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Prostate cancer is now the second most common malignancy in men worldwide, with an increasing incidence in China. Most prostate cancer patients receive whole-gland therapy after diagnosis, but patients with localized prostate cancer may not benefit from the treatment due to side effects. With the development of imaging technology and the theory of "index lesion," focal therapy has been greatly developed, which includs high intensity focused ultrasound, focal laser ablation, cryotherapy, irreversible electroporation and photodynamic therapy. This study reviews the clinical trials in recent years and reveals that high intensity focused ultrasound and focal laser ablation have better failure-free survival and postoperative functional control compared with other focal therapy techniques.
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Objective To evaluate the safety and efficacy of irreversible electroporation (IRE) combined with neoadjuvant chemotherapy in patients with locally advanced pancreatic cancer. Methods We searched PubMed, Embase, Cochrane Library, Web of Science, China Biomedical Literature Database, CNKI, Wanfang, and VIP databases for articles dated from the establishment of each database to March 2022. Meta-analysis was performed using RevMan5.4 software. Results A total of 3970 patients with locally advanced pancreatic cancer were enrolled in eight studies, including one randomized controlled trial, four retrospective studies, and three prospective studies. The patients were divided into the combined therapy group with 344 patients and the chemotherapy-only group with 3626 patients. Meta-analysis showed that the overall survival of patients in the combined therapy group was significantly higher than that in the chemotherapy-only group (OR=4.52; 95%CI: 2.63-7.77; P < 0.00001). However, no significant difference existed in the disease control rate between the combined therapy group and the chemotherapy-only group (OR=0.58; 95%CI: 0.02-18.74; P=0.76). Moreover, no significant difference existed in the disease progression between the two groups (OR=0.49; 95%CI: 0.23-1.02; P=0.06). The combination of neoadjuvant chemotherapy and IRE had no significant effect on the incidence of adverse reactions of gastrointestinal reaction (OR=0.37; 95%CI: 0.10-1.34; P=0.13) and bone marrow suppression (OR=0.61; 95%CI: 0.26-1.40; P=0.24). Conclusion IRE combined with neoadjuvant chemotherapy can remarkably improve the prognosis of patients with locally advanced pancreatic cancer, and significantly prolong the overall survival.
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@#Objective To explore the therapeutic effect and safety of irreversible electroporation (IRE) ablation technique on esophageal cancer. Methods An ECM830 electroporator was used for IRE treatment on esophageal cancer cells EC109 and KYSE30. According to the different electric field intensity, five groups were assigned: a control group, a 500 V/cm group, a 1 000 V/cm group, a 1 500 V/cm group, and a 2 000 V/cm group. After 24 h, methyl thiazolyltetrazolium (MTT) was used to detect the cell proliferation of each group. Western blotting was performed to evaluate the expression of apoptosis proteins in cells before or after IRE treatment. Eight healthy BALB/c nude mice were equally divided into two groups: a control group (n=4) and an IRE group (n=4). EC109 was used to establish subcutaneous transplantation tumors and subsequently the mice in the IRE group were treated with flat electrode. The weight and volume of tumors were measured after 14 days. Ten healthy New Zealand white rabbits were equally divided into two groups: a control group (n=5) and an IRE group (n=5). After exposing the abdominal cavity, the abdominal esophagus of the IRE group was treated with flat electrode. Seven days later, the esophagus was extracted for HE and Masson staining. Results When the electric field intensity was low (500 V/cm), there was no change in esophageal cancer cells proliferation after IRE treatment compared to the control group (EC109: P=0.385, KYSE30: P=0.600). With the increase of electric field intensity, the influence of IRE on the proliferation of esophageal cancer cell gradually increased. When it reached 2 000 V/cm, there was basically no cell viability after IRE treatment (P<0.001). The results of Western blotting showed that the expression of cleaved caspase-3 increased after IRE treatment (P<0.01). Animal experiments indicated that the weight and volume of tumors in nude mice reduced (P<0.05) and the growth of tumors was slowed down after IRE treatment. In addition, the parenchymal cells of rabbit esophagus were largely damaged, while interstitial tissues such as fibers were well preserved. Conclusion IRE ablation has the potential to inhibit the proliferation of esophageal cancer cell and slow down the tumor growth.What’s more, it is safe for the esophagus.
