Technology in Cancer Research and Treatment ISSN 1533-0346 Volume 6, Number 4, August 2007 ©Adenine Press (2007)
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Imaging Guided Percutaneous Irreversible Electroporation: Ultrasound and Immunohistological Correlation
Edward W. Lee, M.D., Ph.D. Christopher T. Loh, M.D. Stephen T. Kee, M.D.*
www.tcrt.org
Department of Radiology
Division of Interventional Radiology
Preliminary results of percutaneous irreversible electroporation (PIE) on swine liver as a novel non-thermal ablation are presented. The goal of this study was to evaluate the feasibility of using irreversible electroporation in more clinically applicable manner, a percutaneous method, and to investigate a possible role of apoptosis in PIE-induced cell death. We performed PIE on four swine livers under real-time ultrasound guidance. The lesions created by PIE were imaged with ultrasound and were correlated with histology data, including pro-apoptotic marker. A total of 11 lesions were created with a mean size of 16.8 cm3 in 8.4 ± 1.8 minutes. Real-time monitoring was performed and a correlation of (+) 2 ± 3.2 mm in measurement comparison between ultrasound and gross pathologic measurements was demonstrated. Complete hepatic cell death without structural destruction, unaffected by heat-sink effect, and with a sharp demarcation between the ablated zone and the non-ablated zone were observed. Immunohistological analysis confirmed complete apoptotic cell death by PIE on Von Kossa, BAX, and H&E staining. In summary, PIE can provide a novel and unique ablative method with real-time monitoring capability, ultra-short procedure time, non-thermal ablation, and well-controlled and focused apoptotic cell death.
University of California-Los Angeles David Geffen School of Medicine 10833 Le Conte Avenue, BL-423 Los Angeles, CA 90095-1721
Introduction Over the last 10-15 years, a number of alternative treatments for cancers have been developed. These include stereotactic radiation therapy, chemoembolization, and several percutaneous ablative techniques. In particular, percutaneous ablation using thermal ablative techniques, such as radiofrequency ablation (RFA), have emerged as novel treatment methods for non-resectable primary cancer and metastasis, as supported by numerous studies validating their efficacy and safety (1-8). However, RFA suffers from a number of limitations; constraints on the maximum size of lesions that can be created, problems with heat sink (dissipation of heat via adjacent vessels), the lack of real-time imaging capability, and poor understanding of the marginal effects of the treatment. There is also a high complication rate, mainly due to soft-tissue thermal injury (6-33%), but also includes infection/abscess, biliary injury, hemorrhage, injury to adjacent organs, and even death (5, 9-15). Research is ongoing to develop a feasible alternative to RFA that would alleviate many of these limitations. One such alterative, percutaneous irreversible electroporation (PIE) is described in this article. Electroporation is a technique that increases the permeability of cell membranes by changing the transmembrane potential and subsequently disrupting the lipid bilayer integrity to allow transportation of molecules across the cell membrane via nano-size pores. This process when used in a reversible fashion, has been
Corresponding Author: Stephen T. Kee, M.D Email:
[email protected] *
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Lee et al. Methods
used in medicine and research for drug or macromolecule delivery into cells (16-21). The use of irreversible electroporation (IE) has been introduced by Rubinsky’s group as a method to induce irreversible disruption of cell membrane integrity subsequently causing cell death (22-24). Previously, the idea of destruction of cell membrane integrity by irreversible electroporation has been applied and demonstrated to effectively exterminate microbial organisms (2527). The use of irreversible electroporation has also been studied in an open fashion with promising results on healthy tissue cells (23, 28). These studies have demonstrated several important points. Unlike established thermal ablation techniques, IE creates tissue death by changing the permeability of the cellular membrane without thermal energy. Therefore, it will not be affected by the “heat sink effect.” Edd et al. (23) observed that IE created a sharp boundary between the treated and untreated area in vivo. This would suggest that PIE will have the ability to sharply delineate the treatment area from the non-treated. In addition, IE can effectively create tissue death in micro- to millisecond ranges of treatment time compared to conventional ablation techniques, which require at least 30 minutes to hours. With non-thermal cell death and a markedly decreased treatment time, IE provides a potential new ablation method that can be operated in a well-controlled and focused manner under image monitoring (such as ultrasound). Additionally, it may be possible to treat a considerably larger lesion with shorter treatment times than available with current techniques. It may also prove that it is possible to reduce the complications associated with conventional ablation while utilizing the advantages of percutaneous methods.
