Evaluation of hepatocellular carcinoma using contrast-enhanced ultrasonography: correlation with microvessel morphology

Ji-Dong Xiao, Wen-Hui Zhu and Shou-Rong Shen

Changsha, China

Evaluation of hepatocellular carcinoma using contrast-enhanced ultrasonography: correlation with microvessel morphology

Ji-Dong Xiao, Wen-Hui Zhu and Shou-Rong Shen

Changsha, China

BACKGROUND:Contrast-enhanced ultrasonography (CEUS) is an important technique for depiction and assessment of tumor vascularity. This study aimed to explore the relationship between the morphological characteristics of tumor microvessels and enhancement patterns on CEUS in hepatocellular carcinoma (HCC).

METHODS:Eighty patients with HCC underwent CEUS using SonoVue before hepatectomy. Contrast-enhanced ultrasonographic enhancement patterns and quantitative parameters were recorded. The tumor tissue sections were immunostained with human CD34 monoclonal antibody. The patients were classified into a point-line type group (n=36) and a loop-strip type group (n=44) according to microvessel morphology. The microvascular density (MVD) in the different types of microvessels was calculated. The relationship between enhancement patterns of HCC lesions and morphological characteristics of tumor microvessels was analyzed.

RESULTS:The mean MVD in HCC was 22.4±3.5 per 0.2 mm2in the point-line group, and 19.6±6.7 per 0.2 mm2in the loop-strip group, and there was no significant difference between them (t=0.948,P=0.354). In the portal vein phase, hypoenhancement was significantly more frequent in HCC (χ2=4.789,P=0.029) in the loop-strip group (40/44, 90.9%) than in the point-line group (26/36, 72.2%). The time to hypoenhancement in the loop-strip group (mean 64.84±26.16 seconds) was shorter than that in the point-line group (mean 78.39±28.72 seconds) (t=2.247,P=0.022). The time to hypoenhancement was correlated with MVD in the loop-strip group (r=-0.648,P=0.001).CONCLUSIONS:The enhancement patterns on CEUS are related to tumor microvascular morphology, and the type of microvascular morphology influences CEUS characterization. CEUS, an important noninvasive imaging technique, is used to evaluate microvascular morphology and angiogenesis, providing valuable information for antiangiogenic therapy in HCC.

(Hepatobiliary Pancreat Dis Int 2010; 9: 605-610)

hepatocellular carcinoma; contrast-enhanced ultrasonography; microvessel morphology

Introduction

Hepatocellular carcinoma (HCC) is a highly vascularized tumor.[1]And tumor angiogenesis is known to play an important role in the progression of HCC,[2,3]and ultrasound is an important technique for imaging the angiogenesis.[4-8]Depiction and assessment of tumor vascularity with ultrasound has advanced progressively from Doppler studies of large vessels to the use of microbubble contrast agents which reveal the microcirculation. The present retrospective study was designed to determine the relationship between the enhancement patterns of HCC on contrastenhanced ultrasonography (CEUS) and the features of microvascular morphology in 80 HCC patients.

Methods

Clinical data

Eighty patients with histopathologically proven HCC (58 males, 22 females; aged 32-75 years, mean 64.0±4.6 years) between January 2006 and October 2009 were included in the study. Patients who had not been operated on or had had no histopathologically identified HCC in this period were excluded. The tumor was solitary in 70 patients. Eight patients had two lesions and two patients had three lesions. In patients with more than one focal lesion,we selected the largest for this study. Lesion size ranged from 1.0 to 8.5 cm (mean 3.2±2.4 cm). Differentiation of these lesions included 18 moderately-differentiated, 30 well-differentiated, and 32 poorly-differentiated HCCs. Seventy-two of the 80 (90%) patients were at high risk for the development of HCC, including chronic hepatitis or cirrhosis related to hepatitis B (48 patients), hepatitis C (8), combined hepatitis B and C (4), liver schistosomiasis (6), alcoholic hepatitis (4), and nonalcoholic steatohepatitis (2). The remaining 8 patients (10%) had HCC in an essentially normal liver on pathologic examination with no identifiable clinical risk factor for HCC.