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A project of control of a high voltage pulse generator based on Arduino and its peripheral pulse sensor and temperature sensor was proposed for cell irreversible electroporation (IRE) test. By programming Arduino board, the analog photoelectric signal and the partial voltage signal of thermistor collected by pulse and temperature sensors were converted into digital signal and temperature value. The threshold of ECG R wave (>550) and temperature threshold (<37 oC) was set as trigger condition to control an 800 W high voltage pulse generator to release a fixed period pulse. Human lung cancer cells cultured in vitro were used to test and verify, and cell staining was used to evaluate the perforation. The results showed that Arduino and its sensors were sensitive to trigger and feedback. When the high voltage pulse generator was controlled to release 100 pulses with the parameters of 600 V, 1 200 V/cm and 100 ms pulse width, more than 95% of the cells showed nonthermal irreversible electroporation.
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Humans , ElectroporationABSTRACT
Pulsed electric field(PEF) provides high-energy instantaneous pulse and release energy to myocardial cell membrane, resulting in irreversible electroporation and causes myocardial cell contents leakage, destruction of intracellular homeostasis, cell death, and slight inflammatory response. PEF as non-thermal energy promotes the design and application of arrhythmia ablation catheter to enter a new stage. There are currently limited clinical studies that have proved the safety and effectieness of Farawave PEF catheter, PVAC GOLD PEF catheter, Lattice-tip Sphere-9 PEF and radiofrequency (RF) catheter used for atrial fibrillation ablation, but still need further discussion. The research of atrial fibrillation ablation with PEF is under study in China. In this paper, the design and application of PEF ablation for tachyarrhythmia are reviewed.
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Humans , Atrial Fibrillation/surgery , Catheter Ablation , Catheters , Pulmonary Veins/surgery , TachycardiaABSTRACT
This study firstly introduced the mechanism, benefits and applications of irreversible electroporation(IRE) for tumor ablation. In addition, this study also introduced the most advanced IRE systems cleared by FDA or CFDA and IRE research equipment. The clinically licensed IRE systems include the Nanoknife 3.0 of Angiodynamics, the Dophi
Subject(s)
Humans , Electricity , Electroporation , Heart Rate , Neoplasms/therapyABSTRACT
Objective: The objective was to evaluate the feasibility and safety of computed tomography (CT)-guided percutaneous irreversible electroporation (IRE) in porcine kidneys. Materials and Methods: Under CT guidance, two monopole probes were used to precisely puncture through the renal parenchyma into the renal hilum in nine anesthetized adult Bama miniature pigs. After which, IRE ablation was performed. Biochemical and pathological examinations were carried out 2 h, 2, 7, and 14 days after the procedure. Results: All procedures were performed successfully without any serious complications such as bleeding, infection, or death. All pigs survived until the end of the study. Pathological examinations showed that cells in the ablation area were dead within 2 days after the procedure, whereas the vascular endothelium showed only slight damage. After 2 days, endothelialization ensued and regrowth of smooth muscle cells was observed after 14 days. Hemogram tests indicated a transient increase but gradually returned to baseline levels 14 days after the procedure. Conclusion: IRE was essentially safe, however further studies on tumor ablation using several different animal models are needed
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Context: The safety and efficacy of irreversible electroporation (IRE) for locally advanced pancreatic carcinoma (LAPC) are well established. However, whether adjuvant chemoradiotherapy after IRE increases, the survival rate remains unknown. Therefore, this study evaluated the effect of chemoradiotherapy combined with IRE in patients with LAPC. Subjects and Methods: We retrospectively analyzed 42 patients with LAPC between July 2015 and December 2016 at PLA General Hospital treated with IRE or IRE combined with radiation and/or chemotherapy. These patients were divided into the IRE group and the combined-therapy group. All patients underwent computed tomography (CT), magnetic resonance imaging, and positron-emission tomography-CT and no signs of metastases were found. The prognosis of these patients was observed. Results: The times after operation and after diagnosis in the combined-therapy group (304.20 ± 118.54) and (334.40 ± 114.07) days, respectively, were better those than in the IRE group (214.36 ± 95.68) and (244.68 ± 110.61) days, respectively. Moreover, patients in the combined-therapy group had a significantly better survival rate than the IRE group (80 vs. 45.45%, P < 0.05). Conclusions: IRE combined with radiotherapy or chemotherapy was superior to IRE alone for the treatment of LAPC, as it prolonged the survival time and improved the survival rate, making it worthy of wide dissemination and clinical application
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Irreversible electroporation (IRE) is an emerging tissue ablation technique. Compared with thermal ablation technique such as radiofrequency, IRE can achieve focal ablation in a shorter time without heat sink effect while sparing the tissue scaffold. IRE has been demonstrated to be a feasible therapeutic modality for the liver, pancreatic, and prostatic cancer. In recent years, several studies regarding of catheter-directed IRE for digestive tract, bronchus, urinary tract, and myocardium have been performed, which preliminarily demonstrated the safety and efficacy of IRE for tissue ablation under endoscopic or interventional technique. This study summarized the research progress of catheter-directed IRE for tissue ablation. The critical technique and future direction of catheter-based IRE are prosp.
