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Treatment of Spontaneous Tumors

With Algorithmically Controlled
Electroporation
Christopher C. Fesmire, Ross A. Petrella, Robert Williamson, Kobi Derks, Jennifer Ruff, Thomas
McParkland, Erin O’Neil, Callie Fogle, Timo Prange, Michael B. Sano

vessels, and minimize collateral damage to surrounding tissues

Abstract— Objective: To study the safety and efficacy of [3-5]. To realize these benefits, the pulse parameters (width,
algorithmically controlled electroporation (ACE) against polarity, amplitude, number, and repetition) must be
spontaneous equine melanoma. appropriately selected. However, there is currently no clinically
Methods: A custom temperature sensing coaxial electrode was
available workflow or technique to prevent excess energy
paired with a high voltage pulse generation system with integrated
temperature feedback controls. Computational modeling and ex delivery and thermal injury [6, 7]. The challenge with current
vivo studies were conducted to evaluate the system’s ability to IRE approaches is that it is difficult to predict the tumor and
achieve and maintain target temperatures. Twenty-five equine tissue macro- and microenvironment as a function of
melanoma tumors were treated with a 2000V protocol consisting temperature, time, and conductivity. Variations in patients and
of a 2-5-2 waveform, 45ºC temperature set point, and integrated tissue type further complicate pre-treatment planning. The
energized times of 0.005 s, 0.01 s, or 0.02 s (2500x, 5000x, and
result is mismatches between pre-treatment planning and actual
10000x 2 µs pulses, respectively). Patients returned 20-50 days post
treatment to determine the efficacy of the treatment. outcomes [8]. Excessive Joule heating can result in undesirable
Results: ACE temperature control algorithms successfully thermal effects [11] and may be responsible for rare IRE related
achieved and maintained target temperatures in a diverse clinical complications including coagulative necrosis [12],
population of spontaneous tumors with significant variation in thrombosis [13], and fistulas [14].
tissue impedance. All treatments were completed successfully To address these drawbacks, algorithmically controlled
without and without adverse events. Complete response rates electroporation (ACE) integrates active sensing, feedback, and
greater than 93% were achieved in all treatment groups. control systems to dynamically adjust pulse delivery parameters
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.

Index Terms—Pulsed Field Ablation, Melanoma Treatment,

Non-Thermal Irreversible Electroporation, Temperature
Feedback, Cancer Therapy
Figure 1: IRE and ACE Treatment Strategies. IRE delivers a series of
monopolar 90-100µs pulses timed with the absolute refractory period of the
heart to prevent accidental arrhythmia. ACE delivers shorter (0.25 – 5 µs)
I. INTRODUCTION alternating polarity waveforms which are repeated at a variable rate to achieve
and maintain a stable tissue temperature during treatment. The duration of the
W HEN compared to thermal-based ablation methods,
irreversible electroporation (IRE, Fig. 1a) has the
potential to preserve the extracellular matrix, major blood
positive pulse (P), negative pulse (N), and inter-pulse delay (D) can all be
adjusted to meet clinical needs.

Research reported in this publication was supported by the National Cancer
Institute of the National Institutes of Health under award numbers
1R01CA276232 and R01CA272550, by the NCSU CMI New Faculty Clinical
Research Award, and by the UNC/NCSU Joint Department of Biomedical
Engineering
MBS, CCF, RAP, RHW are with the UNC/NCSU Joint Department of
Biomedical Engineering, Raleigh, NC
MBS, KD, JR, TM, EO, CF, TP are with the NCSU College of Veterinary
Medicine, Raleigh, NC
MBS is with the Department of Molecular Biomedical Sciences, Raleigh, NC
(e-mail: mike_sano@ncsu.edu)
CCF, RAP, and MBS have intellectual property related to the subject of this
manuscript. MBS has ownership in Gradient Medical Inc., a company
commercializing PEF related technologies and receives royalties from
AngioDynamics Inc. All other authors have no conflicts to declare.

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during treatment (Fig. 1b) [15] to avoid thermal injury.
Previous in vitro [15] and ex vivo [16, 17] investigations have
demonstrated a proportional control algorithm that maintains a
stable treatment temperature in benchtop tissue models. In vitro
studies show that cell death following ACE is temperature
dependent for pulses on the order of one microsecond [15, 18,
19]. Optimal ACE treatment conditions involve mild Joule
heating while preventing tissue temperatures from reaching
levels associated with thermal necrosis. Initial murine model
studies confirmed the preservation of treatment efficacy with
ACE, despite a reduction in ablation speed[20]. Notably,
histopathological staining quality improved, suggesting a
decrease in coagulative necrosis[20]. These findings have
prompted the need for further in vivo testing to determine the
clinical utility of the ACE approach.
