Paulo A. Garcia

Successful Treatment of a Large Soft Tissue Sarcoma With Irreversible Electroporation

Introduction Irreversible electroporation (IRE) is a promising technique for the focal treatment of pathologic tissues that involves placing minimally invasive electrodes within the targeted region. A series of short, intense electric pulses are then applied to destabilize the cell membrane, presumably by creating nanopores, inducing cell death in a nonthermal manner. The unique therapeutic mechanism of IRE does not rely on tissue temperature changes, as with hyperthermic or cryoablative procedures. Therefore, IRE preserves the extracellular matrix, major tissue vasculature, and other sensitive structures. Treated regions resolve rapidly, with submillimeter resolution between treated and unaffected cells, and are predictable with numerical modeling. Treatments promote an immune response, are unaltered by blood flow, can be administered quickly (approximately 5 minutes), and can be visualized in real time. IRE has been studied extensively in healthy tissue, and tumors have been treated with IRE in mice. IRE has been attempted in humans for prostate, lung, kidney, and liver cancers. Human treatments revealed negligible postablation pain and the ability to apply the pulses in proximity to vital structures. Overall, assessment of the therapeutic efficacy of IRE remains in its infancy. We hypothesize that IRE treatments can be designed and implemented to successfully treat soft tissue malignancies, including large and complex tumors, a crucial step for translation of the technology into routine clinical use. Here we report our treatment of a focal histiocytic sarcoma of the coxofemoral joint in a canine patient. Follow-up examinations demonstrated prolonged relief of cancerassociated pain, preservation of pelvic limb function, and complete tumor regression 6 months after initial treatment.

Non-Thermal Irreversible Electroporation (N-TIRE) and Adjuvant Fractionated Radiotherapeutic Multimodal Therapy for Intracranial Malignant Glioma in a Canine Patient

Non-thermal irreversible electroporation (N-TIRE) has shown promise as an ablative therapy for a variety of soft-tissue neoplasms. Here we describe the therapeutic planning aspects and first clinical application of N-TIRE for the treatment of an inoperable, spontaneous malignant intracranial glioma in a canine patient. The N-TIRE ablation was performed safely, effectively reduced the tumor volume and associated intracranial hypertension, and provided sufficient improvement in neurological function of the patient to safely undergo adjunctive fractionated radiotherapy (RT) according to current standards of care. Complete remission was achieved based on serial magnetic resonance imaging examinations of the brain, although progressive radiation encephalopathy resulted in the death of the dog 149 days after N-TIRE therapy. The length of survival of this patient was comparable to dogs with intracranial tumors treated via standard excisional surgery and adjunctive fractionated external beam RT. Our results illustrate the potential benefits of N-TIRE for in vivo ablation of undesirable brain tissue, especially when traditional methods of cytoreductive surgery are not possible or ideal, and highlight the potential radiosensitizing effects of N-TIRE on the brain.

High-frequency irreversible electroporation (H-FIRE) for non-thermal ablation without muscle contraction

BackgroundTherapeutic irreversible electroporation (IRE) is an emerging technology for the non-thermal ablation of tumors. The technique involves delivering a series of unipolar electric pulses to permanently destabilize the plasma membrane of cancer cells through an increase in transmembrane potential, which leads to the development of a tissue lesion. Clinically, IRE requires the administration of paralytic agents to prevent muscle contractions during treatment that are associated with the delivery of electric pulses. This study shows that by applying high-frequency, bipolar bursts, muscle contractions can be eliminated during IRE without compromising the non-thermal mechanism of cell death.MethodsA combination of analytical, numerical, and experimental techniques were performed to investigate high-frequency irreversible electroporation (H-FIRE). A theoretical model for determining transmembrane potential in response to arbitrary electric fields was used to identify optimal burst frequencies and amplitudes for in vivo treatments. A finite element model for predicting thermal damage based on the electric field distribution was used to design non-thermal protocols for in vivo experiments. H-FIRE was applied to the brain of rats, and muscle contractions were quantified via accelerometers placed at the cervicothoracic junction. MRI and histological evaluation was performed post-operatively to assess ablation.ResultsNo visual or tactile evidence of muscle contraction was seen during H-FIRE at 250 kHz or 500 kHz, while all IRE protocols resulted in detectable muscle contractions at the cervicothoracic junction. H-FIRE produced ablative lesions in brain tissue that were characteristic in cellular morphology of non-thermal IRE treatments. Specifically, there was complete uniformity of tissue death within targeted areas, and a sharp transition zone was present between lesioned and normal brain.ConclusionsH-FIRE is a feasible technique for non-thermal tissue ablation that eliminates muscle contractions seen in IRE treatments performed with unipolar electric pulses. Therefore, it has the potential to be performed clinically without the administration of paralytic agents.