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Humans , Catheter Ablation , Catheters , Electroporation , EndoscopyABSTRACT
Objective To investigate the safety and efficacy of irreversible electroporation (IRE) hepatic ablation with high-frequency bipolar pulse in swine.Methods The experimental study was conducted.A total of 18 swines of either gender,aged (6.8+0.8)months with a range of 5.5-8.0 months,were collected from Animal Laboratory Center of Army Medical University.were randomly divided into 15 in experimental group and 3 in control group.The swines in experimental group underwent IRE hepatic ablation with high-frequency bipolar pulse,and 3 swines were chose randomly and underwent enhanced CT examination immediately after ablation,and at 3,7,14,and 28 days after ablation.The liver tissues were taken for histopathological examination.The swines in the control group underwent IRE hepatic ablation with high-frequency monopolar burst,and was performed enhanced CT examination at 3 days after ablation.Liver tissues were taken for histopathological examination.Observation indicators:(1) comparison of muscle contraction of siwnes between two groups;(2) imaging performance on enhanced CT after IRE ablation in the experimental group;(3) hepatic histopathological findings after IRE ablation in the experimental group;(4) comparison of apoptotic index in the ablation zone between two groups.The measurement data with normal distribution were expressed as Mean±SD,and comparison between groups was performed by the independent sample t test.Results (1) Comparison of muscle contraction between two groups:swines in both groups underwent ablation successfully.The degree of muscle contraction was (9.8±0.4)m/s2 and (48.6±0.5) m/s2 in the experimental group and in the control group,respectively,showing statistically significant difference between the two groups (t =-163.50,P＜0.05).(2) Imaging performance on enhanced CT after IRE ablation in the experimental group:the enhanced CT examination of swines immediately after IRE ablation showed a low-density shadow and clear boundary in the ablation zone.There was no obvious abnormality in the ablation zone and its adjacent large vessels.No serious complications occurred after the ablation.The boundary between the ablation zone and the normal liver tissue of the experimental group gradually became blurred over time,and the ablation zone was gradually replaced by normal liver tissue.The ablation zone at the 28 days after ablation was significantly reduced or even disappeared on imaging of enhanced CT examination.The maximum diameter of the ablation zone was (1.81±0.17) cm immediately after ablation,(1.75±0.19) cm at the 3 days after ablation,(1.32±0.22)cm at the 7 days after ablation,(0.65±0.14)cm at the 14 days after ablation,(0.28±0.10)cm at the 28 days after ablation,respectively.(3) Hepatic histopathological findings after IRE ablation in the experimental group:the HE staining of ablated tissue immediately after ablation showed that the cells in the ablation zone were swollen,arranged disorderly,and bleeding was observed around some of the needles.The bile ducts and blood vessels were intact in the ablation zone,and a large number of deeply stained nuclei were seen at 3 days after ablation,some of the nucleus and apoptotic bodies were partially dissolved or cleaved.A large number of inflammatory cell were infiltrated around the ablation zone.Intact vascular and biliary endothelial cells were observed by yon Willebrand factor staining,a larger number of apoptotic cells with deeply stained nuclei in the ablation zone were observed by terminal-deoxynucleoitidyl transferase mediated nick end labeling staining,and partial deposited dark brown calcium salt was seen by Von Kossa staining.More newborn hepatocytes were observed growing from the periphery of the ablation zone to the center at the 7,14,28 days after ablation.Smooth muscle cell proliferation was observed at 14 and 28 days after ablation.The ablation zone was replaced by new cells on 28 days after ablation.(4) Comparison of apoptotic index in the ablation zone between two groups:the apoptotic index of the ablation zone was significantly higher in the experimental group than in the control group on the 3 days after operation (76.67％±0.04％ vs.64.03％±0.05％,t=4.79,P＜0.05).Conclusion IRE hepatic ablation of swine using high-frequency bipolar pulse is safe and reliable,and it has more apoptotic cells than IRE ablation with high-frequency monopolar burst.
