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http://doi.org/10.22214/ijraset.2020.5491

May 2020

Prospects of Irreversible Electroporation in Malignant Tissue or Tumour Ablation Md Sharjis Ibne Wadud1, Rafid Ayman2 1

Department of Biomedical Engineering, Military Institute of Science and Technology 2 Department of EEE, American International University-Bangladesh

Abstract: Irreversible electroporation (IRE) is a novel and promising technique for minimally invasive ablation of solid malignant tumours. IRE uses electric energy to cause cell death by irreversibly destabilizing cell membranes, unlike existing thermal ablation techniques, such cryoablation and radiofrequency ablation. Over the last decade, IRE is being studied as a promising tissue ablation tool utilizing the effect of electric field on cell membrane permeability, without harming the surrounding normal tissue, nerves, vessels and extracellular matrices. IRE is also a non-thermal tissue ablation modality. The potential use of IRE for tissue ablation and cancer treatment has led to considerable amount of studies being conducted to confirm its validity, efficacy, and safety both in in-vitro and in-vivo domain. This work focuses on reviewing the studies that has been conducted so far to analyse the prospects of IRE in tissue and tumour ablation. Keywords: Irreversible Electroporation, IRE, tissue ablation, tumour ablation, non-thermal IRE I. INTRODUCTION Irreversible electroporation (IRE) is a promising novel non-thermal tissue ablation modality which may revolutionize the treatment of local malignant solid tumors [1]–[5]. The growing quest for an alternative minimally invasive for treating localized tumours has steered the investigation and development of several ablation techniques, such as microwave ablation, radiofrequency ablation, and cryoablation. Although these techniques have shown efficacy, they comes with some pitfalls due to their dependence on thermal energy to cause cell death [2], [5]. On the other hand, IRE uses electrical energy to cause focused cell death without thermal damage while keeping the surrounding healthy tissue, vessels and other extracellular matrices unaffected allowing rapid tissue regrowth of normal tissue [1]-[6]. Unlike techniques dependent on thermal energy, IRE requires less consideration for thermal energy dissipation or sinking and has less issues with thermal damage of healthy tissue, thus eliminates a potential cause of treatment failure [1], [3], [5]. Moreover, treatment time for IRE is remarkably shorter than conventional thermal ablation techniques and might allow larger lesions to be treated than other thermal tissue ablation modalities [6]-[7]. Over the last decade, it has drawn considerable attention to carry out continual studies on its safety and efficacy. IRE has been found to demonstrate effective cell death in vitro cell cultures, in normal tissue, in-vivo animal and human clinical studies [8]-[13]. Recently, IRE is being investigated in different organ systems for its applicability. II. BACKGROUND Electroporation is a technique of creating nano-sized pores in cell membrane applying strong electrical fields which increases the permeability of the cell membrane and hampers homeostasis of the cell [7]. The pore formation can be either reversible or irreversible. Although electroporation was invented back in 1750s, it has been used as an important tool in research and medicine since past four decades [7]. Reversible electroporation is being used in various medical applications, such as electro-genotherapy for delivering genes into cell in gene therapy, and electro-chemotherapy for delivering chemotherapeutic drugs which are used as an alternative treatment methods of solid tumours [7], [14]-[15]. When the applied electric field is above a certain threshold, it creates a larger potential gradient, and the cells cannot seal the pores formed. As a result, the cells become dead losing homeostasis. Even a decade ago, IRE was literally considered an unwanted side-effect of reversible electroporation. Until recently, IRE of cell membrane experienced its practical use in food industry for microbial inactivation [16]-[17]. Rubinsky’s group introduced IRE as a viable method to cause cell death by irreversible disruption of cell membrane [1], [3], [4]. Over the past 10 years, IRE is progressing as an important tool for tissue ablation in its own right. IRE has been found to demonstrate effective cell death in normal tissue [1], and in-vitro cancer cell cultures, [18].The first in-vivo study on animal was reported in 2006 by Edd et al [3], and on humans in 2010 by Pech et al [12].

