Irreversible Electroporation of the Pancreas Is ...

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In experimental group 2, a Jackson-Pratt drain (Cardinal Health, Waukegan, IL) was placed in the abdom- inal cavity before closure of the abdomen. The central ...
ORIGINAL ARTICLE

Irreversible Electroporation of the Pancreas Is Feasible and Safe in a Porcine Survival Model Stefan Fritz, MD,* Christof M. Sommer, MD,† Dominik Vollherbst,† Miguel F. Wachter,† Thomas Longerich, MD,‡ Milena Sachsenmeier, MD,* Jürgen Knapp, MD,§ Boris A. Radeleff, MD,† and Jens Werner, MD*||

Objectives: Use of thermal tumor ablation in the pancreatic parenchyma is limited because of the risk of pancreatitis, pancreatic fistula, or hemorrhage. This study aimed to evaluate the feasibility and safety of irreversible electroporation (IRE) in a porcine model. Methods: Ten pigs were divided into 2 study groups. In the first group, animals received IRE of the pancreatic tail and were killed after 60 minutes. In the second group, animals received IRE at the head of the pancreas and were followed up for 7 days. Clinical parameters, computed tomography imaging, laboratory results, and histology were obtained. Results: All animals survived IRE ablation, and no cardiac adverse effects were noted. Sixty minutes after IRE, a hypodense lesion on computed tomography imaging indicated the ablation zone. None of the animals developed clinical signs of acute pancreatitis. Only small amounts of ascites fluid, with a transient increase in amylase and lipase levels, were observed, indicating that no pancreatic fistula occurred. Conclusions: This porcine model shows that IRE is feasible and safe in the pancreatic parenchyma. Computed tomography imaging reveals significant changes at 60 minutes after IRE and therefore might serve as an early indicator of therapeutic success. Clinical studies are needed to evaluate the efficacy of IRE in pancreatic cancer. Key Words: irreversible electroporation, IRE, pancreas, pancreatic fistula (Pancreas 2015;44: 791–798)

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ancreatic ductal adenocarcinoma is the fourth leading cause of cancer death in the United States, with a median survival of 5 months and a 5-year overall survival rate between 3% and 5%.1,2 Pancreatic ductal adenocarcinoma is an aggressive and devastating disease characterized by infiltrating local tumor growth, early manifestation of metastasis, rapid progression, and marked chemoresistance.3,4 In only 10% to 20% of patients with newly diagnosed pancreatic ductal adenocarcinoma can pancreatic resection be performed because, often at this time, the tumor has already systemically spread or is locally too advanced.5 In case of intraoperatively diagnosed, locally advanced pancreatic cancer with infiltration of the celiac trunk or superior mesenteric artery, local ablative interventional techniques can be performed to treat or downstage the tumor to achieve surgical resectability, local tumor control, or reduction of pain.6

From the *Department of General, Visceral, and Transplantation Surgery, †Department of Diagnostic and Interventional Radiology, ‡Institute of Pathology, and §Department of Anesthesiology, University of Heidelberg, Heidelberg; and ||Department of General, Visceral, and Transplantation Surgery LMU, University of Munich, Munich, Germany. Received for publication May 9, 2014; accepted December 3, 2014. Reprints: Stefan Fritz, MD, Department of General and Visceral Surgery, University of Heidelberg, Im Neuenheimer Feld 110, Heidelberg 69120, Germany (e‐mail: [email protected]). This study was technically and financially supported by AngioDynamics Inc (Queensbury, NY). All authors have read the manuscript in its current version and approved its submission. The authors declare no conflict of interest. Copyright © 2015 Wolters Kluwer Health, Inc. All rights reserved.

