CONTRAST-ENHANCED ULTRASONOGRAPHY WITH

PERFLUBUTANE MICROBUBBLES IMPROVES

DETECTION OF EARLY NASH-HCC RELATED IN
EXPERIMENTAL MODEL.

Lima VMR1, Carvalho CF2, Cogliati B3, Stefano JT1, Carrilho FJ1,
Chammas MC2, Oliveira CPMS1

1
Department of Gastroenterology (LIM-07), 2Institute of Radiology - University of São Paulo
School of Medicine, São Paulo, Brazil
3
Department of Pathology, School of Veterinary Medicine and Animal Science - University of
São Paulo School of Medicine, São Paulo, Brazil

2011
 Introduction
• Hepatocellular carcinoma (HCC) is the most common
cancer in human beings and is a recognized complication of
advanced non-alcoholic steatohepatitis (NASH)1.
• Early detection of focal hepatic lesions is a challenge for
both clinical routine and ultrasound (US) scanning, and
becomes even greater in the presence of a diffuse
parenchymal disease.
• In spite of that, US is still the first choice for the screening of
these diseases2.
1
LIMA et al., Journal of Hepatology. v.49, n.6, p.1051-61, 2008.
2
WANG et al., Journal of Gastroenterology and Hepatology. V.23, 1511-1519, 2006
 Introduction
• Elastography has emerged as a new method
to evaluate stiffness of focal lesions and
promises to differentiate their malignancy3.
• Contrast-enhanced ultrasonography (CEUS)
with microbubbles improves the diagnostic
accuracy to differentiate benign from
malignant focal liver lesions4.
3
KONOFAGOU et al., 2003 Summer Bioenfineering Conference, June-25-29, Sonesta Beach Resort in Key Biscayne, Florida.
4
MORIYASU et al., American Journal of Radiology; 193:86-95, 2009.
 Objective

 To evaluate the diagnostic performance of

elastography and CEUS for early detection of

malignant hepatic nodules in an experimental rat

model1 of NASH.

1
LIMA, et al., A rodent model of NASH with cirrhosis, oval cell proliferation and hepatocellular carcinoma. Journal of Hepatology. v.49, n.6, p.1051-61, 2008.
 Methods
• Equipment: model Aplio XG, Toshiba (Tokio, Japan);
• Using 12 MHz linear transducer;
• The exams were performed under anesthesia with
ketamine hydrochloride (0.2 mg/kg intraperitoneal).
• two animal groups were compared (control and
experimental), each one made up of 10 animals. All
data were recorded.
• All animals were euthanized after the last
examination and samples were obtained to
histological analysis. Afterwards we compared the
elastography and CEUS results with histological
samples.
 Methods
Control group: 10 normal adult
Sprague-Dawley rats.
• B-mode was performed in order to
detect abnormalities.
• Color Doppler US was performed
to evaluate the vascular
architecture and exclude any
vascular malformation.
• Elastography and CEUS were
done to analyze liver parenchyma.
 Methods
Experimental group: 10 adult
Sprague-Dawley rats were housed
in a controlled temperature,
humidity and ventilation
environment.
• Provided unrestricted amounts of
food (a coline-deficient and high-fat
diet) and water with
dyethilnitrosamine (135 mg/l)
• B-mode and Doppler
ultrasonography was performed
weekly in all experimental rats until
nodule detection.
 Methods

Elastography
• Tissue stiffness of the nodules on
elastography were classified in negative
(elastic strain) or positive (hard and no
strain) comparing with surrounding liver
parenchyma.
 Methods
CEUS
• Contrast agent was Definity®
(Lantheus) (diluted in 1:5 of saline
solution).
• Internal jugular vein was catheterized
in order to inject contrast agent (0.1 mL
of diluted contrast) .
• In sequence, 1 mL of saline solution
was injected. The criteria studied were:
type of enhancement in arterial (2”- 5”
after injection), portal and late phases
(after 5” seconds).
 Methods

Criteria of focal lesions according to type of

enhancement and wash-out

Type of lesion ARTERIAL PORTAL/ LATE

PHASE PHASE
MALIGNANT EARLY WASH- WASH-OUT
IN (intense or
discrete)
or NOT
BENIGN WASH-IN or WITHOUT
NOT WASH-OUT
Methods
 Methods

• After euthanasia samples of the liver were

assessed by microscopy.
 Methods
- Liver tissue was assessed grossly, samples were fixed in
4% formaldehyde and processed for hematoxylin-eosin (HE)
and Sirius red staining for histological analysis.

