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ORIGINAL ARTICLE
Year : 2019  |  Volume : 15  |  Issue : 1  |  Page : 164-168

An experimental protocol for in situ colorectal liver metastases ablation by radiofrequency toward a standard procedure


1 University of Medicine and Pharmacy ‘Iuliu Hatieganu’ - Surgery Clinic No 1, Cluj-Napoca, Romania
2 University of Medicine and Pharmacy ‘Iuliu Hatieganu’ - Surgery Clinic No 1, Cluj-Napoca, Romania; Assistance Publique–Hôpitaux de Paris, Hôpitaux Universitaires Paris Seine-Saint-Denis, Hôpital Avicenne, Chirurgie Thoracique et Vasculaire, Université Paris 13, Sorbonne Paris Cité, UFR Santé, Médecine et Biologie Humaine, Bobigny, France

Date of Web Publication13-Mar-2019

Correspondence Address:
Dr. Radu Razvan Scurtu
First Surgical Clinic, Iuliu Hatieganu University of Medicine and Pharmacy, 3-5 Clinicilor Street, Cluj-Napoca 400006
Romania
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Source of Support: None, Conflict of Interest: None


DOI: 10.4103/jcrt.JCRT_344_17

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 > Abstract 


Background: Radiofrequency ablation (RF) is already a viable alternative to surgical resection for focal liver tumors treatment. The use of RF ablation in combination with surgery or chemotherapy and the large panel of RF tools need new experimental models to develop new opportunities for this kind of therapy.
Purpose: The purpose of this study was to identify the optimal RF parameters that will allow in situ colic cancer liver metastases destruction with minimal secondary effects.
Materials and Methods: The CC531s colic cancer tumor cells were used to induce liver metastases in 30 synergic Wag/Rij rats. When metastases reached at least 1 cm in diameter, RF generator RITA 1500X, and expandable tip RF probe Starburst SDE (Angiodynamics, USA) was used for the RF ablation. The animal survival rate and the RF-induced lesions have been studied, while only the generator delivered power has been modified (90W, 20W, and 10W, respectively).
Results: Survival was significantly low in the group with 90W-delivered power RF. Moreover, statistically significant differences were revealed between groups with high and low RF power, regarding the morphological changes of the liver parenchyma and the adjacent organs, without significant difference on the RF therapeutically effect.
Conclusions: In an experimental setting, an increased RF generator power induces important lesions of the abdominal organs with subsequently important mortality rate, without improving the RF therapeutic efficiency.

Keywords: Colon cancer, metastasis, radiofrequency ablation, rat model


How to cite this article:
Gonganau-Nitu D, Scurtu RR, Precup CG, Ciuce C. An experimental protocol for in situ colorectal liver metastases ablation by radiofrequency toward a standard procedure. J Can Res Ther 2019;15:164-8

How to cite this URL:
Gonganau-Nitu D, Scurtu RR, Precup CG, Ciuce C. An experimental protocol for in situ colorectal liver metastases ablation by radiofrequency toward a standard procedure. J Can Res Ther [serial online] 2019 [cited 2019 Oct 14];15:164-8. Available from: http://www.cancerjournal.net/text.asp?2019/15/1/164/244210




 > Introduction Top


Radiofrequency tumor ablation (RF) has gained considerable attention in the treatment of focal liver tumors, especially for colorectal metastases. It is a safe procedure (mortality 0.0%–1.4%) with low morbidity (2.2%–12.7%) and with good results in selected cases.[1],[2],[3],[4]

RF devices can use saline-enhanced or/and expandable tip electrodes to cause thermal destruction. RF can be used as a single treatment method or in combination with surgery and chemotherapy. Using electric currents with frequencies between 300 and 500 Hz, different volumes of tissue are heated, and proteins are denatured. The target temperature at the periphery of the tissue volume intended to be destroyed should be >62°C.[5]

Most of the studies are conducted using the internal cooled tip, while those with expandable tip electrodes are only a few. Our approach was to use manufacturer provided expandable tip electrodes, with the possibility of measuring the temperature at the outer borders of the induced lesions.