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Objective@#To investigate the safety and efficacy of irreversible electroporation (IRE) hepatic ablation with high-frequency bipolar pulse in swine.@*Methods@#The experimental study was conducted. A total of 18 swines of either gender, aged (6.8+ 0.8)months with a range of 5.5-8.0 months, were collected from Animal Laboratory Center of Army Medical University. were randomly divided into 15 in experimental group and 3 in control group. The swines in experimental group underwent IRE hepatic ablation with high-frequency bipolar pulse, and 3 swines were chose randomly and underwent enhanced CT examination immediately after ablation, and at 3, 7, 14, and 28 days after ablation. The liver tissues were taken for histopathological examination. The swines in the control group underwent IRE hepatic ablation with high-frequency monopolar burst, and was performed enhanced CT examination at 3 days after ablation. Liver tissues were taken for histopathological examination. Observation indicators: (1) comparison of muscle contraction of siwnes between two groups; (2) imaging performance on enhanced CT after IRE ablation in the experimental group; (3) hepatic histopathological findings after IRE ablation in the experimental group; (4) comparison of apoptotic index in the ablation zone between two groups. The measurement data with normal distribution were expressed as Mean±SD, and comparison between groups was performed by the independent sample t test.@*Results@#(1) Comparison of muscle contraction between two groups: swines in both groups underwent ablation successfully. The degree of muscle contraction was (9.8±0.4)m/s2 and (48.6±0.5)m/s2 in the experimental group and in the control group, respectively, showing statistically significant difference between the two groups (t=-163.50, P<0.05). (2) Imaging performance on enhanced CT after IRE ablation in the experimental group: the enhanced CT examination of swines immediately after IRE ablation showed a low-density shadow and clear boundary in the ablation zone. There was no obvious abnormality in the ablation zone and its adjacent large vessels. No serious complications occurred after the ablation. The boundary between the ablation zone and the normal liver tissue of the experimental group gradually became blurred over time, and the ablation zone was gradually replaced by normal liver tissue. The ablation zone at the 28 days after ablation was significantly reduced or even disappeared on imaging of enhanced CT examination.The maximum diameter of the ablation zone was (1.81±0.17)cm immediately after ablation, (1.75±0.19)cm at the 3 days after ablation, (1.32±0.22)cm at the 7 days after ablation, (0.65±0.14)cm at the 14 days after ablation, (0.28±0.10)cm at the 28 days after ablation, respectively. (3) Hepatic histopathological findings after IRE ablation in the experimental group: the HE staining of ablated tissue immediately after ablation showed that the cells in the ablation zone were swollen, arranged disorderly, and bleeding was observed around some of the needles.The bile ducts and blood vessels were intact in the ablation zone, and a large number of deeply stained nuclei were seen at 3 days after ablation, some of the nucleus and apoptotic bodies were partially dissolved or cleaved. A large number of inflammatory cell were infiltrated around the ablation zone. Intact vascular and biliary endothelial cells were observed by von Willebrand factor staining, a larger number of apoptotic cells with deeply stained nuclei in the ablation zone were observed by terminal-deoxynucleoitidyl transferase mediated nick end labeling staining, and partial deposited dark brown calcium salt was seen by Von Kossa staining. More newborn hepatocytes were observed growing from the periphery of the ablation zone to the center at the 7, 14, 28 days after ablation. Smooth muscle cell proliferation was observed at 14 and 28 days after ablation. The ablation zone was replaced by new cells on 28 days after ablation. (4) Comparison of apoptotic index in the ablation zone between two groups: the apoptotic index of the ablation zone was significantly higher in the experimental group than in the control group on the 3 days after operation (76.67%±0.04% vs. 64.03%±0.05%, t=4.79, P<0.05).@*Conclusion@#IRE hepatic ablation of swine using high-frequency bipolar pulse is safe and reliable, and it has more apoptotic cells than IRE ablation with high-frequency monopolar burst.
