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Non-thermal irreversible electroporation (NTIRE) for cancer therapy is a recent technique introduced by Davalos and Rubinsky1,2 in 2005. The emphasis was on the term, ‘non-thermal’, because membrane electroporation was used to kill cancer cells while protecting lethal heating effects to nearby critical tissue structures.
Lyn Oliver AM MSc PhD
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Introduction
Oliver (1990, 2003) previously showed that the total electrical energy, for a pulse of sufficient combined amplitude and duration, produces reversible electroporation up to an electric field strength of 2kV/cm. Irreversible electroporation occurs at 2 – 3kV/cm and critical electroporation cell breakdown (when the cell membrane structure totally fragments) is at >3kV/cm.
As discussed in The Study of Cell Membranes Treated by Electroporation, each of these electrical breakdown stages depend in a complicated way on the cell’s membrane structure, its protein content, its cytoskeleton support structure and its overall elastic properties (Figure 1). The cell’s membrane elasticity also has a dependency on the amplitude and duration of the pulse applied.
In the research work of Davalos and Rubinsky, they suggested that cancer cells could be killed by electroporation using a 0.5 – 1 kV/cm electric field strength. They described it as non-thermal irreversible electroporation (NTIRE) and said:
“We show through mathematical analysis that irreversible electroporation can ablate substantial volumes of tissue, comparable to those achieved with other ablation techniques, without causing any detrimental thermal effects and without the need of adjuvant drugs. This study suggests that irreversible electroporation may become an important and innovative tool in the armamentarium of surgeons treating cancer.”
The Aycock and Davalos review
Aycock and Davalos later published in 2019 their understanding of the non-thermal irreversible electroporation cancer kill processes (3).
With references that support their findings, they described the non-thermal irreversible electroporation (NTIRE) theory. For their postulation on how NTIRE can kill cancer cells, they said:
“Permeability increase, pore formation and induced biological effects depend on applied parameters, such as the amplitude, number, and length of pulses. ECT treatments typically use eight pulses with electric field magnitudes near 1 kV/cm. However, when a high number of pulses (60–100) of sufficient amplitude (0.5–1.0 kV/cm) are delivered, treated cells lose homeostatic equilibrium and die within minutes to hours.
The exact mechanisms through which IRE causes cell death have not been fully elucidated. But a number of possible pathways such as direct electroconformational denaturation of macromolecules, induced depletion of adenosine triphosphate (ATP), local vascular disruptions, or electrolytic pH changes could contribute to cell injury.
Furthermore, it has been shown that Na+/K+ pumps play a major role in restoring contractility after electroporation of skeletal muscle. This supports the theory that chemical imbalances mediate eventual cell death. Ultimately, high amounts of ATP are required to restore disrupted chemical concentration gradients; depending on the number and lifespan of the pores, this ATP demand can outweigh what can be generated by the cell, leading to high levels of intracellular Ca2+ and eventual cell death. Notably, the temporal scale in which IRE lesions appear seems to vary depending on the tissue type, suggesting a moiety of death mechanisms.“
The 1990 observations and postulates proposed by Oliver (4,5) are, to an extent, consistent with the biological processes described by Aycock and Davalos. However, Davalos described how cancer cells could be killed using 70 – 90 electric pulses of field strength at much less than 2 kV/cm and 100µS pulse duration.
Even though IRE has been thoroughly researched at bench level, the most appropriate electrical parameters to use for IRE and knowledge of how the cancer cells die after non-thermal ablation, is still not well understood.
Nevertheless, the use of the IRE surgical technique has been for some time been going through clinical trials for the treatment of prostate, pancreas and liver cancers.
Non-thermal electroporation (NTIRE)
To protect against heat damage of local nerve and surrounding normal tissue structures, Davalos and Rubinsky (3) used an electric field strength of less than 2kV/cm – which is less than the normal field strength for pore formation.
They copied the electrochemical reversible electroporation technique using <2kV/cm field strength and 1 – 10 x 100µS pulses as a non-thermal condition. Onyk and Rubinsky (2, 6)) reported successful NTIRE prostate treatment in dogs and later experiments when applying an electrical treatment to prostate cancer cells in-vitro. To adequately describe the technique, Davalos, Rubinsky and Onyk called the treatment:
‘non-thermal irreversible electroporation’ (NTIRE).
