ORIGINAL ARTICLE

Contrast-enhanced intraoperative ultrasound for hepatocellularcarcinoma: high sensitivity of diagnosis and therapeutic impact

Yusuke Mitsunori • Shinji Tanaka • Noriaki Nakamura •

Daisuke Ban • Takumi Irie • Norio Noguchi •

Atsushi Kudo • Hiroko Iijima • Shigeki Arii

Published online: 8 March 2012

� Japanese Society of Hepato-Biliary-Pancreatic Surgery and Springer 2012

Abstract

Background Postoperative early recurrence is a crucial

issue in the treatment of hepatocellular carcinoma (HCC)

patients. Some early recurrences seem to occur from

minute tumors which were overlooked during both preop-

erative and intraoperative investigations. Therefore, it is

urgently necessary to increase detectability of minute

HCCs during operation. If they could be detected and

resected during surgery, the prognosis should be improved.

The purpose of this study is to investigate the usefulness of

contrast-enhanced intraoperative ultrasound (CEIOUS) for

the diagnosis and treatment of HCC.

Methods Institutional ethics committee approval and

informed consent were obtained. Fifty-two patients (mean

age 65 years; 38 males and 14 females) who underwent

liver resection with either preoperative computed tomog-

raphy during angiography (CTA) or CEIOUS with Sona-

zoid (perflubutane microbubble contrast agent) were

studied. We determined the presence of HCC on the basis

of the histopathological findings of resected specimens.

Results The sensitivity of CEIOUS [97.6% (95% CI

91.8–99.4)] was higher than that of CTA [89.4% (95% CI

81.1–94.3)]. The positive predictive values of CEIOUS

[91.2% (95% CI 83.6–95.5) and CTA [91.6% (95% CI

83.6–95.9)] were similar. Eight new HCCs from 7 patients,

which accounted for 9.4% (8/85) of the total HCCs, were

correctly detected and diagnosed by CEIOUS, and we

performed an additional partial hepatectomy in 3 of these 7

patients.

Conclusions CEIOUS with Sonazoid provided increased

sensitivity of detection of small HCCs compared with

preoperative CTA, thereby leading to a more appropriate

surgical procedure and contributing to complete tumor

removal.

Keywords Hepatocellular carcinoma � Computed

tomography during angiography � Contrast-enhanced

intraoperative ultrasound � Microbubble contrast agent �Liver resection

Introduction

A high incidence of postoperative recurrence is a crucial

problem in the treatment of hepatocellular carcinoma

(HCC) [1, 2]. A major portion of these recurrences occurs

in the liver remnant, as a result of either intrahepatic

metastasis or multi-centric occurrence [3]. The prognosis

for patients with early recurrences was worse than that for

patients with late recurrences [4]. Some early recurrences

appear to occur from minute tumors which were undetected

during both preoperative and intraoperative conventional

examination. If they could be detected and resected during

surgery, the prognosis should be improved.

Up to now, among the preoperative diagnostic imaging

options for HCC, computed tomography (CT) during

angiography (CTA) is considered to be one of the most

accurate diagnostic modalities because two different pha-

ses of CTA, CT during arterial portography (CTAP) and

Y. Mitsunori � S. Tanaka � N. Nakamura � D. Ban � T. Irie �N. Noguchi � A. Kudo � S. Arii (&)

Department of Hepato-Biliary-Pancreatic Surgery,

Tokyo Medical and Dental University,

1-5-45 Yushima, Bunkyo-ku, Tokyo 113-8519, Japan

e-mail: ars.msrg@tmd.ac.jp

H. Iijima

Division of Hepatobiliary and Pancreatic Diseases,

Department of Internal Medicine,

Hyogo College of Medicine, Hyogo, Japan

123

J Hepatobiliary Pancreat Sci (2013) 20:234–242

DOI 10.1007/s00534-012-0507-9

CT during hepatic arteriography (CTHA) reflect the char-

acteristic blood flow in the HCC nodule accurately [5–8].

Therefore, we perform CTA preoperatively, and plan

treatment strategies for the HCC patients based on the

CTA, even though CTA is a complicated and invasive

technique.

