AIM: To verify whether arterial-phase contrast-enhanced ultrasonography (CEUS) of tumor parenchymal tissue is useful for evaluation of anti-angiogenesis agents.

METHODS: Rabbits with liver tumor were subjected to CEUS, and images of the nodular maximal diameter in vascular phase were recorded. Image analysis was performed to plot the time intensity curve (TIC) at the tumor parenchyma, which set the diameter of the region of interest of intensity measurement. The TIC was calculated to obtain the time to peak intensity (TPI) and the magnitude of PI. Rabbits were randomly assigned to a treatment group with sorafenib and a control group. Two weeks later, the same ultrasound examination was repeated followed by pathological testing to assess the effect of sorafenib on the liver tumor.

RESULTS: In four rabbits in the treatment group, the rate of change of tumor size was decreased compared with that of the control (the rate 2.3 vs 7.9, P = 0.02). The TPI of the treatment group elongated significantly (the rate 3.1 vs 1.1, P = 0.07 for SonoVue, 2.0 vs 0.88, P = 0.09 for Sonazoid). The magnitude of PI showed no significant changes. In pathological examination, capillary diameters in the treatment group were significantly smaller than those in the control group (26.4 vs 42.8 μm, P = 0.013).

CONCLUSION: Analysis of the TIC in the arterial phase of tumor tissue could evaluate the efficacy of anti-angiogenesis drug treatment in liver tumor.

Hepatocellular carcinoma (HCC) is the sixth largest cause of neoplasms worldwide[1]. Although effective treatment for early-stage HCC exists in the form of surgery and locally destructive techniques such as radiofrequency ablation, there are few treatments for advanced HCC. Recent randomized control trials of the anti-angiogenesis drug sorafenib have shown effectiveness for the first time. Time to radiological progression and overall survival were prolonged in patients with advanced HCC who received sorafenib. However, some problems remain for anti-angiogenesis treatment. One of them is how to assess clinically whether the drugs are effective, because some tumors develop tolerance. Oncologists have to change the therapy as soon as they find that the liver tumor has become refractory to anti-angiogenesis therapy[2].

Enhanced computed tomography (CT) and magnetic resonance imaging (MRI) are generally used for this screening. They are convenient to assess the response of anti-angiogenesis therapy, because tumor size, necrotic lesions, vascularity and perfusion are evaluated at the same time. However, these modalities sometimes make incorrect assessments, because tumor perfusion is difficult to evaluate. Perfusion is more important than the size for anti-angiogenesis drug therapy[3].

Ultrasound examination can evaluate tumor size and necrotic lesions. Moreover, contrast-enhanced ultrasonography (CEUS) can evaluate tumor blood flow and perfusion more easily at the bedside than can CT and MRI with contrast agents[4]. Recently, CEUS has been used for differential diagnosis of liver tumor and to determine the degree of differentiation of HCC[5].

Previously, the usefulness of CEUS for the evaluation has been reported. However, a long time is needed for the ultrasound examination[6]. In response to this, we tried to simplify the evaluation method with CEUS. The purpose of this study was to probe whether the perfusion information from tumors at the arterial phase of CEUS is useful for monitoring the early-phase response of anti-angiogenesis treatment.

Two rabbits in the treatment group died of systemic metastasis of the implanted tumor. The other four rabbits were used for evaluation.

Tumor size was calculated by multiplying the longest diameter by the shortest. In the sorafenib group, the tumor grew more slowly than in the control group. Pre-treatment tumor size was a mean 107.5 mm² (range: 58.6-192.9 mm²), and post-treatment size was a mean 255.6 mm² (150.9-547.2 mm²) in the sorafenib group. In the control group, tumor size increased more rapidly. Baseline tumor size was a mean 45.7 mm² (range: 24.5-78.8 mm²) and that at 14 days was a mean 283.2 mm² (range: 210.3-436.6 mm²). Ratio (post-/pre-treatment) of the tumor size was a mean 2.3 (1.4-2.8) in the sorafenib group and 7.9 (3.1-14.2) in the control group (P = 0.02) (Figure 2, Table 1).

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Figure 2 Tumor size variation. Solid lines show the size variation of the sorafenib-treated group. Interrupted lines show the size variation of the control group. The control group showed greater tumor enlargement than the sorafenib group did.

There were no significant differences in variation of PI in the tumor. In SonoVue imaging, variation in PI in the sorafenib group was a mean 2.1 (range: 0.5-5.5) and that of the control group was a mean 1.3 (range: 0.9-1.7) (P = 0.84). In Sonazoid imaging, variation in PI in the sorafenib group was a mean 1.6 (range: 1.2-2.5) and that in the control group was a mean 1.2 (range: 1.0-1.4) (P = 0.28, , Table 2).

Variation in TPI in the tumor was prolonged in the sorafenib group. In contrast, variation in TPI in the tumor did not change or was shortened in the control group. In SonoVue imaging, variation in TPI in the sorafenib group was a mean 3.1 (range: 1.1-4.8) and that in the control group was a mean 1.1 (range: 0.56-1.4) (P = 0.07). In Sonazoid imaging, variation in TPI in the sorafenib group was a mean 2.0 (range: 1.0-3.2) and that in the control group was a mean 0.88 (range: 0.42-1.33) (P = 0.09, Figures 3 and 4, Table 2).

