Ultrasound-guided percutaneous laser and ethanol ablation of rabbit VX2 liver tumors

Abstract

Background

Only a small percentage of patients with hepatocellular carcinoma (HCC) may benefit out of surgical resection. Thus, lots of these patients are in need of local control, such as percutaneous ethanol injection (PEI), percutaneous laser ablation (PLA), or radiofrequency thermal ablation (RF).

Purpose

To investigate the effects of ultrasound-guided PLA combined with PEI on rabbit VX2 liver tumors, using conventional gray-scale ultrasonography (US), color/power Doppler (CD/PD)US, contrast-enhanced (CE) US, and histologic examination.

Material and Methods

VX2 tumors were implanted in the livers of 80 rabbits. Fourteen days after implantation, animals were randomly separated into four groups of 20 rabbits. Treatment of the four groups was with: (i) PLA; (ii) PEI; (iii) combined therapy of PLA immediately followed by PEI; and (iv) combined therapy of PEI immediately followed by PLA. Conventional gray-scale US, CD US, PD US, and CE US were performed before and after ablation. The effects on ablated areas were assessed by histologic examination.

Results

Conventional gray-scale US showed a clear boundary around the ablated area in groups 1, 3, and 4. An isoechoic treated region with an irregular boundary was seen in group 2. On CE US, coagulated areas demonstrated a perfusion defect. Both conventional gray-scale US and CE US showed that the ablated volume in group 4 was larger than that in groups 1, 2, and 3. CD US and PD US demonstrated residual tumor in the periphery of ablated areas in groups 1 and 2, but not in groups 3 and 4. CE US demonstrated no residual tumor in group 4, unlike in groups 1, 2, and 3. Examination of treated tumors demonstrated necrosis in the ablated zones and increasing surrounding fibrous bands in the four treatment groups. Residual viable tissue in group 4 was less than that in groups 1, 2, and 3.

Conclusion

Combined therapy of PEI immediately followed by PLA can coagulate significantly larger volumes of tumor and reduce residual tumor.

Keywords

Hepatocellular carcinoma percutaneous laser ablation animals percutaneous ethanol injection ultrasonography

Hepatocellular carcinoma (HCC) is a common malignancy, with more than 120,000 new cases per year in China. Regional ablative methods are an attractive treatment option since there is a minimal resulting damage to the uninvolved liver parenchyma (1). Ablative methods include radiofrequency ablation (RF), microwave coagulation therapy (MCT), percutaneous laser ablation (PLA), and percutaneous ethanol injection (PEI) (2 –4). PEI has been long used as a method of treating small HCC (5 –7).

PLA is a technique for local tumor destruction within solid organs that can be performed using various imaging methods. Optical fibers deliver high-energy laser radiation to the target tumor lesion. Neodymium: Yttrium Aluminum Garnet (Nd:YAG) lasers, with a wavelength of 1064 nm, are used for PLA because penetration of light is optimal in the near-infrared spectrum. Light scatters within the tumor, generates heat, and coagulates the tumor. The ablated area is predictable. PLA has proven to be an effective modality in treating HCC. Clinical PLA trials have shown promising efficacy in the treatment of small HCC (8 –10). However, similar to other non-operative approaches, PLA could not destroy tumors > 3 cm in diameter (11, 12). An important task in current medical research is to expand the necrosis volume in coagulative therapy. The use of PLA combined with PEI to treat malignant tumors has not been evaluated. We hypothesized that different mechanisms of action would increase the volume of tumor necrosis.

The purpose of this study was to investigate the effects of ultrasound-guided percutaneous laser ablation (PLA) combined with ethanol injection (PEI) in treating highly aggressive VX2 tumors in a rabbit liver tumor model. Results of treatment were evaluated using conventional gray-scale ultrasonography (US), color/power Doppler (CD/PD) US, contrast-enhanced (CE) US, ultrasonography (US), and histologic examination.