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International Journal for Research in Applied Science & Engineering Technology (IJRASET) ISSN: 2321-9653; IC Value: 45.98; SJ Impact Factor: 7.429 Volume 8 Issue V May 2020- Available at www.ijraset.com III. PRINCIPLE OF IRE IRE benefits from the property of electrical potential gradient across cell-membranes to form irreversible pores in the cellmembrane. An IRE generator transmits electric pulses which alters the transmembrane potential of cell membrane, resulting the disruption of lipid bilayer, and creating irreversibly permanent pores that increases the cell membrane permeability as shown in Fig. 1 [1], [3], [4], [7], [19]. As a result the cell loses homeostasis and results apoptotic focused cell-death within few seconds. It also creates a well demarcated ablation region along with sharp separation boundary between untreated and treated areas [3], [6]. IRE’s apoptotic effect leads to its promising features, includes the ability to affect targeted cells, while leaving the surrounding extracellular tissue, bile ducts ,blood vessels, the urethra, and nerves unaffected to function normally [10], [20]–[24]. These surroundings are spared because of their elastic fibre contents doesn’t have cell-membrane where IRE can form pores. Another hypothesis that explains the sparing of these surroundings is that gap-junctions found within the cellular structure of these surrounding structures allow the IRE electric currents to travel through the gap-junctions from one cell to another without altering the integrity of the cell-membrane and connective tissue structures [20]. As IRE is not dependent on heating for ablation, it does not have heat sink effect, the cooling effect caused by blood flow. This effect hampers the effectiveness of thermal ablation techniques [5], [23]. This indicates its potential ability to ablate tumours completely up to the vessels while not losing any cold or heat due to blood flow, as observed in cryoablation and RFA. This in another way means that IRE is able to ablate large hepatocellular carcinoma around hepatic vein and portal vein, which were not possible to ablate in the past [6], [20]-[22]. D i s t an c e b et w ee n E l ect r o d es

N eed l e S h af t (I n su l a t i n g )

Ablation Region Le n g t h o f El e ct r o d e ,L

El ect r o d e

D i am et e r o f E l ec t r o d e , B

Tiss ue

(a) (b) (c) Fig.1: (a) Theoretical representation of pore formation. Transition from intact bilayer followed by a hydrophobic pore and finally the creation and expansion of a hydrophilic pore [42] (b) Example of molecular simulation showing the formation of hydrophilic pores [43]. (c) IRE configuration IV. IRE TREATMENT SYSTEM IRE utilizes a direct current (DC) generator to apply high voltage short pulses of electric current to form irreversible pores in the cell-membrane [21]. The procedure necessitates a medical device designed for medical use, and capable of balancing the high energy electric pulses with operation and patient safety. Bertacchini et al. reported the first ever IRE system approved to be used clinically in 2007 [25]. One of the important concerns of IRE is safety, as irreversible electroporation of a 50 to 70 cm3 target volume requires pulses up to 3000 V and current up to 50 A, [25]. The device should produce an electric field gradient of 800 V/cm in a volume of 40 cm3, which is the threshold for IRE reported by Davalos et al. [1]. The IRE system consists of two principle components: an IRE generator and electrode probes, as shown in Fig 2 (NanoKnife; AngioDynamics, USA). The generator is able to deliver between 100 V and 3000 V of electrical energy in 90-100 pulses having maximum pulse length of 100 ms. The electrodes probes usually have a length of 15 cm and a diameter of 16-19 gauge. The electrode probes are inserted inside or around the target ablation zone. A single bipolar probe or two or mode monopolar probes must be utilized at a time. An electric field is developed between them in a series of microsecond pulses that induces cell-death. The bipolar probes can create an ellipsoid shaped ablation region with axes of 15 mm and 30 mm approximately observer in in-vitro porcine data [12], [25].