However, the problem in the existent literature related to the RF, about using expandable tip electrodes, is the occurrence of two procedures which have the same aim, are used on the same population, but differ dramatically in their assumptions about the level of the power that should be induced.[6],[7] This state of the art is highly problematic because if too low, the intensity of the current has no therapeutic effect, whereas if too high, it can cause extensive burns and organ damage.

Therefore, the present study aims to clarify the debate about the correct parameters for a safe RF procedure and to specify the parameters of an accurate protocol for the use of RF on liver tumors in an experimental setting in rats. To do this, we tested two contradictory protocols reported in the literature in an experimental design, and verified our hypothesis by a third one.


 > Materials and Methods Top


We used a classical RF procedure, described below, the only keying being the level of energy delivered to the liver tissue as follows: (Group 1) 90W versus (Group 3) 10W. A third group (Group 2)-20W was added in the study due to the preliminary important differences observed between the two initial groups. In fact, the initial results (presented further in the results chapter) proved to be far better after RF at 10W and thus we wanted to further test if the 10W value would be an optimal RF value for the liver metastases' in situ destruction. Therefore, we performed the same RF procedure, with a control group, with the same characteristics as Group 1 and 2, while only setting the RF generator power to 20W.

This study was conducted in vivo on 30 male Wag/Rij rats, with a mean weight of 235 g, divided into three equal groups, according to the RF power applied. Rats were anesthetized with a mix of Xylazine and Ketamine. After surgery, they were monitored and treated according to the university's good practice standards in laboratory works, based on the European Convention for the Protection of Vertebrate Animals used for Experimental and other Scientific Purposes and on the Directive no. 2010/63/EU of the European Parliament and of the Council of September 22, 2010 on the protection of animals used for scientific purposes.

Growth of tumors

The liver tumors were induced using colorectal adenocarcinoma (CC531s) synergic Wag/Rij male rats (Charles River, Germany) weighing 210–310 g. Tumor cells were cultured in RPMI 1640 with L-Glutamine 2 mM (Gibco, Grand Island, NY, USA), 10% heat-inactivated FCS, penicillin 100 U/ml, and streptomycin 0.1 mg/ml.

Tumor cells were suspended in EDTA and trypsin 0.25% in HBSS (Sigma, St Louis, MO, USA) and washed in PBS three times. We adjusted the PBS cell suspension to 106 viable cells/ml. For tumor induction, we used 0.1 ml from the cell suspension injected subcapsularly in the liver, in two sites as previously reported.[8]

The RF ablation was performed after 21 days from inoculation on tumors of 1.22 cm in average diameter (standard deviation = 0.24).

Radiofrequency ablation application

The RF ablation was conducted on rat liver tumors using a RF probe (Starburst™ SDE, AngioDynamics, USA) and a RF generator (RITA® Model 1500X RF, AngioDynamics, USA). The probe contains an expandable tip array with the possibility to monitor the temperature near the main tip and at the end of the expandable tips.

Conventionally, monopolar RF was applied using a saline-enhanced probe for 1 min, followed by a device controlled cool down process, with a target temperature set to 62°C. To complete the RF circuit, the animal was placed on a standardized grounding pad (AngioDynamics). Contact was ensured by shaving the animal's back and by the liberal use of electrolytic contact gel. The tip of the electrode was introduced in the middle of the tumor after surgical abdominal opening, and the side electrodes of the array were deployed 1 cm into the tumor. The saline was delivered in small volumes through the RF probe port to ensure a minimum delivery of 50% of proposed power. In the first group of rats, RF was delivered at a power of 90W (Group 1), while in the second group, the RF power was reduced to 20W (Group 2), and the third group underwent RF at a power set to 10W (Group 3). This Group 2 was later added to the initial experiment, due to the significant difference in post-RF survival between the first two groups (Group 1 and Group 3), survival directly correlated to the RF power. The purpose of this study was to identify a minimal threshold of an effective RF power with the lowest rate of side effects.

