Champs Électriques Pulsés

Résumé

Cette information a été composée par le Fonds Anticancer et est basée sur l'information professionnelle. Dernière adaptation: août 2013.

 

L’utilisation de champs électriques pulsés est une technique émergente dans le traitement du cancer. Il s’agit d’une thérapie locale, n’utilisant ni chaleur, ni médicament. Le traitement par champs électriques pulsés (PEF pour Pulsed Electric Fields en anglais) utilise de brèves et intenses impulsions électriques qui provoquent soit la formation de petits trous (nanopores) dans la membrane des cellules cancéreuses, soit une instabilité de la membrane ou des éléments qui composent la cellule. La durée des impulsions électriques varie de pico en microsecondes. L’électroporation irréversible (IRE pour Irreversible Electroporation en anglais) utilise des impulsions relativement longues (microsecondes) de faible intensité. L’ablation par champs électriques pulsés de l’ordre de nanosecondes (nsPEF) utilise en revanche des champs électriques plus intenses pendant une durée plus courte (nanosecondes). Les deux méthodes provoquent des dommages irréparables aux tumeurs et entraînent de ce fait la mort cellulaire. Des aiguilles spéciales (électrodes) sont appliquées directement sur la tumeur. Une étude récente a démontré que même des impulsions plus brèves de quelques picosecondes peuvent entraîner la mort cellulaire. Il ressort d’expériences menées sur des animaux et d’études cliniques de phase I sur l’homme que l’IRE et la nsPEF ralentissent la croissance de la tumeur et induisent la mort cellulaire par apoptose. La sécurité d'utilisation et l'efficacité de ces thérapies font actuellement l’objet d’études. Le but de ce texte est d’offrir un aperçu sur ces techniques en cours de développement.

C'est quoi?

L’ablation par champs électriques pulsés (PEF) traite les tumeurs au moyen de champs électriques intenses, non thermiques et de haute tension, sans avoir recours à la production de chaleur, à des médicaments ou à d’autres substances chimiques. Les techniques basées sur les PEF provoquent des dommages irréversibles aux cellules. Ces techniques sont réparties en trois groupes différents selon la durée des impulsions. L’électroporation irréversible (IRE) est une technique d’ablation qui, comme l’électrochimiothérapie (voir site du Fonds Anticancer), consiste à administrer des impulsions électriques de quelques microsecondes, mais en utilisant des champs électriques plus intenses. De ce fait, de minuscules trous se forment dans la membrane cellulaire et perturbent fortement l’équilibre dans la cellule, jusqu’à provoquer la mort cellulaire. Une caractéristique unique de cette thérapie est que les structures vitales de la zone traitée, comme les vaisseaux sanguins, sont préservées. Les autres techniques d’ablation endommagent ces structures du fait de l’application de températures extrêmement élevées (comme lors de l’ablation par radiofréquence, entre autres techniques) ou de réactions photochimiques (comme lors de la thérapie photodynamique). Il existe un instrument d’ablation des tumeurs disponible sur le marché, appelé NanoKnife® (AngioDynamics). L’ablation par champs électriques pulsés de l’ordre des nanosecondes (nsPEF) utilise des champs électriques plus intenses pendant une durée plus courte (nanosecondes), en comparaison avec l’IRE. Autre différence par rapport à l'IRE, qui provoque avant tout des dommages aux membranes cellulaires, la technologie nsPEF attaque tant les membranes cellulaires que les membranes des structures contenues à l’intérieur des cellules, et mène donc à la mort cellulaire. En outre, la nsPEF perturbe le système vasculaire à l'intérieur de la tumeur, ce qui diminue l’apport en oxygène et en nutriments. Certains instruments, tels que le générateur d’impulsions PulseCure® et l’électrode NanoBlate® (BioElectroMed), ont été mis au point pour administrer des nanoimpulsions localement sur les tumeurs. Récemment, de nouvelles électrodes ont été conçues en combinaison avec des endoscopes pour pouvoir traiter les tumeurs situées plus en profondeur. Ces traitements peu invasifs s’efforcent de limiter le recours à la chirurgie et de réduire les effets secondaires, les cicatrices, la douleur et le risque de décès. Certains chercheurs travaillent sur des champs électriques d’une intensité encore plus élevée et une durée d’impulsion encore plus brève (picosecondes). En réduisant de la sorte la longueur d'impulsion, il devient possible d’utiliser des antennes à haut débit au lieu d’électrodes de contact et ainsi de traiter de manière moins invasive des tumeurs situées en profondeur.

Efficace?

Actuellement, plus de 20 institutions réalisent des études précliniques et cliniques sur l’IRE. Des expériences menées sur des animaux ont démontré que l’IRE provoquait une régression de la tumeur. Les études cliniques de phase I chez l'homme ont démontré que l'IRE pouvait traiter le cancer du foie, du poumon et du rein de manière sûre. Aux États-Unis, environ 30 hôpitaux peuvent proposer ce traitement, au moyen du système NanoKnife®. À l’heure actuelle, les données disponibles sur l’IRE sont insuffisantes pour que cette technique d’ablation soit utilisée en routine. Davantage d'études cliniques sont nécessaires pour résoudre certains problèmes et pour examiner plus minutieusement l’efficacité de l’IRE. L’ablation nanoélectrique (nsPEF) fait également l’objet de recherches comme traitement du cancer. Différentes expériences menées sur des animaux ont démontré que l’utilisation de nsPEF permettait de ralentir la croissance des tumeurs et de provoquer la mort cellulaire par apoptose. Ces études ont surtout été effectuées sur des mélanomes (cancer de la peau). Dans ce cas, les expériences réalisées sur les animaux démontrent en général que la nsPEF est une méthode d'ablation rapide, sûre et puissante et qu'elle assure une destruction totale du tissu. Étant donné que le mécanisme d’ablation grâce à la technologie nanoélectrique est tout simplement physique et non spécifique à certaines cellules, les scientifiques s'attendent à ce que la thérapie soit également efficace chez l'homme. La première étude relative à l'ablation nanoélectrique sur des patients atteints d’un carcinome basocellulaire a débuté récemment.

Sans danger?

Plusieurs caractéristiques uniques distinguent la PEF des thérapies d’ablation thermiques disponibles aujourd’hui, comme l’ablation par radiofréquence, par micro-ondes et la cryoablation. Aucune modification de la température n’intervient dans la tumeur, ce qui évite les dommages thermiques. Le principal avantage de la PEF par rapport à la thérapie photodynamique ou l’électrochimiothérapie est l’absence de substances chimiques, l'effet du champ électrique étant suffisant pour détruire les cellules cancéreuses. Une autre caractéristique majeure de la PEF est qu’elle fonctionne très rapidement. La technique est simple et peut être suivie via des enregistrements vidéo. En général, les complications possibles de l’IRE sont une thrombose d’un vaisseau sanguin, une perforation intestinale, une hémorragie et la formation d’hématomes. Le passage des aiguilles augmente le risque d’infections et de formation de fistules sur le trajet de l’aiguille. La procédure comporte également un certain risque du fait des impulsions électriques intenses. Elles peuvent provoquer des troubles du rythme cardiaque, des mouvements musculaires incontrôlés et une douleur. Il existe certaines mesures de précaution à prendre pour les patients porteurs de dispositifs électriques implantables (pacemaker). Enfin, certains risques sont spécifiques à la localisation de la tumeur. Dans le cas du traitement d’une tumeur au poumon par exemple, il existe un danger de pneumothorax. Bien que les connaissances sur l’utilisation de la nsPEF chez l’homme soient encore limitées, les scientifiques s’attendent à ce que des impulsions électriques correctement paramétrées puissent être appliquées sans effets secondaires importants.

Plus d'info

Plus d’informations

Angiodynamics (http://www.angiodynamics.com)
Pulse Biosciences (http://pulsebiosciences.com/)
Frank Reidy Research Center for Bioelectrics
(http://www.odu.edu/bioelectrics)
National Institute for Clinical Excellence (http://www.nice.org.uk)
Pulsed Power and Bioelectrics (http://www.pulsedpower.eu)

Résumé

This text is written by Erik Cabuy (The Anticancer Fund) and reviewed by Prof. Damijan Miklavčič (University of Ljubljana, Faculty of Electrical Engineering, Slovenia), Prof. Richard Nuccitelli (BioElectroMed Corp., Burlingame, CA, USA), Prof. Thomas Vernier (Center for Bioelectrics, Norfolk, Virginia, USA), and by Prof. Rafael Davalos, Bioelectromechanical Systems Lab, School of Biomedical Engineering and Sciences, Virginia Tech-Wake Forest University, Virginia, USA. Last update: August 2013.
 

The application of pulsed electric fields has emerged as a local, non-thermal and drug-free therapy for cancer. Pulsed electric field (PEF) therapy is a procedure using intense but short electric pulses that provoke either permanent permeabilization of cancer cells or destabilize the cell membranes and intracellular components to which the cells are unable to repair resulting in their death. The length of the electrical pulses may vary from picoseconds to microseconds. Irreversible electroporation (IRE) uses relatively long pulses (100 microseconds) and low electric fields (V or low kV/cm). It induces cell death by creating permanent pores in the cellular membrane. Nanosecond pulsed electric field (nsPEF) ablation uses pulse power with much shorter pulse durations (ns) and higher electric fields (30 kV/cm). Both techniques are applied directly to the tumor through needle electrodes. More recent research has shown that pulses of picoseconds duration may cause cell death as well. Pulse power IRE and nsPEF to solid tumors has shown in in vivo and in phase I clinical trials to result in reduced tumor growth and induction of apoptosis in the treated cells. Its safety and efficacy is currently being investigated more thoroughly. This summary is meant as a general overview and first introduction to the pulsed electric field applications in cancer treatment.
 

C'est quoi?

2.1 Introduction

The effects of pulsed electric fields on biological cells and tissues have been the topic of research for many years. The earliest report of bioeffects arising from the direct application of voltage using contact techniques (as opposed to contactless exposure using electromagnetic radiation) was when Stampfli and Willi reported on electrical induced changes of cell membranes in 1957. In particular they measured changes in membrane conductivity and membrane potential. They further found that membrane damage is irreversible if the applied electrical pulses are longer but otherwise the membrane is restored to its original characteristic. Almost a decade later, damaging effects of strong electric fields on bacteria were reported suggesting nonthermal membrane interactions (Sale and Hamilton, 1967; 1968; Hamilton and Sale, 1967). Subsequent experiments showed that strong electric field pulses caused the increase in permeability of the plasma membrane of a biological cell (Neumann and Rosenheck, 1972), an effect subsequently named electroporation. Kinosita and Tsong also suggested that this transient permeation is due to formation of pores with a diameter in the range of < 1 nm (Kinosita and Tsong, 1977). Electroporation then became a popular method in the laboratory to increase the bacterial or mammalian uptake of drugs, dyes, or DNA in culture. In 1987, Okino and Mohri showed for the first time that electroporation or electro-permeabilization can be applied in vivo to increase the concentration of an anticancer agent (bleomycin) in solid tumors (Okino and Mori, 1987). Similar preclinical investigations in the late 1980s (Orlowski et al. 1988) were followed by the first clinical trials demonstrating its effectiveness over bleomycin alone for the treatment of cutaneous metastases of head and neck carcinoma patients (Mir et al. 1991; Belehradek et al. 1993). Current applications have primarily centered on the use of electrochemotherapy, the combination of reversible electroporation with the administration of otherwise low-permeant cytotoxic drugs (see the Anticancer Fund webpage for a review of this technique).

