| BackgroundHepatocellular carcinoma(HCC)is the sixth most common cancer and the third leading cause of cancer-related death.Specifically,500,000-1,000,000 new cases of HCC are diagnosed annually worldwide,and 85% of these new cases occur in developing countries.This disease is especially prevalent in East Asia,Southeast Asia,and Central and West Africa.The incidence of HCC in China,where this disease is particularly prevalent,accounts for 55% of the worldwide incidence of this disease.Hepatitis B virus(HBV)infection is a major risk factor for HCC in East Asia,and partial hepatectomy and liver transplantation have been considered the primary treatment of HCC.However,most patients are not eligible for surgical treatment because of the anatomical location of the tumor,the tumor size,the number of tumors,an insufficient residual liver volume,or extrahepatic metastasis.Liver transplantation is considered the best treatment option because it removes both the tumor lesions and potentially diseased liver,but this treatment option is limited by a lack of donor livers.Moreover,only 10%-20% of HCCs are resectable.Thus,various local ablation techniques are increasingly being used as alternative treatments for HCC,including radiofrequency ablation(RFA),which has become the most widely used thermal ablation method.RFA is a physical thermal ablation technique that directly delivers energy to tumor tissue via active electrodes,and the current is converted into heat in a close d circuit: the alternating current from the probe tip causes cell vibration,which results in friction between molecules and ions in the cell that generates high heat.The heat(above 60oC)induces coagulation and necrosis in the tumor and surrounding tissue [14].Important factors affecting the success of RFA include the complete ablation of tumor tissue and the capacity to produce a sufficient ablative margin.Theoretically,an ablative margin of 0.5 cm to 1 cm is required around the tumor to ensure that the peripheral part of the tumor and small lesions surrounding the tumor are completely destroyed.In recent decades,RFA has become one of the most promising treatment methods and the most important non-surgical approach for the radical treatment of small HCC(<3 cm).Many studies have shown that RFA is a very safe and efficient procedure.Specifically,the mortality rate and complication rate associated with RFA range from 0% to 1.2% and 3% to 7%,respectively.Thus,RFA is considered to be an effective,repeatable,and safe technique that results in the satisfactory local control of liver tumors.Moreover,RFA avoids the excessive disruption of liver tumors associated with open surgery that may lead to cancer cell diffusion.Moreover,RFA-induced thermal damage results in intravascular coagulation in the surrounding vessels,which may help to reduce tumor metastasis via these vessels.Thus,RFA has become a popular first-line treatment for liver tumors.RFA is considered effective for the treatment of small HCC.For tumor diameters ranging from 3 to 5 cm,the complete ablation rate of RFA ranges from 61.3% to 82.5%,but this result might be outdated because of improvements in RFA technology and equipment as well as the use of clustered probes and modified motors,which provide more powerful and multi-dimensional RFA and result in a sufficient ablative margin.For example,multiple probes can increase the ablation area and result in a higher temperature due to probe synergy,and an internal condensing probe can improve the utilization efficiency of ions.These new RFA techniques may improve treatment of HCC tumors with a diameter ranging from 3 to 5 cm.Thus,RFA treatment strategies need to be modified and revised.At present,tumor recurrence after RFA remains a significant problem that needs to be addressed.We stratify intrahepatic recurrence after RFA into local tumor progression(LTP)and intrahepatic distant recurrence(IDR),which differ in pathogenesis and are thought to occur independently.Typically,LTP is involved in the proliferation of residual tumor cells that are outside the ablative margin and can be visualized under a microscope,whereas IDR is the result of the intrahepatic metastasis of primary HCC or multiple HCC foci.Understanding the mechanisms,patterns,and risk factors of tumor recurrence is of great significance for the development and clinical application of RFA.Study I is a prospective multi-center study to investigate the early risk factors for HCC recurrence and the short-term efficacy of RFA.In study II,we investigated the long-term survival of patients with HCC who meet the Milan criteria after treatment with a multi-probe percutaneous RFA system.In study III,we extended the RFA indication and investigated the efficacy of multi-probe percutaneous RFA for the treatment of medium-sized HCC.In study IV,an in vitro heat-shocked cell model was established to test the mechanism of HCC recurrence after RFA at the cellular level.Part I Multi-center prospective study of risk factors for early recurrence after percutaneous RFA of early-stage HCC*Purpose: To investigate the