On the other hand, intraoperative ultrasound (IOUS),

which is routinely performed for liver surgery, is an

indispensable technique, although it is limitated for diag-

nosis of minute HCC [9–11]. In fact, we have sometimes

found that a small tumor, which was detected by preop-

erative CTA, could not be detected by IOUS. Therefore,

novel techniques, devices and inventions are needed for

greater intraoperative diagnostic accuracy.

Recently, remarkable progress has been recognized in

contrast- enhanced US (CEUS) using microbubble agents

[12–15]. In particular, DD723/NC100100 (Sonazoid:

Daiichi-Sankyo, Tokyo, Japan) is a unique agent approved

for clinical use in Japan since January 2007. Sonazoid,

which consists of perflubutane-based microbubbles, reveals

the tumor clearly in two phases: vascular-phase images and

Kupffer-phase images [16–19]. In the vascular-phase

images, the tumor vasculature is clearly visualized in real

time, and in the Kupffer-phase images, the tumor is

delineated as an area of low intensity, contrasting well with

the non-tumorous parenchyma [20–22]. The innovation in

CEUS described above has led us to employ intraoperative

CEUS (CEIOUS) for liver surgery.

In the present study, we have investigated the degree of

added value of CEIOUS compared with CTA in HCC

patients.

Methods

Patients

This study was performed with the approval of the insti-

tutional ethics committee, and informed consent for the use

of their images and resected specimens was obtained from

all patients. Fifty-two patients with HCC underwent

preoperative CTA between August 2007 and December

2008. All of them also underwent CEIOUS with Sonazoid

as well as resection of the HCC. The 52 patients included

38 males and 14 females with a mean age of 65 years

(range34–81 years).

Preoperative CT during angiography

CTA was performed using a helical CT scanner (Xvision

SP: Toshiba Medical, Tokyo, Japan), enhanced with a non-

ionic contrast medium (Omnipaque: Daiichi-Sankyo). The

scanning was performed at intervals of 7 mm, with pre-

and postcontrast triple-phase (CTAP, early and late

CTHA). On CTAP, after inserting a 4-Fr catheter into the

superior mesenteric artery, 80 ml of contrast medium,

diluted two times with saline, was injected through the

catheter at a rate of 2 ml/s. The scanning was started 35 s

after the injection. On CTHA, after catheter insertion into

the proper hepatic artery, 30–50 ml of the medium diluted

three times with saline was injected at a rate of 0.7–1.2 ml/s.

Scanning was started 20 and 30 s after the injection.

Lesions that showed clear perfusion defects on CTAP,

and early enhancement but late wash out on CTHA, were

diagnosed as HCC with consensus reading. A lesion was

diagnosed as a hypervascular pseudolesion such as an

arterio-portal shunt and was not counted in the present

study when it showed a perfusion defect on CTAP and

enhancement on the early scan of CTHA but did not show

corona enhancement on the late scan [23].

The liver surgery was performed about 1 month (mean

29 days; SD 15.95 days; range 2–72 days) after the CTA

examination.

Intraoperative US and liver resection

Both IOUS and CEIOUS examinations were performed

using a US imaging system (Xario-XG: Toshiba Medical)

and a T-shaped linear probe (PLT-705BTH [7 MHz]:

Toshiba Medical).

On laparotomy, the liver was mobilized away from the

diaphragm and IOUS was performed in a systematic

fashion in baseline fundamental mode scan. On IOUS, low

or high echoic lesions, which had been detected on pre-

operative CTA, were diagnosed as HCC and resected.

CEIOUS was performed with pulse inversion harmonic

(PIH) imaging capability. A bolus intravenous injection of

Sonazoid (0.5 ml/body) was administered via the periph-

eral venous line, followed by a 10-ml normal saline flush.

In vascular phase imaging (0–3 min after the injection),

vascularity of the main tumor was observed. In Kupffer

phase imaging (10 min after the injection), a systematic

scan was performed and defects or low intensity lesions,

which had been detected on preoperative CTA, were

detected as HCC. When new lesions, which had not been

detected on CTA, were revealed by CEIOUS, we per-

formed a defect reperfusion imaging technique with an

additional injection of Sonazoid (0.5 ml/body) [24]. When

arterial vascularity was found inside these lesions, we

designated them as HCC. Furthermore, we performed

fundamental IOUS again to confirm these lesions.

