Contrast-Enhanced Ultrasonography : A Recent Application for the Diagnosis and Treatment of Hepatocellular Carcinoma

Hepatocellular carcinoma has a decisive influence on the prognosis of cirrhotic patients, and the use of imaging modalities is essential for the screening, diagnosis and treatment of hepatocellular carcinoma. Ultrasound plays a major role among them, because it provides real-time and noninvasive observation by a simple and easy technique. In addition, ultrasound-guided needle puncture methods are frequently required for the diagnosis and/or treatment process of hepatocellular carcinoma. The development of digital technology has led to the detectability of blood flow by color Doppler ultrasound, and the sensitivity for tumor vascularity has shown remarkable improvement with the introduction of microbubble contrast agents. Moreover, near real-time three-dimensional ultrasound images are now available. These advancements in the ultrasound field have led to rapid progress in hepatocellular carcinoma management, and continuing advances are expected. This article reviews the current application of contrast-enhanced ultrasound for hepatocellular carcinoma in clinical practice.


INTRODUCTION
2] As the prognosis of cirrhotic patients depends on the occurrence and progression of HCC, diagnosis and treatment of this neoplasm are major issues in clinical practice.
Recent advances in digital technologies have resulted in remarkable developments in the field of imaging modalities.Ultrasound (US) is one of the diagnostic tools that have shown significant improvement within the last decade. 3As for the diagnosis of liver tumors, US examination has the advantages of real-time observation, simple technique and non-invasiveness.It is being used worldwide, and at a high frequency, as a first-step, reliable method for the diagnosis of liver tumors.5] Diagnosis and treatment of HCC have shown remarkable improvement with the clinical usage of contrast-enhanced US.This article reviews the recent application of contrastenhanced US for the management of HCC.

Color Doppler US
[11][12] The waveform analysis was the primary application, and it was frequently used for the characterization of liver tumors.4][15][16][17][18][19][20][21] However, limitations in the detection of slow flow and vessels deeply located from the skin surface have prevented the wider application of Doppler mode in the evaluation of tumor hemodynamics.Furthermore, artifacts caused by respiratory or cardiac motion sometimes affect the precise evaluation of hemodynamic information.At present, the application of Doppler mode alone for detecting tumor blood flow is rare, as the more recent availability of microbubble contrast agents has assisted in overcoming those limits.

Microbubble contrast agents
With the above as a background, there has been considerable expectation that US contrast agents would improve the detectability of blood flow in liver tumors.In fact, since the first report about a US contrast agent by Gramiak et al.3][24][25] While free gas bubbles are efficient scatterers of ultrasound, their utility has been limited because of their immediate removal by the lungs.From the late 1980s to the 1990s, greyscale contrast-enhanced US with carbon dioxide gained broad attention as an echo-enhancing technique, with high sensitivity for detecting tumor vascularity and high performance for the characterization of liver tumors. 26- 27However, the method requires an arteriography procedure because carbon dioxide is easily soluble in blood.The development of microbubble contrast agents available with peripheral venous injection was expected for practical use.
At the end of the 20th century, finally a galactosebased US contrast agent (SHU 508, Levovist) was made available by Schering, Germany.It was a long-awaited material that could provide a stable enhancement effect in abdominal organs with a peripheral injection.Subsequently, many microbubble contrast agents have been developed or are currently under development (Table 1).These intravenously injectable agents improve the detectability of tumor blood flow and the depiction of characteristic flow patterns in Doppler mode or contrast-specific imaging mode with less safety concerns.