Ultrasonographic examination

Prior to the operation, real-time gray scale CEUS was performed in all 80 patients using an Acuson Sequoia 512 scanner (Acuson, Mountain View, CA, USA) with incorporated contrast pulse sequencing (CPS) contrastspecific software and a 4C1 vector transducer with a frequency of 1.0-4.0 MHz. The patients lay in the left lateral decubitus. Before injection of the contrast agent, gray scale ultrasonography was performed to scan the liver thoroughly. The suspected lesion was identified. The settings of the machine parameters were: frame rate, 12 to 15 fps, and dynamic range, 35 dB. A low mechanical index (MI) (0.1-0.2) was selected to avoid the disruption of microbubbles. SonoVue (Bracco SpA, Milan, Italy), the contrast agent used in this study, consists of sulfur hexafluoride microbubbles surrounded by phospholipids. The contrast agent (2.4 ml) was injected via the antecubital vein in a bolus through a 21-gauge cannula within 1 to 2 seconds, followed by a flush of 5 ml of 0.9% normal saline. During the early-contrast enhancement, the patient was asked to take a deep breath followed by breathholding to ensure that the focal lesion remained in the field of view. Measurements in the CEUS process were: 1) degree of lesion enhancement in arterial, portal and late phases; and 2) enhancement time of lesion (the time to become hyperechoic, isoechoic or hypoechoic, and the time to peak intensity from the contrast agent injection). The arterial phase was defined as 10-30 seconds from the contrast agent injection, the portal phase was 31-120 seconds from injection, and the late phase was 121-360 seconds from injection.[9]The lesion was observed continuously for 360 seconds. The degree of enhancement was divided into hypo-enhancement, iso-enhancement and hyper-enhancement compared with the surrounding liver parenchyma. In the portal phase, when the degree of enhancement decreased from hyper-enhancement or iso-enhancement to hypo-enhancement, and the hypo-enhanced area shown in the lesion, the lesion was regarded as portal hypo-enhancement; when the degree of enhancement decreased from hyper-enhancement to isoenhancement, and no hyper-enhanced or hypo-enhanced area showed in the lesion, the lesion was regarded as portal iso-enhancement. The whole CEUS process was stored on the hard disk of the scanner. The imaging data were retrospectively analyzed separately by three radiologists with 4 years of experience in CEUS. Generally, there were no significant differences in observers. In patients with wide differences, the imaging data were reevaluated by all observers until they reached an agreement.

Pathological examination

All nodule specimens were immunostained with human CD34 monoclonal antibody (Santa Cruz Biotechnology, USA). Formalin-fixed, paraffin-embedded cancer tissue were sectioned into 4 μm. Microvessels were stained immunohistochemically using labeled streptavidin-biotin after antigen retrieval for CD34. The sections were incubated in an anti-CD34 monoclonal antibody (dilution 1∶200). The stain was visualized with diaminobenzidine tetrahydroxychloride solution (DAB; DAKO Corp., Carpinteria, USA) as a substrate. The microvessel density (MVD) of HCC was evaluated according to the method described by Weidner et al.[10]Any positively stained endothelial cell or endothelialcell cluster that was clearly separated from adjacent microvessels and connective elements was counted as one microvessel, irrespective of the presence of a vessel lumen. At low power (×100), the tissue sections were screened and five areas with the most intense neovascularization (hot spots) were selected. Microvessel counts in these areas were performed at low power (×200). The mean microvessel count of the five most vascular areas was taken as the MVD. Counts were measured as the number of microvessels per 0.2 mm2. According to microvascular morphology and the staining area of endothelial cells, microvessels were divided into two types, point-line and loop-strip. The point-line microvessel manifested as a spot or line, split the tumor cells, and there was no communication between microvessels. The loop-strip microvessel appeared as a loop or strip, which enclosed the tumor cells, and there was communication between microvessels. Microvessel type was determined by the majority of tumor microvessel morphology in ten areas at high power (×400). Microvessel count and microvessel type were analyzed by the same pathologist (with 5 years of experience in pathology) without knowledge of the CEUS findings.