This study focuses on investigating the in vivo safety and
efficacy of ACE in the treatment of equine melanoma, a
clinically relevant disease state involving large, spontaneous
tumors. Equine melanoma patients were enrolled in this pilot
study. A coaxial applicator, capable of providing visual
indication of the treatment zone and enabling ACE with
temperature control, was developed and validated through finite
element modeling and ex vivo experiments. Equine patients
were treated while awake and standing under mild sedation,
with individual tumors receiving a total active treatment time or
integrated energized (IET) time of either 0.005, 0.01, or 0.02
seconds of electrical energy.
A complete response was found in 93% (14 out of 15) of
tumors treated with 0.01 second integrated time which
increased to 100% (4 out of 4) when the IET was increased to
0.02 seconds. These findings provide evidence of the potential
safety and efficacy of ACE in treating melanoma and other solid
tumors.
II. METHODS
Utilizing ACE in equine patients presented clinical
challenges requiring the development of an application specific
electrode. In particular, the potential for electromagnetic
interference required the use of a fiberoptic temperature sensor
Figure 2: Finite Element Model for Electrode Optimization. (a) A
simplified axial-symmetric geometry representing the center needle electrode
and outer ring electrode was used to predict treatment volumes for a range of
geometric parameters and treatment voltages. (b) Adaptive mesh refinement
was used to produce a highly dense mesh in regions where changes in the
electric field were most substantial.
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2
and the dense fibrous nature of some tumors required this
sensor to be protected inside the electrode rather than in direct
contact with the tissue to avoid routinely breaking the sensor.
The electrode was designed and modeled using computational
electromagnetic simulations. The design was then validated in
an ex vivo liver model using internal and external temperature
sensors to investigate how the sensor location affected
temperature control. The applicator, with only an internal
sensor, was then used clinically to treat a series of equine
patients with spontaneous melanoma tumors.
A. Finite Element Modeling
The electrode’s geometry consists of an inner cylindrical
conductor representing the cathode enclosed within an outer
cylindrical conductor representing the anode. This
classification of electrodes has been previously simulated and
experimentally verified in vitro [15, 18, 19] and validated in
vivo[21].
The non-uniform electric field distribution surrounding the
coaxial ring and pin electrode was calculated using a 2D
axisymmetric finite element model in COMSOL Multiphysics
(V5.5, COMSOL Inc., Los Altos, CA). The simulated geometry
(Fig. 2) consisted of a 5 cm radius, 5 cm tall cylindrical tissue
domain. The central needle had a radius of 2.1 mm (14 gauge).
The needle point was modeled as a 45-degree fillet. A 0.55 mm
radius cylindrical region was modeled representing the internal
region of the needle where the internal temperature sensor was
placed experimentally. The outer ring electrode was modeled as
a 0.88mm x 5 mm rectangle which was swept around the y-axis
to produce a 17mm outer diameter ring matching the final
experimental design.
The Electric Currents, Heat Transfer in Solids, and
Electromagnetic Heating modules were used to calculate the
electric field distribution (E) within the 3D tumors:
𝑬 = −∇𝑉 [1]
where V is the electrical potential. In this model, displacement
currents were assumed to be negligible due to the relatively
high electrical conductivity of tumor tissue. Heat transfer within
all material domains was calculated as:
𝛿𝑇
𝜌𝐶𝑝 − 𝑘∇ ∙ (∇𝑇) = 𝑄𝐸𝑀 [2]
𝛿𝑡
where ρ was the material density, Cp was the material heat
capacity, k was the material thermal conductivity, T was the
element temperature, and QEM was the heating due to resistive
(Joule) heating:
QEM = R(T) J ∙ E [3]
∇ ∙ (−k∇T) = QEM [4]
where J is the current density, T is the element temperature, and
k is the thermal conductivity. QEM was then scaled by R(T), a
factor which was equivalent to the energy delivery rate to
account for the dynamic duty cycle associated with active
temperature control. Thermal conductivity (kT), density (𝜌𝑇 ),
and heat capacity (CPT) of the tissue were modeled using
COMSOL’s built in functions for porcine liver tissue. Tissue
conductivity was modeled using a temperature dependent
function based on impedance changes measured in rapidly
heated tissue [22](Supplemental Fig. 1). The electrical and
material properties for the remaining materials are presented in
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Figure 3: Electrode Design. (a) Overview of electrode needle handle design. (b) Section view of the front portion of the electrode handle showing the location
of the internal temperature sensor. (c) 3D printed electrode handle assembled showing the location of external temperature sensor used in ex vivo validation
experiments. This external sensor was removed for the in vivo studies.
