S. Okolicsanyi

ERK1/2-Dependent Vascular Endothelial Growth Factor Signaling Sustains Cyst Growth in Polycystin-2 Defective Mice

BACKGROUND & AIMS Severe polycystic liver disease can complicate adult dominant polycystic kidney disease, a genetic disease caused by defects in polycystin-1 (Pkd1) or polycystin-2 (Pkd2). Liver cyst epithelial cells (LCECs) express vascular endothelial growth factor (VEGF) and its receptor, VEGFR-2. We investigated the effects of VEGF on liver cyst growth and autocrine VEGF signaling in mice with Pkd1 and Pkd2 conditional knockouts. METHODS We studied mice in which Pkd1 or Pkd2 were conditionally inactivated following exposure to tamoxifen; these mice were called Pkd1(flox/-):pCxCreER (Pkd1KO) and Pkd2(flox/-):pCxCreER (Pkd2KO). RESULTS Pkd1KO and Pkd2KO mice developed liver defects; their LCECs expressed VEGF, VEGFR-2, hypoxia-inducible factor (HIF)-1alpha, phosphorylated extracellular signal-regulated kinase 1/2 (pERK1/2), and proliferating cell nuclear antigen (PCNA). In Pkd2KO but not Pkd1KO mice, exposure to the VEGFR-2 inhibitor SU5416 significantly reduced liver cyst development, liver/body weight ratio, and expression of pERK and PCNA. VEGF secretion and phosphorylation of ERK1/2 and VEGFR-2 were significantly increased in cultured LCECs from Pkd2KO compared with Pkd1KO mice. Inhibition of protein kinase A (PKA) reduced VEGF secretion and pERK1/2 expression. Addition of VEGF to LCECs from Pkd2KO mice increased phosphorylated VEGFR-2 and phosphorylated mitogen signal-regulated kinase (MEK) expression and induced phosphorylation of ERK1/2; this was inhibited by SU5416. Expression of HIF-1alpha increased in parallel with secretion of VEGF following LCEC stimulation. VEGF-induced cell proliferation was inhibited by the MEK inhibitor U1026 and by ERK1/2 small interfering RNA. CONCLUSIONS The PKA-ERK1/2-VEGF signaling pathway promotes growth of liver cysts in mice. In Pkd2-defective LCECs, PKA-dependent ERK1/2 signaling controls HIF-1alpha-dependent VEGF secretion and VEGFR-2 signaling. Autocrine and paracrine VEGF signaling promotes the growth of liver cysts in Pkd2KO mice. VEGF inhibitors might be used to treat patients with polycystic liver disease.

Mammalian target of rapamycin regulates vascular endothelial growth factor-dependent liver cyst growth in polycystin-2-defective mice.

Polycystic liver disease may complicate autosomal dominant polycystic kidney disease (ADPKD), a disease caused by mutations in polycystins, which are proteins that regulate signaling, morphogenesis, and differentiation in epithelial cells. The cystic biliary epithelium [liver cystic epithelium (LCE)] secretes vascular endothelial growth factor (VEGF), which promotes liver cyst growth via autocrine and paracrine mechanisms. The expression of insulin‐like growth factor 1 (IGF1), insulin‐like growth factor 1 receptor (IGF1R), and phosphorylated mammalian target of rapamycin (p‐mTOR) and the protein kinase A (PKA)–dependent phosphorylation of extracellular signal‐regulated kinase 1/2 (ERK1/2) are also up‐regulated in LCE. We have hypothesized that mammalian target of rapamycin (mTOR) represents a common pathway for the regulation of hypoxia‐inducible factor 1 alpha (HIF1α)–dependent VEGF secretion by IGF1 and ERK1/2. Conditional polycystin‐2–knockout (Pkd2KO) mice were used for in vivo studies and to isolate cystic cholangiocytes [liver cystic epithelial cells (LCECs)]. The expression of p‐mTOR, VEGF, cleaved caspase 3 (CC3), proliferating cell nuclear antigen (PCNA), IGF1, IGF1R, phosphorylated extracellular signal‐regulated kinase, p‐P70S6K, HIF1α, and VEGF in LCE, LCECs, and wild‐type cholangiocytes was studied with immunohistochemistry, western blotting, or enzyme‐linked immunosorbent assays. The cystic area was measured by computer‐assisted morphometry of pancytokeratin‐stained sections. Cell proliferation in vitro was studied with 3‐(4,5‐dimethylthiazol‐2‐yl)‐5‐(3‐carboxymethoxyphenyl)‐2‐(4‐sulfophenyl)‐2H‐tetrazolium and bromodeoxyuridine assays. The treatment of Pkd2KO mice with the mTOR inhibitor rapamycin significantly reduced the liver cyst area, liver/body weight ratio, pericystic microvascular density, and PCNA expression while increasing expression of CC3. Rapamycin inhibited IGF1‐stimulated HIF1α accumulation and VEGF secretion in LCECs. IGF1‐stimulated LCEC proliferation was inhibited by rapamycin and SU5416 (a vascular endothelial growth factor receptor 2 inhibitor). Phosphorylation of the mTOR‐dependent kinase P70S6K was significantly reduced by PKA inhibitor 14‐22 amide and by the mitogen signal‐regulated kinase inhibitor U1026. Conclusion: These data demonstrate that PKA‐dependent up‐regulation of mTOR has a central role in the proliferative, antiapoptotic, and pro‐angiogenic effects of IGF1 and VEGF in polycystin‐2–defective mice. This study also highlights a mechanistic link between PKA, ERK, mTOR, and HIF1α‐mediated VEGF secretion and provides a proof of concept for the potential use of mTOR inhibitors in ADPKD and conditions with aberrant cholangiocyte proliferation. (HEPATOLOGY 2010.)

