Pancreatic adenocarcinoma is one of the leading causes of cancer death worldwide with a median survival of 5 months and an overall 5-year survival less than 5%.1 Primary treatment is surgical resection. However, only 15% to 20% of patients have cancers that are limited to the pancreas.2
In patients with unresectable locally advanced pancreatic cancer, palliative chemotherapy consisting of gemcitabine monotherapy has been the accepted standard treatment.3 The benefit of this treatment is marginal with median survival of 9.2 to 11.7 months.4 Therefore, local ablative techniques are being explored as new treatment options for locally advanced pancreatic cancer to improve survival in these patients.
The main local ablative methods currently studied are radiofrequency ablation (RFA), microwave ablation (MWA), and irreversible electroporation (IRE).5–7 Radiofrequency ablation and MWA produce coagulation necrosis through an application of high-frequency alternating currents. Irreversible electroporation is a nonthermal procedure and achieves cell death by nanoscale defects in the cell membranes.8 All techniques are currently used in clinical settings as experimental methods and have limitations. The proximity of vital vessels and duodenum pose a risk for thermal injury during RFA and MWA procedure, and postoperative complications are present in approximately 25% of treated patients.9 Because of the lack of thermal effect, the IRE does not damage the connective tissue matrix, which may preserve vascular structures within the treatment field.10 Complication rates are still high, with series quoting up to 44% of patients experiencing complications Clavien-Dindo grade 3 or higher.11
Electroporation is technique in which an electrical field is applied to cells to increase the permeability of the cell membrane and thus allowing drugs or DNA to be introduced into the cell.12 Electrochemotherapy (ECT) is a form of local treatment modality, which combines the systemic administration of chemotherapy with locally applied electric pulses for reversible cell membrane electroporation.13 The technique already has an established place among other local treatments for the treatment of cutaneous tumors14 and also deep-seated primary and secondary liver tumors.15 The use of ECT on pancreatic carcinoma has been vaguely studied with only 1 published clinical study including 13 patients.16 Up to this date, little is known about potential complications of ECT, especially with regard to the risk of developing acute pancreatitis, pancreatic fistulas, or potential injury to the vessels in the ablative area.
Therefore, the goal of our study was to evaluate the feasibility and safety of the ECT of the pancreas in a porcine survival model. As the first, this study systematically focuses on potential development of the acute pancreatitis, pancreatic fistulas, and injury to the vessels in the ablative area.
MATERIALS AND METHODS
The study was conducted in accordance with ARRIVE (Animal Research: Reporting of In Vivo Experiments) guidelines after approval by the National Ethics Committee and National Veterinary Administration. Pigs were reared according to the European Council directive for minimum standards for the protection of pigs (2008/120/EC) and all procedures complied with the relevant national and European legislation (Permission # U34401-1/2017/4 “Electrochemotherapy with bleomycin induced normal tissue damage in porcine”).
Eight female pigs (Sus scrofa domesticus), aged 10 weeks and weighing 31.74 kg (standard deviation, 2.24 kg), were included in this study. Pigs were under continuous video surveillance. Research staff continuously monitored them for the first 24 hours after the anesthesia; afterward, they were checked by staff 3 times daily. Animals were assessed for general vital signs, food intake, urine, fecal output, and analgesia.
Pigs were divided into 2 study groups. In the first group (n = 4), animals underwent computed tomography (CT)–guided percutaneous ECT of the pancreatic tail. In the second group (n = 4), surgical laparotomy was performed and intraoperative ECT of the pancreatic head and tail was carried out. Electrochemotherapy with bleomycin was performed on 5 pigs, whereas 3 pigs received electric pulses alone to serve as a control group to rule out possible bleomycin cytotoxic effects and thermal damage identification (Table 1). Animals were killed 7 days after the treatment.
TABLE 1 -
Experimental Animal Weights and Data on Type of the Procedure and Applied Treatment
||Time to Killing, d
||ECT with bleomycin
||ECT with bleomycin
||Head and tail
||ECT with bleomycin
||Head and tail
||ECT with bleomycin
||Head and tail
||ECT with bleomycin
||Head and tail
Pigs were premedicated with ketamine (Bioketan, Vetoquinol, France) 10 mg/kg, midazolam (Midazolam Torrex; Torrex Chiesi Pharma GmbH, Vienna, Austria) 0.5 mg/kg, and medetomidine (Domitor; Orion Corporation, Espoo, Finland) 0.02 mg/kg intramuscularly, and induced to anesthesia with isoflurane (Isoflurin; Vetpharma Animal Health S.L., Barcelona, Spain). Antibiotic prophylaxis with single dose enrofloxacin (Enroxil 10%; Krka, Novo mesto, Slovenia) 7.5 mg/kg was administered intramuscularly 20 minutes before the skin incision. A 16-gauge permanent central venous catheter, which served for blood sampling, was surgically placed into external jugular vein and led subcutaneously to the back of the neck.
