HBV viral load and tumor and non-tumor factors in patients with HBV-associated HCC.

Background and Aim: Several tumor and non-tumor factors affect the prognosis of hepatocellular carcinoma (HCC) patients. This study aimed to investigate the effects of hepatitis B virus (HBV) viral load on tumor and non-tumor factors in patients with HBV-associated HCC. Materials and Methods: Patients with hepatitis B and HCC who presented to the HCC council at the Faculty of Medicine, Marmara University Liver Transplantation Institute, were included in our study. Patients were divided into two groups according to the presence or absence of HBV-DNA, and it was determined whether there were differences between these two groups with respect to tumor and non-tumor parameters. Results: Comparison of serum alanine aminotransferase (ALT), gamma-glutamyl transferase (GGT), hepatitis B surface antigen (HBsAg), and C-reactive protein (CRP) levels between HBV-DNA negative and positive patients showed significant differences (respectively p<0.01, p<0.01, p<0.05, and p<0.05). A major finding was a very significant difference between the two patient groups in terms of portal vein invasion (PVI) and venous invasion (p<0.001 and p<0.01, respectively). However, there was no significant difference in metastasis or lymph node involvement between HBV-DNA negative and positive patients. Conclusion: Our findings suggest that HBV viral load plays an important role in PVI in HCC patients, and there is a significant relationship between HBV viral load and inflammation.

Comparison of liver resection and living donor liver transplantation in patients with hepatocellular carcinoma within Milan criteria and well-preserved liver function.

Background and Aim: Liver resection (LR) and liver transplantation (LT) are curative treatments for hepatocellular carcinoma (HCC). The main purpose of this study was to compare the survival of LR and LDLT in patients with HCC within the Milan criteria. Materials and Methods: The results of the LR (n=67) and LDLT (n=391) groups were compared for overall survival (OS) and disease-free survival (DFS). Twenty-six of the HCCs in the LRs met the Milan and Child A criteria. Also, 200 of the HCC patients in the LDLTs met the Milan criteria, of which 70 also met the Child A criteria. Results: Early mortality was higher in the LDLT group (13.9% vs 1.47%; p=0.003). The 5-year OS was higher in the LDLTs than the LRs, but not statistically significant (84.6% vs 74.2%; p=0.287). However, 5-year DFS was better in the LDLT group (96.8% vs 64.3%; p<0.001). When the LRs (n=26) and the LDLTs (n=70) that met both Milan and Child A criteria were compared, 5-year OS was similar (81.4% vs 74.2%; p=0.512), but DFS was better in the LDLTs (98.6% vs 64.3%; p<0.001). Conclusion: LR can be justified as the first-line treatment for HCC patients who meet Milan and Child A criteria in terms of early mortality and OS.

Inflammatory Mechanisms of HCC Development.

HCC (hepatocellular carcinoma) is the second leading cause of cancer deaths worldwide, with several etiologic causes, mostly inflammation-associated. Different inflammatory responses in the liver can be triggered by different etiological agents. The inflammatory process can be resolved or be persistent, depending on the etiology and multiple other factors. Chronic inflammation, tissue remodeling, genetic alterations, and modifications in cellular signaling are considered to be key processes promoting immunosuppression. The progressive immunosuppression leads to the inactivation of anti-tumor immunity involved in HCC carcinogenesis and progression. Tumor cellular processes including DNA damage, necrosis, and ER (endoplasmic reticulum) stress can affect both immune-surveillance and cancer-promoting inflammation, supporting a mutual interdependence. Here, we review the current understanding of how chronic liver injury and inflammation is triggered and sustained, and how inflammation is linked to HCC. The identification of many hepatic microenvironmental inflammatory processes and their effector molecules, has resulted in extensive translational work and promising clinical trials of new immunomodulatory agents.

Ramucirumab and GSK1838705A Enhance the Inhibitory Effects of Low Concentration Sorafenib and Regorafenib Combination on HCC Cell Growth and Motility.