Material and Methods

Animals

This study was conducted at the laboratory in the Department of Ultrasound of Xijing Hospital. On the basis of computer-generated random numbers, 80 animals were assigned to four groups. Approval for this study was granted by the Animal Ethics Committee of Fourth Military Medical University, Shaanxi, China. All animals were cared for in accordance with Chinese legislation guidelines and the international guidelines on protection, care, and handling of laboratory animals.

The tumor was maintained in a rabbit with VX2 tumor implanted in the thigh. The tumor and rabbit were obtained from the laboratory in Department of Hepatobiliary Surgery in Xijing Hospital. Eighty New Zealand White rabbits (Shaanxi, China) were obtained as recipient animals. Rabbits weighing 2500–3000 g were purchased from Laboratory Animal Center of Fourth Military Medical University.

VX2 liver tumor implantation

Under sterile conditions, viable tumor tissue was obtained from the tumor-bearing rabbit and cut into 1 mm³ fragments. The minced tumor tissue was placed in 4°C Hanks solution. The 80 recipient animals were anesthetized using 3% pentobarbital solution (1 mL/kg) intravenous ear injection. The abdomen was shaved and prepared using povi-dine iodine.

A 5–13 MHz linear LA523 probe (My-lab90, Esaote, Genoa, Italy) was used for ultrasound-guided VX2 tumor implantation. The tumor was implanted in liver tissue at least 2 cm in thickness, which was not adjacent to any large vessels visualized using conventional US. During the implantation procedure, an 18-gauge puncture needle consisting of a cannula and a core was employed. Two to three fragments of tumor tissue were placed into the lumen of the cannula, followed by one small piece of gelatin foam. Under ultrasound guidance, the needle was inserted into the liver. The tumor tissue and gelatin foam were injected under ultrasound imaging. The puncture needle was removed after implantation and pressure applied to the abdomen for about 2 min. The selection of the tumor location and the implantation procedure were performed by one radiologist (XJZ), assisted by two other radiologists (QL and XM).

Ablation procedure

An EchoLaser (Esaote, Florence, Italy) was used for tumor ablation. EchoLaser is an innovative integration of the ultrasound imaging device, My-lab90, and four independent laser devices. A fiber test system allows the operator to verify before treatment the energy at the distal tip of the fiber. The My-lab90 ultrasound imaging system can visualize the direction of the needle on the ultrasound screen and consequently, the fiber inside the tissue. The guidance system is designed with different angles of inclination to allow different treatment depths. Guide lines on the ultrasound screen facilitate optimal needle placement for treatment.

Fourteen days after implantation of VX2 tumors, rabbits were randomly divided into four groups of 20 each. Rabbits were fasted for 24 h prior to ablation. The 80 recipient animals were anesthetized and prepped as previously described.

Conventional gray-scale US, CD US, and PD US were performed using the My-lab90 ultrasound imaging system with a 5-13 MHz linear LA523 probe before ablation. CE US was performed using the My-lab90 ultrasound imaging system with a 4-9 MHz linear LA522 probe. CE US was performed using a 0.2 mL dose intravenous ear injection of SonoVue (Bracco, Milan, Italy) was injected in bolus via ear vein, followed by a 2 mL 9% normal saline flush. Image acquisition was performed by two experienced radiologists (GBH and XDZ) who did not attend the tumor implantation. Tumors were measured in the three largest perpendicular diameters (length [L, cm], width [W, cm], and depth [D, cm]) using both conventional gray-scale US and CE US. The volume was calculated according to the formula: V = π/6 × L × W × D (cm³).

Group 1 tumors were treated with PLA

PLA was performed using real-time ultrasound guidance with a 5-13 MHz probe. The energy output was 1800 J with an output power of 5.0 W. A 300-μm plane-cut optic fiber was inserted through a 21G Chiba needle, positioning the fiber 5 mm past the tip of the needle. The optic fiber was connected with a continuous-wave Nd: YAG laser operating at 1.064 nm using an optical beam-splitting device. One needle was placed manually along the longitudinal axis of the tumor. Total illumination time (5 min) was automatically recorded by the laser equipment.

