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1 Digestive Endoscopy 2016; 28: doi: /den Original Article Endoscopic ultrasound-guided ablation of branch-duct intraductal papillary mucinous neoplasms: Feasibility and safety tests using porcine gallbladders Jin-Seok Park, 1 Dong-Wan Seo, 2 Tae Jun Song, 2 Do Hyun Park, 2 Sang Soo Lee, 2 Sung Koo Lee 2 and Myung-Hwan Kim 2 1 Digestive Disease Center, Department of Internal Medicine, Inha University School of Medicine, Incheon, and 2 Division of Gastroenterology, Department of Internal Medicine, University of Ulsan College of Medicine, Asan Medical Center, Seoul, South Korea Background and Aim: Radiofrequency ablation (RFA) and ethanol ablation are accepted methods of tissue destruction for treating cystic tumors. The aim of the present study was to evaluate the feasibility, efficacy, and safety of endoscopic ultrasound (EUS)-guided ablation using normal porcine gallbladders as a substitute model for branch-duct intraductal papillary mucinous neoplasms (BD-IPMN). Methods: Six adult mini pigs were included in this prospective study. EUS-guided RFA with or without ethanol ablation of the gallbladder was carried out using a prototype 18-gauge endoscopic RFA electrode, and RFA and ethanol ablation were done in a single session. Outcomes were assessed in terms of macroscopic and microscopic evaluations of the treated gallbladders. Results: The prototype RFA electrode was used for ablation of the mucosa, aspiration of the internal contents of the gallbladder, and ethanol injection. RFA plus ethanol lavage resulted in a greater mean percentage of denuded gallbladder mucosa (97.5%) than RFA alone (55.8%). Effects of ablation extending to the cystic duct and partially denuded cystic duct mucosae were detected in five of the six pigs. There were no major procedure-related adverse events. Conclusions: EUS-guided ablation of the gallbladder mucosa was feasible and effective in the porcine model. Additional studies will be required to fully assess the risk of procedure-related damage to the main pancreatic duct before using this technique in a clinical setting. Key words: ablation technique, endoscopic ultrasound (EUS), ethanol, intraductal papillary mucinous neoplasm (IPMN), radiofrequency ablation (RFA) Corresponding: Dong-Wan Seo, Department of Internal Medicine, University of Ulsan College of Medicine, Asan Medical Center, Seoul , South Korea. dwseoamc@amc.seoul.kr Received 30 October 2015; accepted 3 February INTRODUCTION INTRADUCTAL PAPILLARY MUCINOUS neoplasm (IPMN) is a mucin-producing cystic neoplasm of the pancreas that is associated with the development of intrapapillary projections of tall columnar epithelium and is commonly regarded as a premalignant lesion. 1 Branch-duct IPMN (BD-IPMN), the most common variant of this disease, has a lower risk of malignancy than other types of IPMN. 2,3 Because it is a slow-growing neoplasm with a good prognosis, clinical treatment of BD-IPMN may vary from watchful observation to surgical resection, 4,5 although complete resection is ultimately recommended because in-situ or minimally invasive carcinoma can occur. 6,7 Fritz et al. recommend early prophylactic resection for patients <65 years of age, including those with small lesions. 8 However, surgical resection of cystic neoplasms of the pancreas is associated with substantial risk of morbidity, ranging from 20 to 40%, as well as 2% mortality, and surgical treatment of BD-IPMN is not appropriate for some patients. 9,10 Radiofrequency ablation (RFA) has become a widely accepted method of tissue destruction for treatment of cystic neoplasms of the liver and for renal cysts. 11,12 RFA causes sustained thermal damage to the epithelium at gradually increasing temperatures, resulting in destruction of the inner epithelial lining of the cyst. 13 Recent studies have also shown that RFA may be useful for treatment of cystic tumors of the pancreas. Endoscopic ultrasound (EUS)-guided RFA caused thermal damage with well-demarcated coagulation in the pancreas in recent experiments using pig models, 14 and successful outcomes were also reported in a recent clinical study of EUS-guided RFA for the treatment of cystic pancreatic neoplasms. 15 bs_bs_banner 2016 The Authors Digestive Endoscopy published by John Wiley & Sons Australia, Ltd on behalf of Japan 599 Gastroenterological Endoscopy Society This is an open access article under the terms of the Creative Commons Attribution-NonCommercial-NoDerivs License, which permits use and distribution in any medium, provided the original work is properly cited, the use is non-commercial and no modifications or adaptations are made.