Animal Care Four female Yorkshire pigs at a weight of 30-60 kg were obtained and maintained by the Division of Laboratory Animal Medicine at the University of California, Los Angeles. All animals received appropriate humane care from properly trained professional staff in compliance with both the Principals of Laboratory Animal Care and the Guide for the Care and Use of Laboratory Animals approved by the Animal Care and Use Committees of UC Regents and in accordance with NIH guidelines. Irreversible Electroporation of Pig Four Yorkshire pigs weighing an average of 35 kg were subjected to general anesthesia: induction was performed using an intramuscular injection of 150 mg of ketamine hydrochloride (Animal Health, Fort Dodge, IA) and 150 mg of xylazine (Bachem, Torrance, CA). The animals then underwent intubation and 0.5-1.5% inhaled halothane (Halocarbon Laboratories, River Edge, NJ) was administered at 5 L/min. The pigs were placed in the supine position after successful anesthesia. The right upper quadrant and epigastrium were shaved and sterilized in the usual fashion. Using ultrasound guidance, we chose sites in all hepatic lobes for ablation. A total of 11 lesions were created: eight lesions with dualprobe system and three with a single-probe bipolar system. Seven lesions were created in the right lobe and four lesions were created in the left lobe.
In our study, we evaluated the applicability of IE in a more clinically practical setting: percutaneous irreversible electroporation (PIE). We present a case series of successful PIE in four swine liver. We also validate the mechanism of irreversible electroporation-induced cell death by using apoptosis specific immunohistological analysis.
For the dual-probe system (Appendix 1), two 18 gauge electrodes (AngioDynamics, Queensbury, NY) made of sintered Ag/AgCl were used for irreversible electroporation. Both electrodes were advanced into the liver parenchyma under ultrasound guidance. A distance of 1.5, 2, 2.5, or 3 cm apart, as summarized in Table I, between the two needles was testTable I
Animal ID
M/F
Wt
Lesions
Probe
1
F
76
2
F
78
3
F
103
4
F
105
Lesion 1 Lesion 2 Lesion 3 Lesion 4 Lesion 5 Lesion 6 Lesion 7 Lesion 8 Lesion 9 Lesion 10 Lesion 11
Bipolar Dual- Probe Dual- Probe Bipolar Dual- Probe Dual-Probe Dual-Probe Dual-Probe Bipolar Dual-Probe Dual-Probe
Probe Distance (cm)
Technology in Cancer Research & Treatment, Volume 6, Number 4, August 2007
n/a 2 3 n/a 1.5 2.5 3 2 n/a 1.5 2
Voltage (kV) 2250 2000 3000 2750 2500 3000 3000 3000 2750 2500 2500
Location Right Lobe Left Lobe Right Lobe Right Lobe Left Lobe Right Lobe Right Lobe Left Lobe Right Lobe Right Lobe Left Lobe
Percutaneous Irreversible Electroporation (PIE) on Swine Liver ed, using a spacer to keep the needles parallel, to compare an actual ablation zone to previously described mathematical models (22). After measuring the frequency-dependent impedance between the electrodes for 1 min, 90 pulses of 2,000 - 3,000 V were applied with a pulse generator (AngioDynamics, Queensbury, NY) across the gap between the electrodes for 100 microseconds (0.1 msec) per each ablation. For single bipolar needle ablation, one 16 gauge electrode (AngioDynamics, Queensbury, NY) was used. The placement of the electrode was performed under ultrasound guidance. Ablation using the same parameters as dual-probe system was performed as described above. A total procedure time for both the dual-probe system and the single bipolar system was summated from the time required to place the probes to the target area, the time required for 90 pulses of ablation with generator recharging time, until the time of removal of the probes. During electroporation, the ablation zone was continuously monitored using ultrasound. Upon completion of the ablation, the electrodes were withdrawn and hemostasis was achieved with manual compression. The ablation zone was again visualized using ultrasound, and the size of ablation zone was measured. The animals were treated with antibiotics and analgesics until sacrificed. Tissue Collection and Immunohistochemistry Twenty-four hours after electroporation, pigs were heparinized with 5,000 units of heparin and then euthanized with an overdose of pentobarbital sodium and phenytoin sodium (Schering-Plough Animal Health, Kenilworth, NJ). The liver was harvested and sectioned in 2-5 mm thickness along the course of the ablation electrode. Gross lesions were photographed (Figure 1A-1C). The sections were fixed in 10% formalin and preserved in 4 °C until further processing by our pathologists. Each section was then stained with Hematoxylin and Eosin (H&E) for histomorphologic analysis, Von Kossa stain for calcium deposition, and BCL-2 oncoprotein for analysis of apoptotic cell death. Data Collection/Analysis and Statistical Analysis SPSS v13 (SPSS Inc., Chicago, IL) and SigmaStat (SPSS Inc., Chicago, IL) were used for all data and statistical analysis. Specifically, one way repeated measure ANOVA, posthoc t-test (P
