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Imaging Guided Percutaneous Irreversible Electroporation: Ultrasound and Immunohistological Correlation (p. 287-294)

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.

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, 2, 3, 4, 5, 6, 7, 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, 10, 11, 12, 13, 14, 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 used in medicine and research for drug or macromolecule delivery into cells (16, 17, 18, 19, 20, 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, 23, 24). Previously, the idea of destruction of cell membrane integrity by irreversible electroporation has been applied and demonstrated to effectively exterminate microbial organisms (25, 26, 27). 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.

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.

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 dual-probe 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.

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 tested, 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, post-hoc t-test (P<0.05) using Dunnett?s t-test were used for experimental data, histological data, and analysis.

Results

Data from a total of 11 ablations in four animals were included for gross and histological observation (Table I). Eight lesions were performed with the dual-probe system and three lesions were created with a single-probe bipolar system. An average total procedure time per ablation was 8.4 1.8 minutes. All four pigs tolerated the procedure well and survived for 24 hours.

The mean ablation size (maximum diameter) measured by ultrasound during the procedure was 38.9 10.5 mm, and measurement comparison demonstrated (+) 2.0 3.2 mm different in average from a gross measurement of the specimen. The measurement correlation in bipolar and dual-probe with 2 cm probe distance, demonstrated the most consistency in measurement comparison with 1 mm and 2 mm differences, respectively. These measurements were also comparable to the predicted mathematical area of ablation created by Rubinsky?s group (22). Figure (7A-F) demonstrates a measurable hypoechogenic lesion surrounding the hyperechoic material (probes) monitored by ultrasound during the ablation procedure. This lesion was measured in gross specimen (Figure 1A-1C). The largest lesion was created using the dual-probe system with a 3 cm distance and voltage of 3 kV, and measured L6.0 D3.1 W4.2 cm (approximate volume of 40.9 cm3). On average, L3.98 ( 1.42) D2.48 ( 0.59) W3.38 ( 0.64) cm (approximate volume of 17.5 cm3) size lesion was created with a two-probe system and L3.80 ( 0.42) D2.65 ( 0.64) W2.95 ( 0.78) cm (approximate volume of 15.6 cm3) size lesion was created with a single bipolar probe.



Figure 1: Gross pathologic images of sectioned specimens containing PIE lesions. Figure 1C demonstrates intact vessels and bile ducts in the area of PIE without structural destruction.
Figure 1(A-C) demonstrates grossly sectioned specimens showing areas of hemorrhagic change with relatively intact hepatic morphology. Each hepatic lobule was visible and intact throughout the ablation area. The vessels and bile ducts appeared intact without apparent structural destruction. The ablated area extended and juxtaposed to the vessels and bile ducts.

In Figure 2A-C, selected images of hematoxylin and eosin (H&E) stained sections showed areas of acute, extensive, and severe cell death. No viable cells were detected within the ablated area. However, the normal hepatic architecture was preserved. The ablation created three zones: (i) central focus with hemorrhage and severe necrosis, which corresponds to the peri-electrode region; (ii) mid zone of coagulative type cell death with evidence of mineralization; and (iii) peripheral zone of lobular cell death with less extensive mineralization. These areas of mineralization were more evident on Von Kossa stains (Figure 3A-3C) as these areas contained intracytoplasmic basophilic materials consistent with dystrophic calcification. Complete cell death was achieved throughout all three zones with a sharply demarcated margin separating the ablated and non-ablated neighboring cells (Figure 2A-2C). The sinusoids in the ablated areas showed congestion filled with neutrophils and eosinophils. The larger vessels and bile ducts in the ablated area appeared well-preserved structurally (Figure 1C) with some of the vessels, arteries and veins, demonstrating signs of vaculitis (multifocal loss of endothelial integrity, edema causing separation of the tunica muscularis layers, and neutrophilic infiltration). The bile ducts showed signs of acute choledochitis with peridochal edema. Some ablated areas showed focally extensive hepatocellular regeneration.