Multiple pulses: the missing parameter
To understand how multiple pulses might more efficiently cause irreparable pulse damage at such a low field strength of 0.5 to 1kV/cm, the 1990 data (Oliver (4)) provides a possible explanation. Figure 2. shows the change in relative light transmission measured after red blood cells, suspended between two electrodes (spaced 1.9mm apart) are electrically treated. The pulse amplitude across the electrodes was 440V (2200V/cm electric field strength). The number of pulses applied to each blood sample increased from 1 to 6 x 50µS duration and separated 1 second between exposures.
A possible explanation of membrane pore formation
At 540V (2.2kV/cm electric field strength), 50µS is the critical pulse duration for pore formation. When extra pulses are applied within 1 second, the cell membrane has insufficient time to reseal the previous pores produced and, as new pores are created by the next electric pulse, osmotic pressure differences exert extra stretch forces on the membrane.
The 30-minute relative light intensities (Figure 1.) show the fate of the RBCs when they are subjected to 1 to 6 x 50µS pulses, delivered over 0 to 6 seconds. The consecutive electric treatment of each pulse compounds the osmotic forces that the cell must endure to remain intact.
As more electric pulses are applied, more pores are formed and the cells become more permeable. The haemolytic results for 4, 5 and 6 pulse treatment indicate how the Na+ K+ pump has worked hard during the first minute to repair osmotic imbalance. But then remaining stresses cause the pores to grow and cells begin haemolysing to form ghost cells.
These results show that the number of electroporated cells produced is proportional to the number of pulses applied.
Discussion
In comparing the independent multiple pulse results previously reported by Oliver (1990), and compare them to Davalos’ (2005) multiple pulse results, the hypothesis for both these authors, are similar.
The Angiodynamics Nanoknife
Claudio Bertacchini et al (7, 2007) published the design of the first Nanoknife manufactured by Angiodynamics (Figure 3.). The US Food and Drug Agency approved the Nanoknife for human clinical trials in 2008.
Nanoknife NIRE clinical trials are now well established in many countries. The electroporation device is capable of providing a maximum voltage of 3kV with needle pairs 1500 volts/cm electric field strength. The patient must be maintained at electrically safe earth potential.
As previously mentioned, the patient receives 90 lots of 100µS pulses as the NTIRE protocol. This is to:
- limit excessive heat; and
- reduce the build-up of hydrolysis (causing increased conductivity and higher currents in the patient’s tissue site).
The pulses are applied in bunches with a very short time-lapse in between. The pulse voltage is reduced if the current approaches the maximum 20 amp current limit.
The needles are normally spaced 1 – 1.5cm apart. Much higher field strengths or more needles are used if the needles have to be spaced any further apart.
The implant needles are expensive and used only once. They are:
- specifically designed to satisfy electrical safety standards;
- specially sterilised for the procedure; and
- made of a suitable metal that can evenly discharge the very high voltage and current along the length of the electrode.
Treatment accuracy and uncertainties
Unfortunately, treatment of a well-defined tumour volume by surgical electroporation still requires further work to develop more accurate calculations of the electrical field dose. Research work is proceeding to overcome limitations in:
- calculations of the electric field distribution in the tissue between the electrodes;
- changes in tissue electrical conductance during treatment;
- the electrode shape; and
- production of tissue hydrolysis during the procedure – this adds uncertainties in the calculations.
Clinical trials
For information on current public clinical trials, click on:
Examples of NTIRE prostate, pancreas and liver cancer trials are listed below. The justification for these clinical trials is quite different for each of the cancer sites.
The NTIRE technique is only effective in directly treating the tumour mass with no metastatic spread. Otherwise, chemotherapy or radiotherapy must be used in conjunction for the treatment.
Prostate Cancer Treatment
In suitable prostate cancer cases, cure rates are over 90% for robotic surgery, radiotherapy and brachytherapy. The NTIRE treatment technique is a close copy of the prostate brachytherapy technique. The method of ultrasound image-guided needle insertion to implant the prostate is the same.
Patients should consult medical specialists who have received special training and participate in the recognised NTIRE trials. The method is limited to a single site and specific clinical protocols defining the patient’s prostate cancer assessment. It’s referred to as ‘focal IRE’ treatment.
Unlike robotic surgery, NTIRE does not cause erectile dysfunction or lack of bladder control side effects. NTIRE also only requires a short treatment time in surgery and recovery after the operation. The patient is admitted in the morning and discharged after surgery by late afternoon to return home.