Electroporation, however, is a dynamic phenomenon that is reversible or irreversible depending on the induced transmembrane voltage at the plasma membrane. When the permeable state is only temporary, the permeabilization is referred to as reversible (ref. electrochemotherapy) as opposed to the irreversible electroporation (IRE) which leads to cell death (Silve et al., 2012). During reversible electroporation, the cancer cell death created by IRE was considered highly undesirable in the past (Lee et al., 2010a) and for many years, IRE was used primarily in the food industry for sterilization of micro-organisms (Hamilton and Sale, 1967). However, in the past few years, the effects of IRE on tissue ablation have been studied more in detail and IRE as a method of permanently destroying cancerous cells have led to its clinical application in cancer treatment. The first studies of IRE involved the exploration of cellular destruction through the application of electrical pulses. Research conducted by Davalos and coworkers tested this concept using a mathematical model and in vivo conditions (Davalos et al., 2005). Results from these studies showed that IRE could indeed induce cell death without using thermal energy. Davalos and colleagues conducted the first successful in vivo IRE procedure on mice tumors (Al-Sakere et al., 2007a).

In 2001 Schoenbach and coworkers reported the cellular response (intracellular effects) due to sub-microsecond pulses with mega-volt per meter magnitudes (i.e. 10 kV/cm) (Schoenbach et al., 2001). Shortly thereafter, Beebe and coworkers first showed that applying ultrashort, nanosecond pulsed electric fields (nsPEF) to mammalian cells and solid tumors results in reduced tumor growth, and induction of apoptosis in the treated cells (Beebe et al., 2002; 2003). A number of studies have confirmed these observations, showing that nsPEFs do induce apoptosis in a number of cancer cell types in vitro and tumors in vivo. Just like for IRE, this has motivated significant interest in nsPEF as a basis for treating cancer tumors without delivering drugs or genes (Garon et al., 2007; Nuccitelli et al., 2006; 2010). With the developments of electrical engineering, researchers could apply electric fields with even higher electric intensity and shorter (subnanoseconds) duration to cells. This push toward further pulse shortening is driven in part by the possibility of using wideband antennas, rather than direct contact electrodes, to deliver such pulses for noninvasive treatment of deep lesions (Joshi and Schoenbach, 2010). However, whereas studies on nanosecond pulse effects have already led to medical applications, there are just a few experimental studies on the bioelectrical effects of subnanosecond pulses with external electric field amplitudes greater than 10 kV/cm. High-intensity ultrashort pulsed-power generated electric fields have been used in a variety of biological applications and have initiated the establishment of a brand new research area called bioelectrics. The growing interest in the research was confirmed when several institutions (including universities in USA, Japan and Germany) came together in an International Consortium on Bioelectrics. Bioelectrics combines two distinct disciplines: pulsed high voltage engineering and cell biology (Schoenbach et al., 2004; 2007; 2008). The field has strong overlap with the field of bioelectromagnetism, which will be the topic of a forthcoming Anticancer Fund review.

The proposals of using pulsed electric fields to pursue the treatment of cancer free of cytotoxic drugs and to achieve selective killing of tumor cells by inducing apoptosis is worthwhile to examine. Many cancers are resistant to current treatments and there is an urgent need for therapeutic innovations and discovery. There are currently three pulsed electric field therapies being tested on various tumor types in animals and preclinical studies. The aim of these minimally invasive treatments is to limit surgery and reduce side effects, scarring, pain, and mortality of patients while remaining cost-effective and safe. This review provides a brief overview of these therapies and their current stage in clinical research.

2.2 Principles of pulsed electric fields

2.2.1 Rationale of using pulsed electric discharges to cancer cells

The cell plasma membrane consists of a lipid bilayer with a thickness of approximately 5 nm. Its function is to protect the cell interior but also to facilitate the flow of selected types of ions and other materials from and to its surroundings. Under physiological conditions, the plasma membrane is subjected to a voltage difference caused by a system of ion pumps and channels in the membrane. Bioelectric fields are present whenever there is a potential difference between two regions in an organism. Every cell in our body generates this electric potential difference across the plasma membrane termed the resting transmembrane voltage that is about 70 mV, inside negative (Alberts et al. 2008). In biophysics, cell membranes represent nonconducting, dielectric barriers and function as a near-ideal capacitor that can easily be charged by applying an external voltage pulse (Joshi and Hu, 2011). Consequently, large localized electric fields can be created across membranes that can then drive a host of bioeffects.

The exposure of a cell to an externally applied electric field results in an additional component of the voltage across the membrane (Kotnik et al., 2010). This component, termed the induced transmembrane voltage, is superimposed onto the resting voltage and exists only as long as the external field is present. With the accumulation of ions along the cell membrane, the potential difference across the membrane increases and the electric field inside the cell is reduced simultaneously (Schoenbach et al., 2007). The induced transmembrane voltage is proportional to the strength of the external electric field, and exposures to sufficiently strong fields can lead to transmembrane voltages far exceeding their physiological range (Hu and Joshi 2009). Such large electric fields will exert a force on charged molecules such as water dipoles and can drive water into the lipid membrane to form a water-filled defect or pore through the bilayer (Delemotte and Tarek, 2012). Such pores will allow the movement of small molecules across the membrane. This phenomenon is termed electroporation, or electropermeabilization (Neumann and Rosenheck 1972; Teissié et al. 2005; Escoffre et al. 2009). This and possibly other intracellular unknown physical insults induce apoptosis in cells. The possibility of using electric pulses in cancer therapy may also rely on the different responses between cancers cells and normal cells. Cancer cells would have a higher apoptosis percentage than normal cells when exposed to the same electric field (Tang et al., 2007) although this statement has not been widely cited.

The electric fields that are required to achieve cell death depend on the duration of the applied pulse, since this process involves the gradual charging of the capacitive sheath followed by the molecular rearrangement of the lipids (Joshi and Schoenbach, 2010). Typical pulses range from tens of microseconds with amplitudes of a few kilovolts per centimeter, to pulses of a few nanoseconds or smaller but requiring fields of tens of kilovolts per centimeter. These parameters of electric pulses play major roles in the type of effect on targeted cells. The PEF-based techniques creating irreversible cellular damage are divided in accordance to the pulse length hereafter. Their medical translations into cancer therapies are named irreversible electroporation (IRE) and nanosecond pulsed electric field (nsPEF) therapy or nanoelectroablation. The use of subnanosecond (i.e. picosecond) pulsed electric fields needs to be further explored as a cancer therapy (sometimes named picoelectroablation hereafter).

2.2.2 Irreversible electroporation

Electrochemotherapy uses conventional electroporation with electric field durations in the micro- to millisecond range (see Anticancer Fund review). This causes transient defects in plasma membranes, allowing the entry of poorly permeable drugs such as bleomycin to ablate tumors. Experimentally, the parameters that mainly influence the size of the ablated zone are the pulse amplitude, pulse duration, number of pulses, and to a lesser extent pulse repetition frequency of pulses. By modulating the parameters of the pulses as suggested by Davalos and coworkers it is possible to obtain a transmembrane potential high enough to ensure permanent permeabilization (Davalos et al., 2005). Hence, conventional electroporation has been extended to irreversible electroporation (IRE), which uses similar pulse durations but electric fields high enough to generate permanent pores in the plasma membrane. IRE uses relatively long pulses (milliseconds or microseconds) and low electric fields (V or low kV/cm) (Edd et al., 2006). A typical IRE procedure for a solid tumor, with a size of approximately 3 cm in diameter, uses 90 pulses with pulse length of 100 microseconds (Lee et al., 2010a). It was demonstrated that short pulses with brief intervals allow for cooling of tissues to avoid any thermal effect. As a result, the electric field created by IRE is devoid of any joule heating and therefore, non-thermal ablation is achieved.Hence, conventional electroporation has been extended to irreversible electroporation (IRE), which uses similar voltages but pulse durations long enough to generate permanent pores in the plasma membrane. It uses relatively long pulses (milliseconds or microseconds) and low electric fields (V or low kV/cm) (Edd et al., 2006). A typical IRE procedure for a solid tumor, with a size of approximately 3 cm in diameter, uses 90 pulses with pulse length of 100 microseconds and voltages between 1.5 and 3kV/cm (Lee et al., 2010a). It was demonstrated that short pulses with brief intervals allow for cooling of tissues to avoid any thermal effect. As a result, the electric field created by IRE produces negligible joule heating and therefore, non-thermal ablation is achieved.

During electroporation, cancer cells are subjected to these short, high-voltage electric pulses. The current understanding of the underlying mechanism in IRE is that the application of a high electric field increases the transmembrane potential of cells, causing structural rearrangements in the lipid bilayer, promoting the formation of semi-permanent to permanent aqueous pores in the cellular membrane (Ivorra et al., 2009; Sahakian et al., 2011). Indeed, the most widely, albeit not universally accepted mechanism underlying permeabilization is the formation of membrane pores, or electroporation. A recent study has shown the existence of nanopores on cell (hepatocyte) membrane surfaces after the application of IRE by scanning electron microscopy (Lee et al., 2012). Nanopores induced by IRE appear to be generally larger than when induced by reversible electroporation i.e. electrochemotherapy, which may have implications for irreversibility or permanency. However, the exact mechanism by which electrical impulses permeabilize the cell membrane is not fully understood. These pores would allow the abnormal flux of ions across the cellular membrane causing a colloid-osmotic imbalance, which disrupts the cellular homeostasis (Al-Sakere et al., 2007b), cell swelling and blebbing leading to necrotic cell death (Nesin et al., 2012). IRE would affect essentially only one component of the tissue, the lipid bilayer, and spares other critical components of the tissue such as major blood vessels, nerves and the extracellular matrix (Edd et al., 2006; Onik et al., 2007; Rubinsky, 2007a; Maor et al., 2009).

2.2.3 Nanosecond pulsed electric fields

Nanosecond pulsed electric fields (nsPEFs) extend conventional electroporation by using electric pulses with shorter durations, in the nanosecond range, and even higher electric fields than IRE, in the tens of kilovolts per centimeter (kV/cm) range (Nuccitelli et al., 2006; Schoenbach et al., 2008; Chen et al., 2009; 2010). These two separate electric field parameters: pulse duration and amplitude determine the efficacy of nsPEF treatment (Nuccitelli et al., 2006). It has been found that the shorter the pulses, the higher the field strength necessary to observe the biological effects (Breton and Mir, 2011). When a voltage gradient is applied long enough to a cell, charges accumulate at the plasma membrane creating an electric gradient across the membrane. This transmembrane potential depends on the electric field amplitude. The most optimal parameters are being determined in in vivo research and may differ per cancer type. For instance, 40 kV/cm electrical pulses have been used to treat melanomas (Nuccitelli et al., 2009; Chen et al., 2010) or 35, 50 and 68 kV/cm to treat hepatocellular cancer (Beebe et al., 2011; Chen et al., 2012). Cell survival studies with 10 ns pulses have shown that the viability of the cells scales inversely with the electrical energy density, which is similar to the dose effect caused by ionizing radiation (Schoenbach et al., 2007). However, the energy transmitted to the treated area by the electric pulses is very low due to the ultrashort pulse duration, and therefore leads to a very weak heating (Schoenbach et al., 2001; Nuccitelli et al., 2006).