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The specific anatomical arrangement of the pancreas makes it challenging to perform any kind of local tissue ablation. Moreover, the pancreas is difficult to handle, and it has been reported that a single needle puncture can induce acinar cell necrosis.7 Because of the high risk of developing severe necrotizing pancreatitis or damage of the main pancreatic duct with consequent pancreatic fistula, established interventional techniques, such as radiofrequency ablation (RFA),8 microwave ablation,9 or cryoablation,10 are not commonly used in the pancreatic parenchyma. A clinically available technique that specifically destroys tumor tissue but spares structures such as the damageable main pancreatic duct, the distal bile duct, and large pancreas-associated vessels (such as the portal vein or superior mesenteric artery) is yet to be defined. Irreversible electroporation (IRE) is a novel nonthermal ablation technique that specifically destroys the parenchyma without damaging surrounding vessels or other structures.11,12 A first theoretical article on IRE was published in 2005 by Davalos et al.13 Using mathematical analysis, they showed that IRE can ablate substantial volumes of tissue with relatively sharp margins. In the future, IRE might become an important and innovative tool in the armamentarium of surgeons who treat cancer.13,14 With this technique, microsecond electrical pulses are applied across the cell to generate a destabilizing electric potential that subsequently leads to permanent nanoscale defects in the lipid layer.15,16 The ongoing permeabilization of the cell membrane with consequent changes in cell homeostasis results in cell death (apoptosis).17 The exact mechanisms responsible for IRE-induced cell death, however, are currently unknown.13 The first long-term IRE in vivo study was performed on liver parenchyma in a porcine model.18 However, to date, only limited data have been published, and certainly more translational research is needed to evaluate the effects and outcomes of IRE under various conditions.11,19 Because IRE induces apoptosis in the parenchyma without destroying ducts of the hepatobiliary system20 or larger vascular structures,16,21 this method is potentially applicable to destroying tumors in the pancreas without inducing pancreatitis or without injuring the main pancreatic duct. Only a few published case reports and series have shown that IRE can locally ablate pancreatic tumors.22–24 The largest series, which used percutaneous IRE for ablation of pancreatic cancer in 14 patients who underwent 15 ablations, was published by the University of Miami.24 These IRE ablations have been performed in patients with locally advanced pancreatic cancer. Pancreatic cancer is associated with extended fibrosis of the pancreatic parenchyma and atrophy of the pancreas distal from the tumor. In most of these cases, the exocrine function of the pancreas is already constrained. Thus, to date, little is known about complications of IRE, particularly with regard to the risk of developing acute pancreatitis or pancreatic fistula in regularly functioning pancreatic tissue. Animal studies are indispensable for analyzing the pathomechanism of IRE in a standardized setting with abdominal imaging follow-up and histology. The present www.pancreasjournal.com

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study aimed to evaluate the feasibility and safety of IRE in the pancreas in a porcine survival model, focusing on the complications and potential risks of this novel technique and on abdominal imaging changes and histology.

MATERIALS AND METHODS Experimental Protocol The present experimental animal study was approved by State Animal Care and by the local Ethics Committee of the University of Heidelberg. Experiments were performed on healthy young female pigs (German domestic pigs) with a mean body weight of 29.4 kg (range, 27.6–31.2 kg). All animals were acclimatized to their environment for 3 to 5 days upon arrival in the experimental unit. Before surgical intervention, the animals were fasted overnight. All experiments were performed under deep general anesthesia. For induction of anesthesia, ketamine (8–10 mg/kg; Ketanest-S; Medistar, Hannover, Germany), azaperone (6 mg/kg; Stresnil; Janssen Animal Health, Beerse, Belgium), and midazolam (0.4 mg/kg; Dormicum; Roche, Basel, Switzerland) were applied intravenously. Animals were subsequently intubated, and anesthesia was maintained using isoflurane (Isofluran CP; CP-Pharma, Burgdorf, Germany) and mechanical ventilation (Servo-Ventilator 900c; Siemens-Elema, Munich, Germany). The animals received continuous cardiopulmonary and temperature monitoring during the whole experiment. Four-lead electrocardiogram was performed throughout the experiment. All procedures were performed under aseptic conditions. Initially, a central venous catheter (1.1  1.7 mm, 45 cm; Braun Melsungen AG, Tuttlingen, Germany) was installed into the external jugular vein to apply intravenous medication and to draw blood during the experiment and on follow-up without causing additional discomfort. For continuous muscle relaxation, intravenous vecuronium was used (Vecuronium Inresa; Inresa Arzneimittel GmbH, Freiburg, Germany). Before surgical incision, the animals received a single shot of cefazolin 40 mg/kg, i.v. After a short ventral midline incision over the length of 8 cm, the pancreas was identified and IRE was performed according to standard protocol. The commercial available NanoKnife Generator (AngioDynamics Inc, Queensbury, NY) was deployed in the present study (Fig. 1A). Electrocardiographically synchronized delivery was used during IRE ablation to prevent cardiac arrhythmias.

