Vito R. Cicinnati

Radioembolization with yttrium‐90 glass microspheres in hepatocellular carcinoma: European experience on safety and long‐term survival

Radioembolization has been demonstrated to allow locoregional therapy of patients with hepatocellular carcinoma not eligible for transarterial chemoembolization or other local therapies. The aim of this study was to validate evidence of the safety and efficacy of this treatment in a European sample of patients with advanced hepatocellular carcinoma (HCC). Therefore, 108 consecutive patients with advanced HCC and liver cirrhosis were included. Yttrium‐90 (Y‐90) microspheres were administered in a lobar fashion over the right or left branch of the hepatic artery. The response to treatment was evaluated by computed tomography (CT) imaging applying Response Evaluation Criteria in Solid Tumors (RECIST) and World Health Organization (WHO) criteria with recent European Association for the Study of the Liver / National Cancer Institute (EASL/NCI) amendments. Time to progression (TTP) and overall survival were estimated by the Kaplan‐Meier method. In all, 159 treatment sessions were performed ranging between one to three treatments per patient. The mean radiation dose per treatment was 120 (±18) Gy. According to EASL criteria, complete responses were determined in 3% of patients, partial responses in 37%, stable disease 53%, and primary progression in 6% of patients. TTP was 10.0 months, whereas the median overall survival was 16.4 months. No lung or visceral toxicity was observed. The most frequently observed adverse events was a transient fatigue‐syndrome. Conclusion: Radioembolization with Y‐90 glass microspheres for patients with advanced HCC is a safe and effective treatment which can be utilized even in patients with compromised liver function. Because TTP and survival appear to be comparable to systemic therapy in selected patients with advanced HCC, randomized controlled trials in combination with systemic therapy are warranted. (HEPATOLOGY 2010;52:1741‐1749)

Right living donor liver transplantation ; An option for adult patients ; Single institution experience with 74 patients

Objective: To present an institutional experience with the use of right liver grafts in adult patients and to assess the practicability and efficacy of this procedure by analyzing the results. Summary Background Data: Living donor liver transplantation (LDLT) for the pediatric population has gained worldwide acceptance. In the past few years, LDLT has also become feasible for adult patients due to technical evolution in hepatobiliary surgery and increased experience with reduced-size and split-liver transplants. Nevertheless, some graft losses remain unexplained and are possibly due to unrecognized venous outflow problems. Methods: From April 1998 to September 2002, we performed 74 right LDLTs (segments 5–8). The 74 donors were selected from 474 candidates according to standard protocol. The median age of the donors was 35 years (range 18–58 years) and 51 years (range 18–64 years) in recipients. Standard and extended indications for transplantation were considered. Over the period reported, technical modifications in the bile duct anastomosis (duct-to-duct, end-to-end, or end-to-side) and a new graft implantation technique that provides maximized venous outflow, leading to outcome improvement, were developed. Results: 64.9% of patients had liver cirrhosis and 35.1% had malignancy. While 44 donors (59.5%) presented an uneventful postoperative course, 27% minor (pleural effusion, pneumonia, venous thrombosis, wound infection, incisional hernia) and 13.5% major (biliary leakage, death of a donor due to unrecognized hereditary liver disease, and consecutive liver insufficiency) complications were documented. In recipients, 23% biliary complications and 6.8% hepatic artery thrombosis occurred. The overall patient and graft survival rate after 1 year was 79.4% and 75.3%, respectively. In cases with extended indication, the patient survival rate was 74% and the graft survival rate 68% at 12 months. Using technical modifications in the last 10 recipients, including 2 critically decompensated cirrhotics, the survival rate was 100% at a median follow-up of 3.5 months. Conclusions: In our transplant program, living donor liver transplantation has become a standard option in the adult patient population. The critical issue of this procedure is donor morbidity. Technical improvements in the harvesting and implantation of right grafts can also offer hope to patients with challenging forms of end-stage liver disease or malignant liver tumors.