Acoustic properties of microbubble and specific imaging mode
The microbubble agents have characteristic acoustic properties that depend on the size and kind of gas and shell. 5,28 The behaviors of microbubbles are related to the acoustic power level (MI; Mechanical Index) of the transmitted ultrasound.As for Levovist, US transmission under a standard acoustic power level for routine US examination provides immediate disappearance of the microbubbles.2] Additionally, the echo signal increases according to lengthening of the interval between frames, as the intermission during US transmission reduces the destruction of microbubbles in the region of interest (ROI) and help microbubble to gather in the US field.With this methodology, an ingenious contrivance of intermittent transmission technique has been developed to obtain strong echo-enhancement in the ROI as a contrast-specific imaging. 33e next-generation contrast agents, such as SonoVue (Bracco Diagnostics, Princeton, NJ, USA) and Definity (Bristol-Myers Squibb, N. Billerica, MA, USA), have characteristic oscillation behavior under very low MI which provides the fewer tissue signals and less microbubble breakdown.[36][37][38]

Accumulation property of microbubble
The diagnostic performance of contrast-enhanced US is not limited to the demonstration of tumor vascularity.7][38][39] Although the precise mechanism remains unclear, the reticuloendothelial system (i.e.phagocytosis by Kupffer cells) may be involved in this phenomenon.Both Levovist and Sonazoid (GE Healthcare, Oslo, Norway) accumulate in the liver, and sonograms in this phase (late liver-specific parenchymal phase) are frequently used for the  [40][41] The improved detectability of tumor vascularity in contrast-enhanced US contributes to the characterization of liver tumors and assessment of the therapeutic response.

Characterization of focal liver lesions
Many studies using contrast-enhanced US have been carried out for the characterization of focal liver lesions with early-phase images and/or delayed phase (liver-specific phase) images (Figure 1, Table 2).3][44] However, evaluation of the enhanced appearance in delayed-phase images is not always simple owing to the fact that the accumulation property of microbubbles affects the enhancement findings in this phase.6][47][48] Despite the various kinds of contrast agents, evaluation of combined multi-phase images would improve the diagnostic ability of contrastenhanced US.
6][47][48][49][50] Furthermore, in a clinical study with two independent image reviewers, Kim et al. described that contrast-enhanced US (agent detecting imaging mode with Levovist) provided a specific diagnosis in 75-79% of 75 patients with focal hepatic lesions, and that the technique was successful as a confirmatory imaging technique in 63-72% of the patients. 516] The recent study reported that intensity analysis of contrast enhancement with Levovist was useful method for the characterization of non-hypervascular small hepatic nodules, regenerative nodule from HCC. 57 However, the diagnostic ability of contrastenhanced US in the early diagnosis of HCC and discrimination of dysplastic nodules from HCC has not been established.At present, percutaneous needle biopsy under US-guidance may be frequently required for the characterization of small hepatic lesions in patients with chronic liver diseases.
Hypervascular hepatic lesions do not always reflect the fact that the final diagnosis of the nodule is HCC in heavy drinkers, since benign hypervascular nodules sometimes occur in their liver. 58A recent report has shown that the ring-shaped appearance on liver-specific contrast-enhanced sonograms with Levovist may be a useful sign for the differential diagnosis of benign nodule from HCC in heavy drinkers. 59Since contrast-enhanced CT hardly differentiates these benign nodules from HCC, this characteristic finding may prevent unnecessary treatments under misdiagnosis.Moreover, it could be expected to lead to a reduction in the application of percutaneous needle biopsy, an invasive procedure, for the precise diagnosis.

Detection of tumor nodules in the liver
Some kinds of hepatic nodules, such as metastatic liver tumors, appear as hypo-enhanced nodules on liver-specific images by accumulated microbubbles in the liver.As a suitable MI for this agent is quite low level which allows less microbubble breakdown, repeated observation for contrastenhancement is possible.However, comparison of detectability of tumor nodules between the agent with accumulation property and the agent without accumulation property remains to be solved.
Recent US systems have provided three-dimensional visualization of the tumor with tumor-associated vessels at any plane from multiple directions. 66- 69Contrast-enhanced 3D ultrasonographies using microbubble contrast agents might become a standard method for the characterization of hepatic tumors (Figure 2).