After that, carprofen (Rycarfa, Krka) 4 mg/kg was administered intravenously (IV) and fentanyl transdermal patch (Durogesic; Janssen Pharmaceutica, Beerse, Belgium) 75 μg/h applied on the lateral aspect of the thorax and left in situ until the end of experiment at day 7 or until the patch fell off. Carprofen (Rycarfa, Krka) 4 mg/kg was given orally once daily for 6 days from the day 2 to day 7.
Animals in the ECT group received 15,000 IU/m2 IV bleomycin (Bleomycinum; Heinrich Mack. Nachf. GmbH, Illertissen, Germany). The commercially available Cliniporator (Igea SpA, Carpi, Italy) was used in both study groups. Two linear 18-G electrodes (Igea SpA) were used to apply electric pulses. The target distance between the electrodes was set to 20 mm. The applicators were installed in a parallel fashion. The distance between the electrodes was confirmed with CT or intraoperative ultrasound and voltage was adjusted accordingly. A test pulse at 10% planned energy output was administered before the procedure to assess whether adequate current was achieved during the procedure. The electroporation device was set to produce 8 electrical pulses with a pulse length of 100 μs and voltage setting 1000 V/cm in accordance with the standard operating procedures of the ECT.14
Percutaneous ECT Procedure
The percoutaneous procedure was performed under the CT guidance (Somatom Scope; Siemens Healthineers, Erlangen, Germany). The pig was set in sternal position on the CT table, and the translumbar approach was used for the electrode positioning, traversing posterior abdominal wall and paravertebral musculature. One standardized ablation zone in the pancreatic tail was used in each animal. Two linear electrodes were positioned parallel in the tail of the pancreas (Fig. 1B). The care was taken to avoid bowel and vessel walls. The crossing of the parenchymal organs (kidney and spleen) was tolerated.
Intraoperative ECT Procedure
A midline laparotomy was used to expose the pancreas. First ablation zone was located in the tail of the pancreas. The electrodes were inserted in craniocaudal direction. Second ablation zone was located in the head of the pancreas. The electrodes were inserted in anteroposterior direction into the pancreatic head. Each of the electrodes was positioned in the pancreatic parenchyma on the different side of the superior mesenteric vein (SMV). Intraoperative ultrasound (Mindray M9 with linear 12 MHz probe; Mindray Co, Shenzhen, China) was used to confirm the appropriate electrode placement.
All animals underwent baseline contrast-enhanced CT scans before the ECT procedure. Noncontrast, the arterial and the portal venous phases, was performed after the IV injection of contrast material (iohexol, 350 mg/mL, Omnipaque; GE Healthcare, Chicago, Ill) at the concentration of 2 mg/kg. The same CT examination was repeated right after the ECT procedure and after 7 days. Images were analyzed by an experienced abdominal radiologist with 8 years of experience in abdominal radiology (R.D.) for the patency of SMV and imaging features of pancreatitis. Pancreatic necrosis, peripancreatic fat stranding, peripancreatic fluid collections, and abdominal free fluid (ascites) were classified as imaging features of acute pancreatitis, in keeping with the Atlanta classification.17
Blood was drawn before the procedure and daily during the postprocedural course of 7 days. Complete blood cell count was first analyzed. For C-reactive protein (CRP), serum amylase and lipase analysis blood was collected into blood collection tubes (BD Vacutainer, Franklin Lakes, NJ) and centrifuged at 1800 rpm for 10 minutes at room temperature. Serum was carefully transferred to a new Eppendorf tube, and samples were stored at −20°C until they were analyzed. Serum amylase and lipase levels were analyzed using routine laboratory tests, whereas CRP levels were analyzed using Pig CRP ELISA Kit (ab205089; Abcam, Cambridge, United Kingdom).
On the day 7, the animals were reanesthetized for CT examination and killed with T61 euthanasia IV solution (Intervet, Boxmeer, the Netherlands). A median laparotomy was performed, and the whole pancreas was removed together with duodenum, large vascular structures that traverse porcine pancreas and surrounding lymph nodes. Identified ablation zone was sectioned perpendicular to the electrodes' tracts and submitted entirely for histological examination. Tissue was fixed in 10% buffered formalin overnight, embedded in paraffin, sectioned to 3- to 4-μm-thick sections, and stained with H&E.