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Figure 3 Contrast-enhanced ultrasonography examination showed that time to peak intensity was delayed in the sorafenib-treated group.

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Figure 4 Time to peak intensity in the tumor was significantly prolonged in the sorafenib group with both SonoVue and Sonazoid imaging. Time to peak intensity (TPI) in the tumor did not change or was shortened in the control group.

Pathological examination showed peritoneal dissemination and multiple metastases outside the liver. Some of the rabbits also showed ascites. Tumors appeared round, yellow/white, and separate from the surrounding parenchyma, with large necrotic lesions inside.

In vascular measurement, tumors in the sorafenib group had smaller vessels than in the control group. In the sorafenib group, the diameter of the tumor vessels was a mean 26.4 μm (range: 23.9-26.7 μm) and that in the control group was a mean 42.8 μm (range: 34.2-50.1 μm) (P = 0.013, Figure 5).

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Figure 5 Pathological examination (HE stain, × 40). A: Sorafenib group: vessels were relatively small and not obvious; B: Control group: dilated vessels were shown in the parenchyma of the tumor.

VEGF immunostaining examination presented no significant difference between both groups.

In the present study, prolonged TPI in arterial phase CEUS was shown during treatment with anti-angiogenesis agent.

Lavisse et al[6] have reported that TPI of tumor was elongated and PI was decreased in an anti-angiogenesis treatment group compared with a control group. Tumors induce new vessels to obtain oxygen and nutrition for their growth, and total blood flow of tumors increases, so-called angiogenesis. According to the study of Wilhelm et al[9], the anti-angiogenesis agent sorafenib inhibits angiogenesis and reduces microvessel density. This should be the reason why PI is reduced and TPI is prolonged in the sorafenib group.

In our study, TPI of tumors in the sorafenib group was similarly prolonged significantly, but no significant difference was detected in PI. In a previous study, entire tumors were estimated as the ROI, but in the present study, part of the viable region of the tumor was estimated as the ROI to simplify the measurement procedure. The analysis software that we used could not cover the entire tumor for the ROI. That was why no significant difference was detected in PI in our study.

The fact that we used rabbits in this study, unlike many previous studies that used mice and rats, is considered to have influenced the difference. First, rabbit liver is sufficiently large to be scanned by ultrasonography and more appropriate than that of mice and rats. The liver of mice is so small that we have to perform ultrasonography under different conditions. We implanted Vx-2 tumor in rabbit liver to satisfy conditions, because intrahepatic tumor and ectopic tumor behave differently in terms of proliferation. The blood supply and the biochemical environment are different in the skin and liver. Furthermore, there are enzymes that metabolize drugs in the liver. As a result, tumor responses to drugs differ depending on its location[10]. Second, we wanted to examine our simplified method, so nodules were implanted in the left lobe of the rabbits, where fixing the scan plane is difficult because of the movement of the heart and lungs. If a significant difference is shown under difficult conditions by a given method, then that method can be performed under most conditions and considered to be useful. As mentioned above, the TPI was significantly prolonged with both ultrasound contrast agents.

Ultrasound examination has less reproducibility and integrity than CT and MRI, because it needs more expertise and stable scanning fields. Our simplified method needs less examination time and no fixing probe or expensive location system. If the tumor has a rich parenchymal vascular network due to increased angiogenic activity, dilation of arterioles in the parenchyma can occur, and increased blood perfusion per unit volume should accelerate onset of tumor enhancement from ultrasound contrast agent. However, accurate blood flow, speed, and flux are difficult to evaluate, and TPI can show them better than other modalities can. When the administrated anti-angiogenesis drug induces a reaction in tumor vessels, the arterioles are not dilated, which results in a different enhancement pattern in CEUS in the arterial phase. TPI is considered to represent flux per unit volume of a scanning lesion. In spite of a lack of samples, the results of this study probably support this hypothesis.

In this study, we compared the dynamics of enhanced signals by a single dose of contrast agent. We did not use more accurate methods, such as the replenish curve or attenuation curve methods, because of complications[11].

In pathological examination, we detected a reduction of tumor vessel density in some parts of the tumors. Sorafenib is an anti-angiogenesis agent that prevents formation of new vessels and affects pre-existing capillaries to cause morphological alterations. As a result, the number of tumor vessels and their area and volume are reduced[12]. This is one of the explanations for prolongation of TPI in the sorafenib group. We could not evaluate endothelial cells because there is no appropriate antibody for CD34 immunostaining in rabbits. No significant difference was observed in VEGF immunostaining between the sorafenib and control groups, which is consistent with inhibition of VEGF signaling by sorafenib at the level of VEGF receptors.

Most HCC occurs in Eastern and Southeastern Asia, Africa and Melanesia. About 70% of patients are not eligible for curative treatment and prognosis is poor[1]. Treatment with anti-angiogenesis drugs such as sorafenib will become widespread and effective assessment of treatment will be needed. Although efficacy of antitumor drugs has previously been evaluated mainly by variation in tumor size, it is complex and demanding to evaluate the size of tumors with necrotic lesions. Tumor markers do not correspond with efficacy of anti-angiogenesis drugs.

It is undeniable that a greater number of samples were required than we used. However, an adequate number of animals were used to allow for proper statistical analysis.

In conclusion, Analyzing the TIC of arterial phase CEUS in tumor parenchyma could be helpful for evaluation of the efficacy of anti-angiogenesis drug treatment of liver tumor.