Combining Radiofrequency Ablation(RFA) With Sorafenib Therapy For Liver Tumor Ablation Efficacy:Study On Mice And Patients

1.BackgroundHepatocellular carcinoma (HCC) is the third leading cause of death in the world. The incidence of HCC is increasing in Europe and the United States, and is currently the leading cause of death amongst cirrhotic patients. Although surgical resection or liver transplantation has been considered as the efficient therapy for HCC, only5%to20%of such tumors have clinical features that are favorable for resection at the time of presentation. Because there are precluded due to the poor liver function from cirrhosis and a shortage of donors, surgical resection remains the gold standard for treatment of hepatic carcinomas..Radiofrequency ablation (RFA) is the most widely used thermal ablation technique for treating hepatic tumors. The advantages of RFA include low morbidity, fast recovery, and the possibility to repeat the treating procedure if tumor recurs. The inflow of relatively cool blood in the tumor vascularity limited the volume of coagulation necrosis obtained with RF ablation. Incompletely ablation of the tumor is a major obstacle for the RFA application. To overcome the limitation, several studies showed that decreasing tumor blood flow and pharmacologic reduction in blood flow could significantly improve the effect of RF ablation on induced coagulation necrosis area. In animal studies, the reduction of blood flow during RF application increased the volume of necrosis In the clinical studies, several methods have been reported a significant increase in the RF ablation-induced coagulation necrosis area obtained with occlusion of the portal or arterial flow. However, these techniques are not convenience because invasive procedures are required. Hines-Peralta et al demonstrated that arsenic trioxide, an antivascular pharmaceutical agents, could reduce the blood flow in the tumor, and increased the size of RF-induced coagulation necrosis. Following the positive randomized controlled trials, sorafenib has emerged as the standard of care systemic therapy for advanced HCC. Sorafenib demonstrates activity against several tyrosine and serine/threonine kinases. Sorafenib blocks tumor-cell proliferation and increases the rate of apoptosis. A randomized controlled phase3trial was conducted to the efficacy and safety of sorafenib in the patients with advanced HCC. So sorafenib demonstrate a statistically significant improvement in OS for patients with advanced HCC.We hypothesized that combining RFA with sorafenib therapy may prove to increase ablation efficacy.2.OBJECTIVE2.1To find if Combination of Radiofrequency Ablation with Sorafenib Therapy can decrease the RFA frequence of advance HCC patients and prolonged the mean survival time.2.2To obtain the function of Sorafenib treated in vitro cell viability of Hep2Cells and HUVEC Cells.2.3To prospectively determine whether modulation of hepatic cell carcinoma (HCC) tumor microvasculature by using the antiangiogenic drug sorafenib could increase the extent of radiofrequency (RF)-induced coagulation in anHCC animal tumor model.3.MATERIALS AND METHODS3.1In Patients' study:From August2008to December2011,23patients with unresectable HCC were administered sorafenib in the Hepatobiliary surgical Department of2nd affiliated hospital of Zhejiang university. Among these patients,8were treated with sorafenib and RFA. All patients involved in this study gave their informed consent. Institutional review board approval of our hospital was obtained for this study. Patients with a diagnosis of HCC based on histology obtained by needle biopsy or European Association for the Study of Liver disease criteria. All the patients had received sorafenib at an initial dose of400mg twice daily for at least12weeks. Relevant data from the patients'clinical records, including history, laboratory results, radiological findings, and follow-up data were recorded prospectively. The median follow-up time was19.2months. The primary endpoint was overall survival(OS), and the secondary endpoint was tumor response, which were assessed every months according to the Response Evaluation Criteria in Solid Tumors.3.2In vitro study:Monolayer cultrures of HepG-2and Huvec cells were maintained in RPMI1640medium with10%fetal bovine serum. All cells were incubated at37℃in a humidified air containing5%CO. We use3-(4,5-dimethylthiazol-2-yl)-2,5-diphenytetrazoliumbromide assay(MTT), flow cytometry and Western-bloting methods to detected the cell function before and after sorafenib treated.3.3In vivo study:Male athymic nude mice were housed and maintained in laminar flow cabinets under specific pathogen-free conditions with free access to food and water of the animal center of Zhejiang Chinese Medical University. Human HCC was implanted subcutaneously into the oxter of the nude mice After3weeks, the mice were randomly assigned to the control or treatment groups. In each experimental group,40or80mg sorafenib per kilogram of body weight were administered daily with oral gavage, respectively. The control group received the solvent only. The compounds were heated to60℃for1minute