The study was designed to report primarily descriptive results; therefore, no valid statistical comparisons were possible between groups. Preprocedural and postprocedural results of laboratory analyses are presented as arithmetic mean ± standard error of the mean and compared using paired-sample t tests (IBM SPSS Statistics Version 20.0; IBM, Armonk, NY). A P value of less than 0.05 was considered significant.
Clinical Observations After the ECT Procedure
All animals recovered well after the procedure. There were no mortalities during the ECT procedure and in the 7-day follow-up period. All animals were in good clinical condition with no signs of pain or distress and had normal food and water consumption. No clinical signs of pancreatitis were present in any animal.
The preoperative levels of amylase and lipase ranged from 22.3 to 42.1 U/L and 0.06 to 0.08 U/L, respectively. An increase of the amylase and lipase of more than threefold of the preoperative value was considered as a sign of acute pancreatitis.17 In 2 animals (no. 6 and 8), amylase and lipase levels increased more than threefold after the procedure and a spontaneous decrease occurred in the following days (Fig. 2). In these animals, a damage of main pancreatic duct due to mechanical injury, caused by electrode insertion, was seen on pathological examination. In other 6 animals, no significant increase of amylase and lipase levels was observed.
The levels of CRP increased on the day 1 after the procedure but returned to normal during the rest of the observational period (Fig. 3).
A significant increase of white blood count (WBC) and monocyte count was recorded on day 7 (Table 2).
TABLE 2 -
Results of Blood Analyses Before and 7 Days After the Procedure
||Before the Procedure
||7 d After the Procedure
Data expressed as mean (standard deviation).
AP indicates alkaline phosphatase; LYMPH, lymphocyte count; MONO, monocyte count; PLT, platelet count; PT, prothrombine time; RBC, red blood cell count; WBC, white blood cell count.
Early (within 30 minutes after the procedure) and late (day 7 after the procedure) follow-up contrast-enhanced abdominal CT scans were performed in all animals. Examinations were reviewed for the patency of the SMV and presence of changes consistent with acute pancreatitis and possible pancreatic fistulae formation. No signs of acute pancreatitis were observed in both CT examinations (Fig. 1), and there were no signs of pancreatic fistula formation.
The SMV patency was checked in animals, which underwent intraoperative ECT procedure (n = 4). The vessel was patent in all animals on both CT examinations with no signs of thrombosis or luminal narrowing.
Histologic changes were similar in all animals but more pronounced in those treated with ECT. No thrombosis of the blood vessels, damage of the duodenal structures, or fistulas have been found.
In pigs treated with pulses only, a fibrotic, starry-shaped scar measuring 7 to 8 mm in the largest diameter was present. The pancreatic tissue between electrodes did not show any morphological changes. In 1 animal (animal 5), ablation zone in pancreatic head surrounded main pancreatic duct leaving it undamaged (Fig. 4B).
In the group treated with ECT, ablation zone was bigger. Fibrotic nidus was already well organized, measuring from 1.2 up to 2.5 cm in the largest diameter. In 2 animals (animals 6 and 8), the damage of main pancreatic duct was present as a consequence of mechanical injury caused by electrode insertion (duct rupture with missing epithelium) Apoptosis of the acinar cells was much more pronounced in cases treated with ECT, producing “vacuolated” appearance of surrounding pancreatic parenchyma (Fig. 4A).
In 2 animals (no. 6 and 8), the damage of main pancreatic duct was present as a consequence of mechanical injury caused by electrode insertion (duct rupture with missing epithelium). Necrosis was present at the site of ablation, surrounded by fibrotic tissue and organization of necrotic area. Interlobular and peripancreatic fat tissue was focally necrotic and fibrotic.
The primary aim of the present study was to determine the safety of the ECT with bleomycin in the porcine pancreas. This is the first systematic study evaluating potential complications and the histological changes of ECT with bleomycin in the pancreas in the porcine survival model. We have shown that in a standardized experimental setting, ECT with bleomycin of the pancreas is feasible and causes no noteworthy adverse effects, including pancreatitis or pancreatic fistula formation. Our study also proved that no damage to the major blood vessels occur when they are included in the ablation area. Because most malignant pancreatic tumors are located in the pancreatic head and in the vicinity of the major vessels, it is essential to evaluate the complications and adverse effects of ECT in this part of the pancreas.