All pulses were administered during the absolute cardiac refractory period. Two monopolar 19-G applicators (AngioDynamics Inc) were used to apply electric pulses (Fig. 1B). The distance between pairs of applicators, as well as tip exposure, was set to 10 mm in all experiments. The applicators were installed in parallel fashion in the exposed pancreas with the help of a 10-mm spacer (Fig. 1B). During the whole ablation period, electrodes were kept still and safely in the pancreatic parenchyma. A 10-mm distance of electrodes was considered ideal in the present experimental model. The IRE device was set up to produce 90 high-voltage electrical pulses with a pulse length of 90 microseconds. The standardized voltage setting for each electroporation was 1900 V/cm. Before each ablation, a test pulse at 10% planned energy output was performed to assess whether adequate current was achieved during the procedure. Typically, pulses were delivered in 9 sets of 10 pulses between 2 paired unipolar electrodes. All IRE procedures were performed by a surgeon (SF) and an interventional radiologist (CMS) who both had previous experience with the use of the IRE device in other organs.25 No system failures occurred. Veterinary staff assessed the animals before and after IRE ablation for general health, vital signs, drinking and eating habits, urine and fecal output, and analgesia. Baseline complete blood cell counts and serum biochemistry levels were obtained before any intervention.

Setup of Experiment Ten animals were divided into 2 experimental groups. In a first group, animals received IRE of the pancreatic tail (pigs 1–5). During this experimental step, IRE was performed in the distal pancreas because we expected less complications (such as damage of the main pancreatic duct with consequent pancreatitis, damage of major vessels, or injuries to the duodenum) in this part of the pancreas. Access to the pancreas was achieved through a ventral midline laparotomy. Sixty minutes later, a computed tomography (CT) scan was performed as described previously. After the CT scan, the animals were killed (acute-phase experiments; group 1) by infusion of hypermolar potassium (20 mL of 7.5% potassium chloride) under deep anesthesia. In a second set of experiments, animals survived for 7 days before the experiments were terminated (survival experiments; group 2). After IRE ablation of the distal pancreas in the first set of experiments, IRE was performed at the head of the pancreas. The pancreatic head was therefore precisely identified within the

FIGURE 1. A, NanoKnife Generator (AngioDynamics Inc). B, Placement of 2 electrodes for IRE at the head of the pancreas in the middle of the duodenal C loop. The electrode distance measured 1 cm, and the exposition length was 1 cm.

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duodenal C loop (pigs 6–10). Two monopolar applicators were positioned within the pancreatic head in the middle of the duodenal C loop (Fig. 1B). In experimental group 2, a Jackson-Pratt drain (Cardinal Health, Waukegan, IL) was placed in the abdominal cavity before closure of the abdomen. The central venous catheter was kept for postoperative blood drawing. During the first 12 hours after IRE, the animals only received water before food and liquid were made available ad libitum. The pigs were followed, with daily assessment of activity, pain, dietary intake, and fecal output. The animal housing environment was maintained at a temperature of 22°C at 60% to 80% humidity with a 12-hour light-dark cycle. Moreover, analgesia was administered daily using carprofen IV (Pfizer Animal Health, Pfizer, NY). Blood serum level was evaluated preinterventionally, postinterventionally, and daily upon the first postoperative day. Furthermore, amount of ascites fluid and amylase and lipase levels in ascites fluid were measured daily. After 7 days, pigs were reoperated, and blood and biopsy samples were taken as described previously. Finally, the animals were euthanized by infusion of hypermolar potassium (20 mL of 7.5% potassium chloride) under deep anesthesia.

Abdominal Imaging All animals underwent baseline nonenhanced and contrastenhanced CT scans before the start of the surgical intervention (Somatom Definition Flash; Siemens Medical Solutions, Forchheim, Germany). After an intravenous injection of 70 mL of iodinated contrast material (iopromide 350 mg/mL; Ultravist, Bayer Schering, Berlin, Germany), arterial, portal-venous, and late phases were followed. Images were reconstructed as multiplanar images in axial planes with a slice thickness of 3 mm and an overlap of 2 mm. To better identify the postinterventional IRE ablation zone in the CT scans, we placed 2 titan clips (medium titanium ligating clips; Aesculap AG, Tuttlingen, Germany) at the margins of the ablation zone in the mesenterium. This method allowed the exact localization of the ablation zone by CT scan. All images were accessed and viewed using the PACS online imaging system of Heidelberg University Hospital (GE Healthcare, Barrington, IL). Imaging analysis was carried out by an experienced pancreatic radiologist (CMS).