Several new multikinase inhibitors have recently been introduced into clinical practice for hepatocellular carcinoma (HCC) therapy. Small increases in survival were reported as well as considerable toxicity. There is thus a need for effective therapies with lower toxicities. We examined whether a combination of sorafenib and regorafenib might also be effective at very low concentrations, with resulting potential for lessened clinical toxicity. MTT test, clonogenic assay, Ki67 staining and cell cycle analysis were assessed for cell proliferation and Annexin V and western blotting analysis relative to the expression of cleaved Caspase-3 and BID for cell apoptosis. In these experimental conditions cell growth and migration were potently inhibited and apoptosis induced even in HCC cells producing high alpha fetoprotein (AFP) levels (clinically worse prognosis). The combination also inhibited levels of the two HCC biomarkers, AFP and des gamma carboxy prothrombin (DCP). Additional inhibition of Vascular Endothelial Growth Factor Receptor (VEGFR) or Insulin-like Growth Factor 1 Receptor (IGF1R) enhanced effects on AFP and DCP levels, cell growth inhibition and MAPK and PI3K/Akt signaling inhibition due to sorafenib/regorafenib combination. These combinations have the potential for decreased toxicity while simultaneously enhancing therapeutic effects. This potential decrease in toxicity is being explored in ongoing studies.

Strong enhancement by IGF1-R antagonists of hepatocellular carcinoma cell migration inhibition by Sorafenib and/or vitamin K1.

PURPOSE: Emerging evidence indicates that combining Sorafenib with vitamin K1 (VK1) may result in a synergistic inhibition of hepatocellular carcinoma (HCC) cell migration and proliferation. Despite this synergy, its benefits may be limited due to drug resistance resulting from cross-talk with the tumor microenvironment. Insulin-like growth factor-1 (IGF1) signaling acts as an important modulator of HCC cell growth, motility and drug resistance. Therefore, we aimed to explore the effects of Sorafenib in combination with VK1 and/or IGF1-R antagonists on HCC cells. METHODS: Scratch wound migration assays were performed to assess the motility of HCC-derived PLC/PRF/5, HLF and Hep3B cells. The synergistic, additive or antagonistic effects of Sorafenib, VK1 and IGF1-R antagonists on HCC cell motility were assessed using CompuSyn software. The effects mediated by these various compounds on HCC cytoskeleton organization were evaluated using DyLight 554 Phalloidin staining. Proliferation and migration-associated signaling pathways were analyzed in PLC/PRF/5 cells using Erk1/2 and Akt activation kits and Western blotting (Mek, JNK, Akt, Paxillin and p38), respectively. RESULTS: The effects of the IGF1-R antagonists GSK1838705A and OSI-906 on HCC cell migration inhibition after Sorafenib and/or VK1 administration, individually or in combination, were evaluated. We found a synergistic effect in PLC/PRF/5, HLF and Hep3B cells for combinations of fixed doses of GSK1838705A or OSI-906 together with different doses of Sorafenib and/or VK1. The levels of synergy were found to be stronger at higher Sorafenib and/or VK1 concentrations and lower or absent at lower concentrations, with some variation among the different cell lines tested. In addition, we found that in PLC/PRF/5 and HLF cells IGF1-R blockage strongly enhanced the reduction and redistribution of F-actin induced by Sorafenib and/or VK1 through alterations in the phosphorylation levels of some of the principal proteins involved in the MAPK signaling cascade, which is essential for cell migration. CONCLUSIONS: Our results indicate that modulation of the efficacy of Sorafenib through combinations with VK1 and/or IGF1-R antagonists results in synergistic inhibition of HCC cell migration.

IGF-1R tyrosine kinase inhibitors and Vitamin K1 enhance the antitumor effects of Regorafenib in HCC cell lines.

The recent RESORCE trial showed that treatment with Regorafenib after Sorafenib failure provided a significant improvement in overall survival in HCC patients. Preclinical and clinical trial data showed that Regorafenib is a more potent drug than Sorafenib. In this study we aimed at improving Regorafenib actions and at reducing its toxicity, by targeting parallel pathways or by combination with Vitamins K (VKs). We investigated the effects of Regorafenib administrated at low concentrations and in combination with either VK1 and/or with GSK1838705A or OSI-906, two IGF1-R inhibitors, on HCC cell growth and motility. Our results showed that both IGF1-R inhibitors potentiated the antiproliferative and pro-apoptotic effects of Regorafenib and/or VK1 in HCC cell lines. Moreover we provide evidence that the combined treatment with IG1-R antagonists and Regorafenib (and/or VK1) also caused a significant reduction and depolymerization of actin resulting in synergistic inhibition exerted on cell migration. Thus, simultaneous blocking of MAPK and PI3K/Akt cascades with IGF1-R inhibitors plus Regorafenib could represent a more potent approach for HCC treatment.

Resistance to multikinase inhibitor actions mediated by insulin like growth factor-1.