2 600 J.-S. Park et al. Digestive Endoscopy 2016; 28: EUS-guided ethanol lavage is another attractive choice for ablation of cystic neoplasms. Ethanol is the most commonly used ablative agent for renal and hepatic cysts. 16,17 The underlying mechanism mainly involves induction of cell death by cell membrane lysis, protein denaturation, and vascular occlusion. 18 Recently, EUS-guided ethanol lavage has been proven to be safe and effective for treating cystic pancreatic tumors. 19 However, these EUS-guided ablation techniques have not been widely studied for treating BD-IPMN as a result of the risks of pancreatitis and damage to the main pancreatic duct. Animal studies evaluating the efficacy and safety of these ablation methods for BD-IPMN are essential before carrying out these procedures in a clinical setting, but BD-IPMN is very rare in healthy animal models and no such studies have yet been conducted. To address this situation, we decided to conduct the current study using the porcine gallbladder. The anatomical features of the gallbladder are similar to those of BD-IPMN. Both are sac-like structures with lumens that are covered with a single layer of epithelial cells and both have connecting ducts, so that the cystic duct can be regarded as the equivalent of the main pancreatic duct. On this basis, we carried out a pilot study using a newly designed RFA electrode to assess the feasibility and efficacy of EUS-guided RFA and EUS-guided RFA plus ethanol lavage for ablation of BD-IPMN in normal porcine gallbladder. We compared the efficacy of RFA alone and that of RFA plus ethanol lavage in terms of the extent of ablation of the gallbladder epithelium. The study was reviewed and approved by the Asan Medical Center Institutional Review Board. Radiofrequency ablation system A newly-designed 18-gauge RFA electrode (STARmed, Seoul, South Korea) and a radiofrequency (RF) generator (VIVA combo System; STARmed) were used for the procedure. The total working length of the RFA electrode including the delivery system is 150 cm. The exposed distal end of the electrode is needle-shaped and echogenic. The active electrode length is 7 mm. The electrode has a unique design featuring two 0.8-mm diameter holes located 5 mm from the tip, permitting RFA as well as aspiration and injection. For the present experiments, the temperature of the electrode was fixed at 90 C and modulated by an automatic power control unit. The RF generator automatically turned off the power when the temperature of the electrode reached 90 C and turned it on again when the temperature fell below 85 C (Fig. 1). Endoscopic ultrasound-guided radiofrequency ablation The 18-gauge endoscopic RFA electrode and linear-array EUS were used to carry out RFA in the porcine gallbladders using an EUS-guided transgastric approach. The electrode was advanced into the center of the gallbladder and the contents of the gallbladder were aspirated through a needle until the diameter of the gallbladder was approximately 5 6 cmto allow for accurate assessment of the effects of RFA. If the gallbladder diameter was <5 cm, additional saline was injected into the gallbladder until the diameter was equal to or slightly above 5 cm. RFA was done using a 7-mm active electrode tip with 50 W of power for 10 min (Fig. 2a). METHODS Animal models SIX MINI PIGS (Sus scrofa; mean age 14 months and mean bodyweight 30 kg) were used in the present study. All procedures involving animals were reviewed and approved by the Institutional Animal Care and Use Committee of the Asan Medical Center (IACUC protocol number ). The animals were fasted from solids and allowed water during the 24 h before the EUS-RFA procedures. Pre-anesthesia sedation consisted of an i.m. injection of tiletamine-zolazepam (7.5 mg/kg, Zoletil 50; Virbac, Carros, France) and xylazine hydrochloride (2 mg/kg; Rompun, Bayer, Germany). The pigs were intubated, general anesthesia was achieved with 1.5% isoflurane (Forane; JW Pharmaceutical Corp., Seoul, South Korea), and the procedures were carried out with the animals in the decubitus position on a fluoroscopy table. Cardiopulmonary status was monitored throughout the procedures. EUS-guided RFA plus ethanol lavage Ethanol lavage was carried out immediately after EUS-guided RFA during the same procedure. After the EUS-guided RFA, the contents of the gallbladder were aspirated using the probe needle until the gallbladder was completely empty. Then, keeping the needle in the nearly collapsed gallbladder, a solution of 100% ethanol was injected into the gallbladder (Fig. 2b). The injected volume was equal to that of the gallbladder aspirate. Following injection, the animal was rotated into the left lateral and right lateral positions every 5 min for three cycles. On completion of the lavage, EUS was repeated and as much residual fluid as possible was removed by aspiration using the RFA needle electrode. Necropsy and pathology assessment The animals were killed immediately after the procedures, and the treated gallbladders, along with the cystic ducts, were removed by laparotomy. The effects of RFA on the gallbladder

3 Digestive Endoscopy 2016; 28: EUS-guided ablation of BD-IPMN 601 (a) (b) (c) Figure 1 Endoscopic radiofrequency electrode and power generator (STARmed, Seoul, South Korea) used for endoscopic ultrasound-guided radiofrequency ablation (RFA) of the porcine gallbladder. (a) 18-gauge endoscopic RFA electrode. (b) Radiofrequency power generator (VIVA combo, RF System; STARmed). (c) Novel design of the RFA electrode features two 0.8-mm holes located 5 mm from the tip of the electrode to allow aspiration and injection.