Supplemental Table 1. (Component → Definitions → Functions → Analytic) to
An initial mesh consisting of 2,237 triangular elements was calculate the integrand (𝑖(𝑇) = 𝜁 ∙ 𝑒 −𝐸𝑎 /(𝑅∙𝑇(𝑡)) ) then creating
generated using the predefined “Finer” settings. Two rounds of a variable specific to the tissue domain (Component →
adaptive mesh refinement were performed by a stationary Variables) which calculates the Ω integral (integrate( i(T), t, 0,
solver using a magnitude of the electric field as the error t ) ) where T is the domain temperature and t is the instantaneous
indicator (ec.normE > 100 [V/cm]) yielding a final mesh which time. Separate integrands and domain variables were used for
consisted of 54,397 elements (Fig. 2b). Excluding a region parameters relative to the induction of blood flow stasis, protein
within 300µm of the sharp electrode tip, the electric field coagulation, and cell death (Table 1). An Ω value of 4.6 [2, 25],
calculation varied by less than 1% between the first and second representing 99% damage probability, was used as the threshold
mesh refinements. This refined mesh was then used for time for each of these processes[24]. To estimate the volume of cell
domain analysis of electric field and temperature distributions death associated with IRE, cell death kinetics were modeled by
[23]. Time domain simulations required approximately 4.4 extrapolating lethal thresholds identified in in vitro 3D tissue
hours for each parameter to solve on a 12-core 3945WX constructs (Supplemental Fig. 2) [26]. This resulted in lethal
processor with 256GB of RAM. thresholds of 873 V/cm, 713 V/cm, and 685 V/cm for IET of
To compare results to standard IRE protocols which use two 0.005s, 0.01s, and 0.02s, respectively.
parallel needle applicators, the identical simulation parameters
B. Development of the Treatment Applicator
were repeated using a 17.3 cm center-to-center electrode
spacing to match the experimental coaxial electrode (i.e. the A custom applicator handle was designed in SolidWorks
distance between the center pin and outer ring electrode). It (SolidWorks, Waltham, MA) to accommodate a 17 mm
should be noted that these simulations required the use of a fully diameter ring, 14 gauge center needle electrode, electrical
3D geometry as the two-applicator configuration lacks axial wiring, and fiber optic temperature sensors (Fig. 3a). Internal
channels were created to accommodate the 10 kV rated silicone
symmetry.
insulated electrical leads (6733-2, Pomona Electronics, Everett,
To assess differences between temperature controlled and
Washington) and the 2.5 mm diameter temperature sensors
uncontrolled treatments in clinical use, thermal damage
(TS4, Micronor Inc., Ventura, CA). A threaded barrel connector
resulting in cell death, protein coagulation, and blood flow
was press-fit into the handle to create a fixed location to secure
stasis were modeled via an Arrhenius type analysis[24, 25]
a threaded luer lock adapter. The depth of the internal
assuming that damage followed first order reaction kinetics:
𝑡 components was selected such a 35 mm length of the disposable
Ω(t) = ∫ 𝜁 ∙ 𝑒−𝐸𝑎 /(𝑅∙𝑇(𝑡)) 𝑑𝑡 [5] 14 gauge needle was exposed. In regions containing exposed
0 metal components, care was taken to maintain wall thicknesses
where 𝜁 is the frequency factor, Ea is the activation energy, R is sufficient for proper electrical isolation. In this design, the
the universal gas constant, and T(t) is the temperature. This was needle electrode is fixed and the penetration depth below the
modeled in COMSOL by defining an analytical function surface is adjusted by increasing or decreasing the length of the
TABLE I
CELL DEATH MODELING PARAMETERS
Symbol Quantity Value Units Reference
𝜁𝐵𝐹𝑆 Frequency Factor - Blood Flow Stasis 1.98x10106 1/s [1, 2]
𝜁𝑃𝐷 Frequency Factor – Protein Denaturation 7.39x1037 1/s [2, 9]
𝜁𝐶𝐷 Frequency Factor – Cell Death 2.984x1080 1/s [2, 10]
𝐸𝑎 𝐵𝐹𝑆 Activation Energy – Blood Flow Stasis 6.670x105 J/mol [1, 2]
𝐸𝑎 𝑃𝐶 Activation Energy – Protein Coagulation 2.577x105 J/mol [2, 9]
𝐸𝑎 𝐶𝐷 Activation Energy – Cell Death 5.064x105 J/mol [2, 10]
𝑅 Universal Gas Constant 8.314 J/(K·mol) -
Parameters used to model cell death processes in COMSOL. Subscripts BFS, PD, and CD represent blood flow stasis, protein denaturation, and cell death,
respectively.