Treatment support and evaluation of therapeutic effect
Treatment of HCC

Percutaneous needle puncture technique
1] In addition, recurrence of HCC is an inseparable companion of post-treatment patients.With such backgrounds, percutaneous ethanol injection (PEI) and radio-frequency ablation (RFA) were developed and came to be widely used in clinical practice as minimally invasive methods. 72- 77They are now a first-line, favored approach that has an efficient therapeutic effect on HCC. 78-84

Problem for US-guided treatments
Although percutaneous US-guided treatments provide sufficient therapeutic effect, recurrence often plagues many HCC patients.According to long-term study results, cumulative recurrence rates of the treated site of post-PEI lesions were 3.4% at 1 year, 7.1% at 2 years, and 10% at 3 years, and those of the untreated sites in liver were 18.7% at 1 year, 62.1% at 3 years, and 81.7% at 5 years, respectively. 80Thus, many HCC patients have to receive repeated treatments during their clinical course.In order to minimize adverse effects to the liver, less invasive treatment such as PEI or RFA is preferable for these patients.8][89][90] Microbubble contrast agents are also useful in such a case.A recent study showed that contrast-enhanced US with Levovist could localize 24/32 (75%) of HCC lesions that were invisible by non-contrast US. 91 Application of the next-generation US contrast agents is expected to improve the ability for tumor localization, because they have acquired stability of microbubble homogenization of particle size distribution in comparison with earlier agents. 5,92Moreover, the combination of second-generation contrast agents with harmonic imaging mode under lower MI may produce US images with improved signal-to-noise ratio, and a higher detection rate of focal lesions in the liver is expected. 36,49The detectability of ultrasonically unrecognizable hypervascular HCC with Sonazoid, a newly developed perflubutane agent, was reported to be 96.4%, which was quite higher than that with Levovist. 93Contrast-enhanced US with Sonazoid would widen the application of percutaneous US-guided treatments (Figure 4).

Assessment of therapeutic response by ultrasound
With high sensitivity and specificity for detecting tumor vascularity, contrast-enhanced US has come to be frequently applied for evaluation of the therapeutic response in HCC nodules (Table 3).As for percutaneous treatment, Bartolozzi et al. reported that contrast-enhanced color Doppler with Levovist showed sensitivity of 92%, specificity of 100%, and accuracy of 98% compared to the results of spiral CT and biopsy, in the detection of viable tumor treated with PEI. 94A study by Choi et al. revealed that the diagnostic agreement between contrast-enhanced power Doppler with Levovist 14-23 hours after ablation therapy and immediate follow-up CT within 30 minutes after ablation therapy was achieved in 100% of the 45 HCC nodules. 95In an assessment of therapeutic response after RFA, Wen et al. compared the result of contrast-enhanced coded harmonic angio mode with Levovist for detecting residual tumor in 91 HCC nodules 5 to 7 days after RFA with that of dynamic CT, and the sensitivity, specificity, and diagnostic accuracy were 95.3%, 100%, and 98.1%, respectively. 96According to the study by Meloni et al., the sensitivity and specificity of contrast-enhanced harmonic US with Levovist were 83.3% and 100%, respectively, for detecting residual non-ablated tumor at 4 months after treatment in 43 HCC nodules, compared with helical CT findings. 97 for assessment of the therapeutic response after transarterial chemoembolization (TACE), it is well known that contrast-enhanced US has the advantage of not being limited by iodized oil  The early-phase image showed positive enhancement and the late-phase image showed negative enhancement in the nodule.These findings provided easy diagnosis of HCC.

(c). Color Doppler US
No findings appeared in the treated area on the image (arrows).

(d). Contrast-enhanced US with Levovist
Contrast-enhanced color Doppler could demonstrate a blood flow which was similar to contrast-enhanced CT finding (arrow).

(e). Contrast-enhanced US with Levovist after PEI
Color flow signal disappeared after the treatment.