Experimental Characterization and Numerical Modeling of Tissue Electrical Conductivity during Pulsed Electric Fields for Irreversible Electroporation Treatment Planning

Irreversible electroporation is a new technique to kill cells in targeted tissue, such as tumors, through a nonthermal mechanism using electric pulses to irrecoverably disrupt the cell membrane. Treatment effects relate to the tissue electric field distribution, which can be predicted with numerical modeling for therapy planning. Pulse effects will change the cell and tissue properties through thermal and electroporation (EP)-based processes. This investigation characterizes these changes by measuring the electrical conductivity and temperature of ex vivo renal porcine tissue within a single pulse and for a 200 pulse protocol. These changes are incorporated into an equivalent circuit model for cells and tissue with a variable EP-based resistance, providing a potential method to estimate conductivity as a function of electric field and pulse length for other tissues. Finally, a numerical model using a human kidney volumetric mesh evaluated how treatment predictions vary when EP- and temperature-based electrical conductivity changes are incorporated. We conclude that significant changes in predicted outcomes will occur when the experimental results are applied to the numerical model, where the direction and degree of change varies with the electric field considered.

A Parametric Study Delineating Irreversible Electroporation from Thermal Damage Based on a Minimally Invasive Intracranial Procedure

BackgroundIrreversible electroporation (IRE) is a new minimally invasive technique to kill undesirable tissue in a non-thermal manner. In order to maximize the benefits from an IRE procedure, the pulse parameters and electrode configuration must be optimized to achieve complete coverage of the targeted tissue while preventing thermal damage due to excessive Joule heating.MethodsWe developed numerical simulations of typical protocols based on a previously published computed tomographic (CT) guided in vivo procedure. These models were adapted to assess the effects of temperature, electroporation, pulse duration, and repetition rate on the volumes of tissue undergoing IRE alone or in superposition with thermal damage.ResultsNine different combinations of voltage and pulse frequency were investigated, five of which resulted in IRE alone while four produced IRE in superposition with thermal damage.ConclusionsThe parametric study evaluated the influence of pulse frequency and applied voltage on treatment volumes, and refined a proposed method to delineate IRE from thermal damage. We confirm that determining an IRE treatment protocol requires incorporating all the physical effects of electroporation, and that these effects may have significant implications in treatment planning and outcome assessment. The goal of the manuscript is to provide the reader with the numerical methods to assess multiple-pulse electroporation treatment protocols in order to isolate IRE from thermal damage and capitalize on the benefits of a non-thermal mode of tissue ablation.

Towards the creation of decellularized organ constructs using irreversible electroporation and active mechanical perfusion

BackgroundDespite advances in transplant surgery and general medicine, the number of patients awaiting transplant organs continues to grow, while the supply of organs does not. This work outlines a method of organ decellularization using non-thermal irreversible electroporation (N-TIRE) which, in combination with reseeding, may help supplement the supply of organs for transplant.MethodsIn our study, brief but intense electric pulses were applied to porcine livers while under active low temperature cardio-emulation perfusion. Histological analysis and lesion measurements were used to determine the effects of the pulses in decellularizing the livers as a first step towards the development of extracellular scaffolds that may be used with stem cell reseeding. A dynamic conductivity numerical model was developed to simulate the treatment parameters used and determine an irreversible electroporation threshold.ResultsNinety-nine individual 1000 V/cm 100-μs square pulses with repetition rates between 0.25 and 4 Hz were found to produce a lesion within 24 hours post-treatment. The livers maintained intact bile ducts and vascular structures while demonstrating hepatocytic cord disruption and cell delamination from cord basal laminae after 24 hours of perfusion. A numerical model found an electric field threshold of 423 V/cm under specific experimental conditions, which may be used in the future to plan treatments for the decellularization of entire organs. Analysis of the pulse repetition rate shows that the largest treated area and the lowest interstitial density score was achieved for a pulse frequency of 1 Hz. After 24 hours of perfusion, a maximum density score reduction of 58.5 percent had been achieved.ConclusionsThis method is the first effort towards creating decellularized tissue scaffolds that could be used for organ transplantation using N-TIRE. In addition, it provides a versatile platform to study the effects of pulse parameters such as pulse length, repetition rate, and field strength on whole organ structures.