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(b)

(a)

(c) Fig. 2: Current IRE system. (a) IRE generator from AngioDynamics Inc. (b) monopolar IRE probes inserted in tumor model. (c) monopolar IRE probe spacer. Depending on the size and shape of the ablation zone, the number of probes to be used. Moreover, the gradient of the field is dependent on the voltage applied and distance between the electrodes. [12], [25]. The treatment using IRE is minimally invasive, and may be performed in association with computed tomography (CT) or ultrasound (US). Magnetic resonance imaging (MRI), US or CT can also be used after an IRE procedure to assess the extent of tissue ablation. The provision of pre- and peri-procedural imaging with CT and US, helps accurately determine the tissue volume to be ablated or treated. It also helps determine appropriate treatment planning and positions of the electrodes to be placed [6], [20]. To minimize the potential risk of arrhythmias, an electrocardiogram (ECG) synchronizer should be utilized to maintain the synchronization of IRE pulses and the cardiac rhythm refractory period [9], [12], [25]. The IRE treatment procedure time is extremely short, lasts minutes because tissue ablation typically takes 90â€“100 pulses, synchronizing with the number of heart beats [12], [25]. Conventional protocols of IRE obtain ablation through a sequence of unipolar electric pulses which result in muscle contractions. IRE procedure is usually administered under general anaesthesia along with neuromuscular blocking agents to prevent muscle contraction, as the treatment requires the patient to be motionless. A method to reduce the untargeted volume which may be vulnerable to muscle contraction have been proposed by Goldberg and Rubinsky [26]. The authors suggested that if a central energized electrode is surrounded with a series of grounded electrodes, the volume of tissue exposed to electric fields is reduced and reduces potential muscle contraction. This novel approach that is based on the concept of Faraday cage. This approach requires one superficially inserted, energized electrode surrounded by at least 16 grounded electrodes. Upon clinical translation, this invention has potential to reduce the amount of muscle relaxants administration to obtain sufficient paralysis [26]. A slight modified approach, termed as high-frequency irreversible electroporation (H-FIRE) has been recently developed by Arena et al [27]. H-FIRE utilizes high frequency bipolar bursts to avoid muscle contraction, without sacrificing the cell death efficiency due to non-thermal electroporation. In an experiment, Rat brain tissue had been subjected to 90 pulses of H-FIRE, and IRE at 250 kHz or 500 kHz. The results showed that IRE led to muscle contractions whereas H-FIRE did not led to muscle contractions. Histopathological examination revealed that H-FIRE at 250 kHz or 500 kHz was indistinguishable from IRE in terms of ablation precision and accuracy, [27]. The clinical application of H-FIRE have the potential to eliminate the necessity to administer neuromuscular relaxants during IRE procedure.