After performing the procedure, the rats were allowed for water and food ad libitum and closely monitored for clinical changes for 7 days. The clinical condition of the rats was scored daily by an independent observer, blinded to the treatment according to a score system [Table 1] published by te Velde et al.[9]
Table 1: Clinical appearance scoring system

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The surviving rats were euthanatized, the morphologic appearance of the internal organs was qualitatively assessed, and liver samples were collected and fixed in 4% buffered formaldehyde and embedded in paraffin. Histological investigation for the necrotized tumor and immunohistochemistry were further conducted to assess RF efficacy and absence of viable tumor cells.

Ethical considerations

The entire experimental procedure was approved by the Ethical Committee of the University of Medicine and Pharmacy “Iuliu Haţieganu” of Cluj-Napoca (code number 155).


 > Results Top


We analyzed the survival curves after applying the RF ablation using the Kaplan–Meier regression and the survival curves, comparing each group to another, and the results are presented in [Figure 1], [Figure 2], [Figure 3]. A statistically significant difference was found between groups survival rates according to the power of the RF delivered. In fact, all tests used (Log-Rank, Breslow, and Tarone-Ware) confirmed a significant better long-term survival for the rats to whom RF was delivered at reduced power, and in particularly for those with a RF power set to 10W.
Figure 1: Survival curves comparing Group 90W and Group 10 W

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Figure 2: Survival curves comparing Group 90W and Group 20 W

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Figure 3: Survival curves comparing Group 20W and Group 10 W

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The mean clinical appearance score (according to te Velde)[9] was 7.41 for the rats in Group 1, 5.37 in Group 2, and 5.1 in Group 3, respectively. Even though, there was no statistical significance difference between groups nor between Group 1 and Groups 2 and 3 (P = 0.207), the clinical appearance was obviously less favorable in the first group compared to the other two in which RF was applied with a reduced power.

After the rats have been euthanized, we qualitatively assessed the internal organs' morphological changes (presence of ascites, peritonitis, fistulae, burns of intestine, and diaphragm). The same assessment was carried out with all rats, including those who had died earlier. The appearance rate of distant morphological changes in the 90 watt group (Group 1) was notably higher than in the 10Wgroup (Group 3). In the first group, 74% of the surviving rats had ascites or intense fibrosis, while only 32% of the third groups' rats had similar lesions.

The inspection of the qualitative indicators showed also that the 10W group had better morphological changes of the internal organs. However, the differences between the 90W group and the 20Wgroup, as well as between the 10Wgroup and the 20W group were not statistically significant (Log-Rank P = 0.061, Breslow P = 0.137, and Tarone-–Ware P = 0.094 for the first comparison, and Log-Rank P = 0.274, Breslow P = 0.187, and Tarone-–Ware P = 0.224 for the second).

The histological studies (Hematoxylin-Eosin coloration) revealed the absence of viable tumor cells, especially on the outer RF zone. Histological findings showed a clear demarcation between RF lesion and normal liver tissue with no signs of remnant tumor in all groups. The only difference between groups was encountered during the RF application, when a longer time was needed to complete the procedure with a reduced power of the generator.


 > Discussions Top


Nowadays, radiofrequency became the most widely used method for in situ destruction of CRC liver metastases, but the local recurrence rate remains very high, independently of the type of generator or RF electrode used during this kind of procedure.[10] The information about the experimental RF liver tumor ablation in rats using RITA generator is scarce in the literature, although a continuously growing number of patients has been reported to be treated by RF ablation using this device. Local recurrence after RF might be explained by an incomplete destruction of the tumor due to an inadequate tissue heating, especially at the lesions edges. The temperature developed in the tumor depends directly on the power produced by the RF generator. Previous studies in both human and animal tissue have shown that RF works optimally when tissue temperatures are >50°C but <110°C. At temperatures >110°C, cavitation and charring severely limit thermal coagulative necrosis.[11]

Wahba et al. used the RITA system to appreciate differences between in vitro and in vivo experimental models on pigs, focusing on the energy delivered to the tissue, and the volume of ablation. They found that in in vivo experiment the larger amount of energy had to be delivered to obtain the same effect on the tissue as seen in the in vitro experiment. Consecutively, all the induced necrosis in living animals largely overlapped the tumor, and in all cases, the liver surface was involved.[12]