If cure rates can be shown to be beyond 90% and the procedure becomes more cost-effective, then NTIRE will become a worthwhile option for clinically suitable patients to discuss with their specialist doctor.
Pancreas Cancer Treatment
The rationale for treating patients with pancreatic cancer, compared to prostate cancer, is different.
Whether surgery, chemotherapy, radiotherapy or a combination of these modes of treatment are used, the 5-year survival rate for pancreatic cancer is approximately 9%. Less than 20% of pancreatic cancer patients are suitable for complex surgery.
With such disappointing survival statistics, any method that can offer improvement in survival is worth considering. Improvements in earlier cancer detection of pancreatic cancer could make NTIRE treatment a suitable method of local treatment of the pancreas cancer mass. Complementary chemotherapy and/or radiotherapy would still be needed to eradicate any unknown spread of the disease.
Any increase in cure rates and length of survival would add substantial quality for these patients.
One of the most prolific publishers of pancreas cancer surgery is Dr Robert Martin, surgical oncologist at the James Brown Cancer Centre, University of Louisville School of Medicine, Louisville, USA. In his most recent 2015 paper (8), he reported the NIRE results of treating 200 locally advanced (stage III) pancreatic adenocarcinoma patients between 2010 – 14. The overall survival for patients Martin operated on during an 18 month period resulted in an average 10.7-month local progression-free survival. Their overall survival was 24.9 months.
Conclusions reached by Dr Martin were that “the addition of IRE to conventional chemotherapy and radiation therapy results in substantially prolonged survival compared with historical controls. These results suggest that ablative control of the primary tumour may prolong survival.“
Liver Cancer Treatment
Information on NIRE for metastatic liver cancer is not so well covered in the literature. The surgeon would need adequate imaging techniques to guide the needle insertion using key-hole surgery methods. The alternative stereotactic ablative radiotherapy technique is a less invasive technique but is still complex to plan and treat. The course of treatment is delivered over a 2 week period. Whereas surgical NIRE requires just one day in surgery and aftercare.
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References
1. R. V. Davalos et al., Tissue ablation with irreversible electroporation, Ann. Biomed. Eng., vol. 33, no. 2, pp. 223–231, Feb. 2005.
2. Rubinsky B, Onik G, Mikus P. Irreversible electroporation: a new ablation modality–clinical implications. Technol Cancer Res Treat. 2007;6:37–48.
3. Kenneth N. Aycock, BS, and Rafael V. Davalos, PhD Irreversible Electroporation: Background, Theory, and Review of Recent Developments in Clinical Oncology, BIOELECTRICITY, Volume 1, Number 4, 2019 Mary Ann Liebert, Inc. DOI: 10.1089/bioe.2019.0029
4. Lyn Oliver, Electromechanical Study of Blood Cells, PhD Thesis (UNSW), 1991.
5. Lyn D Oliver and Hans G. L. Coster, Electrical breakdown of human erythrocytes: a technique for the study of electro-haemolysis, Bioelectrochemistry, Volume 61, Issues 1–2, October 2003, Pages 9-19
6. Onik G, Rubinsky B. Irreversible electroporation: first patient experience focal therapy of prostate cancer. In: Rubinsky B, editor. Irreversible electroporation, series in biomedical engineering. Berlin: Chennai Springer-Verlag; 2010. pp. 235–247.
7. Claudio Bertacchini, Pier Mauro Margotti, Enrico Bergamini, Andrea Lodi, Mattia Ronchetti and Ruggero Cadossi, Electroporation System for Clinical Use, Technology in Cancer Research and Treatment, Volume 6, Number 4, August 2007.
6. Onik G, Rubinsky B. Irreversible electroporation: first patient experience focal therapy of prostate cancer. In: Rubinsky B, editor. Irreversible electroporation, series in biomedical engineering. Berlin: Chennai Springer-Verlag; 2010. pp. 235–247.
8. Martin RCG, Kwon D, Chalikonda S, et al. Treatment of 200 locally advanced (Stage III) pancreatic adenocarcinoma patients with irreversible electroporation safety and efficacy. Ann Surg 2015; 262:486–492.
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For references on non-thermal irreparable electroporation protocols and trials:
L D Oliver AM PhD, 3 May 2021
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