Unlike irreversible electroporation, which relies mainly on increasing cell membrane permeability, nanopulses affect both the external cell membrane and the membranes of intracellular organelles, permeabilizing these structures to ions, water, and small molecules (Vernier et al., 2006; Schoenbach et al., 2008; 2010). This is referred to as supra-electroporation with large numbers of small pores in all cell membranes (Gowrishankar et al., 2006). Plasma membrane resistance decreases and cells are depolarized. Initially, however, it was thought that disruption of intracellular membranes can be achieved with ultrashort electrical pulses without loss of the surface membrane integrity (Schoenbach et al., 2001). Experiments with shorter pulses (10 ns) have confirmed these earlier results (Buescher and Schoenbach, 2003; Tekle et al., 2005). However, recent investigations using more sensitive assays established that the plasma membrane is not exempt from poration by nsPEF (Vernier et al., 2004b, 2006; Pakhomov et al., 2007; 2009; Napotnik et al., 2010). Modeling (Hall et al., 2005; Gowrishankar et al., 2006) and experimental evidence (Vernier et al., 2006; Pakhomov et al., 2007; 2009) indicates that nsPEFs cause plasma membrane permeabilization or transient nanopores through which small molecules and ions can pass. The larger the pulse number and the electric field, the larger will be the permeability increase in the plasma membrane (Frey et al., 2006; Pahhomov et al., 2007). Plasma membrane permeabilization may not be the only primary bioeffect of electric pulses. A number of studies by Chen and co-authors indicated that electric pulses of supra-physiological voltage could cause damage to voltage gated (VG) ion channels (Chen and Lee, 1994a,b; Chen, 2005; Chen et al., 2006) or maybe by inhibition of VG ion channels as suggested in a study by Nesin and colleagues (Nesin et al., 2012). Phosphatidylserine externalization indicating induction of apoptosis has been observed as well (Vernier et al., 2004a).

Nanoelectroablation can also generate transient ion-permeable nanopores in the intracellular membranes in all of the cells in a treated tumor (Buescher and Schoenbach, 2003; Tekle et al., 2005; Kotnik and Miklavcic, 2006; Vernier et al., 2008; Smith and Weaver, 2008). These nanopores allow transmembrane movement of molecules smaller than 300 D (Pakhomov et al., 2009; Andre et al., 2010). The reason that nsPEF can penetrate into cells is that the pulse rise time is faster than the time required for charges to redistribute on the cell’s plasma membrane to block penetration of the electric field into the cell (Schoenbach et al., 2004; Kumar et al., 2011). Molecular modeling and experimental studies in which human cells were exposed to pulsed electric fields of up to 300 kV/cm amplitude, with durations as short as 10 ns, have confirmed this hypothesis (Joshi and Schoenbach, 2010). As long as the pulse rise time is faster than the characteristic cellular membrane charging time of about 100 ns (Tekle et al., 2005), the interior charges will not have sufficient time to redistribute or to counteract the imposed field and it will penetrate into the cell and charge every organelle membrane for a duration, which is dependent on both the charging time constant of the cell’s plasma membrane as well as that of the organelle membrane (Nuccitelli et al., 2009). It has to be noted that the different groups working on nanopulses have used 3-600 ns pulses. So, the duration of the pulses has to be taken into account when interpreting the results. For instance, when 300 ns pulses (or longer) are applied, the pulse is too long to allow the interaction of the electric field with the intracellular organelles. Dependent on pulse duration as well as pulse amplitude and on the number of pulses in a pulse train, various effects have been observed. Effects have been reported on intracellular membranes in the cytoskeleton (Hall et al., 2007), endoplasmic reticulum (Smith and Weaver, 2008), mitochondria (Ford et al., 2010) and small nuclear ribonucleoproteins (nuclear speckles), suggesting changes in RNA-protein complexes (Chen et al., 2007). The ion flow across the organelle membranes also results in an immediate transient mild elevation of intracellular Ca2+, and this is followed by a cascade involving nuclear pyknosis, DNA fragmentation and caspase activation (Schoenbach et al., 2006; Schoenbach, 2010). Moreover, nsPEF application disrupts the vasculature within the tumor and deprives the tumor of oxygen and nutrients (Nuccitelli et al., 2006; Chen et al., 2009) additionally resulting in tumor shrinkage or complete tumor cell death.

All these events generated by nanoelectroablation have shown to induce apoptosis in biological cells (Andre et al., 2010; Pakhomov et al., 2009; Schoenbach, 2010), an effect that has been shown to reduce the growth of tumors.

2.2.4 Subnanosecond pulsed electric fields

An even newer field of research opens up when pulse duration is decreased even further, into the subnanosecond range. By reducing the duration of electrical pulses into the subnanosecond range, the electric field-cell interactions shift increasingly from the plasma membrane to subcellular structures. In this case, the electric field distribution is determined by the dielectric permittivity rather than the resistivity of cell components (Schoenbach et al., 2008). Under these conditions, direct electric field effects at the molecular level will determine the biological outcome, rather than causing charging of the membrane (Xiao et al., 2011). The disruption of the membrane integrity may lead to the change of physiological conditions of the cell and cause cell death (Kumar et al., 2011). The requirement for the dominance of such effects is that the pulse duration be less than the dielectric relaxation time of the cytoplasm. For mammalian cells, this holds for pulse durations of less than 1 ns (Joshi and Schoenbach, 2010; Joshi and Hu, 2011).

This push toward further pulse shortening is driven in part by the possibility of using wideband antennas, rather than direct contact electrodes, to deliver such pulses for noninvasive treatment of deep lesions (Baum, 2007; Xiao et al., 2010; Joshi and Schoenbach, 2010). The preferred pulse duration for subnanosecond pulses is in the range of 100-200 ps, making it possible to focus the radiation on the target efficiently and produce a focal spot of 1 cm size in the tissue (Xiao et al., 2011). So far, a model analysis (Joshi et al., 2009) has shown that subnanosecond pulses can perforate a cell membrane. In addition, subnanosecond pulses (800 ps) have been shown to cause B16 cells to increase trypan blue uptake (Schoenbach et al., 2008), which is a clear indication of the disruption of membrane integrity (Xiao et al., 2011).

2.3 PEF delivery systems

2.3.1 Irreversible electroporation

Recently, a commercially available instrument has been introduced in the United States to perform IRE. The NanoKnife® unit (AngioDynamics) is a device approved by the US Food and Drug Administration for use in ablating soft tissue. Using this technology, a tissue is subjected to an electric field using high-voltage electric pulses of 100 µs (up to 3 kV). This creates multiple pores in the cell membranes and irreversibly damages the cell’s homeostasis mechanism, leading to instant cell death.

The IRE device comprises a generator and 15 or 25 cm long electrodes. The IRE generator produces 100 µs high-voltage (1,500-3,000 V) electric pulses (up to 50 A). The voltage is determined by a standard algorithm that uses factors such as the intended size of the ablation zone, the number of probes, the distance between probes, and the length of the active electrode tip (Thomson et al., 2011). The current flow is determined by tissue resistance and the degree of IRE achieved. Typically, 90 pulses are delivered in nine sets of 10 pulses at each treatment site. The electrodes used with the device are 15 cm long stainless steel needles, partially insulated, with a diameter of 1 mm. The conductive, non-insulated, distal end of the electrodes can be up to 4 cm long. Bipolar needle electrodes have two 7 mm-long electrodes set 8 mm apart on a single needle. The unipolar needle electrodes have an active electrode that can be varied in length from 5 mm to 40 mm by means of a moveable insulator sleeve and must be used in pairs even when an odd number of electrodes is used to encompass a target tumor. Bipolar electrodes are mostly used for difficult access or for small tumors (<2.5 cm). The electrode needles are placed in and adjacent to the target tumor under CT and/or ultrasound (US) image guidance and the distance between the electrodes is confirmed by CT and/or US imaging to ensure that the electrodes were correctly placed parallel to one another and that sufficient current flow would be generated to ensure IRE. The device has been developed to allow the use of up to six independent electrodes. The electrodes are not restricted to a fixed geometry, rather they can be independently positioned based on the tumor size, shape, and position, to ensure that the target is entirely enclosed within the applied electric field, thus ensuring complete tissue ablation. The efficiency of different electroporation protocols for ablating tumors by IRE was assessed by Ivorra and coworkers (Ivorra et al., 2009).

2.3.2 Nanosecond pulsed electric fields

Medical devices are also being developed to apply nanoelectropulse technology for cancer treatment. Compressing the electrical energy by means of pulsed power techniques can generate ultrashort electrical pulses with a power of billions of watt. However, the application of nsPEFs requires electrical pulse generators that provide well-defined, high voltage pulses with fast current rise. The fabrication of these nanopulsers is highly complicated due to the high voltages and ultrashort durations needed. The main challenge is to deliver a repeatable pulse with a minimum of oscillations and reflections in the waveform (Beziuk, 2011; Silve et al., 2011). Most of the nanopulsers currently employed in laboratories use a Blumlein pulse forming network utilizing the propagation of electric signals along a transmission line. The design of these nanopulsers has been reviewed by Sundararajan (Sundararajan, 2009). A more recent paper by Minamitani and co-authors describes the design of a compact high-power pulsed electromagnetic wave generator using a nanosecond pulsed power generator (Minamitani et al., 2010). Earlier, they had proposed a compact high-power pulsed electromagnetic wave generator using a Blumlein line that can output a nanosecond pulse. However, since the output waveform of a Blumlein line is a rectangular pulse that has wide-band frequency elements, the radiation efficiency of an electromagnetic wave from an antenna is low. Therefore, they applied an L-C inversion circuit to the pulsed power generator to obtain a voltage waveform with a single frequency, which is applied to an antenna.
In a study by Nuccitelli and coworkers (2012a) a new pulse generator (PulseCure® Model S-3) was built because there were none commercially available with the rise time and amplitude they require to trigger apoptosis in UV-induced murine melanoma tumors. The PulseCure® 100 ns pulse generator (BioElectroMed) generates 30 kV/cm electric pulses 100 ns long to trigger apoptosis in cells between the electrodes. The NanoBlate® delivery device (electrodes) treats 5 mm wide skin lesions with 100 ns pulses from the PulseCure®. A clinical trial (ID: NCT01463709) started in October 2011 to demonstrate the safety of the PulseCure pulse generator and NanoBlate® electrode and identify the optimal pulse number for treating basal cell carcinomas.
Present developments of nsPEFs are oriented toward the treatment of deep-seated nodules. Therefore, new electrode systems have recently been developed to treat internal tumors in conjunction with endoscopes for intraluminal tumors. BioElectroMed is also developing a medical device called the EndoPulse® that will deliver nanosecond pulsed electric fields (nsPEF) to treat pancreatic carcinomas.