treatment efficacy of multi-probe percutaneous RFA in the treatment of early-stage HCC and assess risk factors for the intrahepatic recurrence of HCC after RFA.Materials and Methods: A total of 139 patients with early-stage HCC were recruited for this multi-center study based on the prospective inclusion criteria.RFA was selected as the first-line treatment.We assessed LTP,the incidence of IDR,cumulative relapse-free survival,IDR-free survival,and overall survival.Results: From January 2014 to December 2014,a total of 139 patients with a mean age of 54 years(range: 19-79 years)were included in this study.The mean follow-up period was 29 months(range: 7-36).The complete tumor ablation rate was 98.9%.overall recurrence rate,LTP rate,and incidence of IDR were 35.9%,4.3% and 31.6%,respectively.The cumulative disease-free survival rates were 73.4%,96% and 75.8% at 1 year,65.4%,95.1% and 68.2% at 2 years,respectively.A multivariate analysis showed that tumor size was the only significant risk factor for LTP,and the ALP level and number of tumors were independent risk factors for IDR.The AFP level and recurrence interval were risk factors for overall survival.Conclusion: MESS-RFA is an effective method for the local control of early liver tumors.Patients with multiple early-stage HCC tumors and high ALP levels have a high recurrence rate,and patients with a high AFP level and short first-time recurrence interval have a poor prognosis.Therefore,these patients should undergo aggressive treatments.Part II Radiofrequency Ablation Using a Multiple-Electrode Switching System for Hepatocellular Carcinoma within the Milan Criteria: Long-term ResultsPurpose: To assess the long-term outcome of 516 consecutive patients treated with multiple-electrode switching system(MESS)radiofrequency ablation(RFA)for hepatocellular carcinoma(HCC)that met the Milan criteria.Materials and Methods: We performed 522 MESS RFAs on 516 patients from 12/2006 to 06/2011.A total of 956 tumors that met the Milan criteria with an average diameter of 2.64 cm(range,0.9-4.6 cm)were treated with MESS RFA,which consisted of bipolar-mode activation of three electrodes inserted just beyond the tumor margins.Ultrasonic contrast and serum alpha-fetoprotein(AFP)were measured every 2 months during the first postoperative year and every 4 months thereafter.Enhanced computed tomography was performed every 6 months.Survival was estimated using the Kaplan-Meier method.Follow-up was censored at 60 months.Multivariate analysis was performed using the Cox proportional hazards model.Results: For the 956 HCC tumors,the complete ablation rate with MESS was 98.83%(510/516).During a median of 34-month(IQR,23-52 months)of follow-up,171 patients died and four were lost to follow-up(15,30,38,and 42 months).The cumulative incidence of local tumor progression at 1,3,and 5 years was 0.39%,4.96%,and 6.66%,respectively,and the 1-,3-,and 5-year overall survival was 99.42%,83.97%,and 68.42%,respectively.Tumor size >30 mm was the only parameter that was predictive of local tumor progression(P<0.0001).Risk factors associated with overall survival included prothrombi n time >14 s,serum AFP levels >200 ng/m L,and tumor abutting vessel diameter <5 mm.The complication rate was 1.74%.Conclusion: MESS RFA is a safe and effective method for HCC treatment.This approach results in a high local progression-free survival for HCC tumors that meet the Milan criteria.Part III Percutaneous radiofrequency ablation with a multiple-electrode switching system for medium-sized hepatocellular carcinomasSummary of Background Data: Conventional monopolar RFA is limited in achieving local control for tumors larger than 3 cm.Therefore,MESS-RFA was developed,and can create a sufficiently large ablation volume including the target tumor and a 5-10 mm safety margin in medium-sized tumors.Purpose: To retrospectively evaluate the safety and short-term therapeutic efficacy of radiofrequency ablation(RFA)with a multiple-electrode switching system(MESS)to treat medium-sized(3.1-5.0 cm)hepatocellular carcinomas(HCCs).Materials and Methods: We performed a total of 168 RFAs with a MESS on 166 patients.The patients were treated under ultrasonographic guidance by percutaneous switching system RFA with a multichannel RF generator and two or three internally cooled electrodes.Technical effectiveness,local progression,and remote recurrence of HCC were determined.Results: For the 166 isolated HCC tumors,the complete ablation rate of MESS-RFA was 98.79%(164/166).Mean ablation time per procedure was 12.33±3.01 min;mean ablation diameter was 5.79±0.61 cm.The complication rate was 2.41%.During follow-up(averaging 16.54 months),local tumor progression occurred in 15/166 patients(9.03%)with technical effectiveness,while new HCCs were detected in 40/166 patients(24.09%).Multivariate analyses revealed that local tumor progression was only associated with serum AFP levels above 100 ng/ml as a risk factor.conclusion: MESS-RFA for achieving sufficient ablation volume is safe and efficient.This method also showed relatively successful therapeutic