Increased Levels of Interleukin-10 in Serum from Patients with Hepatocellular Carcinoma Correlate with Profound Numerical Deficiencies and Immature Phenotype of Circulating Dendritic Cell Subsets

Increased levels of interleukin (IL)-10 have been described as a negative prognostic indicator for survival in patients with various types of cancer. IL-10 exerts tolerogenic and immunosuppressive effects on dendritic cells, which are crucial for the induction of an antitumor immune response. Blood dendritic cell antigen (BDCA)-2 and BDCA-4 are specifically expressed by CD123bright CD11c− plasmacytoid dendritic cells; whereas BDCA-1 and BDCA-3 define 2 distinct subsets of CD11c+ myeloid dendritic cells. In this study, the T-helper cell (Th)1/Th2 cytokine serum profile of 65 hepatocellular carcinoma patients was assessed. We found that serum levels of IL-10 were substantially increased in hepatocellular carcinoma patients as compared with controls. Peripheral blood mononuclear cells from healthy volunteers were exposed to recombinant human (rh)IL-10 in vitro to additionally characterize its impact on distinct blood dendritic cell subsets. A dramatic decrease of all myeloid dendritic cell (MDC) and plasmacytoid dendritic cell (PDC) subsets was detectable after 24 hours of continuous rhIL-10 exposure. Moreover, the expression of HLA-DR, CD80 and CD86, was significantly reduced on rhIL-10-treated dendritic cell subsets. Direct ex vivo flow cytometric analysis of various dendritic cell subpopulations in peripheral blood from hepatocellular carcinoma patients revealed an immature phenotype and a substantial reduction of circulating dendritic cells that was associated with increased IL-10 concentrations in serum and with tumor progression. These findings confirm a predominantly immunosuppressive role of IL-10 for circulating dendritic cells in patients with hepatocellular carcinoma and, thus, may indicate novel aspects of tumor immune evasion.

Role of microRNA-199a-5p and discoidin domain receptor 1 in human hepatocellular carcinoma invasion

BackgroundMicro-ribonucleic acid (miRNA)-199a-5p has been reported to be decreased in hepatocellular carcinoma (HCC) compared to normal tissue. Discoidin domain receptor-1 (DDR1) tyrosine kinase, involved in cell invasion-related signaling pathway, was predicted to be a potential target of miR-199a-5p by the use of miRNA target prediction algorithms. The aim of this study was to investigate the role of miR-199a-5p and DDR1 in HCC invasion.MethodsMature miR-199a-5p and DDR1 expression were evaluated in tumor and adjacent non-tumor liver tissues from 23 patients with HCC undergoing liver resection and five hepatoma cell lines by the use of real-time quantitative RT-PCR (qRT-PCR) analysis. The effect of aberrant miR-199a-5p expression on cell invasion was assessed in vitro using HepG2 and SNU-182 hepatoma cell lines. Luciferase reporter assay was employed to validate DDR1 as a putative miR-199a-5p target gene. Regulation of DDR1 expression by miR-199a-5p was assessed by the use qRT-PCR and western blotting analysis.ResultsA significant down-regulation of miR-199a-5p was observed in 65.2% of HCC tissues and in four of five cell lines. In contrast, DDR1 expression was significantly increased in 52.2% of HCC samples and in two of five cell lines. Increased DDR1 expression in HCC was associated with advanced tumor stage. DDR1 was shown to be a direct target of miR-199a-5p by luciferase reporter assay. Transfection of miR-199a-5p inhibited invasion of HepG2 but not SNU-182 hepatoma cells.ConclusionsDecreased expression of miR-199a-5p contributes to increased cell invasion by functional deregulation of DDR1 activity in HCC. However, the effect of miR-199a-5p on DDR1 varies among individuals and hepatoma cell lines. These findings may have significant translational relevance for development of new targeted therapies as well as prognostic prediction for patients with HCC.

Hepatitis B virus-induced defect of monocyte-derived dendritic cells leads to impaired T helper type 1 response in vitro: mechanisms for viral immune escape

Dendritic cells (DC) are the most potent antigen‐presenting cells and play a central role in the induction of antiviral immune responses. Recently, we have shown that monocyte‐derived DC (MoDC) from patients with chronic hepatitis B virus (HBV) infection are functionally impaired. In our present study MoDC from healthy subjects were propagated in vitro and inoculated with HBV particles to investigate the precise mechanisms that underly MoDC dysfunction. T‐cell proliferation assays revealed an impaired allostimulatory capacity of HBV‐inoculated MoDC (HBV‐MoDC) as well as a lower potential of stimulating autologous T cells against a recall antigen in comparison to control‐MoDC. Interleukin‐2, tumour necrosis factor‐α and interferon‐γ production by T cells in proliferation assays with HBV‐MoDC was significantly lower than with control‐MoDC and correlated with lower IL‐12 production in HBV‐MoDC cultures. The presence of the nucleoside analogue lamivudine (3TC), an inhibitor of HBV replication, restored impaired allostimulatory function of HBV‐MoDC and up‐regulated major histocompatibility complex class II expression. These results show that HBV infection compromises the antigen‐presenting function of MoDC with concomitant impairment of T helper cell type 1 responses. This may play an important role for viral immune escape leading to chronic HBV infection. However, 3TC treatment can overcome HBV‐MoDC‐related T‐cell hyporeactivity and this underscores its important role in enhanced immune responses to HBV.