2.3.3 Subnanosecond pulsed electric fields

At present, electric fields are invasively delivered to the tumor using implanted electrodes. The traditional use of needle or plate electrodes in therapeutic applications that rely on IRE or nsPEFs requires that the electrodes are brought into close contact with the treated tissue. This limits the application to treatments of tissue close to the skin, or tissue close to the surface of the body unless endoscopic ultrasound-guided nsPEF is being used as mentioned in previous chapter. The use of antennas, on the other hand, would allow one to apply such electric fields to tumors that are not easily accessible with needles and may be able to induce apoptosis in tissue (Joshi and Schoenbach, 2010; Joshi and Hu, 2011).

There are several concepts for such a delivery system. A possible configuration that would generate very high electric fields is using a focusing antenna (Baum, 2007; Schoenbach et al., 2008, Xiao et al., 2010). These are mainly based on the use of a prolate spheroidal reflector, where the pulse is launched from one focal point and reflected onto a second focal point which is the target. Impulse radiating antennas (IRAs), as they are called, can deliver a subnanosecond pulse into tissue with a spatial resolution in the centimeter range and even in the millimeter range with the use of a focusing electromagnetic lens (Altunc et al., 2008). In addition to using a reflecting antenna, efforts are underway to utilize lenses in combination with the reflector in order to achieve higher spatial resolution (Altunc et al., 2009; Kumar et al., 2010; 2011). If the power of such ultrashort electrical pulses is very high, it becomes possible to deposit substantial electromagnetic energy into deep-lying tissue, even at the rather high conductivity and consequently strong absorption of most tissues (Schoenbach et al., 2008). An effort at developing systems for delivering electrical energy in pulsing system in this subnanosecond regime is ongoing at Old Dominion University and, for instance, FID GmbH is one of the companies developing pulse generators able to deliver gigawatt peak power and switching times from tens to hundreds of picoseconds. Impulse radiating antennas are now being investigated as a noninvasive pulsed electric field delivery system for skin cancer treatment. Kumar and co-workers (Kumar et al., 2010; 2011) briefly reviews the design of a prolate-spheroidal impulse-radiating antenna system, to launch and focus fast (100 ps) high-voltage (> 100 kV) pulses, as a non-invasive tool for melanoma treatment.

2.4 Treatment planning

Experience with clinical practice of pulsed electric field (PEF) therapy comes from treatments with IRE but very recently a first clinical trial of using nsPEF has started (see below). The clinical application of both PEF therapies requires treatment planning, which mainly requires defining the electrical fields in the treated region. PEF as a technique to treat cancer in a targeted manner requires a good understanding of how electric current flows within biological tissues. Although the PEF procedure is easy to perform, treatment planning is complicated by the fact that the electric field distribution within the tissue, the greatest single factor controlling the extent of PEF, depends non-trivially on the electrode configuration, pulse parameters and any tissue heterogeneities.

Once the patient has been anaesthetized and placed in the appropriate position for best access to the target tumor, an intravenous contrast medium-enhanced computed tomography (CT) scan is carried out. The exact number and position of the probe should be determined based on the geometric appearance of the tumor. Bipolar or unipolar electrode needles are introduced percutaneously and guided into place in and adjacent to the target tumor under CT and/or ultrasound image guidance (Lee et al., 2010b). The proximity to vital structures such as the trachea or large blood vessels must be determined in advance. Not only is it critical to apply the right modality, it needs to be applied at the appropriate dose for maximal benefit to be achieved. Each ablation cycle consists of high-voltage electric pulses delivered in groups (of about 10) with a brief time for recharging between groups (a cycle is usually completed in less than 2 minutes). Cardiac synchronization is used to minimize the risk of arrhythmias. Electrodes are repositioned under imaging guidance to extend the zone of electroporation until the entire tumor and an appropriate margin have been ablated. The number of ablations is determined by the volume of the target tumor. The procedure may also be performed through open surgical or laparoscopic approaches. A neuromuscular blocking agent is administered to prevent muscle spasms. However, investigations are underway to develop new protocols for obviating the use of a neuroblocker (Arena et al., 2011; Golberg and Rubinsky, 2012). When the ablation procedure is completed, another intravenous contrast medium-enhanced CT scan may be carried out to confirm that the entire target region has been ablated.

2.5 Advantages and limitations of PEF therapy

Several unique characteristics of PEF distinguish it from other currently available thermal ablation therapies, such as radiofrequency (RF), microwave (MW) and cryoablation. There is no heat accumulation thereby avoiding thermal injury. PEF spares the extracellular matrix thus allowing a faster recovery (Breton and Mir, 2011). The main advantage of PEF over photodynamic therapy or electrochemotherapy is the possibility to avoid the use of drugs, as it relies only on the effect of the applied electric field to kill the cancer cells. Another important attribute of the PEF modalities is that their time scale is extremely rapid (nanoseconds to milliseconds). The technique is easy to apply and can be monitored and controlled. The low energy input of electric pulses because of the ultrashort duration prevents damage to tissues and cells nearby the tumor. One disadvantage is that the electrodes must encapsulate the entire tumor in order to ablate it with a single treatment.

A unique characteristic of IRE therapy is its capability of preserving vital structures within the ablated zone. In all IRE ablated liver tissues, critical structures, such as the hepatic arteries, hepatic veins, portal veins and intrahepatic bile ducts were all preserved (Lee et al., 2010b; Charpentier et al., 2011; Narayanan, 2011). The preservation of critical structures after treatment with IRE has been shown in other tumor models as well. For instance, in lung tumors blood veins were preserved (Dupuy et al., 2011), and structures such as urethra, vessels, nerves, and rectum were unaffected when treating prostate with IRE (Onik et al., 2007). However, Schoellnast and co-authors found that IRE has the potential to damage nerves and may result in axonal swelling, fragmentation, and distal Wallerian degeneration (Schoellnast et al., 2011) but electrophysiological, histological, and functional results have shown that nerves treated with IRE can attain full recovery after 7 weeks (Li et al., 2011). In general, if the treatment is delivered to avoid thermal damage to the nerves, they are completely preserved (Neal et al., 2011). In other types of tumor ablation, these structures are completely obliterated due to the extreme temperature changes (e.g. RF ablation) or photochemical reactions (e.g. Photodynamic therapy) used by these ablation techniques, causing the destruction of proteins. Some of the essential clinical features of IRE are rapid tissue regeneration because of the large blood vessel scaffolds, rapid activation of the immune system, and no scarring (Rubinsky et al., 2007a). A typical IRE procedure for a solid tumor, with a size of approximately 3 cm in diameter, uses 90 pulses with a pulse length of 100 microseconds (Lee et al., 2010a). A single IRE ablation session takes a minute or two since pulse application must be triggered during the R wave of the ECG to avoid arrhythmias. Therefore, even with three or four overlapping ablations, total IRE treatment time is under 10 minutes. Furthermore, all IRE ablated zones demonstrate sharp margins and sharp demarcation (on the order of 1-2 cell thickness) between the ablated areas and non-ablated areas (Lee et al., 2010b). With its sharp demarcation, IRE ablation can be more accurately evaluated and assessed for effectiveness, tumor treatment outcome, and follow-up. Thus unlike RF and MW ablation, IRE has no grey-zone of ablation. This grey-zone is significant because it is likely another source of incomplete ablation, causing a residual tumor or recurrence (Lee et al., 2010a). Another notable benefit of IRE is its capability of creating complete cell death within the ablation area, regardless of the location, size and shape of the tumor. However, it works best for tumors under 3-4 cm (Narayanan, 2011). Perivascular tumor cells and irregularly shaped or large sized tumors are completely ablated with IRE. Due to immediate changes in the treated tissue’s permeability, the affected regions may be monitored in real-time using ultrasound (Rubinsky et al, 2007b).

Nanoelectroablation therapy has specific advantages. Among its most intriguing characteristics is the incredibly short time that these cells are being exposed to the electric field. All of the tumor regressions have shown to be the result from a total electric field exposure time of 120 μs or less (Nuccitelli et al. 2006; 2009). Another advantage is that it triggers apoptosis which has been shown to stimulate a potent immune response in in vivo (Nuccitelli et al., 2012b). The relatively slow ablation time of about 2-3 weeks allows the immune system to be sensitized to the tumor cells and inhibit the growth of secondary tumors following metastasis. This should reduce the chances for resistances and recurrences, which are common with long acting chemotherapeutic agents. nsPEFs have also well-defined treatment zones with localized effects determined by electrode placement, thereby minimizing adjacent and systemic side effects (Nuccitelli et al., 2010). nsPEF effects are independent of tumor size or shape, equally affecting all heterogeneous tumor cells. Since they do not eliminate cells based on proliferation rates like chemotherapeutic agents, they not only eliminate cancer cells, but also eradicate cancer stem cells and host cells that collaborate with tumors. Unlike IRE, the use of nsPEFs does not require paralytic agents, because muscle contractions are mostly eliminated (Beebe et al., 2011) but as mentioned earlier new research is being conducted by Arena and coworkers and Golberg and Rubinsky to eliminate the paralytic requirement from IRE protocols.

A potential benefit of shorter pulses, besides a more efficient energy system and larger poration for bioapplications, could be the possible use of pulse-driven wideband antennas for allowing access to deeper lying targets within tissues (Joshi and Hu, 2011).

2.6 Combinations of using PEF with other therapies

Several combinations of PEF with other therapies are in research. Experiments were performed on tumors combining nsPEFs with a cytotoxic drug (bleomycin) in which a systematic tumor growth delay was observed but no tumor regression was achieved (Silve et al., 2012). The combination of pulsed electric fields with cryoablation has also been proposed (Daniels and Rubinsky, 2011a). Interestingly, it was shown in a theoretical analysis that lowering the temperature of the PEF electrode, and the consequent local cooling of the tissue, has the effect of confining the electric fields to the cooled region. Thus temperatures may be used to modulate and control electric fields (Daniel and Rubinsky, 2011b). Another example, but this time in the field of nanotechnology, is given by Stacey and coworkers who investigated the increased cell killing of a pancreatic cancer cell line when exposed to nsPEF in the presence of multi-walled carbon nanotubes (MWCNTs) as a potential form of cancer therapy (Stacey et al., 2011). They hypothesized that the electronic properties of MWCNTs would disrupt cell function, leading to cell death, when cells are exposed to nsPEF. An enhanced killing effect was observed when cellsc were previously grown in the presence of MWCNTs.

Efficace?

3.1 Introduction

PEF has been tested and performed in various organs and tumor types. Mainly irreversible electroporation (IRE) but recently also nanosecond pulsed electric field (nsPEF) therapy have reached clinical trial status while subnanosecond electric fields is planning to start tests in animal models. This section mainly highlights the recent clinical outcomes. The purpose is to summarize the clinical feasibility and efficacy of both for each anatomical tumor location. Evidence for its efficacy comes from published research studies and clinical trials. Main focus is on phase 2 and 3 randomized controlled clinical trials if available. Four clinical trials for IRE (liver, pancreas) and one clinical trial for nsPEF (BCC) are registered in ClinicalTrials.gov; two clinical trials for IRE (liver, kidney, lung, and prostate) are registered in the ANZCTR.org.au (access August 2012, conditions: the different cancer types; interventions: irreversible electroporation, nanoelectroablation or nanosecond pulsed electric field, and subnanosecond pulsed electric field). IRE is under review by the National Institute for Health and Clinical Excellence (NICE). The medical literature was searched to identify studies and reviews relevant to pulsed electric fields for the treatment of cancer. No language restrictions were used but conference abstracts were omitted. An electronic search of the Medline, Embase, Cochrane Library, and CancerLit databases was undertaken between July and August 2012. Two sets of keywords were used for the search strategy. One was for the different modalities of PEF interventions; the other set was for each cancer type per anatomical location. Thus these selection criteria would benefit the rationale for rating the efficacy of PEF based on what is available in clinical practice today.