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surface ring. The completed applicator design was fabricated on of being melanoma. Informed consent for this therapy was
a Form2 SLA 3D printer (FormLabs Inc., Somerville, MA) obtained from owners prior to enrollment. Prior to each
using GrayV4 resin and a layer thickness of 100 µm. treatment, tumors were identified, photographed, numbered,
During assembly a 1” long 14 gauge aluminum hub and measured. The length (l), width (w), and depth (d) from the
disposable needle (Monoject 140391, Cardinal Health Inc. tumor apex to the skin were measured with digital calipers.
Dublin, OH) was embedded inside of the electrode handle using Tumor volumes (v) were calculated as an ellipsoid with volume
4
a luer lock threaded connector (51465K21, McMaster-Carr calculation 𝑣 = 𝜋 (𝑙/2 ∙ 𝑤/2 ∙ 𝑑). To confirm malignant
3
Inc., Santa Fe Springs, CA) which was connected to a threaded
disease via histology, a single tumor was resected. If patients
rod hexagonal connector. The hexagonal connector was press
presented with multifocal disease, only a subset of the tumors
fit into the handle using an interference fit of 50 µm. The source
were identified, measured, and accounted for across the trial.
electrical wire was connected by a press fit 14 gauge ring
As this study utilized client owned animals, tumors that were
terminal between the hexagonal connector and the luer lock
likely to be problematic to the patient if left untreated were
threaded connector to establish the electric connection. A 17.3
prioritized. Six patients were admitted into the ACE treatment
mm inner diameter 304 stainless steel pipe (McMaster Part
arm of the trial. All treatments consisted of a 2-5-2 waveform
Number: 4347K321) was used as the outer electrode ring sink.
with an amplitude of 2000V and a temperature set point of
The ring was connected to the internal sink wire via a press fit
45ºC. Tumors were generally categorized as small (mean:
and threaded through a hole drilled in the outer electrode ring
0.18mm3), medium (mean: 0.48 mm3), or large (mean: 2.2
to ensure a mechanical connection (Fig. 1c). Prior to insertion
mm3) and assigned to receive an IET of 0.005 s, 0.01 s, or 0.02
of the internal temperature sensor the 14 gauge needle was filled
s, respectively. This resulted in 2500, 5000, and 10000, pulses
with ultrasound gel to improve heat transfer to the sensor, then
administered in these respective treatments. Patients returned at
the tip was sealed with a two-part epoxy to prevent tissue from
least once following the initial treatment at approximately 20-
entering the needle.
50 day intervals at which time the tumors were re-measured.
C. Evaluation of Thermal Profiles in Ex Vivo Tissue Prior to treatment, patients were sedated with butorphanol
A series of experiments were conducted to characterize the (0.01-0.05 mg/kg IV) and detomidine (0.01-0.02 mg/kg IV),
correlation between the internal temperature sensor and tissue and re-dosed as needed to facilitate treatment application.
temperatures in proximity to the needle applicator. In vivo Treatment sites were prepared with povidone-iodine scrub and
tumor tissue presented a technical challenge as the tissue is alcohol. Treatment sites were desensitized by local injection of
often firm and fibrous with the potential to break the small 2% mepivacaine into the surrounding tissues. During
diameter fiberoptic temperature sensors. Instead, an ex vivo treatments patients were awake and standing.
porcine liver tissue model from a local grocer was utilized.
Tissue was warmed to room temperature (18°C) before
evaluation. An external sensor attached to the applicator
reached a depth of 3mm below the tissue surface matching the
approximate location of the internal temperature sensor.
