These animal model studies demonstrated the potential efficacy of IRE as a targeted ablation technique for the treatment of HCC. MR images showed a significant tumor size reduction within 15 days post-therapy and histology correlation studies showed a clear progression from poorly differentiated viable hepatoma tissue pre-therapy to extensive tumor necrosis and complete tumor regression in 9 out of 10 treated rats 7–15 days after treatment. Our study is the first to demonstrate the efficacy of IRE for targeted treatment of liver tumors in a transplanted rodent hepatoma model.
Relatively early post-IRE therapy (within one day post-treatment), we observed homogeneously necrotizing tissues within treated normal liver parenchyma with clear margins between the treated and untreated tissues. However, we observed somewhat different responses within N1-S1 tumor tissues; specifically, tumor tissues tended to exhibit heterogeneously necrotic characteristics at the early intervals (1–3 days) post-therapy with limited viable tumor trapped within the necrotic tissues. Eventually, all treated tumors progressed from these early interval stages of partial necrosis to essentially complete necrosis with fibrotic scar formations 7–15 days later. One potential explanation for these heterogeneous delays in cell death could be that some of the treated tissues were destroyed due to the alternative mechanisms of ischemia and associated hypoxia (due to entrapment within surrounding necrotic tissue) as opposed to the direct effect of irreversible electroporation. Additional studies will be required to rigorously investigate the mechanism of these observed temporally-dependent necrosis events associated with IRE ablation procedures. For tumor tissues, even though no significant changes were observed on H&E and CD34 staining one-day post-therapy (Group 4), we observed extensive caspase 3 activation which might indicate an alternative underlying cell death mechanism (i.e. tumor cell apoptosis initiation) in addition to solely cell membrane permeablization. Delayed interval results (15 days post-therapy) consistently demonstrated the longitudinal efficacy of this targeted IRE approach; MRI scans depicted significant lesion size reductions for each treated animal whereas significant tumor growth occurred for untreated animals. These imaging results were well correlated to delayed-interval histopathological results that showed no viable tumor tissue within the lesion along with inflammatory cell reaction, fibrotic scar formation, remnant vascular skeleton CD34 positive staining (depicting system of damaged blood vessel walls within the treated tissue region) and an absence of caspase 3 activation.
Our study specifically demonstrated the feasibility of using IRE as a therapeutic modality for the treatment of HCC. All treated tumors demonstrated significant size reductions within two weeks post-therapy and there were no adverse events (i.e. peritoneal bleeding, tumor seeding, liver failure or mortalities) observed for any of the eighteen treated animals. For the IRE protocol selected for our study, we used far fewer pulses than prior cutaneous tumor model studies (8 square wave pulses as opposed to 80 pulses at 0.3Hz) while continuing to achieve effective treatment response. Our study demonstrated the feasibility of using IRE to ablate HCC, however, further studies to optimize IRE parameters are certainly warranted.
The efficacy of conventional RFA approaches is often limited in larger tumors due to perfusion-mediated cooling which can limit thermally-induced coagulation necrosis (39
). The extent of the treated tissue volume can be difficult to control due to blood circulation with heat-sink effects leading to indistinct margins between treated and untreated tissues and/or under-treatment of the targeted tissues ((40
)). IRE results in a distinct margin between ablated and viable tissues at the position where the magnitude of the electrical field falls below a lethal dose threshold (23
). Importantly, IRE does not suffer from the ‘heat-sink’ effect that is commonly problematic for thermal ablation methods (21
). Additional potential advantages for IRE methods include tumor specific immunological reaction (41
), little impact upon the collagen network within treated tissues and the potential to abate tumor tissues near large vessels (42
). Finally, application of the electroporation pulses during IRE procedures requires <1s. This feature contrasts significantly with the time duration required for RFA methods that typically involve application of thermal energy for upwards of 8–20min/ablation to achieve sufficient temperatures for coagulative necrosis (43
). Recently, a commercially developed electroporation device received 510k approval from the FDA (NanoKnife™, AngioDynamics Inc.). Given the promising results of our current IRE ablation studies in the N1-S1 rat hepatoma model, future studies are warranted to further investigate the efficacy of such devices for targeted treatment of liver tumors as well as additional tumor etiologies that can be difficult to treat with conventional ablation methods.
One limitation of our study was the lack of intra-procedural imaging guidance to optimize the placement of IRE electrodes; sub-optimal electrode placement could conceivably have led to the incomplete response observed for one rat in Group 3. During previous studies, ultrasound (US) imaging methods were used for intra-procedural visualization of IRE ablation procedures (44
). In future HCC IRE studies, US, MRI, or CT techniques could be used to optimize placement of IRE electrodes to ensure that the targeted tumor mass is entirely contained within the anticipated IRE ablation zone. Functional imaging methods (dynamic contrast-enhanced CT/MRI and/or diffusion-weighted MRI) may prove useful for immediate or early detection of IRE treatment response.
For these initial studies we did not individually tailor the IRE protocol (voltage, electrode spacing) to produce an ablation zone specific to each individual tumor size. We simply used a single IRE protocol producing an ablation zone size that we anticipated would be sufficiently large to cover all tumors below one given size. For our studies, we did not experience any gross complications due to damage to adjacent liver parenchyma. However, we would anticipate that the use of much larger ablation zones would lead to decomposition and subsequent liver failure. An individualized, patient-specific approach could be important for clinical IRE applications given a desire to spare normal liver tissues to preserve function. As demonstrated during prior studies ((30
)), optimization of the IRE ablation volumes should be possible using pre-procedural FEM simulations. Further studies are warranted to rigorously investigate the potential to individually tailor the size of IRE ablation zones to ensure complete treatment of targeted tumors while sparing as much surrounding normal liver tissue as possible.
In conclusion, this pre-clinical study demonstrated the feasibility of using irreversible electroporation as a novel ablation modality for targeted treatment of hepatoma in the N1-S1 rat model. Follow-up MRI images demonstrated significant tumor size reductions and histology correlation studies demonstrated extensive tumor necrosis within 7–15 days post-therapy. IRE is a promising new approach for liver-directed treatment of HCC and may offer multiple potential benefits over conventional ablation methods.