3.2 In vivo animal studies

Irreversible electroporation

Currently, over 20 institutions are actively involved in pre-clinical/translational research of IRE. Animal studies have been conducted in, for example, liver (Granot et al., 2009; Guo et al., 2010; Beebe et al., 2011; Lee et al., 2010b; 2012), brain (Garcia et al., 2010; 2011; Ellis et al., 2011), breast (Neal et al., 2010), prostate (Onik et al., 2007; Onik and Rubinsky, 2010), small intestine (Phillips et al., 2012), lung (Deodhar et al., 2011b; Dupuy et al., 2011), pancreas (Charpentier et al., 2010; Bower et al., 2011; Bagla and Papadouris, 2012; José et al., 2012) and kidney tumors (Deodhar et al., 2010a; Tracey et al., 2010; Pech et al., 2011). Overall, these studies show that IRE induces tumor regression.

• Nanoelectroablation

Nanoelectroablation is under active investigation as a therapeutic new tool for cancer therapy (Garon et al., 2007; Chen et al., 2009; Nuccitelli et al., 2010). Several in vivo studies have been conducted on tumors since Beebe and colleagues first demonstrated in 2002 that applying nsPEFs to mammalian cells and solid tumors resulted in reduced tumor growth and induction of apoptosis (Beebe et al., 2002). Animal studies have been conducted in, for example, hepatocellular carcinoma (Beebe et al., 2011; Chen et al., 2012), and pancreas (Garon et al., 2007; Nuccitelli et al., 2012c). Nanoelectroablation has also proven efficacy in triggering apoptosis and remission of radiation-induced basal cell carcinomas (BCCs) in mice with a single 6 min-long treatment of 2700 pulses at 30kV/cm (Nuccitelli et al., 2012a). These are remarkable responses to an electric field with a cumulative duration of only a few hundred ms. Every nsPEF-treated BCC began to shrink within a day after treatment and their initial mean volume of 36 mm3 shrunk by 76% over the ensuing two weeks. After four weeks, they were 99.8% ablated if the size of the treatment electrode matched the tumor size. If the tumor was larger than the 4 mm wide electrode, multiple treatments were needed for complete ablation.
It has also been found that treating melanomas in a subdermal allograft model system with nsPEF can stimulate apoptosis and complete remission over a period of weeks (Chen et al., 2009; Nuccitelli et al., 2006; 2009; 2010; 2012b). Nuccitelli’s group developed different protocols and eventually obtained the destruction of nodules with only one sequence of 100 ns pulses (Nuccitelli et al., 2010). A single treatment using the optimal pulse parameters (2000 pulses, 100 ns in duration, 30 kV/cm in amplitude at a pulse frequency of 5-7 pulses/s) eliminated all 17 melanomas treated with these parameters in 4 mice. This was the highest pulse frequency that they could use without raising the treated skin tumor temperature above 40 °C. They also demonstrate that the effects of nsPEF therapy are highly localized to only cells located between electrodes and results in very little scarring of the nsPEF-treated skin. In an experiment, which included more than 300 tumors on 159 mice, tumor cells with nuclear pyknosis and reduced blood flow, as well as red blood cell leakage within a few minutes after treatment was observed (Nuccitelli et al., 2006; 2009). This interruption of the blood supply could also be an important explanation of the observed tumor regression as it will deprive the cells of oxygen and nutrients. The newly developed electrodes used are also compatible with human skin. More recently, Nuccitelli and colleagues applied this therapy to ablate UV-induced murine melanomas (Nuccitelli et al., 2012b). 27 of these melanomas were treated in 14 mice with nsPEF therapy delivering 2000 electric pulses with the same parameters. All nanoelectroablated melanoma tumors began to shrink within a day after treatment and gradually disappeared over a period of 12-29 days. The standard of care is surgical excision and here they have demonstrated that nanoelectroablation is equally effective for eliminating murine melanomas. Overall these studies in animals show that nsPEF proves to be a fast, safe, and potent ablative method, causing complete tissue death.

• Picoelectroablation

Much of the original work on nsPEF has been performed at the Old Dominion University (Norfolk, USA) where currently the application of subnanosecond electric fields is also in research. Together with institutes in Germany and Japan they are actively involved in pre-clinical/translational research of picoelectroablation therapy. No results have been published of in vivo research thus far.

3.3 Clinical applications of IRE and nsPEFs

Preliminary data of irreversible electroporation (IRE) in human clinical trials have been presented at many international conferences over the last years. Most of them are treatments for hepatocellular carcinoma, hepatic colorectal metastasis, renal cell carcinoma, and primary bronchogenic tumor. Approximately 30 hospitals in the United States are offering this treatment option mostly by using the NanoKnife® device (Angiodynamics, USA). One clinical trial on basal cell carcinoma (BCC) using nanoelectroablation has recently started.

3.3.1 Kidney cancer

Two phase I clinical trials of using IRE have been performed on kidney cancer thus far. Pech and co-authors determined the feasibility and safety of ablating renal cell carcinoma (RCC) tissue by IRE in six patients (Pech et al., 2011). They concluded that IRE seems to offer a feasible and safe technique by which to treat patients with kidney tumors and could offer some potential advantages over current thermal ablative techniques. In a single-center prospective nonrandomized cohort study by Thomson and colleagues reporting on thirty-eight volunteers with different tumor types (advanced malignancy of liver, kidney and lung) unresponsive to conventional treatments, the safety of IRE for tumor ablation was investigated as well. However, no details were given for kidney cancer but overall it was concluded that IRE appears to be safe for clinical use provided ECG-synchronized delivery is used (Thomson et al., 2011).

3.3.2 Liver cancer

The National Institute for Health and Clinical Excellence (NICE) has prepared two overviews of using IRE for the treatment of primary liver cancer and liver metastases (“Interventional procedure overview of irreversible electroporation for the treatment of primary liver cancer and liver metastases”). These overviews are based on a rapid review of the medical literature without any restrictions on date of publications of clinical trials. NICE recommendations about the safety and efficacy of IRE for liver cancer are in progress.

A recent study recruited and reported patients with different tumors, not specific to liver cancer (Thompson et al., 2011). Clinical examination, biochemistry, and computed tomography (CT) scans of the treated organ by the NanoKnife® device were performed before, immediately after, and at 1 month and 3 months after the procedure. This case series of 37 patients including 11 patients with primary liver cancer (22 tumors) report a complete response for 64% (14/22) of tumors, progressive disease for 14% (3/22) of tumors and stable disease for 23% (5/22) of tumors at 3-month follow-up. In patients with primary hepatocellular carcinoma, 82% (14/17) of targeted tumors were completely ablated. Patients who did not have severe cirrhosis or previous chemoembolization showed regeneration of liver after the procedure. The number of patients treated with IRE for liver metastases was 32.4% (12/37) with 45 procedures (figures calculated from table 2 of the paper). The IRE response rate in liver metastases was 50% but all patients in this group showed progressive disease from other lesions (actual numbers not reported, response rate was not defined and exact timing of assessment unclear). Liver metastases larger than 5 cm in any dimension showed no response in terms of tumor control.

Another study evaluated the safety and short-term outcomes of IRE to ablate perivascular malignant liver tumors (Kingham et al. (2012). The patients were selected for IRE when resection or thermal ablation was not indicated due to tumor location particularly near portal pedicles and hepatic veins. Twenty-eight patients had 65 tumors treated. Twenty-two patients (79%) were treated via an open approach and 6 (21%) were treated percutaneously. The tumor size ranged between 0.5 and 5 cm. Twenty-five tumors were <1 cm from a major hepatic vein; 16 were <1 cm from a major portal pedicle. Complications included 1 intraoperative arrhythmia and 1 postoperative portal vein thrombosis. Overall morbidity was 3%. There were no treatment-associated mortalities. At median follow-up of 6 months, there was 1 tumor with persistent disease (1.9%) and 3 tumors recurred locally (5.7%). This early analysis of IRE treatment of perivascular malignant hepatic tumors demonstrates safety for treating liver malignancies. Larger studies and longer follow-up are necessary to determine long-term efficacy.

3.3.3 Lung cancer

The National Institute for Health and Clinical Excellence (NICE) has prepared an overview of using IRE for the treatment of primary lung cancer and metastases in the lung (“Interventional procedure overview of irreversible electroporation for treating primary lung cancer and metastases in the lung”) and recommendations about the safety and efficacy of IRE will follow.

Percutaneous IRE of lung tumors is a new minimally invasive technique which has recently been used in the treatment of soft tissue tumors. The case histories are presented of two patients with unresectable malignancies in the lung, who underwent IRE as a treatment attempt (Usman et al., 2012). The procedure was performed under CT guidance and was uneventful. At follow up 6 months later, the tumors both appeared to have recurred. These cases may illustrate a failure of this system (NanoKnife®) within the lungs. In order to ensure complete treatment coverage in these situations, more rigorous treatment planning specific to lung cancer may be required. Previously, Thomson and coworkers reported that most treatment failures occurred in lung (and renal tumors) (Thomson et al., 2011) but that IRE was safe to apply. Although there is potential for IRE for the treatment of lung cancer, future studies must be conducted.

3.3.4 Pancreatic cancer

The National Institute for Health and Clinical Excellence (NICE) has prepared an overview of using IRE for the treatment of pancreatic cancer (“Interventional procedure overview of irreversible electroporation for the treatment of pancreatic cancer”) and recommendations about the safety and efficacy of IRE will be published later.

Martin and colleagues evaluated the safety and efficacy of IRE as a therapy in the treatment of locally advanced pancreatic cancer (Martin et al., 2012). They performed a prospective multi-institutional pilot evaluation of patients undergoing IRE for locally advanced pancreatic cancer. These patients were evaluated for 90-day morbidity, mortality, and local disease control. Twenty-seven patients underwent IRE, with median age of 61 years (range 45 to 80 years). Eight patients underwent margin accentuation with IRE in combination with left-sided resection (n=4) or pancreatic head resection (n=4). Nineteen patients had in situ IRE. All patients underwent successful IRE, with intraoperative imaging confirming effective delivery of therapy. All 27 patients demonstrated nonclinically relevant elevation of their amylase and lipase, which peaked at 48 hours and returned to normal at 72 hour postprocedure. There has been one 90-day mortality. No patient has shown evidence of clinical pancreatitis or fistula formation. After all patients have completed 90-day follow-up, there has been 100% ablation success. The authors concluded that IRE ablation of locally advanced pancreatic cancer tumors is a safe and feasible primary local treatment in unresectable, locally advanced disease. They plan to start with a phase II investigational device exemption (IDE) study to be initiated in 2012.
Another report describes a case of percutaneous IRE in a 78-year-old man with surgically unresectable stage III (tumor/node/metastasis stages, T4N0M0) pancreatic adenocarcinoma (Bagla and Papadouris, 2012). Two ablations were performed for a 4.1 cm mass encasing the celiac and superior mesenteric artery. At 3 months, a solitary liver metastasis developed, which was treated with radiofrequency ablation followed by gemcitabine chemotherapy. At 6-month follow-up, magnetic resonance imaging demonstrated no residual disease and a decreasing cancer antigen 19-9 level. Percutaneous IRE shows promise as a feasible and potentially safe method for local tumor control in patients with surgically unresectable disease.