Gross examination and H&E stain histological analysis were complemented with Von Kossa staining, which demonstrates intra/extracellular calcium deposition, and BAX (BCL-2) staining, which detects intracellular pro-apoptotic oncoprotein. Figure 3B and 3C showed Von Kossa positive, dark-stained cells in the areas of ablation. The Kupffer cells appeared to uptake most of the Von Kossa staining in the ablated area. As noted above, all three zones of ablation contained mineralization but the mid zone appears to uptake more Von Kossa staining, which correlates with marked cell death within the irreversible electroporation area. Von Kossa staining was not observed in normal control hepatic tissue and outside of the ablated area (Figure 3A). Figure 4B and 4C showed cells within the ablated area with positive BAX staining. BAX staining was detected in all slides, predominantly in the cytoplasm. Marked increased BAX positive staining was observed in the some periportal areas. No BAX positive stain was found in normal tissue and outside of the ablation zone (Figure 4A) indicating a specificity of BAX staining in apoptotic cells. Again a clear demarcation between ablated area and juxtaposed non-ablated area was identified in both Von Kossa and BAX stainings.


Figure 2: Hematoxylin and eosin (H&E) staining of PIE lesion. The PIE ablated tissue has markedly increased sinusoidal congestion with neutrophils and eosinophils. A clear demarcation between PIE-ablated tissue (the right side of the image) and normal liver tissue (the left side of the image) is shown.

Figure 3: Von Kossa stain in normal liver tissue (A) and PIE-ablated tissue (B and C).


Figure 4: BCL-2 oncoprotein stain in normal liver tissue (A) and PIE-ablated tissue (B and C).



Figure 5: (A) An image of dual probe system for PIE; (B) Placement of dual probe system using spacer to acheive accurate distance between two probes under ultrasound guidance; (C) An image of appropriately placed dual probe system during PIE.


Figure 6: (A) An image of single bipolar probe system for PIE; (B) Placement of single bipolar probe system under ultrasound guidance; (C) An image of appropriately placed single bipolar probe system during PIE.



Figure 7: Real-time ultrasound images of PIE. (A) Two hyperechoic probe tips of dual probe system (white arrow); (B-F) PIE ablated hypoechoic areas with relatively minimal hyperechoic microbubbles in the close proximity of the probes. A spherical hypoechoic area is well delineated and the ultrasound measurements of the ablated area were well correlated with pathological measurement.

No complications related to hepatic ablation were noted peri- or post-procedure.

Discussion

Electroporation was introduced over 30 years ago and has been utilized in many medical applications including electrogenetherapy and electrochemotherapy as an alternative method to treat skin cancers (16, 17, 18, 19, 20, 21). Recently, a novel concept of utilizing ?electroporation? to effectively kill cancer cells in other locations has been proposed and studied by a number of investigators (22, 23, 24). A higher electric voltage leading to a larger potential gradient to create irreversible electroporation has been studied using in vitro and in vivo studies. The findings have been promising as irreversible electroporation has demonstrated effective cell death in normal tissue and cancer cell cultures (22, 23, 24). Based on these findings, we proposed to evaluate the possibility of translating irreversible electroporation into a more clinically practical form, percutaneous irreversible electroporation (PIE). In addition, the goal of this study was to determine possible pathophysiological pathways of cell death created by PIE: necrosis versus apoptosis.

Our limited study has demonstrated several important points regarding PIE. PIE is a unique and novel ablative technique that can effectively produce controlled cell death without some of the limitations of conventional thermal ablation. Although safety and effectiveness of conventional thermal ablation techniques have been shown in multiple studies (1, 2, 3, 4, 5, 6, 7, 8), several limitations and issues have surfaced. Effectiveness of thermal ablation has been hindered by the ?heat sink? phenomenon as perivascular tissues are not completely ablated and lead to incomplete ablation of tumor and tumor recurrence (29). In our study, we have demonstrated and verified that PIE can effectively cause complete tissue death even in cases where the ablation zone is juxtaposed to a large vessel. The histological analysis of multiple samples has shown complete hepatic cell death regardless of the presence of large blood vessels. An additional important finding was that the vessels within the ablated area were structurally intact. These findings suggest that PIE ablation is likely unaffected by ?heat sink? phenomenon, and, therefore, is capable of producing complete and reproducible areas of ablation of treated regions. However, in neoplastic tissue, the peri-tumoral environments differ significantly in vascularity (e.g., hypervascular) and vascular structure (e.g., leaky vessels) compared to normal tissue. Therefore, further evaluation of PIE in a tumor model is crucial to accurately evaluate the effects of PIE on vessels and the effect of vessels on PIE.