A Numerical Investigation of the Electric and Thermal Cell Kill Distributions in Electroporation-Based Therapies in Tissue

Electroporation-based therapies are powerful biotechnological tools for enhancing the delivery of exogeneous agents or killing tissue with pulsed electric fields (PEFs). Electrochemotherapy (ECT) and gene therapy based on gene electrotransfer (EGT) both use reversible electroporation to deliver chemotherapeutics or plasmid DNA into cells, respectively. In both ECT and EGT, the goal is to permeabilize the cell membrane while maintaining high cell viability in order to facilitate drug or gene transport into the cell cytoplasm and induce a therapeutic response. Irreversible electroporation (IRE) results in cell kill due to exposure to PEFs without drugs and is under clinical evaluation for treating otherwise unresectable tumors. These PEF therapies rely mainly on the electric field distributions and do not require changes in tissue temperature for their effectiveness. However, in immediate vicinity of the electrodes the treatment may results in cell kill due to thermal damage because of the inhomogeneous electric field distribution and high current density during the electroporation-based therapies. Therefore, the main objective of this numerical study is to evaluate the influence of pulse number and electrical conductivity in the predicted cell kill zone due to irreversible electroporation and thermal damage. Specifically, we simulated a typical IRE protocol that employs ninety 100-µs PEFs. Our results confirm that it is possible to achieve predominant cell kill due to electroporation if the PEF parameters are chosen carefully. However, if either the pulse number and/or the tissue conductivity are too high, there is also potential to achieve cell kill due to thermal damage in the immediate vicinity of the electrodes. Therefore, it is critical for physicians to be mindful of placement of electrodes with respect to critical tissue structures and treatment parameters in order to maintain the non-thermal benefits of electroporation and prevent unnecessary damage to surrounding healthy tissue, critical vascular structures, and/or adjacent organs.

A three-dimensional in vitro tumor platform for modeling therapeutic irreversible electroporation.

Irreversible electroporation (IRE) is emerging as a powerful tool for tumor ablation that utilizes pulsed electric fields to destabilize the plasma membrane of cancer cells past the point of recovery. The ablated region is dictated primarily by the electric field distribution in the tissue, which forms the basis of current treatment planning algorithms. To generate data for refinement of these algorithms, there is a need to develop a physiologically accurate and reproducible platform on which to study IRE in vitro. Here, IRE was performed on a 3D in vitro tumor model consisting of cancer cells cultured within dense collagen I hydrogels, which have been shown to acquire phenotypes and respond to therapeutic stimuli in a manner analogous to that observed in in vivo pathological systems. Electrical and thermal fluctuations were monitored during treatment, and this information was incorporated into a numerical model for predicting the electric field distribution in the tumors. When correlated with Live/Dead staining of the tumors, an electric field threshold for cell death (500 V/cm) comparable to values reported in vivo was generated. In addition, submillimeter resolution was observed at the boundary between the treated and untreated regions, which is characteristic of in vivo IRE. Overall, these results illustrate the advantages of using 3D cancer cell culture models to improve IRE-treatment planning and facilitate widespread clinical use of the technology.

A Preliminary Study to Delineate Irreversible Electroporation From Thermal Damage Using the Arrhenius Equation

Intense but short electrical fields can increase the permeability of the cell membrane in a process referred to as electroporation. Reversible electroporation has become an important tool in biotechnology and medicine. The various applications of reversible electroporation require cells to survive the procedure, and therefore the occurrence of irreversible electroporation (IRE), following which cells die, is obviously undesirable. However, for the past few years, IRE has begun to emerge as an important minimally invasive nonthermal ablation technique in its own right as a method to treat tumors and arrhythmogenic regions in the heart. IRE had been studied primarily to define the upper limit of electrical parameters that induce reversible electroporation. Thus, the delineation of IRE from thermal damage due to Joule heating has not been thoroughly investigated. The goal of this study was to express the upper bound of IRE (onset of thermal damage) theoretically as a function of physical properties and electrical pulse parameters. Electrical pulses were applied to THP-1 human monocyte cells, and the percentage of irreversibly electroporated (dead) cells in the sample was quantified. We also determined the upper bound of IRE (onset of thermal damage) through a theoretical calculation that takes into account the physical properties of the sample and the electric pulse characteristics. Our experimental results were achieved below the theoretical curve for the onset of thermal damage. These results confirm that the region to induce IRE without thermal damage is substantial. We believe that our new theoretical analysis will allow researchers to optimize IRE parameters without inducing deleterious thermal effects.

Pilot study of irreversible electroporation for intracranial surgery

Irreversible electroporation (IRE) is a new minimally invasive technique to treat cancer using intense but short electric pulses. This technique is unique because of its non-thermal mechanism of tissue ablation. Furthermore it can be predicted with numerical models and can be confirmed with ultrasound and MRI. We present some preliminary results on the safety of using irreversible electroporation for canine brain surgery. We also present the electric field (460 V/cm – 560 V/cm) necessary for focal ablation of canine brain tissue and provide some guidelines for treatment planning and execution. This preliminary study is the first step towards using irreversible electroporation as a brain cancer treatment.