4 602 J.-S. Park et al. Digestive Endoscopy 2016; 28: Histological examination Specimens were fixed in neutral buffered formalin, serially sectioned (proximal and distal cystic duct and neck, body, and fundus of the gallbladder), and stained with hematoxylin and eosin (HE). Extent of ablation in each section was measured according to the percentage of denuded mucosa. The average extent of ablation in the gallbladder was calculated from the extent of ablation in each section (neck, body, and fundus). (a) RESULTS Feasibility and safety EUS-GUIDED RFA WITH or without ethanol lavage of the gallbladder was successfully carried out in all pigs without technical difficulties. EUS-guided gallbladder puncture with the newly designed 18-gauge RFA electrode was accomplished without difficulty in all cases. The RFA electrode was clearly seen protruding from the working channel of the echoendoscope, and was inserted directly into the gallbladder lumen under real-time EUS guidance. The electrode was visible throughout the entire procedure. No adverse events such as hemodynamic instability, localized bleeding, or tissue damage to the gallbladders were observed. At necropsy, there were no signs of coagulation or other damage to the surrounding tissue, and no bile leakage was detected at thee puncture sites. There were no obvious abnormalities in the tissues on histological examination, only changes that were consistent with needle tract ablation in the gallbladder. (b) Figure 2 Real-time image of endoscopic ultrasound-guided radiofrequency ablation (RFA) and ethanol injection in the porcine gallbladder. (a) RFA electrode within the gallbladder. Ablation is indicated by the hyperechoic cloud. (b) Ethanol injection using the newly designed RFA electrode. The ethanol appears as a hyperechoic haze with small white reflexes. were assessed macroscopically at necropsy and microscopically by histological examination (Fig. 3). EFFICACY OF ULTRASOUND-GUIDED RADIO- FREQUENCY ABLATION ALONE TWO PIGS UNDERWENT EUS-guided RFA without ethanol ablation. In both, depth of ablation around the gallbladder lumen was uneven. Histological examination of specimens obtained from the neck, body, and fundus of the gallbladders revealed areas of complete ablation of the mucosal surfaces combined with remnant mucosa featuring non-pyogenic inflammatory infiltration as well as focal areas of remnant mucosa containing tall columnar cells with basal nuclei (Fig. 4a). The remnant mucosa accounted for an average area of 44.2% of the entire gallbladder mucosa and was mainly found in the fundus. The thermal damage extended to the cystic duct in one of these two cases. The structure of the tall columnar epithelium in the duct was maintained in most of the mucosal layer, but a small portion of mucosa was denuded. It was noted that the thermal lesion in this case was closer to the neck of the gallbladder (Table 1).