A custom pulse generator [27-32]was utilized for all studies.
This system has a built-in data acquisition system that acquired
both voltage and current during pulsing at a rate of 100 million
samples per second and a separate temperature acquisition
system which recorded at a rate of 3 Hz. Pulse parameters
consisted of a 2 µs positive pulse, followed by a 5 µs delay
before a 2 µs negative polarity pulse, referred to as a 2-5-2
waveform. The delay between successive waveform was either
fixed to deliver energy at a constant continuous rate [33] or
controlled algorithmically [34] to achieve a desired tissue
temperature. Treatments with a continuous rate were
administered at 25, 50, 100 and 200 µs/s (6.25, 12.5, 25, and 50
Hz, respectively). Algorithmically controlled treatments were
administered with a target of 25ºC at the internal sensor.
Temperatures at the internal and external sensors were recorded
throughout each treatment. These measurements were used to
fine tune computational models used to predict the extent of
thermal damage anticipated in in vivo treatments.
D. Clinical Trial in Equine Patients Figure 4: Ex Vivo Analysis of Internal (red) and External (black)
Equine patients were recruited into an IACUC approved (19- Temperature Profiles During Pulse delivery. 2000V treatments with a 0.02s
053-O) Phase 1 safety/efficacy study at the North Carolina State IET administered at a constant rate of (a) 25 µs/s and (b) 100µs/s. Shaded
regions represent 1 standard deviation from the mean. (c) Equivalent 0.02s IET
University School of Veterinary Medicine. Inclusion criteria treatment administered with temperature control set to 25ºC. Dotted lines
included: patient health was adequate to handle sedation and represent simulated temperatures measured at the location of the external
presentation of at least three discrete tumor nodules suspected temperature sensor (e.g. blue star in Fig. 2a).

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III. RESULTS
A. Evaluation of the Coaxial Electrode
In ex vivo tissue, the externally measured temperature was
consistently greater than the internally measured temperature
(Fig. 4). The difference between these two measurements
increased as the energy delivery rate increased. A difference of
14% ± 1% in peak temperature observed for treatments
administered at a constant rate of 25 µs/s (Fig. 4a) This
increased to 29% ± 2%, 39% ± 3%, and 56% ± 3% for
treatments administered at 50 µs/s, 100 µs/s (Fig. 4b), and 200
µs/s, respectively. A relatively small difference of 8.3%
between the peak internal and peak external temperatures was
observed when active temperature control was utilized to
maintain a target internal temperature of 25ºC (Fig. 5c).
Without active temperature control, the external temperature
measured adjacent to the needle electrode reached peak
temperatures of 32.2 ±1.3, 48.5 ± 4.8, 61.5 ± 2.0, and 87.2 ±
5.1ºC for treatments administered at 25 µs/s, 50 µs/s, 100µs/s,
and 200 µs/s, respectively. Use of active temperature control
with an internal temperature set point of 25ºC resulted in an
external temperature of 28.2 ± 0.7 ºC. Numerical simulations of
these temperature transients agreed with these experimental
results (Fig. 4, dotted lines).
Figure 5: Numerical Analysis of Coaxial and Two-Electrode
Configurations. Electric field isocountour (685V/cm) showing predicted
treatment volumes for the (a) coaxial and (b) two-electrode configurations. (c-
d) Temperature profiles at the end (t=200s) of a 0.02s 2000V treatment
administered at 50 µs/s. Isocontour lines represent IRE (red), thermal cell
death (yellow, CD), blood flow stasis (orange, BFS), and protein coagulation
(green, PC). (e) Predicted volumes of IRE and thermal cell death at the end
(t=200s) of a 2000V 0.02s treatment administered at a rate of 50 µs/s.
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5
B. Finite Element Modeling Results
It was found that the electrode geometry produced by a
17mm diameter ring and 14 gauge needle provided adequate
coverage for potential tumors between 1 and 2 cm in diameter
(Fig. 5). It was of interest to investigate how the treatment zones
for this coaxial applicator compared to traditional IRE
treatments which use two parallel electrodes. Parametric
analysis of the electrode insertion depth (Supplemental Fig. 3)
indicated that the coaxial electrode configuration produced IRE
volumes which were volumetrically 1.8x larger on average
(range: 2.9-1.3x) than the two-electrode configuration when all
other parameters were held constant.
