E.

Leen1

Ultrasound contrast in clinical applications

Ultrasound remains the most widely used imaging modality in clinical practice, largely because of its relatively low cost, safety and availability. However, there are still fundamental limitations in imaging diseased tissues that have acoustic properties similar to those of normal surrounding parenchyma, as well as in the Doppler assessment of low-velocity blood flows and low volume flow rates. With the current limits on performance, there is clearly a need for ultrasound contrast agents.
Ultrasound contrast agents

and they may enhance the normal parenchyma for up to an hour. The added advantage lies in the fact that lesions, which are deficient of Kupffer cells, do not take up the contrast agent, thereby improving the lesion-to-tissue contrast ratio. Levovist (Schering AG), which was primarily designed as a bloodpool agent, has also been shown to have a delayed liver-specific phase. The mechanism underlying this finding remains unknown.
Clinical applications1

Contrast agents are needed for imaging lowvelocity blood flow and some pathologies.

The ideal ultrasound contrast agent should be safe, stable enough in the vascular system to survive pulmonary capillary circulation, and be capable of modifying the acoustic properties of the tissues of interest. Current contrast agents satisfy these criteria. They consist of microbubbles, which are well recognized to be the most effective backscatterers; they measure between 2 to 8 microns in diameter and contain either air, or perfluorocarbon gas which has prolonged longevity due to its lower solubility. Stability of the microbubbles is provided in the form of a shell, made of denatured albumin, lipid or surfactant layers, or poly-butyl-cyanoacrylate (Table 1). Earlier agents were primarily designed to be bloodpool agents, and have been shown to be highly effective in enhancing spectral/color/ Power Doppler signals, lasting up to 7 minutes following an intravenous bolus administration, and approximately up to 15 - 20 minutes after an infusion. Newer agents such as NAI Investigational Drug (Nycomed Amersham) and SHU 563A (Schering AG) have additional tissue-specific properties; they are selectively taken up by the Kupffer cells of the reticuloendothelial system after the vascular phase (lasting 5 -10 minutes)

Numerous clinical studies have confirmed the usefulness of various bloodpool agents in the assessment of arterial and venous macrovasculature of various systems, including head and neck, limbs, kidneys, collateral circulation and shunts patency. There are consistent reports of improved diagnostic confidence following the use of contrast agents. Of particular relevance is the fact that contrast would convert a previously sub-optimal, non-diagnostic examination into a diagnostic one, thereby deferring referral to more costly and/or invasive procedures. However, it is the field of hepatic oncology which is the model test platform, where the application of these agents remains the most challenging and yet the most promising. Any success in the field could easily be extended to other systems. The characterization of liver tumors has relied on the enhancement pattern and typical morphological appearance of the tumor vascular bed. Early studies using carbon dioxide microbubbles were indeed encouraging. Kudo and colleagues [1] assessed the use of carbon dioxide microbubbles to enhance sonographic examination of hepatic tumors in 184 patients.
1

Current contrast agents consist of microbubbles, which are effective backscatterers.

Newer contrast agents have tissue-specific properties.

Not all applications of contrast agents for ultrasound have

regulatory approval for use in all countries.

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1 University of Glasgow, Royal Infirmary, Glasgow, Scotland.

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Name Albunex* FS-069* MRX-115 AF0150 BR1 Levovist* SHU563A NAI inv. drug Echogen Quantison

Manufacturer Molecular Biosystems Molecular Biosystems ImaRx Pharmaceutical Alliance Bracco Spa Schering AG Schering AG Nycomed Amersham Sonus Pharmaceutical Andaris

Size: microns 4.3 3.9 2.3 5 2.5 2-4 2 ~ 6-8 3.2

Shell Denatured albumin Denatured albumin Lipids & surfactants Surfactant Lipids & surfactants Palmitic acid PBCA Not public information Denatured albumin

Gas Air Air & perfluoropropane Air & perfluoropropane Air & perfluoropropane Air & H2SF6 Air Air ~ Dodecafluoropentane Air

Table 1: Ultrasound contrast agents

*Registered

The carbon dioxide microbubbles were prepared by vigorously mixing 10 ml of carbon dioxide, 10 ml of heparinized normal saline and 5 ml of the patient's own blood. The advantage of such a combination was that blood increases the viscosity and surface tension of the solution, thereby producing smaller microbubbles with improved stability. Furthermore, carbon dioxide is readily cleared from the lungs.
Carbon dioxide allows characterization of hepatic tumors.