BACKGROUND: Blood platelet numbers are correlated with growth and aggressiveness of several tumor types, including hepatocellular carcinoma (HCC). We previously found that platelet lysates (hPLs) both stimulated HCC cell growth and migration, and antagonized the growth-inhibitory and apoptotic effects of Regorafenib, multikinase growth inhibitor, on HCC cell lines. We evaluated the effects of human insulin-like growth factor-1 (IGF1), a mitogen contained in platelets, on the Regorafenib-mediated growth inhibition. METHODS: An Elisa kit was used to evaluate hPL IGF1 concentrations. The effects of IGF1 on cell proliferation were assessed with MTT assay and analysis of cell cycle progression. Apoptosis assays, scratch assay and Transwell assay were performed to measure apoptosis, cell migration and invasion respectively. Western blots were performed by standard protocols. RESULTS: IGF1 antagonized growth inhibition exerted by Regorafenib on HCC cell lines. Moreover the mitogen blocked Regorafenib-induced apoptosis and decreased the rate of cell migration and invasion. The IGF1 effects were in turn antagonized by actions of a potent IGF1 receptor inhibitor, GSK1838705A, showing that the IGF1 receptor was involved in the mechanisms of IGF1-mediated blocking of Regorafenib action. GSK1838705A also partially blocked the effects of hPLs in antagonizing Regorafenib-mediated growth inhibition, showing that IGF1 was an important component of hPL actions. CONCLUSIONS: These results show that IGF1 antagonized Regorafenib-mediated growth, migration and invasion inhibition, as well as the drug-mediated induction of apoptosis in HCC cells and reinforce the idea that microenvironmental factors can influence cancer drug actions.

Modulation of Regorafenib effects on HCC cell lines by epidermal growth factor.

PURPOSE: Blood platelet numbers are correlated to growth and aggressiveness of several tumor types, including hepatocellular carcinoma (HCC). We previously found that platelet lysates (hPLs) also stimulated growth and migration, and antagonized the growth-inhibitory and apoptotic effects of both Sorafenib and Regorafenib, two multikinase inhibitors, on three HCC cell lines. In this study, in vitro function of human epidermal growth factor (EGF) with and without Sorafenib or Regorafenib was investigated. METHODS: An ELISA kit was used to evaluate the EGF concentrations in hPLs. In vitro function of EGF was assessed with proliferation MTT test. Apoptosis assay, scratch assays, and Transwell assays were performed for apoptosis, invasion, and migration, respectively. MAPK Activation Kit was used to explore MAPK phosphorylation. RESULTS: EGF antagonized the growth inhibition of Regorafenib on three HCC cell lines. Regorafenib-mediated growth inhibition was blocked by 70 % when the cells were pre-treated with EGF. EGF also blocked Regorafenib-induced apoptosis, as well as Regorafenib-induced decreases in cell migration and invasion. The EGF effects were in turn antagonized by concomitant addition to the cultures of EGF receptor antagonist Erlotinib, showing that the EGF receptor was involved in the mechanisms of EGF-mediated blocking of Regorafenib effects. Erlotinib also partially blocked the effects of hPLs in antagonizing Regorafenib-mediated growth inhibition, showing that EGF was an important component of hPL actions. CONCLUSIONS: All these results show that EGF antagonized Regorafenib-mediated growth and migration inhibition and apoptosis induction in HCC cells and reinforce the idea that microenvironment can influence cancer drug actions.

Prospective randomized trial of hepatic artery chemotherapy with cisplatin and doxorubicin, with or without lipiodol in the treatment of advanced stage hepatocellular carcinoma.

PURPOSE: This randomized study assessed the responses in hepatocellular carcinoma (HCC) to hepatic arterial doxorubicin plus cisplatin, with or without lipiodol. PATIENTS AND METHODS: Patients with unresectable and biopsy-proven HCC were treated with doxorubicin (30 mg/m) plus cisplatin (100 mg/m) without (Solution) or with (Lipiodol) emulsification every 2 months till tumor progression. Primary end point was response. RESULTS: Partial response rate was 56.8% for Lipiodol and 47% for Solution, P=0.48. Responses were evaluated as tumor size changes without vascularity assessments, due to the lipiodol. There were no complete responses. Survival at 6,12,18, and 24 months was 65, 32, 27, and 10.8% for Lipiodol patients and 70.6, 26.6, 8.8, and 8.8 for Solution patients. Of the total 71 enrolled and evaluable patients, survival at 6, 12, and 18 months was 75.7, 40, and 35% for responders and 58.8, 17.6, and 0 for nonresponders. There were no clear clinical or biochemical profile differences between the 2 treatment groups or between responders and nonresponders. CONCLUSIONS: Lipiodol conferred a minor response advantage, recorded as tumor size change, to hepatic arterial chemotherapy with doxorubicin plus cisplatin for HCC, but no survival advantage.