3.3.5 Skin cancer

Nanoelectroablation therapy has been used to treat skin tumors by Garon and colleagues (Garon et al., 2007). They have found it to be effective for a single case of a human basal cell carcinoma (BCC) that exhibited complete remission after 1 treatment with very little scarring (200 pulses of 20 ns at 43 kV/cm). More recently, nanoelectroablation has also proven efficacy in triggering remission of BCCs in mice (Nuccitelli et al., 2012a). This suggests that this new therapy, which has proven very effective for treating mouse skin cancer, might be equally as effective on humans. Nuccitelli and colleagues are now conducting a pilot clinical trial using nanoelectroablation therapy on human BCCs to determine the safety, tolerability, and efficacy for the primary treatment of BCCs or as an adjuvant to surgery.

As we have seen in 3.2, nsPEF also causes apoptosis and complete remission in subcutaneous murine melanoma (Chen et al., 2009; Nuccitelli et al., 2006; 2009; 2010; 2012b) without affecting the peripheral skin. Depending on electric fields alone, possibly nsPEF therapy could also be used for melanoma treatment in humans without chemical drugs so that the side effects from chemotherapy would be avoided.

Sans danger?

4.1 Does IRE or nsPEFs have any complications or side effects?

In terms of safety relatively little experience has been obtained with IRE regarding cancer stage and location. Potential general complications of IRE include blood vessel thrombosis, intestinal perforation, hemorrhage, and hematoma (Kingham et al., 2012). Other risks are fistula formation and infections any time needles are inserted into the body, especially when 2 or 3 needles are used at once (Narayanan, 2011). Because of the high current used with IRE, the procedure carries some risk of precipitating an irregular heartbeat and causing neuromuscular stimulation entailing uncontrolled movements and pain (Mali et al., 2008). Additionally, precautions should be taken for patients with implantable electrical devices. Ablation of lesions in the vicinity of implanted electronic devices or implanted devices with metal parts should be avoided because unsynchronized IRE close to the heart may cause fatal ventricular arrhythmias. However, it has been found recently that synchronized IRE pulse delivery with absolute refractory period (e.g. by using the Accusync® device) avoids significant cardiac arrhythmias (Deodhar et al., 2011c; Narayanan, 2011). Nevertheless, it is important to ensure that interventions (such as a defibrillator) and people trained to treat cardiac arrhythmias are available. Finally, use of IRE may involve site-specific risks; if a lesion in the lung is treated, for example, there is a risk of pneumothorax, or a collapsed lung, which is usually treated with a chest tube. Similarly, if IRE is used to treat a lesion in the kidney, the procedure carries a risk of injury to the ureter or the blood vessels (Narayanan, 2011). One of the remaining problems is the electrode placing in the case of large tumors because the IRE electrical field must affect the totality of the tumor volume (Breton and Mir, 2011). At the moment, there has not been a long term clinical trial to monitor the possible side effects of IRE.

The safety of nanoelectroablation has to be examined more thoroughly in vivo and in the clinics. Although there is no substantial experience of applying nsPEFs in humans so far, Tang and colleagues predict that electric pulses with defined parameters could be applied to humans without significant side effects (Tang et al., 2009).

4.2 Contraindications of using IRE

In terms of contraindications, patients with pacemakers or patients who have a history of cardiac arrhythmias or irregular heartbeats are currently not being treated, as there are some concerns that IRE might precipitate irregular heartbeats or arrhythmias in these patients. Moreover, ablation of lesions in the vicinity of implanted electronic devices or implanted devices with metal parts should be avoided. IRE is also contraindicated in patients with extensive disease involvement outside a particular organ; if a patient already has metastases in several other organs, he or she would not be a candidate for the procedure. Finally, patients with extremely large lesions are not ideal candidates for IRE (Nararayan, 2011).

5. Conclusions

Patients where the role of resection is limited because of comorbid conditions or because of extensive prior surgery are increasingly being considered for alternative methods of treatment. Various minimally invasive energy-based therapies exist (see the Anticancer Fund website), each with their advantages and disadvantages and particular use. Pulsed electric field (PEF) therapy is a new and safe therapeutic strategy for tumor therapy in development. From the published and experiential evidence, it appears that PEF can be clinically effective. PEF ablation removes tissues by depositing intense, non-thermal, high power electric pulses into tumors instead of heat, drugs or chemical agents and the low energy input of electric pulses prevents damage to tissues and cells nearby the tumor. We have presented in this review the following techniques that use pulsed electrical fields to treat cancers.

Irreversible electroporation (IRE) is a tissue ablation technique that involves delivery of 100 microseconds electric pulses above critical, tissue-specific electric field thresholds which results in permeabilization of cells presumably through formation of nanoscale aqueous pores in cytoplasmic membranes, and resultant death of cells within a treated volume. IRE uses relatively long pulses (microseconds) and low electric fields (kV). IRE has the advantage of preserving important structures and organs, along with uniform and complete tumor destruction, an ultra-short ablation time, and real-time monitoring capability. Results of animal studies suggest that IRE technology can be useful in treating patients with perivascular tumors that cannot be treated safely or effectively by other procedures. Phase I clinical work has shown that IRE can safely ablate liver, lung and kidney cancer in humans. For the time being, the data on IRE is not sufficient to conclude on its potential use as a common tumor ablation technique in the clinic. Further clinical studies are needed to solve some of the problems as well as to investigate the efficiency of IRE for tumor destruction. Currently, an area of development in IRE revolves around targeting the electric field to the desired structure of ablation.

Nanosecond pulsed electric field (nsPEF) ablation or nanoelectroablation uses pulse power with much shorter pulse durations (ns) and higher electric fields (high kV/cm). This causes a range of effects ranging from structural rearrangement of the membrane lipid bilayer (i.e. electroporation), intercellular ionic flows, changes in chemical composition, modulation of conductivities, and triggers apoptosis that leads to cell death. The potential for nsPEFs to ablate tumors was confirmed by showing that they can eliminate liver, pancreas, and basal cell carcinoma in mice. Particularly, extensive research has been conducted in melanoma. Since the mechanism of nanoelectroablation is a physical one that is not cell-specific, scientists in the field expect this therapy to be equally effective in treating human lesions. This highly localized and drug-free physical technique would offer a promising new therapy for tumor treatment. However, the safety and efficacy of nsPEF have to be examined more thoroughly in vivo and as well as in other tumors. A most recent evolution in PEF research is the application of even shorter pulses in the subnanosecond range and use of pulse-driven wideband antennas for allowing access to deeper lying tumors within tissues. We believe that using PEFs in cancer treatment is one of the most appealing techniques in energy-based therapies and a significant effort in basic and clinical research needs to be pursued.

Plus d'info

6.1 Useful links

Angiodynamics (http://www.angiodynamics.com)
Pulse Biosciences (http://pulsebiosciences.com/)
Frank Reidy Research Center for Bioelectrics
(http://www.odu.edu/bioelectrics)
National Institute for Clinical Excellence (http://www.nice.org.uk)
Pulsed Power and Bioelectrics (http://www.pulsedpower.eu)
Electroporation based Technologies and Treatments (http://2013.ebtt.org/)
http://www.electroporation.net/

References

6.2 Scientific publications

Alberts B, Johnson A, Lewis J, Raff M, Roberts K, Walter P. Molecular biology of the cell, 5th edn. Garland Science, New York, 2008;669-71.

Altunc S, Baum CE, Christodoulou CG, Schamiloglu E. Spatially Limited Exponential Lens Design for Better Focusing an Impulse. Proc. URSI General Assembly. August 2008, Chicago, IL.

Al-Sakere B, André F, Bernat C, Connault E, Opolon P, Davalos RV, Rubinsky B, Mir LM. Tumor ablation with irreversible electroporation, Plos ONE 2007a;2:e1135.

Al-Sakere B, Bernat C, Andre F, Connault E, Opolon P, Davalos RV, Mir LM. A study of the immunological response to tumor ablation with irreversible electroporation. Technol Cancer Res Treat. 2007b;6(4):301-6.

Andre FM, Rassokhin MA, Bowman AM, Pakhomov AG. Gadolinium blocks membrane permeabilization induced by nanosecond electric pulses and reduces cell death. Bioelectrochemistry. 2010:95-100.

Arena CB, Sano MB, Rossmeisl JH Jr, Caldwell JL, Garcia PA, Rylander MN, Davalos RV. High-frequency irreversible electroporation (H-FIRE) for non-thermal ablation without muscle contraction. Biomed Eng Online. 2011;10:102.

Bagla S, Papadouris D. Percutaneous irreversible electroporation of surgically unresectable pancreatic cancer: a case report. J Vasc Interv Radiol. 2012;23(1):142-5.

Baum CE. Focal waveform of a prolate-spheroidal impulse-radiating antenna (IRA). Radio Sci. 2007;42(6):RS6-S27.

Beebe SJ, Fox P, Rec LJ, Somers K, Stark RH, Schoenbach KH. Nanosecond pulsed electric field (nsPEF) effects on cells and tissues: Apoptosis induction and tumor growth inhibition. IEEE Transactions on Plasma Science. 2002; 30(1):286-92.

Beebe SJ, White J, Blackmore PF, Deng Y, Somers K, Schoenbach KH. Diverse effects of nanosecond pulsed electric fields on cells and tissues. DNA Cell Biol. 2003;22:785-96.

Beebe SJ, Chen X, Liu JA, Schoenbach KH. Nanosecond pulsed electric field ablation of hepatocellular carcinoma. Conf Proc IEEE Eng Med Biol Soc. 2011;2011:6861-5.

Beziuk G. Subnanosecond pulse generator operating with arbitrary load. Rev Sci Instrum. 2011;82(7):074705.

Bower M, Sherwood L, Li Y, Martin R. Irreversible electroporation of the pancreas: definitive local therapy without systemic effects. J Surg Oncol. 2011;104(1):22-8.

Breton M, Mir LM. Microsecond and nanosecond electric pulses in cancer treatments. Bioelectromagnetics. 2011 Aug 3. doi: 10.1002/bem.20692. [Epub ahead of print]

Buescher ES, Schoenbach KH. Effects of submicrosecond, high intensity pulsed electric fields on living cells - intracellular electromanipulation. IEEE Trans. Dielect. Elect. Insul. 2003;10:788-94.

Charpentier KP, Wolf F, Noble L, Winn B, Resnick M, Dupuy DE. Irreversible electroporation of the pancreas in swine: a pilot study. HPB. 2010;12:348-51.