Cyclic AMP/PKA-dependent paradoxical activation of Raf/MEK/ERK signaling in polycystin-2 defective mice treated with sorafenib.

Mutations in polycystins are a cause of polycystic liver disease. In polycystin‐2 (PC2)‐defective mice, cyclic adenosine monophosphate (cAMP)/protein kinase A (PKA)‐dependent activation of the Rat Sarcoma (Ras)/rapidly accelerated fibrosarcoma (Raf)/mitogen signal‐regulated kinase–extracellular signal‐regulated kinase (ERK) 1/2 pathway stimulates the growth of liver cysts. To test the hypothesis that sorafenib, a Raf inhibitor used for the treatment of liver and kidney cancers, inhibits liver cyst growth in PC2‐defective mice, we treated PC2 (i.e., Pkd2flox/−:pCxCreERTM [Pkd2cKO]) mice with sorafenib‐tosylate for 8 weeks (20‐60 mg/kg/day). Sorafenib caused an unexpected increase in liver cyst area, cell proliferation (Ki67), and expression of phosphorylated ERK (pERK) compared with Pkd2cKO mice treated with vehicle. When given to epithelial cells isolated from liver cysts of Pkd2cKO mice (Pkd2cKO‐cells), sorafenib progressively stimulated pERK1/2 and cell proliferation [3‐(4,5‐dimethylthiazol‐2‐yl)‐5‐(3‐carboxymethoxyphenyl)‐2‐(4‐sulfophenyl)‐2H‐tetrazolium and bromodeoxyuridine assay (MTS)] at doses between 0.001 and 1 μM; however, both pERK1/2 and cell proliferation significantly decreased at the dose of 10 μM. Raf kinase activity assay showed that whereas B‐Raf is inhibited by sorafenib in both wild‐type (WT) and Pkd2cKO cells, Raf‐1 is inhibited in WT cells but is significantly stimulated in Pkd2cKO cells. In Pkd2cKO cells pretreated with the PKA inhibitor 14‐22 amide, myristolated (1 μM) and in mice treated with octreotide in combination with sorafenib, the paradoxical activation of Raf/ERK1/2 was abolished, and cyst growth was inhibited. Conclusion: In PC2‐defective cells, sorafenib inhibits B‐Raf but paradoxically activates Raf‐1, resulting in increased ERK1/2 phosphorylation, cell proliferation, and cyst growth in vivo. These effects are consistent with the ability of Raf inhibitors to transactivate Raf‐1 when a PKA‐activated Ras promotes Raf‐1/B‐Raf heterodimerization, and are inhibited by interfering with cAMP/PKA signaling both in vitro and in vivo, as shown by the reduction of liver cysts in mice treated with combined octreotide and sorafenib. (HEPATOLOGY 2012)

Is the risk of neoplastic recurrence increased after prescribing direct-acting antivirals for HCV patients whose HCC was previously cured?