Validation of putative reference genes for gene expression studies in human hepatocellular carcinoma using real-time quantitative RT-PCR

BackgroundReference genes, which are often referred to as housekeeping genes are frequently used to normalize mRNA levels between different samples in quantitative reverse transcription polymerase chain reaction (qRT-PCR). The selection of reference genes is critical for gene expression studies because the expression of these genes may vary among tissues or cells and may change under certain circumstances. Here, a systematic evaluation of six putative reference genes for gene expression studies in human hepatocellular carcinoma (HCC) is presented.MethodsSix genes, beta-2-microglobulin (B2M), glyceraldehyde-3-phosphate dehydrogenase (GAPDH), hydroxymethyl-bilane synthase (HMBS), hypoxanthine phosphoribosyl-transferase 1 (HPRT1), succinate dehydrogenase complex, subunit A (SDHA) and ubiquitin C (UBC), with distinct functional characteristics and expression patterns were evaluated by qRT-PCR. Inhibitory substances in RNA samples were quantitatively assessed and controlled using an external RNA control. The stability of selected reference genes was analyzed using both geNorm and NormFinder software.ResultsHMBS and GAPDH were identified as the optimal reference genes for normalizing gene expression data between paired tumoral and adjacent non-tumoral tissues derived from patients with HCC. HMBS, GAPDH and UBC were identified to be suitable for the normalization of gene expression data among tumor tissues; whereas the combination of HMBS, B2M, SDHA and GAPDH was suitable for normalizing gene expression data among five liver cancer cell lines, namely Hep3B, HepG2, HuH7, SK-HEP-1 and SNU-182. The determined gene stability was increased after exclusion of RNA samples containing relatively higher inhibitory substances.ConclusionOf six genes studied, HMBS was found to be the single best reference gene for gene expression studies in HCC. The appropriate choice of combination of more than one reference gene to improve qRT-PCR accuracy depends on the kind of liver tissues or cells under investigation. Quantitative assessment and control of qRT-PCR inhibitors using an external RNA control can reduce the variation of qRT-PCR assay and facilitate the evaluation of gene stability. Our results may facilitate the choice of reference genes for expression studies in HCC.

MicroRNA-101 inhibits human hepatocellular carcinoma progression through EZH2 downregulation and increased cytostatic drug sensitivity

BACKGROUND & AIMS Oncogene polycomb group protein enhancer of zeste homolog 2 (EZH2) has been proposed to be a target gene of putative tumor suppressor microRNA-101 (miR-101). The aim of our study was to investigate the functional role of both miR-101 and EZH2 in human hepatocellular carcinoma (HCC). METHODS MiR-101 and EZH2 expressions were evaluated in tumor tissues of 99 HCC patients and 7 liver cancer cell lines by real-time PCR. Luciferase reporter assay was employed to validate whether EZH2 represents a target gene of miR-101. The effect of miR-101 on HCC growth as well as programmed cell death was studied in vitro and in vivo. RESULTS MiR-101 expression was significantly downregulated in most of HCC tissues and all cell lines, whereas EZH2 was significantly overexpressed in most of HCC tissues and all cell lines. There was a negative correlation between expression levels of miR-101 and EZH2. Luciferase assay results confirmed EZH2 as a direct target gene of miR-101, which negatively regulates EZH2 expression in HCC. Ectopic overexpression of miR-101 dramatically repressed proliferation, invasion, colony formation as well as cell cycle progression in vitro and suppressed tumorigenicity in vivo. Furthermore, miR-101 inhibited autophagy and synergized with either doxorubicin or fluorouracil to induce apoptosis in tumor cells. CONCLUSION Tumor suppressor miR-101 represses HCC progression through directly targeting EZH2 oncogene and sensitizes liver cancer cells to chemotherapeutic treatment. Our findings provide significant insights into molecular mechanisms of hepatocarcinogenesis and may have clinical relevance for the development of novel targeted therapies for HCC.