Charpentier KP, Wolf F, Noble L, Winn B, Resnick M, Dupuy DE. Irreversible electroporation of the liver and liver hilum in swine. HPB (Oxford) 2011;13:168-73.

Chen W, Lee RC. Altered ion channel conductance and ionic selectivity induced by large imposed membrane potential pulse. Biophys J. 1994a;67:603-12.

Chen W, Lee RC. Evidence for electrical shock-induced conformational damage of voltage-gated ionic channels. Ann NY Acad Sci. 1994b;720:124-35.

Chen W. Electroconformational denaturation of membrane proteins. Ann NY Acad Sci. 2005;1066:92-105.

Chen W, Zhongsheng Z, Lee RC. Supramembrane potential-induced electroconformational changes in sodium channel proteins: A potential mechanism involved in electric injury. Burns. 2006;32:52-59.

Chen XH, Swanson RJ, Kolb JF, Nuccitelli R, Schoenbach KH. Histopathology of normal skin and melanomas after nanosecond pulsed electric field treatment. Melanoma Res. 2009;19(6):361-71.

Chen X, Kolb JF, Swanson RJ, Schoenbach KH, Beebe SJ. Apoptosis initiation and angiogenesis inhibition: melanoma targets for nanosecond pulsed electric fields. Pigment Cell Melanoma Res. 2010;23:554-63.

Chen X, Zhuang J, Kolb JF, Schoenbach KH, Beebe SJ. Longterm survival of mice with hepatocellular carcinoma after pulse power ablation with nanosecond pulsed electric fields. Technol Cancer Res Treat. 2012;11:83-93.

Davalos R, Mir L, Rubinsky B. Tissue ablation with irreversible electroporation. Ann Biomed Eng. 2005;33(2):223-31.

Deodhar A, Monette S, Single GW Jr, Hamilton WC Jr, Thornton R, Maybody M, Coleman JA, Solomon SB. Renal tissue ablation with irreversible electroporation: preliminary results in a porcine model. Urology. 2011a;77(3):754-60.

Deodhar A, Monette S, Single GW Jr, Hamilton WC Jr, Thornton RH, Sofocleous CT, Maybody M, Solomon SB. Percutaneous irreversible electroporation lung ablation: preliminary results in a porcine model. Cardiovasc Intervent Radiol. 2011b;34(6):1278-87.

Deodhar A, Dickfeld T, Single GW, Hamilton WC Jr, Thornton RH, Sofocleous CT, Maybody M, Gónen M, Rubinsky B, Solomon SB. Irreversible electroporation near the heart: ventricular arrhythmias can be prevented with ECG synchronization. AJR Am J Roentgenol. 2011c;196(3):W330-5.

Dupuy DE, Aswad B, Ng T. Irreversible electroporation in a Swine lung model. Cardiovasc Intervent Radiol. 2011;34(2):391-5.

Edd JF, Horowitz L, Davalos RV, Mir LM, Rubinsky B. In vivo results of a new focal tissue ablation technique: irreversible electroporation. IEEE Trans Biomed Eng. 2006;53:1409-15.

Ellis TL, Garcia PA, Rossmeisl JH Jr, Henao-Guerrero N, Robertson J, Davalos RV. Nonthermal irreversible electroporation for intracranial surgical applications. Laboratory investigation. J Neurosurg. 2011;114(3):681-8.

Escoffre JM, Portet T, Wasungu L, Teissié J, Dean D, Rols MP. What is (still not) known of the mechanism by which electroporation mediates gene transfer and expression in cells and tissues. Mol Biotechnol. 2009;41:286-95.

Ford WE, Ren W, Blackmore PF, Schoenbach KH, Beebe SJ. Nanosecond pulsed electric fields stimulate apoptosis without release of pro-apoptotic factors from mitochondria in B16f10 melanoma. Arch Biochem Biophys. 2010;497:82-89.

Frey W, White JA, Price RO, Blackmore PF, Joshi RP, Nuccitelli R, Beebe SJ, Schoenbach KH, Kolb JF. Plasma membrane voltage changes during nanosecond pulsed electric field exposure. Biophys J. 2006;90:3608-15.

Garcia PA, Rossmeisl JH Jr, Neal RE 2nd, Ellis TL, Olson JD, Henao-Guerrero N, Robertson J, Davalos RV. Intracranial nonthermal irreversible electroporation: in vivo analysis. J Membr Biol. 2010;236(1):127-36.

Garcia PA, Pancotto T, Rossmeisl JH Jr, Henao-Guerrero N, Gustafson NR, Daniel GB, Robertson JL, Ellis TL, Davalos RV. Non-thermal irreversible electroporation (N-TIRE) and adjuvant fractionated radiotherapeutic multimodal therapy for intracranial malignant glioma in a canine patient. Technol Cancer Res Treat. 2011;10(1):73-83.

Garon EB, Sawcer D, Vernier PT, Tang T, Sun YH, Marcu L, Gundersen MA, Koeffler HP. In vitro and in vivo evaluation and a case report of intense nanosecond pulsed electric field as a local therapy for human malignancies. Int J Cancer. 2007;121(3):675-82.

Golberg A, Rubinsky B. Towards electroporation based treatment planning considering electric field induced muscle contractions. Technol Cancer Res Treat. 2012;11(2):189-201.

Gowrishankar TR, Weaver JC. Electrical behavior and pore accumulation in a multicellular model for conventional and supra-electroporation. Biochem Biophys Res Commun. 2006;349:643-53.

Granot Y, Ivorra A, Maor E, Rubinsky B. In vivo imaging of irreversible electroporation by means of electrical impedance tomography. Phys Med Biol. 2009;54(16):4927-43.

Hall EH, Schoenbach KH, Beebe SJ. Nanosecond pulsed electric fields (nsPEF) induce direct electric field effects and biological effects on human colon carcinoma cells. DNA Cell Biol 2005;24:283-91.

Hamilton WA, Sale AJH. Effects of high electric fields on microorganisms. 2. Mechanism of action of the lethal effect. Biochimica et Biophysica Acta. 1967;148:789-800.

Hu Q, Joshi RP. Transmembrane voltage analyses in spheroidal cells in response to an intense ultrashort electrical pulse. Phys Rev E. 2009;79:11901.

Ivorra A, Al-Sakere B, Rubinsky B, Mir LM. In vivo electrical conductivity measurements during and after tumor electroporation: conductivity changes reflect the treatment outcome. Phys Med Biol. 2009;54(19):5949-63.

José A, Sobrevals L, Ivorra A, Fillat C. Irreversible electroporation shows efficacy against pancreatic carcinoma without systemic toxicity in mouse models. Cancer Lett. 2012;317(1):16-23.

Joshi RP, Song J, Schoenbach KH, Sridhara V. Aspects of lipid membrane bio-responses to subnanosecond, ultrahigh voltage pulsing. IEEE Trans. Dielectr. Electr. Insul. 2009;16(5):1243-50.

Joshi RP, Schoenbach KH. Bioelectric effects of intense ultrashort pulses. Crit Rev Biomed Eng. 2010;38(3):255-304.

Joshi RP, Hu Q. Case for applying subnanosecond high-intensity, electrical pulses to biological cells. IEEE Trans Biomed Eng. 2011;58(10):2860-6.

Kingham TP, Karkar AM, D'Angelica MI, Allen PJ, Dematteo RP, Getrajdman GI, Sofocleous CT, Solomon SB, Jarnagin WR, Fong Y. Ablation of Perivascular Hepatic Malignant Tumors with Irreversible Electroporation. J Am Coll Surg. 2012 Jun 15. [Epub ahead of print]

Kinosita K, Tsong T. Formation and resealing of pores of controlled sizes in human erythrocytes membrane. Nature. 1977;268:438-48.

Kotnik T, Miklavcic D. Theoretical evaluation of voltage inducement on internal membranes of biological cells exposed to electric fields. Biophys J. 2006;90:480-91.

Kotnik T, Pucihar G, Miklavcic D. Induced transmembrane voltage and its correlation with electroporation-mediated molecular transport. J. Membrane Biol. 2010;236:3-13.

Kumar P, Altunc S, Baum CE, Buchenauer CJ, Christodoulou CG, Schamiloglu E. A prolate-spheroidal impulse-radiating antenna system to launch and focus 100-ps pulses for melanoma treatment. 5th International Conference on Ultrawideband and Ultrashort Impulse Signals (UWBUSIS), 2010:138-40. 

Kumar P, Baum CE, Altunc S, Buchenauer J, Shu Xiao, Christodoulou CG, Schamiloglu E, Schoenbach KH. A hyperband antenna to launch and focus fast high-voltage pulses onto biological targets. IEEE Transactions on Microwave Theory and Techniques. 2011;59(4): 1090-1101.

Lee EW, Thai S, Kee ST. Irreversible electroporation: a novel image-guided cancer therapy. Gut Liver. 2010a;4 Suppl 1:S99-S104.

Lee EW, Chen C, Prieto VE, Dry SM, Loh CT, Kee ST. Advanced hepatic ablation technique for creating complete cell death: irreversible electroporation. Radiology. 2010b;255(2):426-33.

Lee EW, Wong D, Tafti BA, Prieto V, Totonchy M, Hilton J, Dry S, Cho S, Loh CT, Kee ST. Irreversible Electroporation in Eradication of Rabbit VX2 Liver Tumor. J Vasc Interv Radiol. 2012 Apr 23. [Epub ahead of print]

Li W, Fan Q, Ji Z, Qiu X, Li Z. The effects of irreversible electroporation (IRE) on nerves. PLoS One. 2011 Apr 14;6(4):e18831.

Mali B, Jarm T, Corovic S, Paulin-Kosir MS, Cemazar M, Sersa G, Miklavcic D. The effect of electroporation pulses on functioning of the heart. Med Biol Eng Comput. 2008;46(8):745-57.

Maor, E., Ivorra, A., Rubinsky, B. Non thermal irreversible electroporation: novel technology for vascular smooth muscle cells ablation. PLoS ONE. 2009;4:e4757.

Martin RC 2nd, McFarland K, Ellis S, Velanovich V. Irreversible Electroporation Therapy in the Management of Locally Advanced Pancreatic Adenocarcinoma. J Am Coll Surg. 2012 Jun 20. [Epub ahead of print]

Minamitani Y, Ueno T, Ohe Y, Kato S. Intensity of electric field radiating from high-power pulsed electromagnetic wave generator for use in biological applications. IEEE Transactions on Dielectrics and Electrical Insulation. 2010;17(6):1895-00.

Mir LM, Belehradek M, Domenge C, Orlowski S, Poddevin B, Belehradek J Jr, Schwaab G, Luboinski B, Paoletti C. Electrochemotherapy, a novel antitumor treatment: first clinical trial. C R Acad Sci III. 1991;313:613-18.

Napotnik TB, Rebersek M, Kotnik T, Lebrasseur E, Cabodevila G, Miklavcic D. Electropermeabilization of endocytotic vesicles in B16 F1 mouse melanoma cells. Med. Biol. Eng. Comput. 2010;48(5):407-13.

Narayanan G. Irreversible electroporation for treatment of liver cancer. Gastroenterol Hepatol (N Y). 2011;7(5):313-6.