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Pancreatic cancer has a very poor prognosis. Complete surgical resection remains the only current curative treatment. Locally advanced pancreatic cancer (LAPC) is considered as unresectable because of involvement of celiac and/or mesenteric vessels. The treatment of LAPC is a challenge. Current guidelines suggest systemic therapy. However, the majority of patients will never experience conversion to surgical resection. Thus, in these patients, ablation is an alternative therapy for local control, which causes local destruction while ideally avoiding injury to surrounding healthy tissue. Irreversible electroporation (IRE) is an energy delivery system, effective in ablating tumors by inducing irreversible membrane destruction of cells. IRE demonstrated to be safe in previous studies. However, it is not free from complications, even serious. Here, we reported two cases of the IRE in LAPC patients.
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Humans , Electroporation , Membranes , Pancreatic Neoplasms , PrognosisABSTRACT
Objective To investigate the CT-guided percutaneous irreversible electroporation (IRE) in treating locally advanced pancreatic cancer (LAPC) and providing guidance for its prevention and treatment. Methods We retrospectively analyzed the clinical and imaging data of 17 patients (17 lesions) of LAPC treated with CT-guided IRE in our hospital from July 2015 to June 2016. Complications were documented and reviewed at day 7,30 and 90 follow up as well as during the procedure. The Clavien?Dindo was used for classification. The reasons that induced complications were summarized and to further discuss the prevention and treatment approaches. Results Of 17 patients, 2 patients suffered a transient tachycardia during the procedure. Eleven patients (65%) showed complications at day 7, of which gradeⅠcomplications occurred in 6 cases, including abdominal pain, nausea, vomiting, or a few of inflammatory exudations around the pancreas; four patients have grade Ⅱ complications, along with portal vein thrombosis;one patient showed grade Ⅲ a complications for retroperitoneal infection. With 30 days follow up, the grade Ⅰ complications disappeared, gradeⅡcomplications have not getting better, while grade Ⅲ a complications have been improved. With 90 days follow up,patients with grade Ⅲ a complications getting better; two patients with grade Ⅱ complications didn't show any changes;2 cases progressed to grade Ⅴ, and died of digestive tract bleeding at 82 days and 98 days after procedure. Conclusion CT-guided irreversible electroporation for treating LAPC is a safe ablation approach. Strict patient selection before procedure and make a reasonable prevention and treatment measures can reduce the complications.
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Locally advanced pancreatic cancer remains to be a disease with dismal prognosis,despite the improvements in chemotherapy or chemoradiotherapy,which was recognized as standard treatment.A heterogeneous armamentarium of locoregional ablative therapeutic options has been successfully applied for other solid organ malignancies.Recently,they were gradually applied in pancreas and commenced to show benefits.However,there are still many problems to be discussed.Here we reviewed the recent publications on the feasibility,safety and efficiency of various ablation treatments that have been applied to pancreatic cancer.
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Objective To summarize and analyze the perioperative complications of irreversible electroporation (IRE) ablation in treating tumors at different locations and to discuss their managements. Methods A total of 200 patients with tumors, including pancreatic tumor (n=71), liver tumor (n=64) and other tumors (n =65), were enrolled in this study. All patients received IRE ablation treatment. The perioperative complications were recorded and the data were statistically analyzed. The causes of severe complications and the treatment of complications were discussed. Results None of the patients died during the course of IRE ablation procedure. Severe postoperative complications occurred in the patients with pancreatic tumor or liver tumor, including duodenal artery bleeding in 3 patients with pancreatic tumor, which occurred at 10 days, 11 days and 15 days after IRE ablation respectively, and gastrointestinal bleeding (n =1) and biliary septic shock (n=1) in 2 patients with liver tumor, which occurred at 9 days after IRE ablation, the clinical symptoms were controlled after interventional embolization and/or vascular ligation together with anti-infective therapy. All minor complications were relieved after symptomatic treatment within 14 days. Conclusion IRE ablation has less systemic inflammatory response, and both the intraoperative and postoperative adverse reactions can be easily controlled, besides, IRE ablation has higher clinical safety. Although IRE ablation procedure may damage the internal or peripheral vessels of the pancreatic tumor, this severe complications can be effectively avoided if proper measures are adopted based on the causes of complications. (J Intervent Radiol, 2018, 27: 223-227)
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Pancreatic ductal adenocarcinoma (PDAC) is one of the most lethal tumor worldwide.Emerging evidence has shown that loco-regional therapy such as stereotactic body radiotherapy (SBRT) and radiofrequency ablation might be a rational choice for unresectable PDAC patients.Meanwhile,immunotherapy alone for PDAC have rarely achieved encouraging results.Accumulating data have revealed prominent tumor microenvironment change after loco-regional therapy in both target and remote lesions which might sensitize immunotherapy.It is thrilling to envisage the potential benefits of the combination therapy.We will focus on the underlining mechanism and clinical studies of the combination therapy.