5 Digestive Endoscopy 2016; 28: EUS-guided ablation of BD-IPMN 603 Figure 3 Porcine gallbladder and cystic duct after the procedure. The gallbladder and cystic duct were removed by laparotomy and evaluated. Variable degrees of discoloration are seen throughout the gallbladder mucosa. Arrow indicates needle tract ablation. Endoscopic ultrasound-guided radiofrequency ablation plus ethanol lavage The original aim of the present study was to assess RFA as an alternative technique for treating BD-IPMN. However, as the results of RFA alone in the first two pigs were not as satisfactory as expected, we decided to modify the study to see if we could achieve more effective ablation by adding ethanol lavage after RFA. Ethanol lavage added a total of min to the total procedure time. Aspiration of the gallbladder contents and injection of the ethanol took approximately 20 min, position changes 30 min, and aspiration of residual fluid from the gallbladder 10 min. On post-procedure histology, the gallbladder mucosa of the four pigs that underwent ethanol lavage after EUS-guided RFA was diffusely denuded (Fig. 4b). We achieved nearly complete, diffuse, and uniform ablation of the gallbladder mucosa in all cases. In two of the four pigs, focal areas of mucosa representing <7% of the entire area of the gallbladder mucosa remained. The remnant mucosa was mainly observed in the gallbladder fundus. Patchy mucosal destruction as a result of thermal and chemical injury was noted in the cystic duct in all four cases (Fig. 5); the extent of this ablation increased when the thermal lesions were closer to the neck of the gallbladder. DISCUSSION THIS PILOT STUDY has shown that EUS-guided RFA and ethanol lavage of the normal porcine gallbladder using the newly designed RFA electrode is safe and effective. Ethanol lavage after RFA resulted in an average of 97.5% mucosal ablation and was found to be more effective than RFA alone (55.8% ablation) in terms of uniform ablation of the entire gallbladder mucosa. RFA is known to be effective in the treatment of various types of malignant tumors, but RFA alone is not appropriate for large cystic tumors. RFA induces damage in tissues through propagation of heat energy from the tip of the electrode. 20 The temperature of the targeted tissues should be increased by C, which will result in nearly immediate tissue coagulation leading to irreversible damage. 21 In this study, the temperature of the RFA electrode was fixed at 90 C, but the actual fluid temperature in the gallbladder, as measured with a thermometer on the electrode tip in one of the experimental animals, ranged between 50 C and 90 C. The measured temperature tended to decrease as the distance from the electrode increased. In particular, the temperature of the gallbladder fundus (50 C) was lower than that of other locations (55 90 C), which might have contributed to the suboptimal ablative outcomes during RFA alone. This finding suggests that RFA could be useful for reliable eradication of small BD-IPMN, but treatment of larger lesions might be associated with an increased risk of residual and recurrent neoplasia. This risk could be minimized by aspiration of the cystic fluid before RFA in cases of larger lesions. This would reduce the distance between the electrode and the mucosa and improve heat conduction. RFA followed by ethanol ablation seems to be another reasonable strategy for improved outcomes in the treatment of BD-IPMN, as ethanol can increase the size of the ablation zone. This finding agrees with the results of a previous study by Kim 22 demonstrating that treatment of non-ablated thyroid nodules with RFA followed by ethanol ablation had a high success rate (62.5%) and was effective in reducing the volume of benign solid thyroid nodules over a 6-month follow-up

6 604 J.-S. Park et al. Digestive Endoscopy 2016; 28: (a) (b) Figure 4 Photomicrographs of the ablated gallbladder wall. (a) Partially denuded epithelium and focal areas of remnant mucosa resulting from radiofrequency ablation (RFA) (hematoxylin and eosin [HE] stain, 40 original magnification). (b) Completely denuded epithelium resulting from RFA followed by ethanol ablation (HE stain, 40 original magnification.) period. Based on these data, it has been recommended that ethanol ablation should be used to remove any non-ablated components of benign solid thyroid nodules that persist after RFA. Histological findings in the present study showed % ablation of the cystic epithelium in the four animals that underwent ethanol ablation after RFA. Direct comparison of ablation efficacy between RFA and ethanol lavage is impossible with the results of this study because we did not carry out ethanol lavage alone for ablation of the gallbladder mucosa. However, the results of a previous study by DeWitt et al. 23 showed that EUS-guided ethanol lavage in three patients with mucinous neoplasms of the pancreas resulted in % epithelial ablation, which is superior to the result of RFA alone in our study. It is not clear why the epithelium of some gallbladders was completely ablated while it remained intact in others. A possible cause of the incomplete ablation is that the volume of ethanol used for irrigation was less than the actual gallbladder volume. For chemical ablation of the gallbladder, an amount of ethanol that exceeded the actual gallbladder volume would have damaged the cystic duct and the common bile duct. To prevent this, the appropriate amount of ethanol was determined by measuring the volume of fluid aspirated from the gallbladder prior to ethanol injection. Because the volume of the aspirate might have been smaller than the actual volume of the gallbladder, it is possible that not all parts of the organ came into contact with the ethanol. Additional animal and human studies will be needed to develop methods for accurate determination of the appropriate amount of chemical to use for ablation. The rate of adverse events associated with treatment of BD-IPMN is similar to that of the treatment of other types of cysts. 