The pancreas, in contrast to other organs, is composed of parenchyma that is very sensitive to all kinds of injury, leading to inflammation and consequent pancreatitis.18 In addition, the pancreatic duct and blood vessels in the vicinity of structures are vulnerable to and in danger of being harmed by all kinds of ablation techniques. In our study, none of the animals developed clinical or imaging signs of pancreatitis and most animals (6/8) showed no increase in serum amylase and lipase levels. The histological examination in 2 animals with a transient increase in the serum amylase and lipase levels (Fig. 2) demonstrated damage of the main pancreatic duct due to mechanical injury caused by electrode insertion, an inadvertent event. Necrosis was present at the site of ablation, surrounded by fibrotic tissue and organization of necrotic area. However, changes were limited to the pancreas with no pancreatic fistula formation, and none of these animals showed clinical or imaging signs of pancreatitis. Furthermore, no increase of the serum CRP levels (Fig. 3) was detected after the procedure, including the animals no. 6 and 8, marking the absence of systemic inflammatory response. A small-scale increase in the WBC and monocyte counts was noted on day 7 in comparison with preprocedural values. The increase was not clinically relevant, as both parameters remained well in the normal reference intervals.19 These findings show that ECT with bleomycin does not cause acute pancreatitis. However, caution should be used to avoid the insertion of the electrode in the main pancreatic duct, although this does not produce catastrophic complications. This contrasts the results obtained by thermal ablative techniques (RFA, MWA) that may produce pancreatitis and injury to vascular structures.20
Furthermore, the ablation zone in four of the animals also encompassed the SMV, with electrodes inserted in the pancreatic parenchyma on the opposite sites of the vessel. Imaging and histological findings presented a completely normal vessel with intact lumen and absence of thrombosis or vessel wall injury. These findings are in accordance with previous studies that also found that large vessels in the liver are not damaged by the ECT.21 This is an important advantage in comparison with the other thermal ablation techniques such as RFA and MWA, which were shown to produce injury and thrombosis of the vascular structures in the vicinity of the ablation area.22 The acute thrombosis of SMV could result in a potentially catastrophic outcome for the patients and SMV thrombosis with fatal outcome has been described in the IRE of the pancreatic adenocarcinoma.6 Electrochemotherapy could therefore be used as an alternative technique for these patients.
Procedures in our study were performed with 2 different approaches, intraoperatively with median laparotomy and percutaneously using CT guidance. Pancreatic tail was treated in both groups, but the head of the pancreas was not treated in the percutaneous group because of the inaccessibility. The interposing colon prohibited the anterior percutaneous approach to the head of the pancreas and the porcine anatomical characteristics did not allow for the posterior percutaneous approach. Both, intraoperative and percutaneous approaches are used in clinical practice with ablative treatment of pancreatic and other neoplasms.20 Percutaneous approach is gaining popularity because of the low invasiveness and potential faster recovery of the patient, and reports on percutaneous IRE of pancreatic adenocarcinoma have already been published.23 The technique of electrode placing for IRE and ECT procedures is similar, and percutaneous ECT procedure in humans should therefore be feasible.
Small number of animals and different locations of the ablation zones prevent us from accurately comparing both approaches in our study. However, we have shown that the both approaches are feasible and safe in the porcine model, and this should be taken into account when designing the clinical studies.
In contrast to the liver parenchyma in which bleomycin did not cause additional damage to the use of electrical pulses alone,21 pancreatic parenchyma was affected and cells in the vicinity of the electrodes were apoptotic and necrotic. The fibrosis was already present, indicating the healing process.
Finally, the limitations of the study need to be addressed. After the principle of the 3 Rs (replacement, reduction, and refinement), we performed ECT on only a limited number of animals, which precluded more statistical analysis of data and comparison of the intraoperative and percutaneous approach. The other main limitation of the study is that ECT with bleomycin was performed in healthy porcine pancreas, because pancreas tumor cell line, which can create pancreatic tumors, does not exist in pigs. A pancreatic adenocarcinoma has a different content and structure compared with normal pancreatic parenchyma, and this could produce different results. Pancreatic tumor in mice models do exist; however, the anatomical conditions and size differential between mice and humans are too far apart for realistic result. Future studies should be directed in assessing feasibility and safety of the use of ECT with bleomycin in pancreatic cancer tissue and to determine its effectiveness in the treatment of pancreatic cancer.
The present study shows that the ECT with bleomycin of the pancreas is feasible and does not cause relevant complications such as acute pancreatitis, pancreatic fistula, or vascular injury. Consequently, this promising ablative technique might be implemented in clinical practice in the future to treat locally advanced pancreatic cancer or other pancreatic tumors.