Liver transplantation for patients with metastatic endocrine tumors: Single‐center experience with 15 patients

In contrast to other secondary liver malignancy, orthotopic liver transplantation (OLT) is considered as a treatment modality for nonresectable endocrine liver metastases in selected patients. However, only few series have assessed patient selection criteria and long‐term results, and no reports have focused on the impact of new technologies in this regard. Between 1992 and 2004, 28 patients with malignant endocrine tumors underwent evaluation for OLT according to our protocol. Data were entered into a prospective database. During pretransplant evaluation, somatostatin receptor scintigraphy detected extrahepatic metastases not diagnosed in standard imaging in 10 patients. Of them, 3 showed aberrant Ki67 labeling results. One patient was excluded from further evaluation due to severe carcinoid heart. Thus far, 15 patients, 10 men and 5 women, aged 37 to 67 years, were subjected to the transplant procedure (11 deceased donor OLT, 3 living donor liver transplantations, and 1 cluster transplantation). Four patients died during the hospital treatment. The median follow‐up of the discharged patients was 60.8 months. The actuarial patient survival was 78.3% at 1 year and 67.2% at 5 years. The actuarial 1‐, 2‐, and 5‐year tumor‐free survival amounted to 69.4%, 48.3%, and 48.3%, respectively. Two patients underwent surgery for isolated tumor recurrence. In 2 patients, peptide receptor radiotherapy was carried out because of multilocular recurrent disease. In conclusion, liver transplantation is a realistic therapeutic option for highly selected patients with hepatic metastases of endocrine tumors. Our strategy, which implements strict pretransplant selection and aggressive surgical approach, in case of disease recurrence, in addition to systemic radiopeptide treatment, led to an excellent long‐term survival cure, however, is unlikely to be achieved. Liver Transpl 12:1089–1096, 2006.

Efficacy, safety, and immunosuppressant adherence in stable liver transplant patients converted from a twice-daily tacrolimus-based regimen to once-daily tacrolimus extended-release formulation

The aim of this study was to determine the efficacy, safety, and immunosuppressant adherence in 125 stable liver transplant (LT) patients converted from twice‐daily tacrolimus (TAC BID) to once‐daily TAC (TAC OD). Tacrolimus trough levels, laboratory parameters, metabolic disorders, selected patient reported outcomes, and adverse events were assessed. Mean TAC trough level concentration was 6.1 ± 2.3 ng/ml at study entry, decreased to 5.5 ± 2.1 ng/ml (P = 0.016) and 5.5 ± 2.2 ng/ml (P = 0.019) after 1 and 2 weeks, respectively, and tended to equal the baseline value during further follow‐up. At week 1, TAC concentrations were lower in 62.4% of patients and higher in 36.0% when compared with baseline. Renal and cardiovascular risk factors remained stable and no rejection episodes occurred over 12 months. Adverse events were consistent with the safety profile known from previous studies with TAC BID. Nonadherence measured by the “Basel Assessment of Adherence Scale to Immunosuppressives” was evident in 66.4% at study entry and decreased to 30.9% postconversion (P < 0.0001). Prevalence of nonadherence at baseline was significantly higher in patients converted >2 years after LT and in those ≤60 years of age. Conversion to TAC OD is safe, enhances immunosuppressant adherence and should be accompanied by a close TAC level monitoring during the initial period.

Assessment of allograft fibrosis by transient elastography and noninvasive biomarker scoring systems in liver transplant patients.

Background. This prospective, monocentric study was designed to assess the efficacy of transient elastography (TE), biochemical tests, and more complex scores in determining fibrosis stage in 157 patients transplanted for hepatitis C virus (HCV) infection or non–HCV-related liver diseases. Methods and Results. The optimal TE cutoff values for HCV patients and non-HCV patients were 4.7 and 5.0 kPa for F≥1, 7.1 and 7.3 kPa for F≥2, 10.9 kPa and 9.9 kPa for F≥3, and 17.3 and 12.6 kPa for F=4, respectively. The corresponding area under the receiver operating characteristic (AUROC) curves for F≥1, F≥2, F≥3, and F=4 were 0.95 and 0.86, 0.89 and 0.85, 0.97 and 0.88, and 0.99 and 0.97 for HCV and non-HCV patients, respectively. On the basis of the logistic regression equation, we created a model (FibroTransplant score) to identify advanced fibrosis (F≥3). The accuracy of this model was tested in a validation group (n=74). AUROCs for diagnosis of F≥3 in HCV patients and non-HCV patients of the training group were 0.89 and 0.83 (FibroTransplant score), 0.86 and 0.66 (Benlloch score), 0.81 and 0.71 (aspartate aminotransferase-to-platelet ratio index), 0.80 and 0.77 (Hepascore), 0.79 and 0.70 (FibroTest), 0.78 and 0.71 (FIB-4), 0.75 and 0.60 (Forns index), 0.73 and 0.69 (FibroIndex), and 0.70 and 0.59 (Lok score). Among the validation group, AUROCs of the FibroTransplant score for F≥3 were 0.90 and 0.91, respectively. Conclusions. TE and the FibroTransplant score can be reliably used for diagnosing advanced fibrosis in transplanted patients.