Variable Total cohort (n = 31) (%) Antitumoral treatment Resection 13 (42%) Resection and percutaneous ablation 1 (3%) Resection and TACE and percutaneous ablation 1 (3%) Resection and TACE 3 (10%) Percutaneous ablation and TACE 3 (10%) Percutaneous ablation 6 (19%) TACE 4 (13%) Worst BCLC stage prior to DAA therapy BCLC-0 8 (26%) BCLC-A1 4 (13%) BCLC-A2 9 (29%) BCLC-A3 2 (6%) BCLC-A4 3 (10%) BCLC-B 4 (13%) BCLC-C 1 (3%) Bilirubin (mean ± SD) 1.1 ± 0.7 Albumin (mean ± SD) 3.7 ± 0.5 Prothrombin time (INR) (mean ± SD) 1.1 ± 0.1 Alanine aminotransferase (mean ± SD) 71 ± 30 Pre-DAA HCV RNA levels (Log10), (median, range) (IU/ml) 5.6 (3.8-7.5) Pre-DAA serum AFP levels (median, range) (ng/ml) 10 (2-278) Treatment regimens SOF/LDV ± RBV for 12 or 24 weeks 15 (48%) SIM/SOF for 12 weeks 6 (19%) SOF/DCV ± RBV for 24 weeks 2 (6%) PrOD or the 3D regimen ± RBV for 12 or 24 weeks 3 (9.6%) SOF/RBV for 24 weeks 3 (9.6%) End of treatment response 31 (100%) Post-DAA serum AFP levels (median, range) (ng/ml) 6 (1-44) Interval between start of DAA therapy and last radiological assessment (months) (median, percentile 25-75) 8 (5-10.9)

Diagnostic value of dynamic contrast-enhanced CT with perfusion imaging in the quantitative assessment of tumor response to sorafenib in patients with advanced hepatocellular carcinoma: A feasibility study

PURPOSE To investigate the feasibility of perfusion-CT (p-CT) measurements in quantitative assessment of hemodynamic changes related to sorafenib in patients with advanced hepatocellular carcinoma (HCC). MATERIALS AND METHODS Twenty-two patients with advanced HCC underwent p-CT study (256-MDCT scanner) before and 2 months after sorafenib administration. Dedicated perfusion software generated a quantitative map of arterial and portal perfusion and calculated the following perfusion parameters in target liver lesion: hepatic perfusion (HP), time-to-peak (TTP), blood volume (BV), arterial perfusion (AP), and hepatic perfusion index (HPI). After the follow-up scan, patients were categorized as responders and non-responders, according to mRECIST. Perfusion values were analyzed and compared in HCC lesions and in the cirrhotic parenchyma (n=22), such as between baseline and follow-up in progressors and non-progressors. RESULTS Before treatment, all mean perfusion values were significantly higher in HCC lesions than in the cirrhotic parenchyma (HP 47.8±17.2 vs 13.3±6.3mL/s per 100g; AP 47.9±18.1 vs 12.9±10.7mL/s; p<0.001). The group that responded to sorafenib (n=17) showed a significant reduction of values in HCC target lesions after therapy (HP 29.2±23.3 vs 48.1±15.1; AP 29.4±24.6 vs 49.2±17.4; p<0.01), in comparison with the non-responder group (n=5) that demonstrated no significant variation before and after treatment of HP (46.9±25.1 vs 46.7±24.1) and AP (43.4±21.7 vs 43.5±24.6). Among the responder group, HP percentage variation (Δ) in target lesions, during treatment, showed a significantly different (p=0.04) ΔHP in the group with complete response (79%) compared to the group with partial response or stable disease (16%). CONCLUSIONS p-CT technique can be used for HCC quantitative assessment of changes related to anti-angiogenic therapy. Identification of response predictors might help clinicians in selection of patients who may benefit from targeted-therapy allowing for optimization of individualized treatment.

Directly acting antivirals combination for elderly patients with Chronic Hepatitis C: a Cost-Effectiveness Analysis

Chronic hepatitis C (CHC) has been undertreated among elderly patients. Interferon‐free treatment represents an opportunity for these patients. The aim of this study was to assess the cost‐effectiveness of directly acting antivirals (DAAs) in CHC elderly patients.

Optimising the clinical strategy for autoimmune liver diseases: Principles of value-based medicine.

BACKGROUND Autoimmune hepatitis, primary biliary cholangitis, and primary sclerosing cholangitis represent the three major autoimmune liver diseases (AILDs). Their management is highly specialized, requires a multidisciplinary approach and often relies on expensive, orphan drugs. Unfortunately, their treatment is often unsatisfactory, and the care pathway heterogeneous across different centers. Disease-specific clinical outcome indicators (COIs) able to evaluate the whole cycle of care are needed to assist both clinicians and administrators in improving quality and value of care. Aim of our study was to generate a set of COIs for the three AILDs. We then prospectively validated these indicators based on a series of consecutive patients recruited at three tertiary clinical centers in Lombardy, Italy. METHODS In phase I using a Delphi method and a RAND 9-point appropriateness scale a set of COIs was generated. In phase II the indicators were applied in a real-life dataset. RESULTS Two-hundred fourteen patients were enrolled and followed-up for a median time of 54months and the above COIs were recorded using a web-based electronic medical record program. The COIs were easy to collect in the clinical practice environment and their values compared well with the available natural history studies. CONCLUSIONS We have generated a comprehensive set of COIs which sequentially capture different clinical outcome of the three AILDs explored. These indicators represent a critical tool to implement a value-based approach to patients with these conditions, to monitor, compare and improve quality through benchmarking of clinical performance and to assess the significance of novel drugs and technologies. This article is part of a Special Issue entitled: Cholangiocytes in Health and Diseaseedited by Jesus Banales, Marco Marzioni, Nicholas LaRusso and Peter Jansen.