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International Journal for Research in Applied Science & Engineering Technology (IJRASET) ISSN: 2321-9653; IC Value: 45.98; SJ Impact Factor: 7.429 Volume 8 Issue V May 2020- Available at www.ijraset.com V. STUDIES, CLINICAL PROGRESS, AND POTENTIAL IRE is recently being extensively studied in several organ systems as a minimally invasive, non-thermal tissue ablation technique for ablating tumours, and other undesired tissue. After Davalos et al [1] demonstrated that IRE can be utilized to destroy substantial amount of tissue sparing thermal damage, several researchers investigated and confirmed these findings in large and small animal models in the liver, [1], [3], [6], [20], [28] pancreas,[29]-[30], breast [31], brain [32]–[33], prostate [21], lung [34], kidney [35], and in mouse sarcomas [36]-[37] by applying various pulse parameters. Over the past few years, in clinical in vivo human trials of IRE various data have been presented at different literature concentrating on the treatment of malignant tumours within the liver, pancreas, prostate, lung, and kidney. These studies have evaluated the efficacy and safety of performing IRE for malignant tissue ablation in human patients, presenting a favourable safety profile and suggests the possibility of conducting further human studies. The following is a summary of preclinical and clinical human studies performed by IRE tumour ablation technique. A. Studies on Liver In a study by Lee et al [38] which consists of 35 New Zealand white rabbits with VX2 tumours transplanted within their livers, the efficacy of IRE has been demonstrated in treating large hepatic tumours. In the study, 10 rabbits were applied single IRE, 10 rabbits were subjected to multiple IRE applications, and 15 rabbits were used as a control group. After IRE ablation to each treatment group, the respective livers had been evaluated with CT, US, and immune-histochemical analysis. The study found that multiple IRE applications were able to induce complete ablation of tumour without any issues [38]. Kingham et al [10] performed a ten month retrospective study on 28 human patients who were treated with IRE for 65 hepatic malignant tumours. The patients underwent MRI or contrast-enhanced CT postoperatively, at 1 to 3 months, and at 6 months after the procedure. At 6 months, a recurrence occurred for four tumours. Only one postoperative portal vein thrombosis was observed despite 41 tumours being present within 1 cm of portal pedicle or a major hepatic vein. There was no mortality reported associated with the treatment at 6 months. Hence, the researchers concluded that IRE may be used as a safe treatment option of perivascular hepatic malignant tumours which might not be treated with conventional techniques [10]. Cannon et al [13] reported on effect of IRE of hepatic tumours in proximity of vital structures. In this study, 44 patients underwent 46 IRE procedures, includes 14 hepatocellular, 20 colorectal metastases, and 10 other metastases. The study reported primary success in all 46 (100%) of the treatments, along with no local recurrence survival at 3 months, 6 months, and 12 months of 97.4% , 94.6%, and 59.5%, respectively. They observer a trend toward higher recurrence rates during the ablation of tumours having size over 4 cm (HR 3.236, 95% CI: 0.585–17.891; P ¼ 0.178). Five patients experienced nine adverse events, of which three were assumed to be related to the IRE procedure, which includes abdominal pain, neurogenic Bladder, and flank pain. But all the complications were resolved within 30 days. B. Studies on Pancreas Before IRE, the options for the non-invasive treatment of locally progressed pancreatic cancer was literally non-existent due to the damage to critical adjacent vessels and high risk of pancreatitis, [30]. In a study conducted by Charpentier et al [29], IRE was applied on normal pancreas of four domestic female swine, using two monopolar probes keeping spaced 9–15 mm apart. 90 pulses of 1500 V/cm were applied for each ablation. Three animals in which probes were separated buy 10 +/– 1 mm demonstrated evidence of irreversible ablation by triphenyltetrazolium chloride (TTC) staining and gross appearance. The only animal for which the probes were spaced at 15 mm did not show irreversible ablation even after 2 weeks. This was expected as the space was wider with relatively low voltage, which created reversible electroporation. These observations indicates that IRE appears to be a safe procedure for ablation of pancreas tissue. And with staining by TTC, it is possible to predict the extent of the IRE ablation zone within 2 hours of IRE treatment [29]. In a recent study, José et al [39] showed the potential of IRE in pancreatic ductal adenocarcinoma (PDAC) treatment. The first report of application of IRE for human pancreatic cancer treatment, a prospective multi-institutional pilot had been conducted by Martin et al [11] and they evaluated the applicability of IRE in locally progressed pancreatic cancer treatment. 27 patients were subjected to treatment, of them 26 were invasively treated and one was percutaneously treated as the patient had a history of multiple past surgeries in the same region. Nineteen patients underwent in situ IRE, whereas the others’ treatment consisted of IRE along with either pancreatic head (n ¼ 4) or left-sided (n ¼ 4) resection. No postoperative complications were observed except one mortality. All patients who were under 90-day follow-up had successful ablation of all tumours with no recurrences [11]. The success of the study suggests that IRE is one of only a few options for patients with locally progressed pancreatic cancer. Following the previous study, Bagla and Papdouris [8] demonstrated the utility of percutaneous IRE for surgically unresectable, nonmetastatic, pancreatic adenocarcinoma treatment in a 78-year-old patient.