1. Ferlay J, Soerjomataram I, Dikshit R, et al. Cancer incidence and mortality worldwide: sources, methods and major patterns in GLOBOCAN 2012. Int J Cancer
2. Morganti AG, Massaccesi M, La Torre G, et al. A systematic review of resectability and survival after concurrent chemoradiation in primarily unresectable pancreatic cancer. Ann Surg Oncol
3. Hu J, Zhao G, Wang HX, et al. A meta-analysis of gemcitabine containing chemotherapy for locally advanced and metastatic pancreatic adenocarcinoma. J Hematol Oncol
4. Tu C, Zheng F, Wang JY, et al. An updated meta-analysis and system review: is gemcitabine+fluoropyrimidine in combination a better therapy versus gemcitabine alone for advanced and unresectable pancreatic cancer?Asian Pac J Cancer Prev
5. Fegrachi S, Molenaar IQ, Klaessens JH, et al. Radiofrequency ablation of the pancreas
: two-week follow-up in a porcine
model. Eur J Surg Oncol
6. Martin RC 2nd, McFarland K, Ellis S, et al. Irreversible electroporation
in locally advanced pancreatic cancer: potential improved overall survival. Ann Surg Oncol
. 2013;20 Suppl 3:S443–S449.
7. D'Onofrio M, Beleù A, De Robertis R. Ultrasound-guided percutaneous procedures in pancreatic diseases: new techniques and applications. Eur Radiol Exp
8. Verloh N, Jensch I, Lürken L, et al. Similar complication rates for irreversible electroporation
and thermal ablation in patients with hepatocellular tumors. Radiol Oncol
9. Pandya GJ, Shelat VG. Radiofrequency ablation of pancreatic ductal adenocarcinoma: the past, the present and the future. World J Gastrointest Oncol
10. Pedersoli F, Ritter A, Zimmermann M, et al. Single-needle electroporation
and interstitial electrochemotherapy
: in vivo safety and efficacy evaluation of a new system. Eur Radiol
11. de Liguori Carino N, O'Reilly DA, Siriwardena AK, et al. Irreversible electroporation
in pancreatic ductal adenocarcinoma: is there a role in conjunction with conventional treatment?Eur J Surg Oncol
12. Tanihara F, Takemoto T, Kitagawa E, et al. Somatic cell reprogramming-free generation of genetically modified pigs. Sci Adv
13. Campana LG, Clover AJ, Valpione S, et al. Recommendations for improving the quality of reporting clinical electrochemotherapy
studies based on qualitative systematic review. Radiol Oncol
14. Gehl J, Sersa G, Matthiessen LW, et al. Updated standard operating procedures for electrochemotherapy
of cutaneous tumours and skin metastases. Acta Oncol
15. Djokic M, Cemazar M, Popovic P, et al. Electrochemotherapy
as treatment option for hepatocellular carcinoma, a prospective pilot study. Eur J Surg Oncol
16. Campana LG, Edhemovic I, Soden D, et al. Electrochemotherapy
– emerging applications technical advances, new indications, combined approaches, and multi-institutional collaboration. Eur J Surg Oncol
17. Banks PA, Bollen TL, Dervenis C, et al. Classification of acute pancreatitis—2012: revision of the Atlanta classification and definitions by international consensus. Gut
18. Mueller P, Miketic L, Simeone J, et al. Severe acute pancreatitis after percutaneous biopsy of the pancreas
. AJR Am J Roentgenol
19. Cooper CA, Moraes LE, Murray JD, et al. Hematologic and biochemical reference intervals for specific pathogen free 6-week-old Hampshire-Yorkshire crossbred pigs. J Anim Sci Biotechnol
20. Shah R, Ostapoff KT, Kuvshinoff B, et al. Ablative therapies for locally advanced pancreatic cancer. Pancreas
21. Zmuc J, Gasljevic G, Sersa G, et al. Large liver blood vessels and bile ducts are not damaged by electrochemotherapy
with bleomycin in pigs. Sci Rep
22. Kim AY, Rhim H, Park M, et al. Venous thrombosis after radiofrequency ablation for hepatocellular carcinoma. AJR Am J Roentgenol
23. Narayanan G, Hosein PJ, Beulaygue IC, et al. Percutaneous image-guided irreversible electroporation
for the treatment of unresectable, locally advanced pancreatic adenocarcinoma. J Vasc Interv Radiol