The numbers of HCCs, identified by CTA, IOUS and

CEIOUS, were counted and mapped on a liver schematic

chart, according to the Couinaud classification. In short, we

resected all the lesions as below: (1) detected as HCC by

CTA, and detected on IOUS or CEIOUS; (2) not detected

J Hepatobiliary Pancreat Sci (2013) 20:234–242 235

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by CTA but detected as HCC on CEIOUS. The IOUS and

CEIOUS scans and image analyses were reached by con-

sensus between the surgeons.

Histological diagnosis

After resection, we scanned the specimens with US and

finger palpation carefully, and sliced through the center of

the tumors. Hematoxylin and eosin staining of the tissue

slices was performed for all liver specimens. The histo-

logical diagnosis was made by experienced pathologists at

our institution.

Statistical analysis

We made a flowchart of resected lesions which were

detected as HCC on CTA, IOUS and CEIOUS, respec-

tively, then investigated the number of lesions detected on

each of the modalities and the existence of HCC diagnosed

on the basis of the histopathological findings of the

resected specimens. Using the results of these data, tables

of detection and diagnosis by CTA and CEIOUS were

made. We determined the true positives and false positives

on the basis of the histological findings of resected speci-

mens. It was impossible to determine the complete true

negatives and false negatives because the histological

findings of the liver remnant were unknown, but the true

and false negatives on the resected specimens could be

calculated. Therefore, we defined the false negatives and

calculated the sensitivity based on the resected specimens.

We calculated the sensitivity and positive predictive value

for CTA and CEIOUS, respectively, and compared them.

However, comparison between the diagnostic capability of

CTA and CEIOUS was difficult because the surgeon could

not be blinded to the results of preoperative CTA. In

summary, the present study indicates a comparison

between the diagnostic capability of CTA with CEIOUS

and that of CTA without CEIOUS. The 95% confidence

interval was considered to indicate statistical significance.

Results

The number of lesions detected as HCC by preoperative CTA

was 83 nodules. On IOUS, 78 of these 83 nodules were

detected and 5 were not detected. On CEIOUS, 76 out of 78

lesions were detected on IOUS, and 5 nodules, which were not

detected on IOUS, were detected. Figure 1 shows a flow chart

of lesions detected by preoperative CTA and their histopa-

thological results. A typical case of HCC detected on CTA and

CEIOUS, but not detected on IOUS, is shown in Fig. 2.

We detected 10 new lesions as HCC in 9 patients by

CEIOUS. These 10 lesions had not been detected by CTA.

We could confirm only one of these 10 lesions on the

fundamental IOUS performed after CEIOUS. Figure 3

shows a flow chart of new lesions detected on CEIOUS and

their histopathological results. Finally, we intraoperatively

detected and resected 93 nodules as HCC, and one nodule

was incidentally detected and diagnosed as HCC on the

resected specimen. Out of 94 resected lesions, 85 were

histopathologically confirmed as HCC. Of the 9 resected

lesions which were misdiagnosed as HCC, 1 was von

Meyenburg complex, 1 was malignant lymphoma, and 7

were cirrhotic nodules.

Fig. 1 Flowchart of the lesions

detected on preoperative CTA.

?, lesions detected as HCC; -,

lesions not detected as HCC; *a,

five lesions which were not

detected on IOUS were detected

on CEIOUS. Of these lesions, 3

HCCs were diagnosed

histopathologically. *b, Only 1

HCC was not detected on

CEIOUS

236 J Hepatobiliary Pancreat Sci (2013) 20:234–242

123

Fig. 2 Images in a 70-year-old man with hepatitis C and HCC. a A

large HCC in the right lobe and a small one at S3 on preoperative

CTA. b On IOUS with fundamental mode, no nodule was detected at

S3. c A small defect detected on the Kupffer phase image at S3.

d Vascularity inside the lesion on the defect re-perfusion image.