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International Journal for Research in Applied Science & Engineering Technology (IJRASET) ISSN: 2321-9653; IC Value: 45.98; SJ Impact Factor: 7.429 Volume 8 Issue V May 2020- Available at www.ijraset.com C. Studies on Prostate Onik et al [21] investigated IRE ablation in six males’ dogs normal prostate and observed total cellular destruction within the IRE zones, and within 2 weeks rapid resolution of the zones with marked shrinkage. Structures, such as vessels, urethra, rectum, and nerves were unaffected by the IRE application [21]. Rubinsky et al [18] studied ablation by IRE of prostate adenocarcinoma cells in vitro in order to determine the length, number, and field strength of IRE electric pulses needed to cause total ablation of human cancer cell. The group found that the lower and upper limits of pulse number and length in a field range of 2000 V/cm to 2500 V/cm with 90 pulses of 100 ms at 2500 V/cm separated by 100 ms off-time could solely ablate prostate cancerous cells without thermal damage [18]. These findings indicates that IRE ablation of the prostate is possibly safe and have advantageous in the clinical setting than the thermal ablation modalities, also preservation of surrounding important structures is a plus in this delicate part of the body. D. Studies on Breast In a recent study conducted by Neal et al [31], human breast cancer tumours were implanted orthotopically in the mammary fat pad of 11 female Nu/Nu mice. The mice were divided into control and treatment groups. IRE were applied on seven tumour-bearing mice and regression of tumour was noticed in five out of seven of the human mammary tumours in 4 weeks after the treatment, along with continued growth in controls [31]. These findings suggest that IRE might be an alternative to surgical resection used for breast conserving therapy. E. Studies on Lung The proximity of the heart to the lungs can cause a contraindication to ablative modalities such as RFA. Because RFA results in thermal sinks within the mediastinum. As IRE is a non-thermal modalities, its clinical application would present hope for thoracic tumours patients who are presently not candidates for surgical procedures [40] Dupuy et al [40] first studied to evaluate the efficacy of IRE for the lung tissue ablation. They created fifteen percutaneous fluoroscopy-guided IRE lesions in nine anesthetized domestic swine lungs. High-resolution CT and Radiographs were used to evaluate the animals prior to each lung was harvested to perform histological examination. Microscopically, the parenchyma which was subjected to IRE demonstrated a well-demarcated damage of alveoli with inflammatory infiltration and fibrosis. There was no observed damage to blood vessels and bronchioles within the ablative zone [40]. Deodhar et al [34] performed a similarly designed study, within months of the initial study, also involved swine. The study was conducted to re-confirm the potential use of IRE for lung tissue ablation [34]. F. Studies on Sarcoma Al-Sakere et al [23] investigated the application of IRE for the treatment of aggressive cutaneous sarcoma tumors carefully implanted in mice. Six mice of 6–8 weeks old, had been inoculated subcutaneously with cells from an LPB cell-line, a methylcholanthrene-induced C57Bl/6 mouse sarcoma cell line, resulting tumours of diameter 4–5 mm in 9 days. They demonstrated that successful result of the IRE technique is associated with the applied electric field strength, temporal pulse delivery mode, and the total duration of the pulse. The best outcomes were achieved using plate electrodes to deliver 80 pulses of 100 ms at rate 0.3 Hz, and with an electrical field amplitude of 2500 V/cm across the tumour. Histological studies were performed to confirm tumour regression, with total regression in 12 out of 13 tumours without tissue heating. G. Studies on Cervix The efficacy of IRE treatment at inducing death of human cervical adenocarcinoma (HeLa) cells has been investigated by Zhou et al [41]. The researchers observed that either a high number of low-voltage pulses or a low number of high-voltage pulses were able to cause HeLa cell death. When the number of High voltage pulses were low, necrosis of targeted HeLa cells. In contrast, higher number of low-voltage IRE pulses induced apoptotic cell death, which is a preferred method due to the lack of associated inflammation and capability of post therapy cell regeneration [41]. VI. CONCLUSIONS In conclusion, Irreversible Electroporation (IRE) is a promising novel minimally invasive tissue or tumour ablation modality with several demonstrated advantages: (1) preservation of surrounding vital structures while treating a targeted region;(2) no thermal damage; (3) non-necrotic, apoptotic cell death along with a rapid tissue regeneration; (4) not affected significantly by the heat-sink effect; (5) procedure time is shorter; and (6) able to perform with imaging modalities and real-time monitoring is possible. However, the research and development of IRE ablation is still at its infancy. Hence, more and more investigations are needed for further understanding and to optimize this modality to improve its efficacy and safety.

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International Journal for Research in Applied Science & Engineering Technology (IJRASET) ISSN: 2321-9653; IC Value: 45.98; SJ Impact Factor: 7.429 Volume 8 Issue V May 2020- Available at www.ijraset.com REFERENCES [1] [2] [3] [4] [5] [6] [7] [8] [9] [10] [11] [12] [13] [14] [15] [16] [17] [18] [19] [20] [21] [22] [23] [24] [25] [26] [27] [28] [29] [30] [31] [32] [33] [34] [35]

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