Group 2 tumors were treated with PEI

A 21G needle was placed in the tumor using US guidance; 99% ethanol was slowly injected into the liver tumor. The entire tumor was injected with ethanol. The volume of ethanol injected was approximately equal to the volume of the tumor. The injection time was about 2 min.

Group 3 tumors were treated with PLA followed by ethanol injection

PLA was performed similar to group 1. After PLA ablation, PEI was performed similar to group 2.

Group 4 tumors were treated with ethanol injection followed by PLA

PEI was performed similar to group 2. After PEI ablation was completed, PLA was performed similar to group 1.

The ablation procedure was performed by one radiologist (XJZ), assisted by two other radiologists (QL and XM).

Images evaluation

Conventional gray-scale US, CD US, PD US, and CE US were performed 3 days after ablation. Image acquisition was performed by either of two experienced radiologists (GBH and XDZ) who did not attend the ablation. Conventional gray-scale US and CE US were used to measure the three largest perpendicular ablation diameters. Cine loop records for CE US and still images for conventional US stored on the hard disk were subsequently reviewed by radiologists (MY and ZHH) blinded to the treatment information. A consensus regarding necrosis and residual tumor was achieved.

Statistical analysis

Data were presented as mean ± standard deviation. SPSS 11.0 software (SPSS Inc, Chicago, IL, USA) was used to evaluate statistical differences. Comparison of the depth, length, width, and volume of tumors and coagulative areas was made using variance analysis. P < 0.05 was considered statistically significant.

Results

Conventional gray-scale US

Ascites was detected in four rabbits and abdominal wall metastases were found in four rabbits. The VX2 tumors were unevenly hypoechoic or isoechoic, and had no sharp boundary from surrounding normal liver parenchyma (Fig. 1a). Anechoic areas of tissue necrosis were found in the center of 10 tumors. The ablated region in groups 1, 3, and 4 had a clear boundary. There was a central core of anechoic vaporization, hyperechoic carbonization, and a surrounding hypoechoic area. The irregular ablation region was isoechoic in group 2.

Fig. 1

Images on conventional gray-scale US before and after ablation in group 4. (a) Before ablation VX2 tumors were unevenly isoechoic and had poorly defined margins, (b) ring-like vessels were found in the periphery of the tumor. (c) After ablation, a clear boundary was seen between a central core of anechoic vaporization and hyperechoic carbonization, and the surrounding hypoechoic areas. No residual vessels were visualized within the ablated area. Arrowhead indicates the margin of the ablated area

The length, depth, width, and volume of tumors and ablated regions are shown in Fig. 2a and Table 1. There was no significant group difference in VX2 tumor length, width, depth, or volume (P > 0.05). The ablated length, width, depth, and volume in group 4 was larger than that of groups 1, 2, and 3 (P < 0.05). The ablated length, width, depth, and volume in groups 3 and 4 were larger than that of their tumor (P < 0.05).

Table 1

Summary of tumor volume and ablated areas, measured by conventional gray-scale US and CE US (n = 20)

	Group 1	Group 2	Group 3	Group 4
Tumor volume (cm³)
Conventional gray-scale US	0.83 ± 0.45	0.82 ± 0.50	0.84 ± 0.41	0.87 ± 0.41
CE US	0.82 ± 0.48	0.81 ± 0.45	0.82 ± 0.42	0.84 ± 0.43
Volume of ablated areas (cm³)
Conventional gray-scale US	0.83 ± 0.39^*	0.83 ± 0.58^†	1.82 ± 0.47^‡	2.71 ± 1.21
CE US	0.92 ± 0.45^*	0.93 ± 0.61^†	2.18 ± 0.63^‡	3.59 ± 1.44

All the data were presented as mean ± standard deviation

vs. Group 4 (P < 0.05)

†

vs. Group 4 (P< 0.05)

‡

vs. Group 4 (P < 0.05)

Fig. 2

Comparison of depth, length, and width of tumors and ablated areas (a) using conventional gray-scale US and (b) CE US. (a, b) Compared with group 4, groups 1, 2, and 3 had significantly less depth, length, and width of ablated areas (P < 0.05). There was no group difference in VX2 tumor size before ablation (P > 0.05). In groups 3 and 4, the depth, length, and width of tumors and ablated areas were significantly different (P < 0.05)