Sonographic angiography was performed by injecting 5 - 20 ml of carbon dioxide microbubbles (at a rate of 1-2 ml/s) through a catheter placed within the hepatic artery (proper, left or right hepatic artery) using the same technique as conventional selective hepatic angiography. It allowed characterization of the hepatic tumors, based on their vascular anatomy. Hepatocellular carcinomas displayed a homogeneous or mosaic hypervascular pattern with peripheral arterial blood supply (sensitivity: 90%, specificity: 89%). In contrast, focal nodular hyperplastic nodules were characterized by an early central arterial hypervascular supply giving a uniform or lobulated enhancement (sensitivity and specificity :100%). Adenomatous hyperplasia, hemangioma and metastasis displayed hypovascularity, spotty pooling and peripheral hypervascularity patterns respectively. Veltri and colleagues [2] reported distinctive enhancement patterns in hepatomas, compared with focal nodular hyperplasias and hemangiomas, during the wash-in and delayed phases. Using the same technique, Kudo at al. [3] confirmed its ability to detect small hepatocellular carcinomas measuring less than a centimeter in diameter, which had been missed by the more

Levovist gives highly significant enhancement of color Doppler signals in liver tumors.

conventional techniques. Furthermore it was reported that the precise assessment of tumor vascularity also enabled determination of appropriate therapeutic strategy for these tumors. In a more recent study, the use of carbon dioxide alone in the sonographic examination of 45 patients with hepatocellular carcinomas was compared with digital subtraction angiography (DSA), conventional ultrasound, CT and CT arterio-portography [4]. Sonographic examination was performed during the injection of 10 - 20 ml of carbon dioxide (at a rate of 1 ml/s) via a catheter placed within the hepatic artery proper. This technique allowed the detection of more lesions than any of the other conventional modalities, and no side effects had been reported. Arawaka and colleagues [5] confirmed the higher rate of detection of HCC's (85%) using carbon dioxide enhanced sonography, when compared with digital subtraction angiography (DSA), dynamic CT or Lipiodol CT (68% - 77%). The invasive nature of the technique is a major disadvantage, and it has not gained support in routine clinical practice. However the results of the carbon dioxide enhanced Doppler studies have provided the motivation for using ultrasound contrast agents, with trans-pulmonary capillary stability, as a less invasive method of improving liver tumor detection and characterization. Earlier results of clinical phase trials of Levovist (Schering AG) were promising in the characterization of focal liver tumors. In a study of 28 patients with histologically proven liver tumors (2 hepatocellular carcinomas, 2 carcinoid and 24 colorectal cancer metastases), highly significant enhancement of the color Doppler signals was observed in 25 cases, lasting for a

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mean period of 180 (sd: 45) seconds [6]. No enhancement was observed in three colorectal cancer patients undergoing regional chemotherapy. Characteristic patterns of color Doppler scans were demonstrated in colorectal hepatic metastases with predominantly increased tumor rim enhancement, while in the hepatocellular carcinomas an infiltrating enhanced basket like pattern of hypervascularity was observed. Increased homogeneous color Doppler signals (which had been absent on baseline scans) were observed throughout carcinoid lesions after the injection of Levovist. In another study of 38 patients with hepatocellular carcinomas, a similar enhanced pattern of hypervascularity was also demonstrated following the injection of Levovist [7]. The characteristic hypervascular nature of focal nodular hyperplasia and adenomas was clearly demonstrated following Levovist. In contrast, there was little or no enhancement of Doppler signals in the case of hemangiomas [8]. Improved differentiation between hepatomas, metastases and benign hemangiomas were subsequently confirmed by other workers [9]. Tanaka and colleagues [10]showed improved visualization of the vascularity of the hepatomas, thereby increasing the incidence of a positive finding from 29% to 86% following Levovist administration. The subjective assessment of tumoral vascularity by non-enhanced or enhanced color Doppler ultrasound, as a method of differentiating between benign and malignant lesions, has been widely criticized and seen as an oversimplification, since there was overlap of the findings which appeared to be dependent upon the size of lesions. Exploitation of other characteristic features of malignancy was therefore required, and led to the development of quantitative methods to measure functional indices of the microcirculation including relative changes in transit time and tissue perfusion. In that respect, ultrasound contrast agents are ideal tracers to assess tissue perfusion in both temporal and spatial domain. The advent of power Doppler imaging and subsequent demonstration of the linear relationship

