Quantitative Assessment Of Angiogenesis And Blood Perfusion In Hepatocelluar Carcinomas With Real-time Contrast-enhanced Gray-scale Ultrasonography

Since 1971, when Folkman first hypothesized that tumor growth depends on angiogenesis, considerable researches have validated that tumor angiogenesis are correlated with tumor invasion, metastasis, stage and prognoses. Currently, histologic determination of the mean intratumoral microvessel density (MVD) is the golden standard used for assessing angiogenesis. Studies have demonstrated a direct correlation between MVD and patients survival, suggesting a prognostic implication of MVD counts in hepatic carcinoma. On the other hand, antiangiogenic agents which target the tumor vasculature for therapy are in the process of clinical trial and MVD is used to assess the efficiency of antiangiogenic therapy in most researches of antiangiogenic therapy.However, histological evaluation of MVD may necessitate an invasive procedure, which is hard to make a clinical assessment for patients before or without operation. There is a crucial need of imaging technology for accurate and noninvasive methods to evaluate tumor angiogenesis in vivo. Several functional imagings or perfusion imaging modalities which may reflect early haemodynamic changes in tumors, for instance blood velocity, blood volume and blood flow are being investigated to evaluate tumor angiogenesis and antiangiogenic therapy, including dynamic computer tomography (CT), magnetic resonance (MR), positron emission tomography (PET) and ultrasonography.Conventional color Doppler ultrasonography is poor to display vasculature in hepatic carcinoma. Current contrast-enhanced ultrasonography with low mechanical index techniques has advantages in characterizing focal liver lesions and assessing the outcome of interventional therapy, including real-time demonstration of continuous hemodynamic changes in both the liver and the liver lesions. Quantitative analysis of contrast emhancement is potential to evaluate angiogenesis in tumors and to monitor the efficacy of antiangiogenic theraphy. In this study, we investigated the clinical value of real-time contrast-enhanced gray-scale ultrasonography in the quantitative assessment of angiogenesis and blood perfusion in hepatic carcinomas.The First Part: Assessment Research of Antiangiogenic Therapy in a Nude Hepatocellular Carcinoma Model with Real-time Contrast-enhanced Gray-scale UltrasonographyMethods: Human hepatocellular carcinoma xenograft model was established in thirty nude mice subcutaneously. When tumors were approximately 30 mm~3, fifteen nude mice were treated with a potent and selective inhibitor of vascular endothelial growth factor receptors (pazopanib) 40mg/kg once daily by oral administration and fifteen nude mice were set as control. Contrast-enhanced ultrasonography was performed by using a commercially available ultrasound system Philips iU22 with a low MI contrast specific imaging technology (Pulse Inversion Harmonic Imaging) and a bolus injection of 20μL of SonoVue in order to determind tumor volume and blood perfusion 3 days before treatment, 7 days, 14 days and 21 days after treatment, respectively. Regions of interest within tumors were analyzed off-line to perform time-intensity curves and determine US perfusion parameters. Changes of tumor volume, enhancement pattern and US perfusion parameters in the treatment group were compared with the control group.Result: 1. Pazopanib showed significant growth inhibition of the tumor xenograft. The relative tumor proliferation rate in the treatment group was 41% on 21 days after treatment.2. After the injection of contrast agent, tumors in the treatment group showed peripheral hypervascular enhancement followed by a rapid wash out in the centre of tumor and blood perfusion of tumors in the treatment group was low.3. The ISI, AUC and BF were lower in treatment tumors than in control tumors, and a2 was higher in treatment tumors than in control tumors (P<0.05).4. The a3, ISI, AUC and BF correlated with the continuous volume growth rate in treatment tumors, respectively (P<0.05).Conclusion: Low mechanical index real-time contrast-enhanced gray-scale ultrasonography reflects blood flow changes of microvascular perfusion in tumors, being potential in monitoring tumor vascular response to antiangiogenic therapy.The Second Part: Quantitative Assessment with Real-time Contrast-enhanced Gray-scale Ultrasonography and Microvessel Density in Hepatocellular Carcinomas: A Comparative Study Methods: Twenty-nine patients with hepatocellular carcinoma confirmed by pathology were examined by using a commercially available ultrasound system Technos MPX DU8 with a low MI contrast specific imaging technology (Contrast Tuned Imaging) and a bolus injection of 0.6ml of SonoVue. Regions of interest within tumors and hepatic parenchyma were analyzed off-line to perform time-intensity curves and determine US perfusion parameters. Repeat examinations were performed in four patients after at least 15 minutes to test reproducibility of US perfusion analysis. The mean intratumoral endothelial cell (CD34) counts at immunohistochemical analysis were evaluated by both manual and computer-assisited analysis under different magnification (of×200,×100,×50). The relation between US perfusion parameters and MVD was investigated.Results: 1. A good correlation of the first and second US perfusion parameters from both tumor lesion and hepatic parenchyma was obtained (r=0.9, P<0.001).2. There was a significant difference in US perfusion parameters of hepatic parenchyma with different depth to the probe (P<0.05).3. US perfusion parameters objectively showed a quick wash in/wash out enhancement pattern in hepatocellular carcinomas.4. There was a better correlation between US perfusion parameters and computer-assisted microvessel counts. The sISI, sAUC and sBF standardized by hepatic parenchyma were positively correlated with MVD, respectively (all P<0.05).5. US perfusion parameters (sISI, sAUC and sBF) and MVD were higher in well-differentiated than that of poor-differentiated tumors (P<0.05).Conclusion: US perfusion parameters can reflect the activity of angiogenesis in hepatocellular carcinomas, which is promising in the quantitative assessment of tumor angiogenesis in vivo.In summary, quantitative analysis with real-time contrast-enhanced gray-scale ultrasonography offers potentially useful markers for evaluating angiogenesis and blood perfusion in hepatocelluar carcinomas, providing an accurate, noninvasive and repeatable imaging method to evaluate patient prognosis and tumor antiangiogenic therapy clinically.