers, all of B cell origin (7 non-Hodgkin's lymphoma, 2 multiple myeloma, and 1 chronic lymphocytic leukemia). Thus, in this series, B-lymphocyte-derived neoplasms were the most frequent PMNs associated with HCC. These 10 patients showed no difference for age, male:female ratio, HCC cytotype, presence of cirrhosis, alcohol abuse, markers related to hepatitis B and C virus, and serum level of alpha-fetoprotein when compared with the 22 patients with HCC and other PMNs and the 285 with HCC alone. B cell neoplasms constitute half of the synchronous or metachronous cancers, and must, therefore, be kept in mind in the management of HCC patients.

A 50-year-old man was diagnosed with non-Hodgkin's lymphoma (NHL) in 1980; he was treated with chemotherapy and achieved complete remission. Six years later he reported upper abdominal pain. Ultrasound (US) showed a 3-cm mass in the right lobe of the liver. Needle aspiration showed hepatocellular carcinoma (HCC). The patient was treated with radical resection of the tumor. Three years later (June 1989), abdominal US showed two lesions in the right lobe of the liver. Needle aspiration and tissue core biopsy showed NHL in one lesion and HCC in the other. The lymphomatous lesion resolved after chemotherapy. The patient died 30 months later (January 1992) from a gastrointestinal hemorrhage; the NHL was in complete remission. This case of the simultaneous presence of HCC and hepatic lymphoma is, to our knowledge, the first diagnosed in vivo.

From January 1984 to December 1991 we carried out a prospective surveillance of the development of hepatocellular carcinoma (HCC) in 200 cirrhotic patients, monitored from the first histologic diagnosis of cirrhosis. Real-time ultrasonography (US) and serum alpha-fetoprotein (AFP) assays were performed every 3-6 months. During this period we detected HCC in 38 patients. The overall cumulative incidence of HCC in the 8 years was 41%, with a yearly incidence of 5.1%. Eighteen of 38 patients (48%) had a tumor < 5 cm in diameter. AFP reached diagnostic levels (> 500 ng/ml) in eight cases only (21%). Patients with initial AFP values > 20 ng/ml developed HCC more frequently than patients with values < or = 20 ng/ml; the percentage of HCC was statistically higher (p < 0.01) in patients in Child's B and C than in Child's A class. A periodic follow-up, using US and AFP, is suggested for the early diagnosis of HCC in cirrhotic patients.

From June 1981 to June 1989 we diagnosed 174 cases of hepatocellular carcinoma (HCC) at our institution (Piacenza, Northern Italy). Average age was 65.6 years; male to female ratio 3.4. 149 patients were cirrhotic (85.6%); alcohol abuse was present in 88/169 (52.1%); in 53/145 patients all hepatitis B virus markers were negative. Alpha-fetoprotein showed a low diagnostic sensitivity (values above 500 ng only in 49/169 or 29.0%). We used ultrasound (US) examination with a very high identification rate in all cases; pathological diagnosis was achieved by US guided fine-needle aspiration biopsy in 135 patients; in 13, by laparoscopy-histology. Metastases were found in 24/169 cases (14.2%); a second malignancy was diagnosed in 13/169 (7.7%): the most common association was HCC-non-Hodgkin lymphoma. Only 14 patients could be referred to surgery, which significantly improved prognosis.

In 170 cases of hepatocellular carcinoma, ultrasound showed a high sensitivity in identifying focal liver lesions. Fine needle aspiration biopsy guided by ultrasound yielded a pathological diagnosis in the majority of cases. The advantages of this technique, its high diagnostic yield and low cost, render the older technique of blind percutaneous biopsy using a coarse needle obsolete. Laparoscopy retains its essential role in selected cases. Complementary use of fine needle aspiration biopsy under ultrasound guidance and laparoscopy assures the highest rate of diagnostic accuracy in hepatocellular carcinoma. We confirm the poor sensitivity of alpha fetoprotein.

In 481 patients, with sonographically suspected neoplastic involvement of the liver, we performed an ultrasonically guided fine-needle aspiration biopsy with 22-gauge Chiba needle. In the 441 controlled cases, we obtained 68.5% true positive, 26.5% true negative, and 5% false negative, with a sensitivity of 93.2%, specificity of 100%, and overall accuracy of 95%. The true positive include 42.1% hepatocellular carcinomas, 28.1% metastases from known extrahepatic malignancy, 24.5% metastases from unknown primary site, 5% hepatic involvement by non-Hodgkin lymphomas, and 0.3% undetermined malignancy. Also, in a larger series, ultrasonically guided fine-needle biopsy confirms its high specificity and overall accuracy. The cytologic assessment of the obtained material is almost always adequate. The use of immediate cytologic staining reduces the number of passes in each lesion with absence of insufficient sampling. Biopsy with fine cutting needles should be reserved only for cases not diagnosed by cytology.

Antibodies against hepatitis C virus (anti-HCV) were detected in 60.8% of 78 patients with hepatocellular carcinoma (HCC). Cirrhosis, present in most of the patients, as well as alcohol abuse, age, sex, and alpha-fetoprotein were equally distributed in the anti-HCV-positive and -negative groups. HBsAg positivity was significatively higher in negative anti-HCV group. By contrast, hepatitis B virus (HBV) antibodies were detected more frequently in positive anti-HCV patients than in the negative anti-HCV group. These data must be considered with caution because of the small number of HBsAg-positive patients. It is concluded that the high prevalence of anti-HCV in patients with HCC may suggest an etiological role of the hepatitis C virus, although in relationship to age, alcohol abuse and cirrhosis, the similarity in the two groups questions this hypothesis.