between contrast concentration and the Power or Spectral Doppler Signal intensity were to extend contrast application into a functional imaging tool. Preliminary studies assessing the temporal changes of power Doppler signal intensity in liver tumors, during the wash-in and wash-out phase, had shown improvement in the differentiation between metastases, hepatomas and hemangiomas; the gradient of the arterial phase curves and peak enhancement were also reported to be significantly higher in the presence of malignancy [11]. The interaction between the insonating ultrasound beam and microbubbles is indeed very complex. Nevertheless, understanding the underlying process has been key to the development of improved methods of visualizing and displaying the contrast. With increasing peak pressure of the incident ultrasound field, microbubbles respond within three broad regimes of scattering: linear, nonlinear and transient scattering. Development of harmonic/pulse inversion imaging and transient/ flash echo imaging utilize the microbubbles non-linear and transient scattering properties respectively, to enhance signals from the contrast over those from tissue. The combination of these new modalities with liver-specific agents showed, for the first time, real promise in improving detection and characterization of overt liver tumors along the same pathway as contrast-enhanced MR. Flash/transient echo imaging using Levovist has been shown, in a small series, to improve conspicuity and detection of lesions not seen on contrast-enhanced CT scans [12]. However, there are limitations, such as transience of the effect and lack of penetration to show deeper areas of interest. Unfortunately, the transient effect does not allow biopsy to be performed to confirm presence of these lesions. Preliminary results from the same group of workers have also shown that benign tumors scored more color Doppler signals from stimulated acoustic emission (SAE) than metastases. This may reflect the presence of functioning liver tissue in the case of focal nodular hyperplasia, and regenerating nodules or delayed clearance in the case of hemangiomas. In a small study of 38 patients with focal liver tumors, designed to evaluate the liver-specific
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Levovist gives improved differentiation between hepatomas, metastases and benign hemangiomas.

Contrast application has become a functional imaging tool.

Flash/transient echo imaging using Levovist improves conspicuity and detection of lesions not seen on CT scans.

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Fig. 1. Multi-phasic Pulse Inversion Harmonic Imaging of colorectal liver metastasis at: baseline, 23 s (hepatic arterial phase), 40 s (mid phase) and 77 s (portal venous phase).

Fig. 2. SHU 563 A: Multi-phasic Pulse Inversion Harmonic Imaging of hepatocellular carcinoma invading portal vein at: baseline, 23 s (arterial phase showing tortuous feeding vessels, 60 s (portal venous phase) and 60 s (intermittent delay scanning showing as much perfusion in HCC as normal liver).

Studies demonstrate the superiority of harmonic imaging over the fundamental mode.

NAI Investigational Drug from Nycomed Amersham, harmonic imaging was shown to be superior to the fundamental imaging mode, providing significantly improved delineation at lower contrast doses [14]. The detection of lesions as small as 4 mm in diameter was also demonstrated. Uniform grayscale enhancement of the normal liver parenchyma was also achieved at relatively low contrast doses. Of particular interest is the fact that metastases were characterized by a surrounding brighter rim, replacing the halo sign seen at baseline. This is believed to reflect the higher concentration of Kupffer cells around metastases. Preliminary data of pulse inversion imaging studies suggest the same will be true, with the added benefit of improved resolution, penetration and the use of even lower contrast doses.