e Small, well-differentiated HCC confirmed by histopathological

diagnosis

Fig. 3 Flowchart of the new

lesions detected on CEIOUS

with Sonazoid. ?, lesions

detected as HCC; -, lesions not

detected as HCC; *a, only 1 of

these 10 lesions was confirmed

on IOUS; *b, Eight HCCs

which accounted for 9.4%

(8/85) of the total HCCs were

correctly detected and

diagnosed by CEIOUS

J Hepatobiliary Pancreat Sci (2013) 20:234–242 237

123

The 8 HCC nodules not detected by CTA, accounting for

9.4% (8/85) of the total HCCs, were detected and diagnosed

by CEIOUS. These new nodules were detected as minute

defects on the Kupffer phase imaging and vascularity inside

the lesions on defect re-perfusion imaging. A typical example

of a new HCC detected on CEIOUS, but not detected on CTA

and IOUS, is shown in Fig. 4. Table 1 shows a summary of the

HCCs which were detected by these three modalities.

On the other hand, 2 new lesions from 2 patients were

over-diagnosed by CEIOUS. These lesions were detected

as defects on Kupffer phase imaging and also showed

slight vascularity inside the lesions. The eventual patho-

logical diagnoses of these lesions were 1 von Meyenburg

complex and a cirrhotic nodule. The sizes of these lesions

were 0.4 and 0.3 cm respectively, on the CEIOUS images.

Only 1 HCC was detected on CTA but not detected on

CEIOUS. This lesion was observed as a high-echoic lesion

on IOUS, but as iso-echoic on the Kupffer phase image of

CEIOUS (Fig. 1).

The numerical analysis is summarized in Tables 2, 3

and 4. The sensitivity of CEIOUS [97.6% (95% CI

91.8–99.4)] was higher than that of CTA [89.4% (95% CI

81.1–94.3)]. The positive predictive values of CEIOUS

[91.2% (95% CI 83.6–95.5)] and CTA [91.6% (95% CI

83.6–95.9)] were similar.

Fig. 4 Images in a 63-year-old man with hepatitis C and HCC. a No

HCC was detected at lateral segment on preoperative CTA. b A small

defect detected on the Kupffer phase image at S2. c Vascularity inside

the lesion on the defect re-perfusion image. d On IOUS with

fundamental mode, no lesion was detected at S2. e Small, moderately

differentiated HCC confirmed by histopathological diagnosis

Table 1 All detected HCC among the 3 modalities

CTA IOUS CEIOUS Number Histological size,

cm (median)

Positive Positive Positive 72 0.5–14.0 (3.0)

Positive Positive Negative 1 0.3

Positive Negative Positive 3 0.5–0.8 (0.8)

Positive Negative Negative 0 ––

Negative Positive Positive 1 1.0

Negative Positive Negative 0 ––

Negative Negative Positive 7 0.4–1.3 (0.7)

Negative Negative Negative 1 0.7

238 J Hepatobiliary Pancreat Sci (2013) 20:234–242

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As an alternative surgical procedure for the 10 new

lesions on CEIOUS, we added partial hepatectomy for 5 of

the 9 patients. Three of these 5 patients received appro-

priate additional hepatectomy to resect the newly discov-

ered HCCs (Table 5).

Discussion

With recent advances in diagnostic imaging, the complete

range of examinations for HCC has been gradually

changing. For the diagnosis of HCC, dynamic multidetec-

tor computed tomography (MDCT) and dynamic magnetic

resonance imaging (MRI) have been supported by the

American Association for the Study of Liver Diseases

(AASLD) guidelines recently. Moreover, it has been

reported that MRI with gadolinium-ethoxybenzyl-diethy-

lenetriamine pentaacetic acid (Gd-EOB-DTPA MRI) has a

high differential diagnostic capacity for HCC [25, 26].

CTA was considered the most sensitive test before the

introduction of MDCT and Gd-EOB-DTPA MR, and it is

still one of the most sensitive modalities for HCC [7]. On

the other hand, it has been reported that small lesions might

be missed frequently despite the remarkable progress in

diagnostic modalities [27, 28].