In this study, we aimed to assess in an experimental design the level of the RF power needed to induce irreversible changes in liver tumors, without harming the adjacent tissues and organs. In one group of animals, RF was done according to the manufacturer recommended parameters, but despite a well-controlled temperature inside the liver the survival in this group was very poor, with most of the deaths occurring in the first 48 h after the procedure. De Heer et al. also suggested that RF ablation for liver tumors in rats may be done using the standard settings of the RITA generator, as recommended by the manufacturer.[6] However, they reported a high incidence of intra-abdominal abscess formation. This might be explained by microscopically RF-induced lesions of the bowel. In this study, we found that in situ ablation of liver metastases in rats using standard settings for the RITA generator, induces large lesions to the normal rat liver, and also an important inflammatory reaction of the peritoneum and the adjacent organs (small and large bowel especially), and consecutively the survival rate was very poor. Since the majority of deaths occurred in the first 48 h after RF, we did not find the significant incidence of intra-abdominal abscess at the necropsy.

To limit the RF action only to the tumor, we reduced the generator power to 10W. The temperature recorded at the tumor periphery did not dropped below 62°C, and the animals' survival was significantly improved. Moreover, the histology examination of the destroyed liver identified a clear demarcation between normal and scared tissue but did not find any viable tumor cells after this procedure. Nijkamp et al. also performed RF in rats for colorectal liver metastases with a generator power set at 2W for 150s, but they used a bipolar electrode (Celon AG), with a saline-cooled diffuser tip. They also performed a RF on mice with the same power generator this time with a noncooled electrode. In both of these animal groups, there was no post-RF mortality reported.[13] Similar results were reported by Macatula et al. in a clinical study on hepatocellular carcinoma RF ablation. They concluded that low-power RF had a comparable clinical outcome compared to maximal power RF but induced fewer adverse effects.[14]

Since histology did not revealed viable tumor cells in the 1st days after RF, we tried to identify the presence of the vascular endothelial growth factor (VEGF), one of the factors involved in tumor recurrence. Even if the VEGF is not directly related to the tumor recurrence, it plays a pivotal role in the angiogenesis stimulation.[15] Angiogenesis has been long recognized as a prerequisite for metastatic growth, and was already incriminated for the surgery-induced metastatic recurrence.[16]

The staining for VEGF revealed a thin outer positive area for the vascular growth factor at the periphery of the RF lesions in all animals. However, we did not find any significant differences between groups regarding the VEGF level due to the fact that the blood samples were collected only from the rats that survived at the end of the study, and therefore, the sample size was not suitable for statistical analysis since very few of the rats which underwent RF with the generator power set at 90W survived the procedure. However, to the best of our knowledge, there are no data in the literature on the RF treatment for colorectal liver metastases.


 > Conclusions Top


An increased power delivered to the liver metastases during the RF tumor ablation in small animals may cause important damages to the surrounding normal liver tissue and to the adjacent organs. This results in a low survival rate, even when compared to natural evolution of the tumor. This study suggest that setting the RF generator to deliver a power of only 10W, to ensure the increase of the tissue temperature at 62°C is sufficient to acquire a complete lesion ablation with a very low risk of adverse effects. However, further tests are needed to investigate the tumor recurrence rate related to the power delivered to the tissue.

Financial support and sponsorship

This work was supported by the Executive Unit for Higher Education, Development and Innovation grant number ID 336.

Conflicts of interest

There are no conflicts of interest.