24 However, ablation of BD-IPMN is still controversial because of concerns about the injection of ablating agents and exposure of the main pancreatic duct to thermal injury. In the current study, we evaluated mucosal injury in the cystic duct to assess any likely injury to the main pancreatic duct as a result of BD-IPMN ablation. To the best of our knowledge, this is the first attempt to evaluate damage to this duct associated with ablation of BD-IPMN. Histological examination of specimens obtained from the cystic duct demonstrated that the ablation effect extended some way towards the proximal and distal cystic portions of the duct. This suggests that there is some risk of injury to the main pancreatic duct during endoscopic ablation. Our results also suggest that this risk increases as the distance between the area of ablation and the duct decreases. However, it is also possible that the viscous mucins that fill BD-IPMN could prevent the spread of the ablative agents into the main pancreatic duct, thus limiting unwanted damage. Our results cannot exclude the inherent risk of pancreatitis associated with endoscopic ablation therapies. Further studies to identify techniques that can limit extravasation of ablative agents and the propagation of energy into ductal structures are essential to minimize the risk of pancreatitis. The present study had several limitations. First, the results of RFA and ethanol lavage would apply only to cysts >5 cm. Small cysts tend to be more responsive to ablative treatments than large cysts, 11 but the potential adverse effects of these ablation methods for smaller cysts could not be thoroughly

7 Digestive Endoscopy 2016; 28: EUS-guided ablation of BD-IPMN 605 Table 1 Histological examination of the ablated areas in the gallbladder and cystic duct mucosa Cystic duct Gallbladder Distal (%) Proximal (%) Neck (%) Body (%) Fundus (%) RFA alone Case Case RFA + EL Case Case Case Case EL, ethanol lavage; RFA, radiofrequency ablation. CONFLICTS OF INTEREST AUTHORS DECLARE NO conflicts of interest for this article. Figure 5 Photomicrograph of the cystic duct showing the partially denudated epithelium after ablation (arrows; hematoxylin and eosin stain, 200 original magnification). evaluated in the present study. Furthermore, the risk of continuous thermal damage to the surrounding tissues including liver and portal vein were not evaluated in this study. Next, this study evaluated the effect of endoscopic ablation in the gallbladder, which is only representative of a unilocular cyst. Most BD-IPMNs are multilocular. Therefore, the present experimental evidence may not be sufficient to support the clinical use of endoscopic ablation methods for BD-IPMN. Lastly, long-term outcomes such as tumor seeding could not be evaluated by the present experimental model. In conclusion, EUS-guided RFA and ethanol lavage shows promise as a local treatment for patients with BD-IPMN. However, the risk that endoscopic ablation might damage the main pancreatic duct and cause pancreatitis cannot be ruled out. ACKNOWLEDGMENT THIS STUDY WAS supported by a grant (14 201) from the Asan Institute for Life Sciences, Asan Medical Center, Seoul, South Korea. REFERENCES 1 Sohn TA, Yeo CJ, Cameron JL, et al. Intraductal papillary mucinous neoplasms of the pancreas: an updated experience. Ann. Surg. 2004; 239: Salvia R, Fernandez-del Castillo C, Bassi C, et al. Main-duct intraductal papillary mucinous neoplasms of the pancreas: clinical predictors of malignancy and long-term survival following resection. Ann. Surg. 2004; 239: Sugiyama M, Suzuki Y, Abe N, et al. Management of intraductal papillary mucinous neoplasm of the pancreas. J. Gastroenterol. 2008; 43: Kang MJ, Jang JY, Kim SJ, et al. Cyst growth rate predicts malignancy in patients with branch duct intraductal papillary mucinous neoplasms. Clin. Gastroenterol. Hepatol. 2011; 9: Levy P, Jouannaud V, O Toole D, et al. Natural history of intraductal papillary mucinous tumors of the pancreas: actuarial risk of malignancy. Clin. Gastroenterol. Hepatol. 2006; 4: Nair RM, Barthel JS, Centeno BA, et al. Interdisciplinary management of an intraductal papillary mucinous neoplasm of the pancreas. Cancer Control 2008; 15: Grutzmann R, Post S, Saeger HD, et al. Intraductal papillary mucinous neoplasia (IPMN) of the pancreas: its diagnosis, treatment, and prognosis. Dtsch. Arztebl. Int. 2011; 108: Fritz S, Klauss M, Bergmann F, et al. Small (Sendai negative) branch-duct IPMNs: not harmless. Ann. Surg. 2012; 256: Roch AM, Ceppa EP, Al-Haddad MA, et al. The natural history of main duct-involved, mixed-type intraductal papillary mucinous neoplasm: parameters predictive of progression. Ann. Surg. 2014; 260: discussion Allen PJ, D Angelica M, Gonen M, et al. A selective approach to the resection of cystic lesions of the pancreas: results from 539 consecutive patients. Ann. Surg. 2006; 244: Kim PN, Lee Y, Won HJ, et al. Radiofrequency ablation of hepatic cysts: evaluation of therapeutic efficacy. J. Vasc. Interv. Radiol. 2014; 25: 92 6.

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