Treatment of Spontaneous Tumors With Algorithmically Controlled Electroporation.

OBJECTIVE: To study the safety and efficacy of algorithmically controlled electroporation (ACE) against spontaneous equine melanoma. METHODS: A custom temperature sensing coaxial electrode was paired with a high voltage pulse generation system with integrated temperature feedback controls. Computational modeling and ex vivo studies were conducted to evaluate the system's ability to achieve and maintain target temperatures. Twenty-five equine melanoma tumors were treated with a 2000V protocol consisting of a 2-5-2 waveform, 45ºC temperature set point, and integrated energized times of 0.005 s, 0.01 s, or 0.02 s (2500x, 5000x, and 10000x 2 µs pulses, respectively). Patients returned 20-50 days post treatment to determine the efficacy of the treatment. RESULTS: ACE temperature control algorithms successfully achieved and maintained target temperatures in a diverse population of spontaneous tumors with significant variation in tissue impedance. All treatments were completed successfully without and without adverse events. Complete response rates greater than 93% were achieved in all treatment groups. CONCLUSION: ACE is a safe and effective treatment for spontaneous equine melanoma. The temperature control algorithm enabled rapid delivery of electroporation treatments without prior knowledge of tissue electrical or thermal properties and could adjust to real time changes in tissue properties. SIGNIFICANCE: Real time temperature control in electroporation procedures enables treatments near critical structures where thermal damage is contraindicated. Unlike standard approaches, ACE protocols do not require extensive pretreatment planning or knowledge of tissue properties to determine an optimal energy delivery rate and they can account for changes in tissue state (e.g. perfusion) in real time to simultaneously minimize treatment time and potential for thermal damage.

A novel in vitro model of clinical cryoablation to investigate the transition zone for focal tumor ablation.

Cryoablation (CA) of solid tumors is highly effective at reducing tumor burden and eliminating small, early stage tumors. However, complete ablation is difficult to achieve and cancer recurrence is a significant barrier to treatment of larger tumors compared to resection. In this study, we explored the relationship between temperature, ice growth, and cell death using a novel in vitro model of clinical CA with the Visual-ICE (Boston Scientific) system, a clinically approved and widely utilized device. We found that increasing the duration of freezing from 1 to 2 min increased ice radius from 3.44 ± 0.13 mm to 5.29 ± 0.16 mm, and decreased the minimum temperature achieved from -22.8 ± 1.3 °C to -45.5 ± 7.9 °C. Furthermore, an additional minute of freezing increased the amount of cell death within a 5 mm radius from 42.5 ± 8.9% to 84.8 ± 1.1%. Freezing at 100% intensity leads to faster temperature drops and a higher level of cell death in the TRAMP-C2 mouse prostate cancer cell line, while lower intensities are useful for slow freezing, but result in less cell death. The width of transition zone between live and dead cells decreased by 0.4 ± 0.2 mm, increasing from one to two cycles of freeze/thaw cycles at 100% intensity. HMGB-1 levels significantly increased with 3 cycles of freeze/thaw compared to the standard 2 cycles. Overall, a longer freezing duration, higher freezing intensity, and more freeze thaw cycles led to higher levels of cancer cell death and smaller transition zones. These results have the potential to inform future preclinical research and to improve therapeutic combinations with CA.

Integrated Time Nanosecond Pulse Irreversible Electroporation (INSPIRE): Assessment of Dose, Temperature, and Voltage on Experimental and Clinical Treatment Outcomes.

OBJECTIVE: This study sought to investigate a novel strategy using temperature-controlled delivery of nanosecond pulsed electric fields as an alternative to the 50-100 microsecond pulses used for irreversible electroporation. METHODS: INSPIRE treatments were carried out at two temperatures in 3D tumor models using doses between 0.001 s and 0.1 s. The resulting treatment zones were quantified using viability staining and lethal electric field intensities were determined numerically. Computational modeling was then used to determine parameters necessary for INSPIRE treatments to achieve equivalent treatment zones to clinical electroporation treatments and evaluate the potential for these treatments to induce deleterious thermal damage. RESULTS: Lethal thresholds between 1109 and 709 V/cm were found for nominal 0.01 s treatments with pulses between 350 ns and 2000 ns at physiological temperatures. Further increases in dose resulted in significant decreases in lethal thresholds. Given these experimental results, treatment zones comparable to clinical electroporation are possible by increasing the dose and voltage used with nanosecond duration pulses. Temperature-controlled simulations indicate minimal thermal cell death while achieving equivalent treatment volumes to clinical electroporation. CONCLUSION: Nanosecond electrical pulses can achieve comparable outcomes to traditional electroporation provided sufficient electrical doses or voltages are applied. The use of temperature-controlled delivery may minimize thermal damage during treatment. SIGNIFICANCE: Intense muscle stimulation and the need for cardiac gating have limited irreversible electroporation. Nanosecond pulses can alleviate these challenges, but traditionally have produced significantly smaller treatment zones. This study suggests that larger ablation volumes may be possible with the INSPIRE approach and that future in vivo studies are warranted.