Time dependent simulations of the coaxial electrode indicate
a moderate change in the electric field distribution
(Supplemental Fig. 4a-d) over time as temperature transients
affect the electrical conductivity of the tissue (Supplemental
Fig. 4e-h) with the most significant changes in the electric field
occurring within the first few seconds of the simulation.
The resulting coaxial treatment zones had a parabolic
inverted bell shape which extended approximately 5mm from
the tip of the electrode up to the surface of the tissue (Fig. 5a).
In contrast, the traditional two-applicator configuration resulted
in a substantially smaller oval shaped treatment zone (Fig. 5b)
which extended from the tissue surface to approximately 5mm
past the distal ends of the electrodes. With a baseline 1 cm
electrode penetration into the tissue the coaxial electrode
resulted in an IRE treatment volume of 2.4 cm3 (Fig. 5c), while
Figure 6: Effect of Dose Exposure on Treatment Volume. Temperature
distribution for 2000V treatments with temperature control set to 45℃. The
red line indicates the volume of cell death due to IRE for a dose of (a)
0.00125s, (b) 0.005s, (c) 0.01s, (d) 0.02s. (d) Predicted volumes of protein
coagulation (PC), blood flow stasis (BFS), thermal cell death (CD), and IRE
for 45℃ temperature controlled treatments versus uncontrolled treatments
delivered at a rate of 50µs/s.
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the two-applicator configuration produced an IRE treatment

volume of 1.4 cm3 (Fig. 5d). Both applicator configurations
resulted in predicted thermal cell death and blood flow stasis
when 2000 V treatments were administered at a rate of 50 µs/s.
However, the coaxial configuration resulted in greater tissue
heating (Fig. 5c) than the two-applicator configuration (Fig.
5d), yielding greater predicted volumes of thermal injury (Fig.
5e) for the coaxial electrode when simulated treatments were
administered without temperature control.
Implementation of temperature control with a set point of
45℃ significantly reduced the volume of tissue likely to
undergo any form of thermal damage with the coaxial electrode
(Fig. 6). IRE induced cell death is a function of the electrical
dose administered[26] with smaller doses requiring higher
electric fields to induce cell death than larger doses. For very
small doses (0.00125s) the coaxial electrode produced
simulated treatment volumes of 1.1cm3 (Fig. 6a). This
increased to 1.4 cm3, 1.9 cm3 (Fig. 6b), 2.3 cm3(Fig. 6c), and
2.4 cm3(Fig. 6d) for doses of 0.0025 s, 0.005 s, 0.01 s, and 0.02
s, respectively. In these temperature-controlled simulations
(45℃) there was no predicted thermal damage for doses
between 0.00125 and 0.01 s (Fig. 6e). The 0.02 s dose 45℃
simulation predicted a thermal cell death (CD) volume of 0.1
cm3 and no predicted blood flow stasis (BFS) or protein
coagulation (PC). In contrast, simulated 0.02s dose treatments
without temperature control (50µs/s, Fig. 5c) predicted
volumes of 2.1 cm3, 1.9 cm3, and 0.04 cm3 for CD, BFS, and
PC, respectively.
Thermal injury kinetics are a function of temperature and
exposure time. Simulations predicted that CD, BFS, and PC
volumes can be modulated without the use of temperature
control by decreasing or increasing the fixed rate of energy
delivery (Supplemental Fig. 5a-e). Reducing the fixed rate to
25 µs/s resulted in predicted volumes of 0.6 cm3, 0.4 cm3, and
0.0 cm3 for CD, BFS, and PC, respectively while maintaining
the IRE volume (2.4 cm3) at the expense of doubling the
treatment time (800s). Alternatively, increasing the fixed rate to
200 µs/s significantly reduced the treatment time (100s) at the
expense of increasing predicted thermal damage volumes to 2.7
cm3, 2.6 cm3, and 0.9 cm3 for CD, BFS, and PC, respectively.
Thermal damage volumes can similarly be modulated with
temperature control via adjustment of the target temperature
(Supplemental Fig. 5f-j). Simulated treatments with a
temperature set point of 65℃ resulted in thermal damage
volumes of 2.4cm3, 2.0 cm3, and 0.0 cm3 for CD, BFS, and PC,
respectively. In most cases, the predicted treatment times with
temperature control enabled were longer than those required for
fixed delivery rates. These decreased from 1202s to 905 s, 724
s, 549 s, and 491 s as the temperature set point was adjusted
from 45℃ to 50℃, 55℃, 60℃, and 65℃, respectively.