Laboratory In the acute-phase experiment (group 1), blood was drawn before and 60 minutes after IRE ablation. In survival groups, animals underwent laboratory tests before IRE, 60 minutes after IRE, and daily during the postoperative course of 7 days. Blood was obtained via the central venous line placed under general anesthesia during the day of surgery. Besides a complete blood cell count, biochemistry levels, including serum amylase and lipase levels, were measured. In addition to blood serum analysis, amylase and lipase levels in the ascites fluid obtained by the Jackson-Pratt drain that had been placed during the initial surgical procedure in survival experiments (group 2) were measured.

Pathology and Histopathology After euthanasia, the IRE ablation zone with sufficient margins (including the healthy pancreas) was harvested, serially sectioned at 5-mm intervals, placed in formalin, and embedded in paraffin blocks for further histological evaluation. Before lamination, photographs of the surgical specimen were made, and a qualitative description of the gross section of the electroporation zone was performed. Moreover, the height and width of the ablation zone were measured. After embedding the tissue in paraffin, we made serial slices of 5 μm thickness. Subsequently, © 2015 Wolters Kluwer Health, Inc. All rights reserved.

Irreversible Electroporation of the Pancreas

hematoxylin-eosin stains were evaluated in a standardized way by an experienced pancreatic pathologist (TL).

Statistical Analysis Statistical analysis was performed using SAS version 9.1 for Windows (SAS Institute, Cary, NC) and expressed as mean (SD). Differences between subgroups were calculated by Student t test or Fisher exact test, as indicated. Statistical significance was acceptable at the 5% level (P < 0.05).

RESULTS Clinical Observations After IRE Ablation All animals survived IRE ablation, and no hemodynamic cardiac effects were noted during the procedure. Moreover, no ventricular arrhythmia occurred in the animals during or after the ablation. Because IRE leads to strong stimulation of skeletal muscles and the diaphragm, adequate quantities of vecuronium as muscle relaxant were used. The dose of vecuronium was considered sufficient when muscular twitches were exclusively confined to the treatment area. In none of the animals was there evidence of adjacent organ damage related to electroporation. All animals recovered quickly after the procedure. No signs of cerebrovascular events, such as paraplegia or paraparesis, were noted during the follow-up period. All animals survived until the designated endpoint. The animals showed normal activity and normal feeding habits on day 1 after IRE ablation, indicating minimal pain level. There was no clinical evidence of acute pancreatitis in any animal. One animal (pig 7) showed surgical site infection with subcutaneous abscess formation. However, this animal was clinically unremarkable and did not show fever or increased white blood cell count.

Laboratory Results Before IRE ablation, the mean (SD) white blood cell count was 14.8 (1.9) cells/nL. Sixty minutes after IRE ablation, no significant change was observed (mean [SD], 14.6 [3.0] cells/nL). On day 1 after surgery and IRE ablation, the mean (SD) leukocyte count increased to 26.1 (9.9) cells/nL but decreased to reference range on day 2 (12.3 [1.2] cells/nL) and remained unremarkable in all animals throughout the 7-day follow-up period. Besides leukocytes, serum amylase and lipase levels were measured at different time points (Fig. 2A). The mean (SD) serum amylase and lipase levels were 2425 (231) and 23.6 (5.8) U/L, respectively. Neither in the acute-phase experimental group nor in the survival group were significant changes observed at 60 minutes after IRE. On day 1, mean (SD) serum amylase and lipase levels increased significantly (P < 0.01) to 4334 (1350) and 114.3 (40.9) U/L, respectively, but went back to reference range on day 2 (Fig. 2A). In addition to serum laboratory tests, amylase and lipase levels in the ascites fluid of animals in the survival group were assessed. Every 24 hours, the amount of fluid in the Jackson-Pratt drain was measured, and samples were sent for laboratory analysis. All 5 animals in the survival group developed ascites after the surgical procedure and IRE ablation. The maximal amount of ascites fluid observed during 24 hours was 100 mL on day 1 after surgery. In all animals, ascites fluid was found to be serous (just a little tinged with blood) during the first postoperative day. On days 1 and 2 after surgery, the mean (SD) amount of ascites fluid was 49.6 (35.8) and 52.4 (30.3) mL, respectively (range, 2–100 mL per 24 hours). The quantity of fluid continuously decreased on day 3 in all animals and stayed consistent between 20 and 30 mL up to www.pancreasjournal.com