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The incidence of liver cancer has been increasing year by year.In China,the incidence of liver cancer is 25.7/10 million.However,when the important organs have been involved or when the malignant liver tumors are located adjacent to the hepatic portal vital organs,the lesions are usually difficult to be surgically removed,and the curative results of chemotherapy and radiotherapy for such lesions are often poor.In recent years,along with the gradual rise of ablation techniques,the irreversible electroporation (IRE),regarded as a new ablation method that uses non-thermal energy,has been widely employed in clinical practice.Through generating instantaneous high voltage electrical pulses,IRE produces irreversible nanoscale damage to the cell membrane.Both domestic and foreign researches have indicated that for the treatment of inoperable primary or metastatic liver tumors IRE can effectively inactivate the tumor tissue while the lumen architecture of the vessels can be preserved and large vessel or bile duct will not be damaged.This paper aims to make a comprehensive review about IRE device structure,mechanism of therapeutic action,preclinical studies,clinical application methods and clinical efficacy in order to provide reference for the application of this technology in clinical practice.(J Intervent Radiol,2017,26:285-289)
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Hepatocellular carcinoma is a commonly-seen malignant tumor with high morbidity and mortality all over the world.With the rapid development of molecular biology and scientific technology,in treating HCC the use of nano knife technology,which is developed on the principle of irreversible electroporation,has come into clinicians' consideration.The authors are hereby making a comprehensive review about nano knife,focusing on the principles of nano knife therapy for liver cancer,the advantages of nano knife,and the latest developments in clinical practice and researches,etc.
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Objective To evaluate the overall survival in patients with locally advanced pancreatic cancer (LAC) treated with irreversible electroporation (IRE) and chemotherapy.Methods A retrospective study on the overall survival of 30 patients with LAC treated with IRE,and 30 patients with LAC treated with chemotherapy from July 2015 to October 2016 in the PLA General Hospital was conducted.Results For the 30 patients with LAC who underwent IRE successfully,there were 21 women and 9 men.The median age was 59 (36 ～81) years.Twenty-four patients had primary pancreatic head cancer and 6 had body cancer.Twelve (40.0％) of these patients had chemotherapy after the IRE ablation.The 90-day mortality in the IRE treated patients was 3 (10.0％).For the 30 patients with LAC who were treated with chemotherapy,the 90-day mortality was 6 (20.0％).In comparison of the IRE treated patients with the chemotherapy treated patients,improvements on disease-free survival (6 months vs.4 months,P ＜ 0.05) and overall survival (11 months vs.5.6 months,P ＜ 0.05) were observed.Conclusion IRE ablation of LAC was safe and could potentially improve overall survival when compared with the standard chemotherapy treatment.
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BACKGROUNDS/AIMS: Resection or enucleation is currently the treatment of choice for small pancreatic neuroendocrine tumors (NETs). Irreversible electroporation is a novel ablative method that is used for locally advanced pancreatic adenocarcinoma, but little data exists for its use for pancreatic NETs. We report an early experience of IRE for early pancreatic NETs. METHODS: Between April 2014 and March 2015, 3 patients with small (<2 cm) pancreatic NETs were treated with percutaneous IRE. RESULTS: There were no adverse effects during the procedure. Mean hospital stay was 2.6 days. All patients remained disease free on 12-19 months follow up. One patient developed recurrent pancreatitis with pseudocyst formation. CONCLUSIONS: IRE for small tumors of the pancreas is practical and may offer advantages over other thermal ablative techniques, since it preserves vital structures such as blood vessels, bile and pancreatic ducts. Further data regarding the long term disease free interval is required to establish efficacy.