C. Clinical Trial Results
Treatments were well tolerated in awake and standing horses Figure 7: In Vivo Treatment of Spontaneous Tumors. (a) Treatments were
under mild sedation (Fig. 7a-b). Occasionally, muscle accomplished by placing the needle electrode at the center of the tumor and
applying pressure to advance the outer ring into contact with the skin.
stimulation was observed in direct proximity to the applicator. Photographs and measurements were taken (b) Pretreatment and (c) at follow
This typically diminished over the duration of the treatment up 20-50 days post treatment. 2000V 0.02s IET treatments were then
without the need for additional intervention. If necessary, local administered via active temperature control with a set point of 45ºC. (d)
Current and (e) resistance measurements were acquired in real time along with
injections of 2% mepivacaine were found to substantially temperature data. (f) The energy delivery rate was adjusted in real time to
achieve and maintain a stable temperature of 45ºC.

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Figure 8: Tumor response to ACE treatments. (a) Small (mean: 0.18cm3),
(b) medium (mean: 0.48 cm3), and (c) large (mean: 2.2 cm3) tumors were
treated with doses of 0.005s, 0.01s, and 0.02s, respectively. All groups resulted
in a mean reduction of tumor volume at the first (black) or second (red) visit.
Only a subset of patients returned for a second follow up. Some tumors,
marked with a *, were observed to increase in volume at the first follow up,
but had reduced in volume by the second visit.
reduce stimulation associated with treatments and this has
become standard practice in our ongoing clinical workflow. Via
analysis of owner provided photographs, tumors were observed
to reduce in size over the first 1-5 days following treatment.
Treated tissue typically formed an eschar overlaying a region of
healthy regenerative tissue and in some cases tissue
depigmentation occurred (Fig. 7c)
Active temperature control achieved stable tissue
temperatures across a diverse tumor population with an average
initial temperature of 31.1 ± 2.9 ºC and average peak
temperature of 42.2 ± 5.2 ºC. The average pulse delivery rate
was 31.7 ± 16.7 µs/s. Representative temperature, current, and
resistance profiles are shown in in Fig. 6 b-c.
There was significant variability in the initial resistance of
the tumors which measured 175 ±86 Ω across all tumors (62 –
306Ω). In all cases the resistance decreased throughout the
treatment by an average of 30% to 115±49Ω (47-205 Ω). This
corresponded to an increase in treatment current from an
average initial value of 14.6 ± 8.3A (3.3-31.4A). to an average
final value of 20.4 ± 9.2A (9.6 – 41.1A)
All treatment IETs administered (0.005, 0.01, and 0.02s)
were capable of achieving complete responses when applied to
appropriately sized tumors. At the last follow up visit tumors
were found to have reduced in volume by an average of 100%,
98%, and 100% for the 0.005s (Fig. 8a), 0.01 (Fig. 8b), and
0.02s (Fig. 8c) IET groups, respectively.
IV. DISCUSSION
This study validated the use of ACE with a coaxial electrode
via numerical simulations and ex vivo experimentation. In both
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7
approaches, when using a standard constant delivery rate
method we found that regions of the tissue were elevated from
room temperature to temperatures above 60ºC (Fig. 4e and Fig.
5b); where thermal injury is nearly instantaneous [35-37]. This
provides evidence for the need of temperature control in IRE
treatments.
Thermal injury can be avoided at the expense of increased
treatment times by reducing the pulse delivery rate
(Supplemental Fig. 5a-e). There is therefore a clinical
challenge in balancing the potential for thermal damage and
treatment durations. There are risks associated with each, and
the potential for thermal injury to critical nerves and blood
vessels must be balanced against risk concomitant with
increased time under sedation. Complicating matters, Joule
heating is dependent on tissue conductivity which is
challenging to assess a priori and tissue conductivity has a
positive temperature dependence leading to a feed forward
thermal response. As observed in this study, initial tumor
impedance (a proxy for tissue conductivity) varied significantly
from 47 to 205Ω even though all tumors were of the same type
and the applicator geometry was fixed.
The use of thermal control algorithms helps to balance these
factors by automatically selecting the highest energy delivery
rate possible without exceeding a prescribed temperature limit.