Ultrasound is the first option for screening liver nodules

as it is convenient and noninvasive, although it is not

appropriate for making a final diagnosis of HCC. IOUS is

superior to transcutaneous US in higher spatial resolution

and small blind spots, though it lacks detailed information

about blood flow in the tumor. In this regard, the advan-

tages of CEIOUS are high spatial resolution, small blind

spots and the visualization of small-scale blood flow by

microbubbles [29–31]. Furthermore, CEIOUS with Sona-

zoid shows high and sharp contrast between a tumor and

the non-tumorous liver parenchyma in the Kupffer phase

images, as Kupffer cells in a tumor are generally reduced in

number [32–34]. These characteristics of the Kupffer phase

images of CEIOUS with Sonazoid are expected to con-

tribute to the detection of small lesions and its high sen-

sitivity. As mentioned above, we have focused on the

usefulness of CEIOUS in this study, and therefore this

might explain why there was no detection of new lesions

on IOUS, regardless of previous articles, which have

reported that new HCCs were detected not only on CE-

IOUS, but also on IOUS [30, 35].

With respect to the introduction of CEIOUS, as IOUS is

already routinely performed and has assumed an indis-

pensable role in liver surgery already, only additional

Table 2 The number of lesions on preoperative CT during

angiography

HCC Not HCC Total

CTA positive 76 7 83

CTA negative 9 2 11

Total 85 9 94

Table 3 The number of lesions on contrast-enhanced intraoperative

US with Sonazoid

HCC Not HCC Total

CEIOUS positive 83 8 91

CEIOUS negative 2 1 3

Total 85 9 94

Table 4 Statistical analysis of sensitivity and positive predictive

value

CTA CEIOUS

Sensitivity (%) 89.4 97.6

(95%CI) (81.1–94.3) (91.8–99.4)

Positive predictive value (%) 91.6 91.2

(95%CI) (83.6–95.9) (83.6–95.5)

Table 5 Alteration of the surgical procedure after the discovery of new HCCs on CEIOUS

Patient Location of the

new lesions

Planed operation Modification

of operation

Histopathology of the new lesions

1 Different segment S8 partial hepatectomy ?S4 partial hepatectomy 0.7 cm well-differentiated HCC

2 Different segment Left hepatectomy No 0.7 cm poorly differentiated HCC

3 One of multiple Extended right hepatectomy S5/6/8 hepatectomy 0.9 cm well-differentiated HCC

4 Same segment S8 segmentectomy No 1.0 cm poorly differentiated HCC

5 Same segment Posterior segmentectomy ? S4

partial hepatectomy

No 0.5 cm moderately differentiated HCC

6 Different segment Right hepatectomy ?S2 partial hepatectomy 0.4 cm moderately differentiated HCC

7 One of multiple Anterior and medial segmentectomy ?

S2/3 partial hepatectomy

?S3 partial hepatectomy 0.5 cm well-differentiated HCC; 1.3 cm

moderately differentiated HCC

J Hepatobiliary Pancreat Sci (2013) 20:234–242 239

123

contrast agents would be required. The implementation of

routine CEIOUS is a feasible and cost-effective approach.

The present study has shown that the sensitivity of

CEIOUS is superior to that of CTA and that the positive

predictive value is similar. Although CEIOUS has an

advantage in the detection of nodules owing to the already-

known information from preoperative CTA, 8 new HCCs

were detected on CEIOUS. This detection resulted in a

higher sensitivity measurement for CEIOUS. We per-

formed CTA at 7-mm intervals although 0.3–0.5-cm HCCs

were detected on CTA (Table 1). Of new HCCs on CE-

IOUS, although the width of the CTA slice intervals might

result in some minute foci being overlooked, 5 out of the 8

tumours detected were more than 7 mm in diameter

(Table 5). The largest one was 1.3 cm in diameter, which

was small nodular type with an indistinct margin. It is

thought that the main reason why 8 new HCCs were

detected on CEIOUS is that these tumors were clearly

delineated with Kupffer phase imaging as shown in

Fig. 4b. A focal liver lesion showing hypo-intense Kupffer

images such as SPIO-MRI and CEUS is potentially HCC

regardless of vascularity [34]. Namely, in the detection of

some HCCs, Kupffer phase imaging might be superior to

CTA which depends on the hemodynamics of the tumors,

in spite of their histopathological differentiation. The

Kupffer phase imaging of CEIOUS with Sonazoid seems to

be useful for HCC detection.