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FIGURE 2. A, Concentrations of amylase and lipase in serum. B, Concentrations of amylase and lipase in ascites fluid.

day 7. In 1 animal (pig 8), the drain clotted on day 4. Nevertheless, in this animal, just a little ascites fluid was found during reoperation, indicating that the clotting of the drain did not affect the health status of the animal. Measurements of mean (SD) amylase and lipase levels in ascites fluid revealed a significant but only transient increase on the first postoperative day compared with baseline: from 2992 (562) to 14,942 (4978) U/L and from 234.3 (130.6) to 2551.8 (903.3) U/L, respectively (Fig. 2B). In summary, the course of amylase and lipase levels in blood and ascites fluid showed transient elevation 24 hours after IRE. The clinical course was uneventful, and the small amounts of

postoperative ascites fluid indicated that neither clinically evident acute pancreatitis nor pancreatic fistula occurred.

Abdominal Imaging Before any manipulation, nonenhanced and contrast-enhanced CT imaging was performed on each animal to visualize the normal pancreas. Figure 3A (arrows, pig 4) shows the late-venous phase of a normal porcine pancreas. At 60 minutes after IRE ablation at the tail of the pancreas (acute-phase experiments), a hypodense lesion with sharp margins

FIGURE 3. Contrast-enhanced CT showing IRE of the pancreas at different time points. A, Acute-phase experiments, pig 4. Control CT of the normal pancreas before IRE, late-venous phase. Arrows indicate the head and the tail of the pancreas. B, Acute-phase experiments, pig 4. Irreversible electroporation lesion at the tail of the pancreas, 60 minutes after IRE, portal-venous phase (arrow). For better identification of the ablation zone on CT scan, the area was marked using 2 titan clips. C, Acute-phase experiments, pig 4. Ablation zone at the tail of the pancreas (arrow), 60 minutes after IRE, late phase. D, Survival experiments, pig 7. Ablation lesion at the head of the pancreas (arrow), 60 minutes after IRE, arterial phase. The ablation zone is hardly visible in this early-arterial phase. In all animals in the survival group, a Jackson-Pratt drain was put in place (arrowheads). E, Survival experiments, pig 7. Ablation lesion at the head of the pancreas (arrow), 60 minutes after IRE, late-arterial phase. The ablation zone demarcates in the late-arterial phase. F, Survival experiments, pig 7. Seven days after IRE ablation at the head of the pancreas (arrow), portal-venous phase. The ablation zone is marked with clips and is visible. G, Survival experiments, pig 6. Seven days after IRE ablation at the head of the pancreas (arrow), arterial phase. A pseudocyst measuring 1.0 cm in diameter marks the ablation zone. A titan clip indicates the margin of the ablation. H, Survival experiments, pig 6. Seven days after IRE ablation at the head of the pancreas (arrow), portal-venous phase. In the venous phase, the pseudocyst demarcates.

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Irreversible Electroporation of the Pancreas

was detected. None of the other animals showed cystic formation. Although the cystic lesion was already visible in the arterial phase (Fig. 3G), it demarcated in the portal-venous phase (Fig. 3H, arrow, pig 6). In summary, at 60 minutes after IRE, the ablation zone was already detected as a hypodense lesion with sharp margins between 2 titan clips (Fig. 3B). Computed tomography morphology remained unchanged after 7 days. Although the lesions were visible in the early-arterial phase (Fig. 3D), they started to demarcate in the portal-venous and late-venous phases (Figs. 3B, C, respectively). Overall, the portal-venous phase best visualized the ablation zone.