Dynamic contrast enhanced perfusion CT imaging: A diagnostic biomarker tool for survival prediction of tumour response to antiangiogenetic treatment in patients with advanced HCC lesions

PURPOSE To investigate whether perfusion-CT (p-CT) imaging could depict the inhibition of tumor neoangiogenesis induced by Sorafenib in advanced hepatocellular carcinoma (HCC), and whether it could be useful in predicting survival during treatment. MATERIALS AND METHODS Ninety-eight p-CT examinations were performed among 29 cirrhotic patients, with advanced HCC, before and every 2 months after Sorafenib administration, on a 256-slice MDCT scanner. Perfusion parameters were considered and statistically compared, at baseline and follow-up, between non-progressor (complete response, stable disease or partial response) and progressor (progressive disease) group. Kaplan-Meier analyses estimated the time-to-survival in overall population, after stratifying patients according to mRECIST. RESULTS The group that responded to Sorafenib showed a significant reduction of values in HCC target lesions after anti-angiogenic therapy (p < 0.01), in comparison with progressor group that demonstrated an increase or no significant variation. When patients were stratified into mRECIST, higher survival rate was observed in the non-progressor group compared to the progressor (48.6% vs 28.6%), and statistically significant correlation (p=0.01) was found between percentage variation of perfusion parameters, from baseline to follow-up, and overall survival rate. CONCLUSION Quantitative analysis of perfusion parameters, represents prognostic indicators useful in assessment of response to anti-angiogenic therapy, allowing for optimization of individualized treatment.

Liver-allocation policies for patients affected by HCC in Europe

The main goal of organ-allocation systems is to guarantee equal access to the limited resource of liver grafts for every patient on the waiting list, striking a balance between the ethical principles of equity, utility, benefit, need, and fairness. The European healthcare scenario is very complex, as it is essentially decentralized, and each nation—and region inside the nation—operates with a significant degree of autonomy. Furthermore, the epidemiology of liver diseases and hepatocarcinoma (HCC) differs between European countries and clearly affects indications and priorities. The aims of this review were to analyze the liver-allocation policies for HCC in different European countries. The European area considered for this analysis included five macro-areas or countries with similar liver-sharing and allocation policies: Centro Nazionale Trapianti (CNT) in Italy; Eurotransplant (Germany, the Netherlands, Belgium, Luxembourg, Austria, Hungary, Slovenia, and Croatia); Organizacion Nacional de Transplantes (ONT) in Spain; Etablissement français des Greffes (EfG) in France; NHS Blood & Transplant (NHSBT) in the UK and Ireland; and Scandiatransplant (Sweden, Norway, Finland, Denmark, and Iceland). Each area identified as a network for organ sharing in Europe adopts an allocation system based on either a center-oriented or a patient-oriented policy. Worldwide, two primary principles dominate the priorization of patients with HCC on the waiting list for deceased-donor liver transplant: urgency and utility. Although no common organ-allocation policy for European countries exists, long-term survival rates for patients with HCC on the transplant waiting lists are comparable to those reported in the United Network for Organ Sharing (UNOS) register. However, as allocation principles are discussed, new proposals emerge, and the epidemiology of liver disease changes, we strongly recommend steps are taken toward a common system.

P1169 : Outcome indicators in primary sclerosing cholangitis: Interim analysis of the value-based medicine in hepatology study

L. Fabris1,2, A. Ciaccio3, S. Okolicsanyi3, M. Rota4, P.A. Cortesi5, M.R. Buonocore3, M. Gemma3, P. Giani6, L.S. Belli 7, S. Fagiuoli 6, L. Scalone5, M.G. Valsecchi4, L.G. Mantovani5, M. Strazzabosco2,3 1 Department of Molecular Medicine, University of Padua, Padua, Italy 2 Liver Center & Section of Digestive Diseases, Department of Internal Medicine, Yale University School of Medicine, New Haven, CT, United States 3 Department of Surgery and Translational Medicine, University of Milano-Bicocca, Milan, Italy 4 Department of Health Sciences, Centre of Biostatistics for Clinical Epidemiology, University of Milano-Bicocca, Milan, Italy 5 Research Centre on Public Health (CESP), University of Milan-Bicocca, Milan, Italy 6 Department of Gastroenterology, Papa Giovanni XXIII Hospital, Bergamo, Italy 7 Department of Hepatology and Gastroenterology, Liver Unit, Niguarda Hospital, Milan, Italy