Another significant finding of this study was to demonstrate a significantly shorter procedure time compared to any existing thermal ablation technique. We were able to perform a full session of ablation creating mean size of L3.92 ( 1.12) D2.53 ( 0.54) W3.23 ( 0.64) cm (approximate volume of 16.8 cm3) lesion in 8.4 1.8 minutes. This validates prior findings and mathematical models, which demonstrated the feasibility of performing irreversible electroporation in micro- to millisecond intervals. This compares with conventional thermal ablation techniques that require more than 30-60 minutes to ablate a lesion of similar size (30, 31). A shorter procedure time using PIE should contribute to less complications, better patient experience, and improved outcome.

In our pathological analysis, we have demonstrated complete cell death in the area of ablation. We confirmed the previous findings of the in vivo study and mathematical models, identifying a clear demarcation zone between the ablated area and the non-ablated area. Regardless of the margin of ablation being interlobular or intralobular, we observed a sharp demarcation between the ablated areas from non-ablated areas. This is another advantage of PIE as this can create well-controlled and focused ablation of undesirable tissue without damaging bordering healthy tissue.

An interesting finding of our study compared to the previous irreversible electroporation studies, is the additional immunostaining analysis using antibody to BAX (BCL-2) oncoprotein. In our study, we demonstrated for the first time that cell death created by PIE has increased BAX staining compared to normal adjacent tissue. This indicates the role of apoptosis in cell death created by electroporation rather than coagulative thermal necrosis created by radiofrequency ablation. A recent study by Rubinsky et al. (28) demonstrated marked cellular tissue repair, which is seen 14 days post-irreversible electroporation, and our pathologic analysis showed hepatocellular regeneration as early as 24 hours after ablation. These findings also support a possible role of apoptosis in PIE-induced cell death where apoptotic cells are rapidly removed by immune cell derived phagocytosis and replaced by innate cellular regeneration. Necrosis-induced cell death does not get replaced by intrinsic cellular regeneration but rather by fibrosis and scarring of cellular and tissue remnant. Further molecular and biochemical investigations, such as TUNEL assay, Caspase-3 staining or gene array for apoptotic pathways, of this phenomenon will be important in understanding the pathophysiological pathways of PIE in more depth.

We also demonstrated that PIE can be performed as a ?real-time? image-guided intervention. We were able to visualize the effect of PIE under direct real-time ultrasound. Conventional thermal ablation techniques create hyperechoic micro-bubbles from thermally injured tissue in ultrasound images, which significantly hinders the opportunity for real-time monitoring. However, with PIE, a spherical hypoechoic area of ablation was detected during and immediately after PIE in ultrasound images. This hypoechogenicity is likely due to increased intra/extracellular water molecules after opening of transmembrane pores by the high voltage of electroporation. No lesion-obscuring hyperechoic gas is formed in PIE, which allows for direct and real-time monitoring. We have demonstrated that observation and measurements of the treated area acquired during real-time monitoring correlated well with pathological measurement of the lesion.

Our limited study has investigated a short term outcome of PIE in normal swine liver. Although no definite complication is noted peri- and post procedure, a long term outcome study will be useful to further develop this technique into clinical application. In addition, further studies investigating a comparison of PIE with RFA, analyzing systemic effects of PIE, effects of PIE on tumor, analyzing other imaging modalities such as MRI or CT on PIE, and molecular and biochemical properties of PIE on tissue will be important in understanding the efficacy, applicability, and toxicity of PIE in more depth.

With real-time monitoring capability and non-thermal, well-controlled cell death of the target tissue, percutaneous irreversible electroporation can provide a novel and unique ablative method; providing a solution to the problems of existing conventional thermal ablation techniques.



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TCRT August 2007

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Volume 6
No. 4 (p 255-360)
August 2007
ISSN 1533-0338

Edward W. Lee, M.D., Ph.D.
Christopher T. Loh, M.D.
Stephen T. Kee, M.D.*

Department of Radiology
Division of Interventional Radiology
University of California-Los Angeles
David Geffen School of Medicine
10833 Le Conte Avenue, BL-423
Los Angeles, CA 90095-1721
*SKee@mednet.ucla.edu

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