and sonicated for20-30minutes to suspend the sorafenib. Sorafenib was weighed and stored in dry form away from light and dissolved to liquid form immediately prior to administration. The mice were checked daily and tumors were measured twice a week. Drug administration was terminated after10days. At the time of measure, the tumors were measured in all three dimensions. The volumes of the tumors were calculated twice aweek. We used an internally cooled RF ablation system (Radionics, Burlington, MA) with a200-W RF generator. We placed the mice in the supine position after adequate anesthesia was achieved. A standard RF ablation needle with a1-cm active-tip electrode was inserted into the tumor under direct visualization. The temperature was maintained at70℃±5for5minutes. The mice were sacrificed after RFA. The tumors were sliced in2-3-mm pieces perpendicular to the RF needle axis and the section of the largest diameter one was pick out. Ablation size was visualized by staining for mitochondrial activity with2%triphenyltetrazolium chloride (TTC) The images were photographed with a digital camera. The area of coagulation area was quantified by measuring tained (red, viable tissue) and unstained(white, ablated tissue) regions using Image J for all samples in blinded fashion. Histopathologic examination was also performed in all ablated tumors with hematoxylin-eosin staining.In our study, additional tumor specimens harvested from the mice without RF ablation were stained with DAPI and CD31to determine microvascular density (MVD). Tumor sections were embedded in O.C.T. matrix and snap frozen in liquid, stored at-80℃. Tumor sections were fixed with cold acetone for10min in4℃, and blocked with goat serum, then the nuclei were stained with DAPI. MVD was then correlated with tumor growth and ablation outcome. Observation and image acquisition were performed with a Zeiss confocal microscope.4.RESULTS4.1In PatientThe median age for the patients was58.2years (range38-78years). The men to women ratio was19:4. The etiology of cirrhosis was related to hepatitis B infection in every patients.19(82.6%) patients had experienced Child-Pugh grade A and4(17.4%) patients had underlying Child-Pugh grade B.17patients(73.9%) had BCLCstage b as compared with6patients (26.1%) with BCLC stage C.26.1%of patients (6/23) had portal vein thrombosis and the mean index tumor size was5.3cm. All patients had advanced HCC at the time of commencement of sorafenib, and13%(3/23) had extrahepatic metastasis.21patients (91.3%) had previously undergone at least1different therapy.15patients (65.2%) had undergone liver resection,9patients(39.1%) had undergone transarterial chemoembolization (TACE) and RFA was carried out on14patients (62.9%).8patients (34.8%)received RFA treatments after sorafenib therapy and15patients(65.2%) were not. After a mean follow-up duration of11.78months, the overall median survival was9months (95%CI,7.547to10.453). The overall median survival for the study group receiving RF+sora was28.3months (95%CI18.5-38.2).The overall mean survival for the study group receiving RF+sora was28.3(95%CI,18.5-38.2) months.When sub-stratifying by BCLC stage, median survival for stage B was11(95%CI,5.213-16.787)months and for stage C was9(95%CI,3.342-14.658) months. Median survival of Child-Pugh A patients was11months (95%CI:6.743-15.257) versus8months (95%CI:4.733-15.257) in Child-Pugh B patients.4.2In vitroSorafenib Inhibits the In vitro cell viability of Hep2Cells and HUVEC Cells. We first examined the antiproliferative effects of sorafenib on Hepg2Cells and HUVEC cells. Sorafenib significantly inhibited the proliferation of HUVEC and HepG2. In vitro experiments confirmed Sorafenib decreased the expressionn of viability associated protein in both Hep G2and HUVEC, meanwhile, it can also induces the cell apoptosis and celll cycle blocking.4.3In vivo:Mice were devide to3groups and treated with different dose of sorafenib (0,40and80mg/kg) for10days. Among the three groups,Tumor growth was significantly different (P<0.01). The extent of RF-induced tumor necrosis area was significantly greater in the experimental groups treated with sorafenib. RF coagulation in control group measured57.29mm2±3.39in area. For tumors in mice pretreated with40to80mg/kg sorafenib, RF coagulation area significantly increased in a dose-dependent manner.Compared with that of control group, tumors in mice treated with sorafenib(40and80mg/kg) demonstrated differences in tumor vascularity with CD31staining. The control group tumors showed grade Ⅲ or Ⅳ vascularity and the mice treated with80mg/kg sorafenib had nearly absent vascularity (grade Ⅰ). The tumors of the mice treated with40mg/kg sorafenib had grade Ⅱ. All the tumors in mice treated with sorafenib showed great reduction in vascularity in the center of the tumor.Conclusion1. Combination of Radiofrequency Ablation with SorafenibTherapy can prolone the os of advance HCC patients.2. Sorafenib Inhibits the In vitro cell viability of Hep2Cells and HUVEC Cells. 3. Our results suggest a potential role for combining RF ablation with sorafenib therapy in human HCC in nude mice can enlarge the necrosis area.