The temperature control algorithm was able to achieve and
maintain stable target temperatures (Fig. 6f). This control was
achieved by increasing the delay between successive bipolar
waveforms (Fig. 1b). The temperature can be set in real time by
clinicians and can vary case-by-case or tumor-by tumor. For
example, near a major blood vessel clinicians may select a
temperature below 42ºC for a purely non-thermal treatment.
This is especially beneficial if surgical / procedural time is not
an issue. Alternatively, for tumors far from critical structures
clinicians may a select higher target temperature (e.g. 60-99ºC)
if some thermal damage to the core of the tumor is acceptable
or treatment times must remain short.
In general, ACE was well tolerated by the patients. No
adverse events were reported by clinicians or owners. In some
cases, muscle contraction was observed during treatment. There
was an observed relationship between the pulse parameters and
the observed muscle stimulation. As the delivery rate reduced
(due to increased temperature), there was a greater probability
of visible muscle contraction. This is in agreement with ex vivo
[31] and in vivo [38, 39] studies. In most cases, administration
of additional local anesthetic (2% mepivacaine, 15-50 mL SQ
total volume) into the surrounding tissues was sufficient to
alleviate local muscle activation. It was also noted that some
patients had muscle contraction in response to the initial 2000V
pulses, then the number and magnitude of contractions
decreased as the treatment progressed. In response to this
observation, we have modified our typical clinical practice.
Treatments are now started at 250V to condition the patient and
steadily increased to 2000V over 5-10 seconds before
beginning full treatment.
Prolonged electrical stimulation of muscle induces a state of
fatigue during which contractile force declines and the muscle
becomes resistant to further stimulation [40]. The mechanism
for this is generally considered to be due to depression of
contractile protein force generation due to the presence of
metabolites [41] and the alteration in sarcoplasmic reticulum
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calcium release resulting in reduced activation of contractile
proteins [40, 42]. The reduction in muscle contraction over time
observed in this study is likely due to this form of muscle
fatigue in response to continuous tetanic stimulation. Slowly
ramping from 250V may act to gradually fatigue a growing
region of tissue reducing the overall stimulus experienced when
the final voltage is achieved.
This study examined only a single waveform (2-5-2)
administered at a single voltage (2000V). However, three IETs
were selected (0.005s, 0.01s, and 0.02s) and assigned to small,
medium, and large tumors. This selection was informed by in
vitro[18, 43] and ex vivo[44] data showing that treatment zone
sizes increase with increasing IET and that IETs beyond 0.02s
yield diminishing returns. Tumor sizes varied significantly,
with the smallest and largest diameters being approximately
5mm and 32mm, respectively. All IETs achieved complete
responses, highlighting the need for appropriate treatment
planning to balance over/under treatment and overall treatment
times. In general, 0.02s IET treatments resulted in a treatment
zone which extended from the center needle to just beyond the
diameter of the outer ring. The bell-shaped treatment zone (Fig.
5a) extended into the tissue approximately 5mm beyond the tip
of the center needle. Lower IET resulted in smaller treatment
zones originating at the center pin, but not fully reaching the
diameter of the outer ring.
This study has several important limitations. Only a single
tumor type was investigated using a new applicator design with
a single waveform and voltage. There may be variability in the
lethal response between cell / tumor types for pulses on the
order of one microsecond [30, 45] and the voltage or IET may
need to be adjusted when treating different forms of cancer. The
geometry of the treatment zones, while not quantified directly,
appeared somewhat smaller than those predicted numerically
(Fig. 6 a-d) for 0.005s IET doses indicating that the lethal
electric field threshold for melanoma cells may be higher than
values derived from in vitro models. Care should be taken when
using in vitro data for treatment planning purposes and models
should be refined with clinical data to prevent over or under
treatment with this approach. A limited number of follow up
visits were conducted (1-2x) and microscopic margins are
unknown leading to some potential for disease recurrence.
Future clinical studies should consider expanding the number
of patients, diversity of tumor types, and duration of follow up
as well as exploring the application of ACE in deep seated
tumors.
V. CONCLUSION
This study demonstrates the safety and efficacy of ACE
treatments against equine melanoma. All treatments were
completed without complications and no adverse events were
reported. Significant reductions in tumor volume were achieved
for all parameters investigated with an overall 93% complete
response rate. ACE treatments successfully achieved and
maintained a target temperature in a complex spontaneous
tumor population. This protocol successfully balances the need
to minimize treatment times while avoiding deleterious thermal
damage in tissues. Future investigation in complex deep-seated
tumors is warranted.
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8
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