Statistical analysis

Statistical analysis was performed using the SPSS program (version 13.0 for Windows; SPSS Inc, Chicago,IL, USA). Statistical comparisons were made by the Chisquare test for significanct differences between the tumor echogenicity and the type of microvascular morphology in the portal phase . Student'sttest was used to compare the mean times of tumor enhancement in the point-line and loop-strip groups. Simple regression analysis was performed to test the correlation between the MVD of the loop-strip type and the time to hypo-enhancement in HCC.Pvalues less than 0.05 were considered statistically significant.

Results

Classification of microvascular morphology

All of the 80 HCC patients were classified into a point-line group and a loop-strip group according to microvascular morphology. The point-line group was observed in 36 patients and the loop-strip group in 44. The mean MVD of HCCs was 22.4±3.5 in the point-line group, and 19.6± 6.7 in the loop-strip group. Microvessel density by CD34 expression was not significantly different between the two groups (t=0.948,P=0.354).

Correlation between enhancement patterns and microvascular morphology in HCC

Table 1 shows the relationship between the patterns of tumor enhancement and microvascular morphology. The degree of enhancement on CEUS was significantly different between the two groups in the portal phase. Hypo-enhancement was significantly more frequent in the loop-strip than point-line group in the portal phase (χ2=4.789,P=0.029) (Figs. 1 and 2).

Table 2 shows the mean time of tumor enhancement in the point-line and loop-strip groups. There was a significant difference between the two groups in the time to hypo-enhancement (t=2.247,P=0.022), but no difference in the time to peak (t=1.437,P=0.152), the time of commencement of hyper-enhancement (t=1.043,P=0.236) and commencement of iso-enhancement (t=0.758,P=0.425), which indicated that tumors with the loop-strip wash out faster than the point-line one.

Fig. 1. Ultrasound images and point-line microvessels in a 68-year-old man with HCC. A: Baseline ultrasound image shows a hypoechoic HCC nodule 2.0 cm in diameter in the right lobe of the liver. B: Arterial phase image obtained at 14 seconds after contrast agent administration shows a hyper-enhancement of the lesion. C: Portal phase image obtained at 72 seconds. The nodule is isoechoic with respect to the surrounding liver. D: Portal phase image obtained at 120 seconds. The nodule is isoechoic with respect to the surrounding liver. E: Late phase image obtained at 150 seconds. The HCC is slightly hypoechoic with respect to the surrounding liver. F: Photomicrograph showing point-line microvessels (anti-CD34 stain, original magnification ×400).

Fig. 2. Ultrasound images and loop-strip microvessels in a 70-year-old woman with HCC. A: Baseline ultrasound image shows a hypoechoic HCC nodule 1.5 cm in diameter in the right lobe of the liver. B: Arterial phase image obtained 18 seconds after contrast agent administration shows a hyper-enhancement of the lesion. C: Arterial phase image obtained at 26 seconds. The nodule is isoechoic with respect to the surrounding liver. D: Portal phase image obtained at 35 seconds. The nodule is slightly hypoechoic with respect to the surrounding liver. E: Late phase image obtained at 130 seconds. The nodule is remarkably hypoechoic with respect to the surrounding liver. F: Photomicrograph showing loop-strip microvessels (anti-CD34 stain, original magnification ×400).

Table 2. Relationship between mean time of enhancement and microvascular classification in patients with HCC

Fig. 3. Correlation between MVD of loop-strip type and the time to hypo-enhancement in patients with HCC.

Correlation of the MVD of the loop-strip type with the time to hypo-enhancement in HCC

The results of correlation studies are shown between the MVD of the loop-strip type and the time to hypoenhancement (Fig. 3). The MVD of loop-strip in HCC was correlated with the time to hypo-enhancement (r=-0.648,P=0.001). The time to hypo-enhancement tended to increase along with reduction of MVD in the loop-strip type.