Pathology and Histopathology

FIGURE 4. Transverse gross section of the pancreas 7 days after IRE (centimeters scale). The electroporation zone (arrows) shows a red-white aspect indicating tissue damage, hemorrhage, and necrosis.

was already detected in the portal-venous phase (Fig. 3B, arrow, pig 4). For better identification, the margins of the ablation zone were tagged by 2 titan clips placed during the surgical procedure. Figure 3C (arrow, pig 4) demonstrates the same conditions in the late-venous phase. Figure 3D shows the IRE ablation zone at the head of the pancreas after 7 days (survival experiments). In all survival experiments, we installed a Jackson-Pratt drain, which can be distinctly identified by CT imaging (arrowheads). In the early-arterial phase, the ablation zone is hardly visible (Fig. 3D, pig 7). In contrast, in the late-arterial phase, the ablation zone demarcates at the head of the pancreas (Fig. 3E, arrow, pig 7). Figure 3F (pig 7) shows the ablation zone at the head of the pancreas in the portal-venous phase. One of the 2 clips and a cross-section of the drain can be identified in this section. In 2 of the animals in the survival group (pigs 6 and 9), cystic lesions were found by CT imaging. In one of the animals, a pancreatic cyst measuring 1.0 cm (Fig. 3G, arrow, pig 6) was detected; in another animal, a cyst 2.8 cm in diameter

Macroscopic findings during autopsy included localized adhesions and small amounts of serous ascites fluid in all animals. On evaluation of the pancreas, the ablation zone could be precisely identified between 2 titan clips, which were found to be in the correct location in all animals. Sixty minutes after IRE ablation (acute-phase experiments), the ablated pancreatic tissue was characterized by gross evidence of edema and hemorrhage (Fig. 4). The maximal diameter of the macroscopically visible ablation zone varied between 12 and 22 mm. On evaluation of histology in acute-phase experiments, the ablation zone was characterized by edematous swelling of the interstitium, necrobiosis of acinar pancreatic tissue, and minor foci of interstitial hemorrhage (Fig. 5B) compared with normal pancreas (Fig. 5A). In all animals, a sharp margin between the ablation zone and normal pancreatic tissue was found (Fig. 5B, arrows). Seven days after IRE (survival experiments), a sharp transition zone between normal tissue and necrotic tissue was still observed. The lesions remained similar in size compared with the cohort of animals killed 60 minutes after IRE ablation. From a histopathological point of view, the ablated area was characterized by cellular eosinophila, beginning fibrosis, and glandular atrophy. Histology of the ablation zone showed that the connective matrix of the main pancreatic duct and blood vessels remained intact, whereas the pancreatic parenchyma showed signs of apoptosis and necrosis. Histology of the 2 cystic lesions that were also visualized by CT scan (pigs 6 and 9) revealed pseudocysts associated with local pancreatic tissue damage and consequent

FIGURE 5. Histological comparison of untreated pancreatic parenchyma (A) with areas that have undergone IRE (B). A, Untreated normal exocrine and endocrine pancreatic parenchyma. B, Ablation zone 60 minutes after IRE. Arrows indicate the transition margin between normal and electroporated (lower part) pancreatic parenchyma. The ablation zone is characterized by edematous swelling of the interstitium, necrobiosis of acinar pancreatic tissue, and minor foci of interstitial hemorrhage. Hematoxylin-eosin staining; original magnification: 100. © 2015 Wolters Kluwer Health, Inc. All rights reserved.

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pancreatitis. The pancreatic parenchyma outside the ablation zone appeared normal without any signs of acute pancreatitis. In summary, IRE of the pancreas resulted in reliable necrosis and scarring, with a sharp transition zone between targeted and adjacent parenchyma. In none of the animals were histological signs of acute pancreatitis observed outside the ablation zone.