effectiveness on short-term follow up in the treatment of medium-sized HCCs.Part IV Effect and mechanism of mi R-103 target-regulating PTEN in the ablative margin after RFA*BackgroundPrimary liver cancer,particularly HCC,is the world’s fifth most comm on cancer and the third leading cause of cancer-related death.Moreover,the incidence of HCC continues to increase.Early surgical resection is the primary treatment for liver cancer,but its efficacy is compromised by hepatic decompensation and portal hypertension.Alternatively,liver transplantation is considered the best option,but the shortage of donors limits its application.Moreover,only 10-20% of patients are eligible to receive a transplant or surgical resection worldwide.RFA is a local thermal ablation technique that delivers high-frequency alternating current into tumor tissue via an electrode to cause coagulation and necrosis in the tumor and its surrounding tissue,resulting in a therapeutic effect.Many studies have shown that RFA is a safe and efficient procedure,and RFA has become the most widely used local thermal ablation technique in recent years due to several advantages,including the convenience,safety,minimally invasive nature,and repeatability of the procedure.However,tumor recurrence after RFA remains a significant problem,which primarily presents as intrahepatic HCC recurrence.Our previous prospective study showed that the 2-year recurrence rate is 24.1%,and tumor recurrence after RFA is reportedly related to residual cancer cells in the ablative margin.Moreover,these residual cancer cells undergo heat shock.Thus,elucidating the biological properties of residual cancer cells in the ablative margin after RFA is important to understand the high incidence of HCC recurren ce after RFA.Phosphatase and tensin homolog gene(PTEN)is a tumor-suppressor gene that exhibits both lipid and protein phosphatase activity.PTEN is widely distributed in a variety of tumor cells and can be used to monitor disease recurrence and(or)pro gression.Thus,PTEN has been gradually accepted as an independent prognostic factor and plays important regulatory roles in cell growth,cell signaling,migration,and apoptosis.Moreover,the downregulation,deletion,or mutation of PTEN can activate PI3 K/Akt signaling to result in tumor progression and invasion.mi RNAs are non-coding RNAs that are transcribed from DNA and usually inhibit gene expression after transcription by binding to the targeted m RNA.Thus,they play a very important role in regulating the cell cycle,gene expression,and development.Moreover,one mi RNA can regulate many genes.Specifically,non-coding RNAs,such as mi RNAs,have been shown to play an important role in the pathogenesis of various diseases.For example,Yu et al.found that mi R-103 may down-regulate TIPM3 to regulate endometrial cancer invasion and metastasis.Furthermore,Chen et al.found that mi R-103 is regulated by hypoxia-inducible factor-1α and targets the protein Argonaute 1(AGO1)gene to promote tumor angiogenesis.Therefore,further studies are needed to understand the mechanism of action of mi R-103 in residual cancer cells in the hypoxic ablative margin after RFA.Purpose: This study investigated the effect of the PTEN gene on the ablative margin after RFA and the role of mi R-103,which directly regulates the PTEN gene,in this effect.This study also preliminarily explored the mechanism by which the PTEN gene and mi R-103 affect tumor growth and invasion in the ablative margin after RFA.Materials and Methods:The cells in the ablative margin after RFA were transfected with a plasmid to selectively overexpress mi R-103.Then,3-(4,5-dimethylthiazol-2-yl)-2,5 diphenyltetrazolium bromide(MTT)and cell invasion assays were used to measure the proliferation and invasion of cells in the ablative margin after RFA.Quantitative real-time PCR(q PCR)was used to measure gene expression levels of mi R-103,Cyclin D1,p21,Bim,Fas ligand(FASL),and PTEN in cells or tissuesWestern blotting was used to measure the protein expression of Cyclin D1,p21,Bim,FASL,matrix metalloproteinase 9(MMP9),protein kinase B(AKT),phosphorylated AKT(p-AKT),retinoblastoma protein(p Rb),and phosphorylated p RB(p-p Rb)in the cells.A luciferase assay was used to examine the interaction between mi R-103,the PTEN gene,and the 3’ end of mutant PTEN based on fluorescence.Resultss: 1.mi R-103 is up-regulated in recurrent HCC tissue,the heat-shocked cells,and in HCC cells in the ablative margin after RFA.2.mi R-103 can promote the proliferation and migration of heat-shocked cells in the ablative margin of HCC after RFA.3.mi R-103 inhibits residual cancer cells in the ablative margin after RFA via PTEN,which is involved in the PIK/Akt signaling pathway.Conclusion: mi R-103 up-regulation can promote the proliferation and migration of residual cancer cells in the ablative margin after RFA by inhibiting PTEN via the PIK/Akt signaling pathway.This study may help elucidate the pathogenesis of HCC recurrence after RFA and provide a basis for developing treatments. |
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