 
 > References Top

1.
Curley SA, Izzo F, Ellis LM, Nicolas Vauthey J, Vallone P. Radiofrequency ablation of hepatocellular cancer in 110 patients with cirrhosis. Ann Surg 2000;232:381-91.  Back to cited text no. 1
    
2.
de Baère T, Risse O, Kuoch V, Dromain C, Sengel C, Smayra T, et al. Adverse events during radiofrequency treatment of 582 hepatic tumors. AJR Am J Roentgenol 2003;181:695-700.  Back to cited text no. 2
    
3.
Livraghi T, Solbiati L, Meloni MF, Gazelle GS, Halpern EF, Goldberg SN, et al. Treatment of focal liver tumors with percutaneous radio-frequency ablation: Complications encountered in a multicenter study. Radiology 2003;226:441-51.  Back to cited text no. 3
    
4.
Mulier S, Mulier P, Ni Y, Miao Y, Dupas B, Marchal G, et al. Complications of radiofrequency coagulation of liver tumours. Br J Surg 2002;89:1206-22.  Back to cited text no. 4
    
5.
Gazelle GS, Goldberg SN, Solbiati L, Livraghi T. Tumor ablation with radio-frequency energy. Radiology 2000;217:633-46.  Back to cited text no. 5
    
6.
De Heer P, Sandel MH, Speetjens FM, Koudijs MM, Putter H, Ensink GN, et al. Cutaneous and intra-abdominal abscess formation in rats following radio frequency [corrected] ablation of liver tumors in combination with celecoxib treatment.In Vivo 2006;20:373-5.  Back to cited text no. 6
    
7.
Zardi EM, Picardi A, Borzomati D, Coppola R, Caricato M, Galeotti T, et al. Radiofrequency ablation of hepatic tissue: A new experimental animal model. Hepatogastroenterology 2004;51:570-4.  Back to cited text no. 7
    
8.
Precup CG, Gonganau-Nitu D, Scurtu RR, Dindelegan G, Biro A, Soritau O, et al. Assessement by laser doppler of the peripheral tumour perfusion after radiofrequency ablation for colorectal liver mestasis – Experimental study. Chirurgia (Bucur) 2010;105:71-6.  Back to cited text no. 8
    
9.
te Velde EA, Vogten JM, Gebbink MF, van Gorp JM, Voest EE, Borel Rinkes IH, et al. Enhanced antitumour efficacy by combining conventional chemotherapy with angiostatin or endostatin in a liver metastasis model. Br J Surg 2002;89:1302-9.  Back to cited text no. 9
    
10.
Curley SA. Radiofrequency ablation of malignant liver tumors. Ann Surg Oncol 2003;10:338-47.  Back to cited text no. 10
    
11.
Pereira PL. Actual role of radiofrequency ablation of liver metastases. Eur Radiol 2007;17:2062-70.  Back to cited text no. 11
    
12.
Wahba R, Bangard C, Kleinert R, Rösgen S, Fischer JH, Lackner KJ, et al. Electro-physiological parameters of hepatic radiofrequency ablation – A comparison of an in vitro versus an in vivo porcine liver model. Langenbecks Arch Surg 2009;394:503-9.  Back to cited text no. 12
    
13.
Nijkamp MW, van der Bilt JD, de Bruijn MT, Molenaar IQ, Voest EE, van Diest PJ, et al. Accelerated perinecrotic outgrowth of colorectal liver metastases following radiofrequency ablation is a hypoxia-driven phenomenon. Ann Surg 2009;249:814-23.  Back to cited text no. 13
    
14.
Macatula TC, Lin CC, Lin CJ, Chen WT, Lin SM. Radiofrequency ablation for hepatocellular carcinoma: Use of low vs. Maximal radiofrequency power. Br J Radiol 2012;85:e102-9.  Back to cited text no. 14
    
15.
Bruns CJ, Liu W, Davis DW, Shaheen RM, McConkey DJ, Wilson MR, et al. Vascular endothelial growth factor is an in vivo survival factor for tumor endothelium in a murine model of colorectal carcinoma liver metastases. Cancer 2000;89:488-99.  Back to cited text no. 15
    
16.
Abdalla EK, Vauthey JN, Ellis LM, Ellis V, Pollock R, Broglio KR, et al. Recurrence and outcomes following hepatic resection, radiofrequency ablation, and combined resection/ablation for colorectal liver metastases. Ann Surg 2004;239:818-25.  Back to cited text no. 16
    


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