DISCUSSION The present study shows that, in a standardized experimental setting, IRE of the pancreas is feasible without causing noteworthy adverse effects, including severe acute pancreatitis. Our study is the first to prove that no pancreatic fistula was provoked by IRE even though the ablation was performed at the head of the pancreas in the anatomical neighborhood of the main pancreatic duct. Because most malignant pancreatic tumors occur in the pancreatic head, it is essential to evaluate the complications and adverse effects of IRE in this part of the pancreas. Moreover, to our knowledge, this study is the first to specifically evaluate how IRE ablation affects CT signaling at different time points and various contrast-enhanced phases. At 60 minutes after IRE ablation, a hypodense lesion was already observed in the portal-venous phase, indicating the ablation zone. Histological examination of the samples revealed that the effect of IRE was evident and persistent after 7 days. Moreover, it became clear that IRE is able to ablate cells of the pancreatic parenchyma without harming the main pancreatic duct or major vessels. In the present study, larger blood vessels and the main pancreatic duct showed an intact cellular matrix in all animals. The ability of IRE to ablate undesirable tissue near large blood vessels and damageable structures, such as the main pancreatic duct, is unique among known tissue ablation methods. These remarkable effects highlight the potential clinical relevance of IRE as a future treatment modality for pancreatic diseases. Recently, IRE has been shown to be a novel method for renal tissue ablation that can be safely performed in a porcine model.26,27 In 2012, Kingham et al20 reported the first clinical series of 28 patients who underwent IRE of malignant hepatic tumors. However, to date, only few reports on IRE of the pancreas exist. In 2010, Charpentier et al28 reported in a pilot study that IRE of the pancreas in a porcine model was performed in 4 animals. In 2012, José et al29 showed that IRE was feasible in a mouse model without causing relevant systemic toxicity. Meanwhile, the first clinical series of IRE in locally advanced pancreatic cancer is available.23,24 However, these studies are in the first evaluation phase and use heterogeneous study populations, including metastatic diseases, resectable pancreatic tumors, and irresectable pancreatic tumors. Moreover, extended locally advanced pancreatic tumors are frequently associated with marked fibrosis and atrophy of the parenchyma. Thus, these studies do not reflect ablation effects on normal pancreatic tissue, which has the ability to react by developing inflammation and consequent acute pancreatitis. In contrast to kidney and liver parenchyma, the pancreas is composed of tissue that is very sensitive to all kinds of manipulation and injury, leading to inflammation and consequent pancreatitis.7 Moreover, blood vessels, the pancreatic duct, and distal biliary ducts are structures that are vulnerable to and in danger of being harmed by all kinds of ablation techniques. Particularly the thermal effect of established ablation techniques, such as RFA or microwave ablation, certainly affects and potentially injures these structures.30 However, if applied with less energy, the thermal effect of blood flow in larger blood vessels reduces ablation efficacy because intensive blood flow leads to thermal sinks.31,32 The so-called heat-sink effect was also observed with

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microwave ablation.33 This is one of the reasons that RFA or other ablation techniques are not commonly used in the pancreas.34 Although electrodes are usually needed to apply energy to the target in RFA or microwave ablation, it is conceivable to use IRE with plate electrodes. because of the sensitive tissue structure of the pancreas, this technique might help to induce the electric field without harming the pancreatic parenchyma. However, the use of plate electrodes for IRE of the pancreas is disabled by the fact that the pancreas is covered by a connective tissue capsule. This thin layer displays high electrical permittivity that limits the amount of inner pancreatic tissue that can be treated by IRE.35 In the present study, we show that the application of electric field by electrodes stuck into the pancreatic parenchyma is safe without causing pancreatic fistula, even when needles were placed in the direct anatomical neighborhood of the main pancreatic duct at the head of the pancreas. On final histological examination, a sharp transition zone between normal tissue and necrotic tissue was observed 7 days after IRE. This is in accordance with reports describing the pathomechanism of IRE, which induces irreversible structural changes in the cell membrane of the target tissue that leads to cell death.36 The extent of the IRE-treated area is determined by the extent of the electric field to which the tissue is exposed.13 Radiofrequency ablation has been used in locally advanced pancreatic cancer in a combined therapeutic plan. Improvement in quality of life caused by achievement of pain relief has been reported.37,38 However, to date, significant treatment results or increase in survival has been missing.39 A recent systematic review concluded that RFA in an open surgical approach is feasible, but the complication rate seems to be unacceptably high without a clear benefit for survival.8 The anatomical complexity of the pancreas and peripancreatic region (which pancreatic tumors frequently infiltrate) makes RFA in the pancreas more challenging to perform than RFA in other organs. In addition to RFA, cryoablation has been reported to treat unresectable pancreatic cancer locally.40 Cryoablation is generally performed intraoperatively under ultrasound guidance. Small lesions (