(b). B-mode US
Hepatic lesion was not recognized on B-mode sonogram.

treatment. Morimoto et al. compared the results
of contrast-enhanced US with histologic findings, and sensitivity and specificity for discerning viable and nonviable HCC after TACE in 29 HCC nodules were 100% and 81%, respectively. 99With the use of SonoVue, Pompili et al. described that contrastenhanced US resulted in diagnostic agreement in 53/56 cases (94.6%), with sensitivity and specificity of 87.0% and 98.4%, respectively, after nonsurgical treatments for HCC (PEI, RFA, TACE, TACE followed by PEI, RFA followed by PEI), compared with contrast-enhanced CT findings. 100 mentioned above, the diagnostic ability with the assessment of therapeutic response in HCC is now sufficient, and is equivalent to contrast-enhanced CT findings.Although there are some limitations that the performance of US examination depends on the operator's skill, location of the tumor and system capability, contrast-enhanced US would play a major role in evaluation of the therapeutic effect of HCC.The recent developments in this technology would allow contrast-enhanced US to be positioned as the standard method for evaluation of the therapeutic effect in many HCC patients.
In conclusion, US has made amazing strides in the last decades because of digital technology progress, and it will continue to grow.The advancement of imaging methods is expected to support the clinical management of patients with HCC.

(c). Contrast-enhanced US with Sonazoid
Hepatic lesion which corresponded to CT finding was demonstrated on contrast-enhanced US image with Sonazoid (arrow).deposition that affects the evaluation of contrastenhanced CT findings.Minami et al. reported that contrast-enhanced harmonic US with Levovist had a significantly higher sensitivity in depicting residual blood flow in HCC about one week after TACE than dynamic CT and dynamic MR imaging: contrastenhanced US: 38 of 44 (86%) lesions, dynamic CT: 19 of 44 (43%) lesions, dynamic MR imaging: 10 of 20 (50%) lesions. 98They also added that the contrast-enhanced US findings are predictive for local recurrence of HCC in the clinical course after

Figure 3 (
Figure 3 (a).HCC (S8, 29.4 mm), local recurrence of HCC after PEI B-mode USTreated area of HCC after PEI (arrows)There was no identification whether the tumor was viable or not.

Figure 4 (
Figure 4 (a).Detection of ultrasonically invisible hypervascular HCC using contrast-enhanced US with Sonazoid a) Contrast-enhanced CT Hypervasuclar lesion which was local recurrence of HCC was observed on contrast-enhanced CT image (arrow).
Maruyama et al.Contrast-Enhanced Ultrasonography: A Recent Application for the Diagnosis and Treatment... detection or characterization of liver tumors.In contrast, Definity and SonoVue do not accumulate in the liver.The characterization of liver tumors by contrast-enhanced US has been carried out using accumulation images as well as vascular enhancement images (Figure 2A, B).
Maruyama et al.Contrast-Enhanced Ultrasonography: A Recent Application for the Diagnosis and Treatment...Meanwhile, as neither Definity nor Sonovue accumulate in the liver, they do not provide liverspecific phase which is useful to detect occult tumor nodules (blood-pool contrast agents).However, previous study showed that contrast-enhanced US with Definity improved the detection rate of hepatic tumors in rabbit liver.

Table 1 . US contrast agents (quotation from World J Gastroenterol 2008;21:1710-9)
Diameter of microbubble (μm).Numbers in parentheses are mean diameters.†: Prior manufacturer ‡: Echo Gen is the first phase shift US contrast agent which has a boiling point significantly below body temperature.It has two different conditions, a liquid at room temperature (non echogenic particles with a mean diameter of approximately 0.4μm) and a gas at body temperature (echogenic microbubbles with a diameter of 2-5μm) *:

Table 2 . Characterization of focal hepatic lesions by contrast-enhanced US
: Discrimination of benign and malignant lesions †: Specific diagnosis: Correct diagnosis was obtained by contrast-enhanced US ‡: Confirmatory imaging technique: The reader judged that no further imaging for lesion characterization was needed and that the lesion concerned had been correctly diagnosed.§: Evaluation by two different readers Maruyama et al.Contrast-Enhanced Ultrasonography: A Recent Application for the Diagnosis and Treatment... *

Table 3 . Assessment of therapeutic response after radiofrequency ablation (RFA) for HCC using contrast-enhanced US
*: Comparison with contrast-enhanced helical CT †: 1-month follow-up CT