Polymer Nanoparticles Enhance Irreversible Electroporation In Vitro.

Expanding the volume of an irreversible electroporation treatment typically necessitates an increase in pulse voltage, number, duration, or repetition. This study investigates the addition of polyethylenimine nanoparticles (PEI-NP) to pulsed electric field treatments, determining their combined effect on ablation size and voltages. U118 cells in an in vitro 3D cell culture model were treated with one of three pulse parameters (with and without PEI-NPs) which are representative of irreversible electroporation (IRE), high frequency irreversible electroporation (H-FIRE), or nanosecond pulsed electric fields (nsPEF). The size of the ablations were compared and mapped onto an electric field model to describe the electric field required to induce cell death. Analysis was conducted to determine the role of PEI-NPs in altering media conductivity, the potential for PEI-NP degradation following pulsed electric field treatment, and PEI-NP uptake. Results show there was a statistically significant increase in ablation diameter for IRE and H-FIRE pulses with PEI-NPs. There was no increase in ablation size for nsPEF with PEI-NPs. This all occurs with no change in cell media conductivity, no observable degradation of PEI-NPs, and moderate particle uptake. These results demonstrate the synergy of a combined cationic polymer nanoparticle and pulsed electric field treatment for the ablation of cancer cells. These results set the foundation for polymer nanoparticles engineered specifically for irreversible electroporation.

Electro-Thermal Therapy Algorithms and Active Internal Electrode Cooling Reduce Thermal Injury in High Frequency Pulsed Electric Field Cancer Therapies.

Thermal tissue injury is an unintended consequence in current irreversible electroporation treatments due to the induction of Joule heating during the delivery of high voltage pulsed electric fields. In this study active temperature control measures including internal electrode cooling and dynamic energy delivery were investigated as a process for mitigating thermal injury during treatment. Ex vivo liver was used to examine the extent of thermal injury induced by 5000 V treatments with delivery rates up to five times faster than current clinical practice. Active internal cooling of the electrode resulted in a 36% decrease in peak temperature vs. non-cooled control treatments. A temperature based feedback algorithm (electro-thermal therapy) was demonstrated as capable of maintaining steady state tissue temperatures between 30 and 80 °C with and without internal electrode cooling. Thermal injury volumes of 2.6 cm3 were observed for protocols with 60 °C temperature set points and electrode cooling. This volume reduced to 1.5 and 0.1 cm3 for equivalent treatments with 50 °C and 40 °C set points. Finally, it was demonstrated that the addition of internal electrode cooling and active temperature control algorithms reduced ETT treatment times by 84% (from 343 to 54 s) vs. non-cooled temperature control strategies with equivalent thermal injury volumes.

Algorithmically Controlled Electroporation: A Technique for Closed Loop Temperature Regulated Pulsed Electric Field Cancer Ablation.

OBJECTIVE: To evaluate the effect of a closed-loop temperature based feedback algorithm on ablative outcomes for pulsed electric field treatments. METHODS: A 3D tumor model of glioblastoma was used to assess the impact of 2 µs duration bipolar waveforms on viability following exposure to open and closed-loop protocols. Closed-loop treatments evaluated transient temperature increases of 5, 10, 15, or 22 °C above baseline. RESULTS: The temperature controlled ablation diameters were conditionally different than the open-loop treatments and closed-loop treatments generally produced smaller ablations. Closed-loop control enabled the investigation of treatments with steady state 42 °C hyperthermic conditions which were not feasible without active feedback. Baseline closed-loop treatments at 20 °C resulted in ablations measuring 9.9 ± 0.3 mm in diameter while 37 °C treatments were 20% larger (p < 0.0001) measuring 11.8 ± 0.3 mm indicating that this protocol induces a thermally mediated biological response. CONCLUSION: A closed-loop control algorithm which modulated the delay between successive pulse waveforms to achieve stable target temperatures was demonstrated. Algorithmic control enabled the evaluation of specific treatment parameters at physiological temperatures not possible with open-loop systems due to excessive Joule heating. SIGNIFICANCE: Irreversible electroporation is generally considered to be a non-thermal ablation modality and temperature monitoring is not part of the standard clinical practice. The results of this study indicate ablative outcomes due to exposure to pulses on the order of one microsecond may be thermally mediated and dependent on local tissue temperatures. The results of this study set the foundation for experiments in vivo utilizing temperature control algorithms.