Results of a more recent study of 15 patients with focal liver tumors, evaluating the use of SHU 563A, another liver-specific agent, confirm the superiority of pulse inversion harmonic imaging over tissue harmonic imaging [15]. The characteristic rim enhancement of colorectal liver metastases was evident in the delayed liver phase, and detection of lesions as small as 3 mm in diameter was reported. The encouraging results following the use of pulse inversion harmonic imaging in combination with these agents have led to an on-going multi-center trial. Results so far appear to support the findings described above. More recently, Burns and colleagues [15] developed a simple method of interval delay

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imaging using Optison (Mallinckrodt) enhanced harmonic or pulse inversion mode to clearly differentiate between benign and malignant hepatic tumors. This is based on the relative differential re-perfusion of the tumor tissue compared with normal liver parenchyma. With interval delay imaging, metastases known to be hypovascular appeared as focal filling defects, smaller than their original size, in a background of brighter liver. In contrast, hypervascular hepatomas appeared as bright as the surrounding liver, reflecting the same degree of perfusion. In hemangiomas, characteristic peripheral nodular enhancement as seen on CT and MR could be demonstrated in the early vascular phase. In our own experience, this new technique is simple and reproducible.
Detection of occult liver metastases

results showed significant increase in the CEDPI values of patients with liver metastases, with clear separation of the values between those with and without liver metastases. The time delay in the first appearance of contrast in the portal vein, relative to its first appearance in the hepatic artery, was also significantly elevated in the liver metastases group. However, there was some overlap of the values between the two groups which would limit its usefulness as an independent index. Nevertheless, the technique is relatively simple to perform, and can be carried out within 3 - 5 minutes, compared with the conventional DPI technique which can take between 10 to 30 minutes. The preliminary data also suggest that contrast-enhanced measurement of CEDPI may provide an alternative and easier method for the detection of occult liver metastases. Based on the same principle, using spectral Doppler intensitometry, Blomley and colleagues [20] showed that the transit time required for Levovist to arrive in the hepatic veins, following an intravenous bolus administration, was significantly reduced in patients with metastases when compared with controls. The mechanism underlying these hemodynamic changes remains unknown, but it is believed that the arteriovenous shunting known to be associated with liver metastases may play a major role. This new technique may provide another simple alternative method for identifying patients with occult liver metastases. Comparative as well as reproducibility studies of these two techniques are currently in progress.

Levovistenhanced power Doppler imaging clearly differentiates between patients with and without liver metastases.

It has long been known that the presence of even micro-metastases is associated with changes in liver blood flow. Previous studies using duplex/ color Doppler ultrasound have shown that measurement of the Doppler Perfusion Index (DPI), defined as the ratio of hepatic arterial to total liver blood flow, can identify colorectal cancer patients and gastric cancer patients with occult liver metastases, i.e. those which had not been detected on CT scan or intra-operative ultrasound [16,17]. A new technique using Levovist-enhanced power Doppler imaging was recently developed to determine the same hepatic arterial contribution to total liver blood flow, in an attempt to identify patients with liver metastases [18]. Power Doppler imaging of the hepatic artery proper and the portal vein was performed before and after an intravenous bolus injection of 5 ml of Levovist (dose: 400 mg/ml). The power Doppler signal intensity (PDSI) - time curves for the hepatic artery and portal vein were obtained following quantification of the PDSI changes in each vessel. Analogous to previous Doppler Perfusion Index (DPI) and dynamic scintigraphic studies, the hepatic arterial contribution to total liver blood flow was calculated as the Contrast-Enhanced Doppler Perfusion Index (CEDPI). This is the ratio of G1/G1 + G2 where is G1 represents the gradient for hepatic arterial inflow, and G2 the gradient for portal venous inflow. Preliminary

Spectral Doppler intensitometry may provide another simple method for identifying patients with occult liver metastases.