Discussion

The development of CEUS techniques has overcome the limitations of conventional ultrasonography and enabled the evolution of CEUS from vascular imaging to imaging of perfused tissue at the microvascular level.[11]SonoVue, a new contrast agent, contains microbubbles of different sizes, ranging from 1 to 10 μm, with a mean size of 2.5 μm. The sizes of SonoVue microbubbles are large enough to retain the agent within the vascular system, but small enough to allow free passage through the capillaries. Thus, it acts as a blood-pool agent which makes calculation of perfusion indices much more straightforward. It radiates sufficient harmonic signals by low MI transmission power to allow continuous real-time imaging. Real-time examination of HCC withSonoVue provides detailed hemodynamic information about hepatic microcirculation and also is time-efficient for the examination itself.[12]

The sinusoid is a specialized network of capillaries, composed of microvessels, endothelial cells, Kupffer cells and sinus pericytes, of which the main constituent is sinusoidal endothelial cells (SECs). SECs of the liver play important roles in maintaining normal liver functions through interaction with hepatocytes and other nonparenchymal cells. The morphology of SECs in early HCC is similar to that of normal liver, with almost no CD34 antigen expression. With tumor dedifferentiation, fenestrae in SECs disappear, and the CD34 phenotype changes. SECs begin to express CD34 antigen and participate in hepatic sinusoidal capillarization.[13-15]The CD34 marker shows microvessel morphology in HCC, which contributes to understanding the occurrence and development of sinusoidal capillarization.

The relationship between conventional dynamic multi-detector computed tomography or CEUS findings and angiogenesis in cancer has been reported.[16-24]The studies have found that the attenuation value of the peak enhancement of the tumor and the enhancement ratio are correlated positively with the extent of angiogenesis. However, exploration of the relationship between microvascular morphology and the imaging characteristics of tumor angiogenesis has not been reported. In the current study, the correlation between microvascular morphology and the patterns of CEUS was analyzed in the 80 patients with HCC. The results showed that the incidence of hypo-enhancement in the portal phase was higher in the loop-strip group than in the point-line group. The time to become hypo-enhancing in the loop-strip group was shorter than in the point-line group. This may be due to the difference in microvascular morphology. Communication between microvessels occurred in the loop-strip group, so contrast agent clearance could occur via the liver microcirculation and by abnormal access, such as arteriovenous fistulae. Therefore, the loop-strip group showed a higher incidence of a quick wash-out clearance pattern than the point-line group. The MVD of the loop-strip group in HCC was correlated with the time to become hypo-enhancing; this probably reflects the number of abnormal clearance channels to a certain extent. The shorter the time to hypo-enhancement, the more abnormal clearance channels in the HCC. Therefore, the time to become hypo-enhancing, enhanced manifestations in the portal phase, and the pattern of ultrasound contrast agent clearance are correlated with microvascular morphology, which is one of the morphological bases of CEUS characterization. The correlation between CEUS pattern and microvascular morphology suggested that it is an important noninvasive imaging technique to provide information about microvascular morphology and angiogenesis, which is valuable for antiangiogenic therapy in HCC.

In conclusion, the contrast-enhancement patterns on CEUS are related to the microvascular morphology of tumors. The type of microvascular morphology influences CEUS characterization. The present study contributes to a thorough understanding of angiogenesis on ultrasound imaging in HCC and also is of clinical value.

Funding:This work was supported by grants from the Natural Science Foundation of Hunan Province (10JJ5041) and the Medical Research Foundation of Hunan Province (B2010-023), China.

Ethical approval:The protocol of this study was approved by the Ethics Committee for Clinical Studies of the Third Xiangya Hospital, Central South University, China.

Contributors:XJD and SSR designed the research. XJD wrote the paper. All authors contributed to the design and interpretation of the study and to further drafts. XJD is the guarantor.

Competing interest:No benefits in any form have been received or will be received from a commercial party related directly or indirectly to the subject of this article.

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July 12, 2010

Accepted after revision October 27, 2010

Author Affiliations: Department of Diagnostic Ultrasound (Xiao JD and Zhu WH), and Department of Gastroenterology (Shen SR), Third Xiangya Hospital, Central South University, Changsha 410013, China

Wen-Hui Zhu, MD, Department of Diagnostic Ultrasound, Third Xiangya Hospital, Central South University, Changsha 410013, China (Tel: 86-731-88618401; Fax: 86-731-88618403; Email: wenhuizhu@sohu.com)

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