Conventional CD US and PD US

CD US showed blood flow in 60 of 80 tumors, while PD US demonstrated blood flow in 70 tumors. Distinct ring like vessels were seen in the periphery, along with bar-like or spot-like vessels in the central regions (Fig. 1B). Most of the ablated tumors in groups 1 and 2 had no blood flow using CD US and PD US. CD US demonstrated a few dot-like blood flow signals in the periphery of five lesions in group 1 and six lesions in group 2. PD US demonstrated dot-like blood flow signals in five lesions and bar-like blood flow signals in one tumor in group 1. Dot-like blood flow signals were seen in six lesions and bar-like blood flow signals in two lesions in group 2. No blood flow signals were seen using CD US (Fig. 1C) and PD US in 40 rabbits in groups 3 and 4.

CE US

Good tumor enhancement with tortuous intratumoral vessels was visualized in 80 tumors, 7–9 s after the injection of SonoVue. Rapid homogeneous or heterogeneous tumor enhancement was seen (Fig. 3A). Between 40 and 50 s after injection, there was decreased microbubble density within the tumor. A perfusion defect in the tumor was detected, The length, width, depth, and volume of the tumors, as measured by CE US, are shown in Fig. 2b and Table 1. There were no significant differences between the four groups (P > 0.05). After treatment, the ablated areas were anechoic, compared to the enhanced surrounding parenchyma (Fig. 3b). In groups 1, 2, and 3, 8, 10, and two tumors, respectively, demonstrated peripheral vessels (Fig. 4). In group 4, no vessels were detected. The length, width, depth, and volume of ablated areas in group 4 were significantly larger than in groups 1, 2, and 3 (P < 0.05) (Fig. 2b; Table 1). For groups 3 and 4, the length, width, depth, and volume of ablated areas were larger than those of their tumors before ablation (P < 0.05) (Fig. 2). In groups 3 and 4, the volume of ablated areas measured by CE US was larger than that using conventional gray-scale US (P < 0.05). No significant difference in tumor volume was found between measurements by conventional gray-scale US and that with CE (Table 1).

Fig. 3

Images on CE US before and after ablation in group 4. (a) The tumor before ablation; (b) perfusion after ablation. Arrowheads point to the margin of the tumor

Fig. 4

Conventional CD US and CE US after ablation in group 3. (a) The ablated region had no blood flow. (b) The ablated region was visualized using CE US. The arrow indicates residual tumor in the ablated region

Pathologic analysis

Pathological examination of treated tumors was performed after standard hematoxylin and eosin (HE) staining. Coagulative necrosis was visualized using light microscopy. Lysed cell membranes and nuclear fragmentation were seen in normal and tumor tissue. Three days after ablation, fibrous bands consisting of fibrocytes and capillary vessels were seen in the transient zone. From day 3 to day 14 after ablation, island like residual tumors with unorderly cells and nuclear atypia were found in 55% (11/20) of rabbits in group 1, 65% (13/20) of rabbits in group 2, 30% (6/20) of rabbits in group 3, and 10% (2/20) of rabbits in group 4. There was less residual viable tumor in group 4 than in groups 1, 2, and 3 (Fig. 5).

Fig. 5

Histopathological examination (hematoxylin-eosin staining) 3 days after ablation in group 4. (a) A central core of vaporized cells (V) was seen 3 days after ablation. Surrounding carbonization (C), coagulative necrosis (CN), untreated liver (L), and a surrounding zone of inflammatory response (I) was also seen. (Scale bar = 2.5 μm). (b) Fibrous band in the transient zone: ablated zone (A), transient zone (T), normal tissue (N). (Scale bar = 300 μm)