References
[1] Kudo M, Tomita S, Tochio H et al.. Sonography with Intraarterial Infusion of Carbon Dioxide Microbubbles: Value in Differential Diagnosis of Hepatic Tumors. AJR 1992; 158: 65-74. [2] Veltri A, Capello S, Faissola B et al.: Dynamic Contrast Enhancedultrasound with Carbon Dioxide Microbubbles as an Adjunct to Arteriography of Liver Tumors. Cardiovasc Intervent Radiol 1994; 17: 133-137. [3] Kudo M, Tomita S, Tochio H et al. Small Hepatocellular Carcinoma: Diagnosis with US Angiography with Intraarterial CO2 Microbubbles. Radiology 1992; 182: 155-160.
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[4] Garbagnati F, Milella M, Spreafico C et al. US Contrast Enhancement with Intraarterial CO2 Injection in Staging Hepatocellular Carcinomas. Radiol Med 1994; 87(Suppl. 1 Al N.5): 65-70. [5] Arakawa A, Nishiharu T, Matsukawa T et al. Detection of Hepatocellular Carcinoma by Intraarterially Enhanced Ultrasonography with CO2 Microbubbles. Acta Radiologica 1993, 187: 353-356. [6] Leen E, Angerson WG, Warren H et al. Improved Colour Doppler Flow Imaging of Colorectal Hepatic Metastases using Galactose Microparticles: a Preliminary Report. Br J Surg. 1994; 81: 252-254. [7] Angeli E, Carpanelli R, Crespi G et al. Efficacy of SH U 508 a in Color Doppler Ultrasonography of Hepatocellular Carcinoma Vascularization. Radiol Med 1994; 87(Suppl. 1 Al N.5): 24-31. [8] Maresca G, Barbaro B, Summaria V et al. Color Doppler Ultrasonography in the Differential Diagnosis of Focal Hepatic Lesions. The SHU 508a Experience. Radiol Med 1994; 87(Suppl. 1 Al N.5): 41-49. [9] Tano S. Ueno N. Tomiyama T. Kimura K. Possibility of Differentiating Small Hyperechoic Liver Tumors using Contrast-Enhanced Color Doppler Ultrasonography: a Preliminary Study. Clinical Radiology 1997. 52(1): 41-5. [10] Tanaka S, Kitamra T, Yoshioka F et al. Effectiveness of Galactose based Intravenous Contrast Medium on Color Doppler Sonography of Deeply Located Hepatocellular Carcinoma. Ultrasound In Med Biol 1995; 21: 157-160. [11] Leen E, Ramnarine K, Oppo K et al. Dynamic Power Doppler Imaging of Focal Liver Tumors using NC100100, a new Organ Specific Echo Enhancer. Radiology 1998 (Abstract RSNA 1998 Proceedings).

[12] Blomley MJK, Albrecht T, Cosgrove DO et al. Improved Imaging of Liver Metastases Using Stimulated Acoustic Emission in the Late Enhancement Phase of Ultrasound Contrast Agent Levovist, Early Experience. Radiology 1999 (In Press). [13] Leen E, Needleman L, Kyriakopoulou K et al. Comparison between Fundamental and Harmonic Imaging of Focal Liver Tumours using NC100100, a new Liver-specific Echo-enhancer. Eur J Rad 1999 (Abstract ECR99 Proceedings). [14] Leen E, Bauer A, Oppo K et al. Imaging of Focal Liver Lesions in Phase II Clinical Study with SHU563a as Ultrasound Contrast Agent. Ultrasound Contrast Research Symposium 1999 Proceedings. [15] Burns PN, Wilson S, Muradali D & Lai X. Liver Mass Characterization using Harmonic Interval Delay Imaging. Ultrasound Contrast Research Symposium 1999 Proceedings. [16] Leen E, Angerson WG, Cooke TG & Mcardle CS. Prognostic Power of Doppler Perfusion Index In Colorectal Cancer. Correlation with Survival. Annals of Surgery 1996. 223(2): 199-203. [17] Leen E, Anderson JR, Robertson J, O' Gorman P, Cooke TG & Mcardle CS. Doppler Index Perfusion in the Detection of Hepatic Metastases Secondary to Gastric Carcinoma. American Journal of Surgery. 1997; 173(2): 99-102. [18] Leen E, Anderson JH, Horgan P et al. Contrast Enhanced Doppler Perfusion Index: Detection of Colorectal Liver Metastases. Radiology 98 (Abstract:RSNA 1998 Proceedings). [19] Blomley MJK, Cosgrove DO, Albrecht T et al. Liver Vascular Transit Time Analyzed using Microbubble Injections: How Helpful is it in Imaging Liver Tumor? Ultrasound Contrast Research Symposium 1999 Proceedings.

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