Electro-thermal therapy: Microsecond duration pulsed electric field tissue ablation with dynamic temperature control algorithms.

Electro-thermal therapy (ETT) is a new cancer treatment modality which combines the use of high voltage pulsed electric fields, dynamic energy delivery rates, and closed loop thermal control algorithms to rapidly and reproducibly create focal ablations. This study examines the ablative potential and profile of pulsed electric field treatments delivered in conjunction with precise temperature control algorithms. An ex vivo perfused liver model was utilized to demonstrate the capability of 5000 V 2 µs duration bipolar electrical pulses and dynamic temperature control algorithms to produce ablations. Using a three applicator array, 4 cm ablation zones were created in under 27 min. In this configuration, the algorithms were able to rapidly achieve and maintain temperatures of 80 °C at the tissue-electrode interface. A simplified single applicator and grounding pad approach was used to correlate the measured ablation zones to electric field isocontours in order to determine lethal electric field thresholds of 708 V/cm and 867 V/cm for 45 °C and 60 °C treatments, respectively. These results establish ETT as a viable method for hepatic tumor treatment with ablation profiles equivalent to other energy based techniques. The single applicator and multi-applicator approaches demonstrated may enable the treatment of complex tumor geometries. The flexibility of ETT temperature control yields a malleable intervention which gives clinicians robust control over the ablation modality, treatment time, and safety profile.

Irreversible electroporation is a thermally mediated ablation modality for pulses on the order of one microsecond.

Irreversible electroporation (IRE) is generally considered to be a non-thermal ablation modality. This study was designed to examine the relative effect of temperature on IRE ablation sizes for equivalent dose treatments with constitutive pulses between 1 and 100 µs. 3D in-vitro brain tumor models maintained at 10 °C, 20 °C, 30 °C, or 37 °C were exposed to 500 V treatments using a temperature control algorithm to limit temperature increases to 5 °C. Treatments consisted of integrated energized times (doses) of 0.01 or 0.1 s. Pulse width, electrical dose, and initial temperature were all found to significantly affect the size of ablations and the resulting lethal electric field strength. The smallest ablations were created at 10 °C and ELethal were calculated to be 1729, 1359, 929, 777, 483 V/cm for 0.01 s treatments with 1, 2, 4, 8, and 100 µs pulses, respectively. At 37 °C these values decreased to 773, 614, 507, 462, and 394 V/cm, respectively. Increasing the dose from 0.01 to 0.1 s at 37 °C resulted in statistically significant decreases (p < 0.001) in ELethal for all treatments except for the 100 µs group. This study found that IRE is a thermally mediated, dose-dependent ablation modality for pulses on the order of one microsecond. Tissue temperatures are not accounted for when determining ablative boundaries in treatment planning algorithms. This work demonstrates that data generated at room temperature may not be predictive of ablation volumes in-vivo and that local temperatures should be accounted for in treatment planning.

Temperature Dependence of High Frequency Irreversible Electroporation Evaluated in a 3D Tumor Model.

Electroporation is a bioelectric phenomenon used to deliver target molecules into cells in vitro and irreversible electroporation (IRE) is an emerging cancer therapy used to treat inoperable tumors in situ. These phenomena are generally considered to be non-thermal in nature. In this study, a 3D tumor model was used to investigate the correlation between temperature and the effectiveness of standard clinical IRE and high frequency (H-FIRE) protocols. It was found for human glioblastoma cells that in the range of 2 to 37 °C the H-FIRE lethal electric field threshold value, which describes the minimum electric field to cause cell death, is highly dependent on temperature. Increasing the initial temperature from 2 to 37 °C resulted in a significant decrease in lethal electric field threshold from 1168 to 507 V/cm and a 139% increase in ablation size for H-FIRE burst treatments. Standard clinical protocol IRE treatments resulted in a decrease in lethal threshold from 485 to 453 V/cm and a 7% increase in ablation size over the same temperature range. Similar results were found for pancreatic cancer cells which indicate that tissue temperature may be a significant factor affecting H-FIRE ablation size and treatment planning in vivo while lower temperatures may be useful in maintaining cell viability for transfection applications.

High-Frequency Irreversible Electroporation Using 5,000-V Waveforms to Create Reproducible 2- and 4-cm Ablation Zones-A Laboratory Investigation Using Mechanically Perfused Liver.