With respect to the postoperative follow up, it was only

posible to follow up 34 patients at our institution. We

identified early recurrences in the remnant liver in 2

patients out of the 34, and assumed that they were residual

tumors. These recurrences were diagnosed on days 88 and

84 post-operatively respectively by dynamic CT. Although

the beneficial effects on recurrence-free and overall sur-

vival cannot be clarified yet because of the short observa-

tion period, more postoperative early recurrences have

developed in patients who did not receive CEIOUS.

The only HCC detected by CTA and IOUS but not

detected by CEIOUS was observed as a high-echoic lesion

on IOUS and iso-echoic on the Kupffer phase imaging.

These images might be the result of fatty degenerative

changes and the abundant Kupffer cells in the well-dif-

ferentiated HCC. Novel methods of suppression of high-

echoic signals from fatty tissue are required for more

accurate diagnosis on Kupffer phase imaging but we do not

have such methods yet.

We misdiagnosed 8 lesions as HCC on CEIOUS. In

general, differentiation of HCC from cyst, hemangioma or

arterio-portal shunt is made easy by visualizing the

behavior of the blood flow in the indicated foci using a

defect re-perfusion imaging method. However, it is diffi-

cult in some cases to evaluate precisely the blood flow

inside a minute lesion. In the present study, we tentatively

detected such minute lesions as HCC and resected them.

However, 6 out of these 8 lesions were cirrhotic nodules

with no tumor. Due to experiencing these false-positive

cases described above, we have recognized that some cir-

rhotic nodules are seen as low intensity on Kupffer phase

imaging of CEIOUS, and the main reason for the misdi-

agnoses of these minute foci as HCCs was an overesti-

mation of the blood flow in the foci. Greater experience

will lead to better differential diagnoses, so that the number

of false-positive cases will decrease over time. We have

resected all such lesions to obtain histopathological con-

firmation; however, it may be possible to adapt radiofre-

quency ablation for the treatment of these minute foci

depending on the location of the tumors. In this study, there

was no nodule which did not show apparent vascularity in

the early phase but did show hypo-intensity in the Kupffer

phase on CEIOUS. As mentioned above, an incorrect or

overestimation of the vascularity might produce this result.

We could not calculate the true negative ratio or the

specificity of CEIOUS for the whole liver because it is

impossible to obtain histological confirmation of the rem-

nant liver. In the present study setting, we could not cal-

culate the false negatives for the whole liver either, but the

false negatives on the resected specimens could be calcu-

lated. In fact, 2 HCCs not detected on CEIOUS and 8

HCCs not detected on CTA, were diagnosed histopatho-

logically as false negatives. We calculated the sensitivity in

this way, because it is obvious that a higher sensitivity

leads to better treatment results for HCC.

One of the drawbacks of the present study is its retro-

spective nature. It would be ideal to perform CTA or

dynamic CT and CEIOUS in all consecutive patients, and to

decide the eligible criteria strictly. Furthermore, it would be

favorable for the study if the surgeon using CEIOUS did not

have any information regarding the preoperative CT study.

However, CEIOUS is usually performed in patients who

have undergone CT study. Therefore, this kind of prospec-

tive study is not possible in the ordinary clinical situation.

The outstanding advantage of CEIOUS is to provide an

immediate impact on surgical procedures, in addition to

improved detection of small tumorous lesions [35].

Because there is usually a time lag between the preopera-

tive examination and surgery, it is beneficial to confirm

minute tumors at surgery. The important aim of CEIOUS in

the clinical setting is the prolongation of recurrence-free

survival and overall survival of HCC patients.

In conclusion, CEIOUS with Sonazoid is an indispensable

diagnostic modality for HCC patients, especially in con-

junction with another modality such as CTA. It contributes to

complete tumor resection, and may also lead to a better

prognosis for HCC patients, although further study is needed.

240 J Hepatobiliary Pancreat Sci (2013) 20:234–242

123

Conflict of interest We have received financial support from Health

and Labour Sciences Research Grants under the study ‘‘Development

of early detection systems of liver cancer using molecular markers

and diagnostic imagings’’. We have no commercial sponsorship

sources to disclose.

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