Predictive Value Of ZNF545Epigenetic Change To Hepatocellular Carcinoma Recurrence After Thermal Ablation And Randomized Controlled Trial Of Microwave And Radiofrequency Ablation For Liver Cancer

Purpose:(1) To explore the predictive value of epigenetic changes in human hepatocellular carcinoma (HCC), the methylation status of ZNF545was analyzed by methylation specific PCR (MSP) and bisulfite sequence. The function was studied in liver cancer cell lines to understand the mechanism of ZNF545in liver cancer development. The association of ZNF545methylation and the survival or recurrence of early HCC after thermal ablation was analyzed to find prediction and prognosis marker.(2) According to randomized controlled trial (RCT) requirement, the intermediate term therapeutic effectiveness of cooled-probe microwave ablation (MWA) and radiofrequency ablation (RFA) on early-stage HCC was compared to find a better approach for minimally invasive thermal ablation.(3) In a prospective study, the intermediate term therapeutic effectiveness was compared between hepatectomy and minimally invasive thermal ablation (cooled-probe MWA and RFA) on early-stage HCC to find a proper therapeutic method for liver cancer.Materials and methods:1. Epigenetic study:Semi-quantitative RT-PCR was used to examine ZNF545mRNA expression in HCC cell lines before and after5-aza-deoxycytidine (5-aza-dc) treatment. MSP was employed to detect ZNF545methylation in7liver cancer cell lines,1immortalized liver cell line,8cases of normal liver tissue,10cases of liver cirrhosis and101cases of primary HCC. Expression of ZNF545was detected by immunohistochemistry in20cases of available matched HCC and adjacent tissue samples. Colony formation, MTT, and flow cytometry analysis were employed to analyze the function of ZNF545in HCC. The association of ZNF545methylation and clinical factors (including age, sex, Child class, AFP, tumor number, size, pathological differentiation, viral load, hepatitis type, local tumor progress, intrahepatic metastasis and survival) were analyzed by SPSS16.0software, P<0.05was regarded as significant difference.2. Clinical comparative study:(1) From October2008to January2013, according to following enrollment standard,290cases of biopsy-proved HCC patients were involved in a randomized control study,146(192nodules) cases were treated with percutaneously MWA and144(172nodules) cases were treated with percutaneously RFA. The indications of patients enrolled in the study were as follows:tumor size≤5cm in diameter, tumor number≤3, Child-Pugh class A or B classification, no evidence of extrahepatic metastasis, vein or bile duct tumor embolus, and no any other anti-cancer treatment was accepted. Written informed consents for participation in this study were obtained from all the patients.(2) From August2008to January2013, according to above mentioned enrollment standard,290patients (364nodules) were treated by percutaneously MWA and RFA,306patients (339nodules) were treated by hepatectomy. They were designated as ablation group and resection group, respectively. The efficiency of these two groups was compared prospectively, including following items:the treatment time, post-ablation hospitalization, hospital expense, technique effectiveness rate (TER), local tumor progression (LTP), intra-hepatic metastases, extra-hepatic metastases, complications and intermediate term survival.Results:1. Epigenetic study:Complete promoter region methylation and expression loss of ZNF545were found in6of8liver cell lines (HepG2, SMMC7721, SUN449, PLC-PRF-5, SK-Hepl and LO2). Re-expression of ZNF545was induced by5-aza-dc in these cell lines, but no expression changes was found before and after5-aza-dc treatment in HBXF344and BEL7402cell lines, which is unmethylated and partial methylated, respectively. ZNF545methylation was found in56.4%(57/101) of HCC, and no methylation was detected in normal liver and cirrhosis. Univariate analysis showed ZNF545methylation took a larger percentage in elders more than65years old (P<0.05). Multivariate regression analysis showed no statistical association of ZNF545methylation with the12factors (P>0.05). However, the increasing tendency of intrahepatic metastasis and death was showed in ZNF545methylated patients. Re-expression of ZNF545in HepG2and SUN449cell line inhibited colony formation and cell proliferation, and induced cell apoptosis.2. Clinical comparative study:The follow-up period was2-51.8months (median19.8months).(1) Comparing between percutaneously cooled-probe MWA and RFA for HCC treatment both under ultrasound guidance, MWA needed less applicator number, ablation session, puncture number, ablation time and expense(P<0.05). Two groups showed similar TER, LTP, intra-hepatic metastases, extra-hepatic metastases, intermediate term cumulative and disease-free survival rate and complications (P>0.05). However, MWA showed slight advantage in locally completely ablating the tumors adjacent to vessels, gastrointestinal tract, diaphragm and3cm-5cm size tumors.(2) Comparing between percutaneously cooled-probe thermal ablation and hepatectomy for HCC treatment, two groups showed similar TER, LTP, intermediate term cumulative and disease-free survival rate (P>0.05). However, hepatectomy showed slight advantage in local tumor control of tumors with the size of3cm-5cm. In major complication incidence, variety and severity, MWA showed less invasion and needed less post-treatment hospitalization and hospital expense than hepatectomy (P<0.05).Conclusion:1. Epigenetic study:ZNF545was silenced by promoter region hypermethylation in HCC. ZNF545methylation promoted colony formation and cell apoptosis, and inhibited cell proliferation in HCC cells. Increasing tendency of intrahepatic metastasis and death for early HCC after thermal ablation was showed in ZNF545methylated patients. ZNF545may serve as a potential HCC detection and prognosis marker. The study laid the foundation for the combination treatment of thermal ablation and epigenetic therapy. Larger sample and longer follow-up were mandatory to define the predictive value of ZNF545methylated in prognosis of different stage HCC.2. Clinical comparative study:For the early-stage HCC treatment:(1) Percutaneous cooled-probe MWA and RFA achieved similar intermediate term effectiveness, however, MWA showed slight advantage in local tumor control for tumors adjacent to important structures and larger tumors, and MWA needed less ablation time, session and hospital expense, which reduced the invasion and saved the medical resources.(2) Percutaneous cooled-probe thermal ablation and hepatectomy achieved similar intermediate term effectiveness. Though hepatectomy showed better local tumor control for larger tumor, ablation showed the advantages of lower expense, minimal invasion and repeatability.