PURPOSE: To investigate if high-frequency irreversible electroporation (H-FIRE) treatments can be delivered at higher voltages and with greater energy delivery rates than currently implemented in clinical irreversible electroporation protocols. MATERIALS AND METHODS: Treatments using 3,000 V and 5,000 V were administered to mechanically perfused ex vivo porcine liver via a single applicator and grounding pad (A+GP) as well as a 4-applicator array (4AA). Integrated energized times (IET) 0.01-0.08 seconds and energy delivery rates 25-300 µs/s were investigated. Organs were preserved at 4°C for 10-15 hours before sectioning and gross analysis using a metabolic stain to identify the size and shape of ablation zones. RESULTS: A+GP ablations measured between 1.6 cm and 2.2 cm, which did not increase when IET was increased from 0.02 seconds to 0.08 seconds (P > .055; range, 1.9-2.1 cm). Changes in tissue color and texture consistent with thermal damage were observed for treatments with energy delivery rates 50-300 µs/s, but not for treatments delivered at 25 µs/s. Use of the 4AA with a 3-cm applicator spacing resulted in ablations measuring 4.4-4.9 cm with energy delivery times of 7-80 minutes. CONCLUSIONS: H-FIRE treatments can rapidly and reproducibly create 2-cm ablations using an A+GP configuration. Treatments without thermal injury were produced at the expense of extended treatment times. More rapid treatments resulted in ablations with varying degrees of thermal injury within the H-FIRE ablation zone. Production of 4-cm ablations is possible using a 4AA.

Simplified Non-Thermal Tissue Ablation With a Single Insertion Device Enabled by Bipolar High-Frequency Pulses.

OBJECTIVE: To demonstrate the feasibility of a single electrode and grounding pad approach for delivering high frequency irreversible electroporation treatments (H-FIRE) in in-vivo hepatic tissue. METHODS: Ablations were created in porcine liver under surgical anesthesia by adminstereing high frequency bursts of 0.5-5.0 µs pulses with amplitudes between 1.1-1.7 kV in the absence of cardiac synchronization or intraoperative paralytics. Finite element simulations were used to determine the electric field strength associated with the ablation margins (ELethal) and predict the ablations feasible with next generation electronics. RESULTS: All animals survived the procedures for the protocol duration without adverse events. ELethal of 2550, 1650, and 875 V/cm were found for treatments consisting of 100x bursts containing 0.5 µs pulses and 25, 50, and 75 µs of energized-time per burst, respectively. Treatments with 1 µs pulses consisting of 100 bursts with 100 µs energized-time per burst resulted in ELethal of 650 V/cm. CONCLUSION: A single electrode and grounding pad approach was successfully used to create ablations in hepatic tissue. This technique has the potential to reduce challenges associated with placing multiple electrodes in anatomically challenging environments. SIGNIFICANCE: H-FIRE is an in situ tumor ablation approach in which electrodes are placed within or around a targeted region to deliver high voltage electrical pulses. Electric fields generated around the electrodes induce irrecoverable cell membrane damage leading to predictable cell death in the relative absence of thermal damage. The sparing of architectural integrity means H-FIRE offers potential advantages compared to thermal ablation modalities for ablating tumors near critical structures.

Burst and continuous high frequency irreversible electroporation protocols evaluated in a 3D tumor model.

High frequency irreversible electroporation (H-FIRE) is an emerging cancer therapy which uses bursts of alternating polarity pulses to target and destroy the membranes of cells within a predictable volume. Typically, 2 µs pulses are rapidly repeated 24-50 times to create a 48-100 µs long energy burst. Bursts are repeated 100× at 1 Hz, resulting in an integrated energized time of 0.01 s per treatment. A 3D in vitro tumor model was used to investigate H-FIRE parameters in search of optimal energy timing protocols. Monopolar IRE treatments (100 × 100 µs positive polarity pulses) resulted in a lethal electric field threshold of 423 V cm-1. Baseline H-FIRE treatments (100 × 100 µs bursts of 2 µs pulses) resulted in a lethal threshold of 818 V cm-1. Increasing the number of H-FIRE bursts from 100× to 1000× reduced the lethal threshold to 535 V cm-1. An alternative diffuse H-FIRE protocol, which delivers 4 µs pulse cycles (one positive and one negative 2 µs pulse) continuously at 100 Hz, resulted in the lowest H-FIRE lethal threshold of 476 V cm-1. Finite element simulations using 5 kV pulses predict an IRE ablation volume of 3.9 cm3 (1.7 cm diameter) and a maximum H-FIRE ablation volume of 5.3 cm3 (2.4 cm diameter) when a clinical electrode and grounding pad configuration is used. Ablations as large as 15.7 cm3 (3.3 cm diameter) are predicted for H-FIRE treatments with 10 kV pulses. These results combine to demonstrate the importance of electrode geometry, pulse timing, and clinical delivery protocols for the creation of large clinically meaningful ablations.