Neal RE 2nd, Singh R, Hatcher HC, Kock ND, Torti SV, Davalos RV. Treatment of breast cancer through the application of irreversible electroporation using a novel minimally invasive single needle electrode. Breast Cancer Res Treat. 2010;123(1):295-301.

Nesin V, Bowman AM, Xiao S, Pakhomov AG. Cell permeabilization and inhibition of voltage-gated Ca2+ and Na+ channel currents by nanosecond pulsed electric field. Bioelectromagnetics. 2012;33(5):394-404.

Neumann E, Rosenheck K. Permeability changes induced by electric impulses in vesicular membranes. J Membr Biol. 1972;10:279-90.

Nuccitelli R, Pliquett U, Chen X, Ford W, James Swanson R, Beebe SJ, Kolb JF, Schoenbach KH. Nanosecond pulsed electric fields cause melanomas to self-destruct. Biochem Biophys Res Commun. 2006;343(2):351-60.

Nuccitelli R, Chen X, Pakhomov AG, Baldwin WH, Sheikh S, Pomicter JL, Ren W, Osgood C, Swanson RJ, Kolb JF, Beebe SJ, Schoenbach KH. A new pulsed electric field therapy for melanoma disrupts the tumor's blood supply and causes complete remission without recurrence. Int J Cancer. 2009;125(2):438-45.

Nuccitelli R, Tran K, Sheikh S, Athos B, Kreis M, Nuccitelli P. Optimized nanosecond pulsed electric field therapy can cause murine malignant melanomas to self-destruct with a single treatment. Int J Cancer. 2010;127(7):1727-36.

Nuccitelli R, Tran K, Athos B, Kreis M, Nuccitelli P, Chang KS, Epstein EH Jr, Tang JY. Nanoelectroablation therapy for murine basal cell carcinoma. Biochem Biophys Res Commun. 2012a Jul 4. [Epub ahead of print]

Nuccitelli R, Tran K, Lui K, Huynh J, Athos B, Kreis M, Nuccitelli P, De Fabo EC. Non-thermal Nanoelectroablation of UV-Induced Murine Melanomas Stimulates an Immune Response. Pigment Cell Melanoma Res. 2012b Jun 11. [Epub ahead of print]

Nuccitelli, R, Huynh J, Lui K, Wood R, Kreis M, Athos B, Nuccitelli P. Nanoelectroablation of human pancreatic carcinoma in a murine xenograft model without recurrence. Int. J. Cancer. 2012c [Epub ahead of print]

Okino M, Mohri H. Effects of a high-voltage electrical impulse and an anticancer drug on in vivo growing tumors. Japanese Journal of Cancer Research. 1987;78:1319-21.

Onik G, Mikus P, Rubinsky B. Irreversible electroporation: implications for prostate ablation. Technol Cancer Res Treat. 2007;6:295-300.

Onik G, Rubinsky B. Irreversible Electroporation: First Patient Experience Focal Therapy of Prostate Cancer, in: B. Rubinsky (Ed.), Irreversible Electroporation, Springer-Verlag, Berlin Heidelberg, 2010, pp. 235-47.

Orlowski S, Belehradek J Jr, Paoletti C, Mir LM. Transient electropermeabilization of cells in culture: increase of the cytotoxicity of anticancer drugs. Biochem Pharmacol. 1988;37:4727-33.

Pakhomov AG, Kolb JF, White JA, Joshi RP, Xiao S, Schoenbach KH. Long-lasting plasma membrane permeabilization in mammalian cells by nanosecond pulsed electric field (nsPEF). Bioelectromagnetics. 2007;28:655-63.

Pakhomov AG, Bowman AM, Ibey BL, Andrei FM, Pakhomova ON, Schoenbach KH. Lipid nanopores can form a stable ion channel-like conduction pathway in cell membrane. Biochem. Biophys. Res. Commun. 2009;385:181-86.

Pech M, Janitzky A, Wendler JJ, Strang C, Blaschke S, Dudeck O, Ricke J, Liehr U-B. Irreversible electroporation of renal cell carcinoma: A first-in-man phase I clinical study. Cardiovasc Intervent Radiol. 2011;34(1):132-38.

Phillips MA, Narayan R, Padath T, Rubinsky B. Irreversible electroporation on the small intestine. Br J Cancer. 2012;106(3):490-5.

Rubinsky B, Onik G, Mikus P. Irreversible electroporation: a new ablation modality - clinical implications. Technol Cancer Res Treat. 2007a;6:1-11.

Rubinsky, B. Irreversible Electroporation in Medicine. Technol Cancer Res Treat. 2007b;6:255-60.

Sale AJH, Hamilton WA. Effects of high electric fields on microorganisms. 1. Killing of bacteria and yeasts. Biochimica et Biophysica Acta. 1967;148:781-88.

Sale AJH, Hamilton WA. Effects of high electric fields on microorganisms. 3. Lysis of erythrocytes and protopasts. Biochimica et Biophysica Acta. 1968;163:37-43.

Schoellnast H, Monette S, Ezell PC, Deodhar A, Maybody M, Erinjeri JP, Stubblefield MD, Single GW Jr, Hamilton WC Jr, Solomon SB. Acute and subacute effects of irreversible electroporation on nerves: experimental study in a pig model. Radiology. 2011;260(2):421-7.

Schoenbach KH, Beebe SJ, Buescher ES. Intracellular effect of ultrashort electrical pulses. Bioelectromagnetics. 2001;22(6):440-48.

Schoenbach KH, Joshi RP, Kolb JF, Chen N, Stacey M, Blackmore PF, Buescher ES, Beebe SJ. Ultrashort electrical pulses open a new gateway into biological cells. Proc IEEE. 2004; 92(7):1122-37.

Schoenbach KH, Nuccitelli R, Beebe SJ. Extreme voltage could be a surprisingly delicate tool in the fight against cancer, IEEE Spectr. 2006;43:20-6.

Schoenbach KH, Hargrave B, Joshi RP, Kolb JF, Osgood C, Nuccitelli R, Pakhomov A, Swanson RJ, Stacey M, White JA, Xiao S, Zhang J, Beebe SJ, Blackmore PF, Buescher ES. Bioelectric effects of intense nanosecond pulses. IEEE Trans. Dielect. Electr. Insul. 2007;14(5):1088-1119.

Schoenbach KH, Xiao S, Joshi RP, Camp JT, Heeren T, Kolb JF, Beebe SJ. The effect of intense subnanosecond electrical pulses on biological cells. IEEE Trans. Plasma Sci. 2008;36(2):414-22.

Schoenbach KH. Bioelectric effect of intense nanosecond pulses, in: A.G. Pakhomov, D. Miklavcic, M.S. Markov (Eds.), Advanced Electroporation Techniques in Biology and Medicine, Taylor and Francis Group, Boca Raton, 2010, pp. 19-50.

Silve A, Villemejane J, Joubert V, Ivorra A, Mir L. Nanosecond pulsed electric field delivery to biological samples: Difficulties and potential solutions. In: Markov M, Miklavcic D, Pakhomov A, editors. Advanced electroporation techniques in biology and medicine. Boca Raton, FL: CRC Press, Taylor & Francis Group. 2011;pp 353-68.

Silve A, Leray I, Al-Sakere B, Mir LM. Nanopulses and their applications: Permeabilisation to bleomycin molecules by 10 ns duration electric pulses in a tumor model in vivo. 6th European Conference on Antennas and Propagation (EUCAP). 2012;348-50.

Smith KC, Weaver JC. Active mechanisms are needed to describe cell responses to submicrosecond, megavolt-per-meter pulses: cell models for ultrashort pulses. Biophys J. 2008;95:1547-63.

Stacey M, Osgood C, Kalluri BS, Cao W, Elsayed-Ali H, Abdel-Fattah T. Nanosecond pulse electrical fields used in conjunction with multi-wall carbon nanotubes as a potential tumor treatment. Biomed Mater. 2011;6(1):011002.

Stampfli R, Willi M. Membrane potential of a Ranvier node measured after electrical destruction of its membrane. Experientia. 1957;13:297-98.

Sundararajan R. Nanosecond Electroporation: Another look. Mol Biotechnol. 2009;41(1):69-82.

Tang LL, Sun C, Liu H, Mi Y, Yao CG, Li CX. Steep pulsed electric fields modulate cell apoptosis through the change of intracellular calcium concentration. Colloids Surf. B Biointerfaces. 2007;57:209-14.

Tang L, Yao C, Sun C. Apoptosis induction with electric pulses - a new approach to cancer therapy with drug free. Biochem Biophys Res Commun. 2009;390(4):1098-101.

Teissié J, Golzio M, Rols MP. Mechanisms of cell membrane electropermeabilization: a minireview of our present (lack of?) knowledge. Biochim Biophys Acta. 2005;1724:270-80.

Tekle E, Oubrahim H, Dzekunov S, Kolb J, Schoenbach K, Chock P. Selective field effects on intracellular vacuoles and vesicle membranes with nanosecond electric pulses. Biophys J. 2005;89(1):274-84.

Thomson KR, Cheung W, Ellis SJ, Federman D, Kavnoudias H, Loader-Oliver D, Roberts S, Evans P, Ball C, Haydon A. Investigation of the safety of irreversible electroporation in humans. J Vasc Interv Radiol. 2011;22(5):611-21.

Usman M, Moore W, Talati R, Watkins K, Bilfinger TV. Irreversible electroporation of lung neoplasm: A case series. Med Sci Monit. 2012;18(6):CS43-47.

Vernier PT, Sun Y, Marcu L, Craft CM, Gundersen MA. Nanoelectropulse-induced phosphatidylserine tranlocation. Biophys. J. 2004a;86:4040-80.

Vernier PT, Sun YH, Marcu L, Craft CM, Gundersen MA. Nanosecond pulsed electric fields perturb membrane phospholipids in T lymphoblasts. FEBS Lett. 2004b;572(1-3):103-08.

Vernier PT, Sun Y, Gundersen MA. Nanoelectropulse-driven membrane perturbation and small molecule permeabilization. BMC Cell Biol. 2006;7(37):1-16.

Vernier PT, Sun Y, Chen MT, Gundersen MA, Craviso GL. Nanosecond electric pulse-induced calcium entry into chromaffin cells. Bioelectrochemistry. 2008;73:1-4.

Xiao S,  Altunc S, Kumar P, Baum C, Schoenbach KH. A reflector antenna for focusing in the near field. IEEE Antennas Wireless Propag. Lett. 2010;9(1):12-15.

Xiao S, Guo S, Nesin V, Heller R, Schoenbach KH. Subnanosecond electric pulses cause membrane permeabilization and cell death. IEEE Trans Biomed Eng. 2011;58(5):1239-45.

6.3 Books

Advanced Electroporation Techniques in Biology and Medicine. Pakhomov AG, Miklavcic D, Markov MS. Boca Raton, FL: CRC Press, 2010.

Bioelectromagnetic Medicine. Paul J. Rosch and Marko S. Markov (Editors) Informa Healthcare 2007

 

Champs Électriques Pulsés

PEF

Impulsions électriques ultra-courtes

Irreversible electroporation therapy

IRE

Non-thermal irreversible electroporation

NTIRE

Nanosecond pulsed electric field

nsPEF

Nanoelectroablation

Pulsed electric field therapy

PEF

Picoelectroablation