Discussion

Since the approval of RF ablation in the 1990s, percutaneous tumor ablation has been proposed as palliative care for patients with widespread metastatic disease (13). Ablative techniques include RF, MCT, cryotherapy, and PLA. The mechanism of tumor destruction in these different modalities is very similar. During the past decade these modalities have improved the treatment of small HCCs. Among the ablation treatments available for HCC, PLA has been less extensively studied. The wavelength and power of the Nd:YAG laser is well suited to medical applications. The tissue scatter of light and subsequent absorption create an ablation zone of approximately 1.6 cm, allowing multiple percutaneous punctures to reach a tumor ablation zone of approximately 4 cm. According to Pacella et al. (11), the rate of effectiveness was 89% for tumors < 3.0 cm, and 74% for tumors 3.0–4.0 cm. Late local tumor progression was observed in 18/122 patients (15%). In a recently published multicenter study, complete necrosis was achieved in 81.1% of 387 HCCs < 3 cm (14).

Single treatment PLA did not completely treat tumors > 2 cm in diameter. There are some reasons for this limitation. When using PLA, the abundant blood flow carried part of the energy away, especially in malignant tumors with increased vascularity (15).

Pacella et al. (16) reported complete ablation of large HCCs in 21 of 30 patients using laser ablation followed by transarterial chemoembolization (TACE). TACE has been shown to reduce systemic toxicity and increase local effects, thus improving the therapeutic results (17). However, the anticancer effect of TACE is offset by its adverse effect on liver function. Its treatment effect is limited by the lack of an ideal embolic agent, and in hypo-vascular or infiltrative tumors (18). The principle behind combining more than one technique is to take advantage of the merits of each of the modalities, to overcome their shortcomings, and reduce side-effects. Preservation of liver function and immunity are advantages of such minimally invasive techniques (19).

We observed the effects of PLA combined with PEI on highly aggressive rabbit VX2 liver tumors. Combined treatment (Group 4) gave significantly larger treatment length, width, and depth, volume of coagulation area, complete necrosis rate of tumors, and volume of ablated tissues (3–4 times) compared with PLA alone. This may occur because tumor tissue destroyed by ethanol could have increased thermal conduction (20). In addition, PEI is associated with alcohol absorption, which could sclerose blood vessels. Occlusion of small blood vessels may reduce heat emission and enhance the thermal effect of the laser (21). Kurokohchi et al. (22) used ethanol ablation immediately prior to RF in treating small HCC (< 5 cm). They found the combination to be beneficial in achieving larger areas of necrosis in fewer sessions compared with RF ablation alone.

Our study shows that all techniques are safe. No rabbits died due to the ablation procedure. No abdominal wall or peritoneal metastases were observed after ablation. ALT increased on the first day after ablation and decreased to normal by 14 days in all four groups.

When assessed by CE US, a clear treatment margin was observed because of the perfusion defects in necrosis areas. Volume of the ablated areas measured by CE US was larger than that measured by conventional gray-scale US. This suggests that some areas that appear to be normal liver were ablated. CD US and PD US were limited by the depth of the tumor and speed of blood flow. This may explain the lack of difference in residual vessels between groups 3 and 4. After injection of the micro-bubble contrast agent, CE US demonstrated the tumor blood flow, overcoming the shortage of conventional CD US and PD US. With its high sensitivity to blood flow, CE US visualized residual vessels not seen by CD US and PD US.

Conventional US is not considered a reliable modality for the evaluation of extent of necrosis. CE US, which was sensitive to the non-linear behavior of microbubbles, has been identified as an accurate modality in the assessment of ablative therapies (23, 24). Unfortunately, pathological examination has shown small remnants of viable carcinoma are still present. This may be because available imaging methods tend to overestimate the treatment volume.

There are some limitations to this study. PLA combined with PEI has proven itself to be a safe and effective local therapy, but is a short-term result. Randomized trials are required to establish long-term survival rate to validate this method of treatment. More studies are needed to establish the dose-volume effects of the laser and ethanol.

In conclusion, this report verifies that PLA preceded by intratumoral ethanol injection enlarged the ablated volume of tissue and reduced the amount of residual viable tumor. The combination of PLA and intratumoral ethanol injection is an effective treatment method worthy of further study.

Footnotes

Conflict of interest: None.

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