- Variables were blindly scored by an experienced

hepatopathologist using a modified scoring system adapted
from the NASH activity score: macrosteatosis (0-3), lobular
inflammatory changes (0-3), hepatocyte ballooning (0-2)
and fibrosis scored as portal and perivenular (stages 0-4)5.

- Hepatocellular carcinoma was classified according to

Edmondsin and Steiner as recently re-assessed by Kojiro6,7.
5
Kleiner et al. Hepatology 2005; 41:1313-1321.
6
EdmondonH, Steiner PE. Cancer 1954;7:462-503.
7
KojiroM. Advanced hepatocellular carcinoma. In: Pathology of hepatocellularcarcinoma. Malden, USA: Blackwell Publishing;2006.p.77-96, Chapter6.
 Methods

Statistical Analysis
• All data were expressed as median and
within a range.
• All p values were two-tailed, and p < 0.05
was considered statistically significant.
• Sensitivity and specificity were calculated.
 Results

• In the control group samples from all rats

were obtained.
• Experimental group: 3 rats died before the
end of study (during anesthetical procedure).
• We studied 10 nodules in 7 rats.
 Results
TABLE 1: US results compared with histological analysis

Elastograph
Samples y Wash-in Wash-out Histology
A + + + +
B + - + +
C - - - -
D - - - -
E + + + +
F + + + +
G + + - +
H + - + +
I + + + +
J - - + -

(+) positive for malignancy; (-) negative for malignancy

 Results
TABLE 2: sensitivity and specificity of malignancy detection of imaging methods

Sensitivity Specificity
Method
Estimat Estimat Confidence
e Confidence interval e interval
value 95% Value 95%

Elastograph
y 1.00 0.47-1.00 0.90 0.19-1.00

Wash-in 0.71 0.29-0.96 1.00 0.19-1.00

Wash-out 0.86 0.42-1.00 0.67 0.09-0.99

 Results

Method Estimated Confidence interval

Accuracy value 95%

Elastography 0.90 0.59 - 1.00

Wash-in 0.80 0.44 - 0.97

Wash-out 0.80 0.44 - 0.97

 Results

B-mode shows increased hepatic echogenicity and hypoechogenic and

heterogeneous hepatic nodule.
 Results

Color Doppler mapping shows vascularization of the suspicious nodule.

 Results

Late vascular phase, with wash out pattern suggesting malignancy

 Results

HCC vascularized nodule

Congested and dilated vessels in the nodule;

Classified as moderately differentiated HCC (type II)
 Results

HCC nodule

Pseudo-capsule of collagen;
Well-differentiated nodule of HCC (type I)
 Discussion
• As in all diffuse parenchymal disease, the diagnosis of focal
lesions in the advanced non-alcoholic steatohepatitis
(NASH) is a challenge for B-mode US.
 At first, nodule detection was searched by B-mode in our
study, but elastography and CEUS were decisive to confirm
the presence of nodule and to characterize malignancy.
 Elastography presented high sensitivity and specificity in this
experimental model. As far as we know there is no study in
the literature demonstrating the application of this type of
elastography in liver.
 Discussion
• In our research, considering the animal size and the facility
of compression on the liver, the results obtained applying
the qualitative real-time US elastography were very
promising. These successful results are not likely to be the
same whether performed in humans or in big animals.
• In these cases, the literature shows good results using
others technologies as transient elastography with acoustic
radiation force impulse (ARFI) and Fibroscan® to assess
diffuse liver disease8.
• On the other hand elastography provided promising
perspectives for the assessment of malignancy of focal
hepatic lesions in our sample.
8
Takahashi, et al. Liver International 2010. 30:538-545
 Discussion
• The early wash-in in the arterial phase and overall
wash-out in the late vascular phase were strongly
suggestive of malignancy, as shown in many
studies9,10.
• Our results showed accuracy of 80% using CEUS in
order to differentiate between benign and malignant
nodules.
• In this casuistry, we observed 2 cases did not
present wash-in in the arterial phase, although they
showed wash-out in the late phase, which classified
as suspicious for malignancy.
9
Wilson S, Greenbaum LD, Goldberg B. AJR 2009; 193:55–60
10
O'Brien et al., VRU 2004; 45(6): 547–553.
 Discussion

• We had 1 false negative and 1 false positive

case.
• We can explain these vascular patterns
through 2 causes: vascular alterations
promoted by experimental model involved in
this study or because the false negative
nodule was a well differentiated HCC.
 Conclusion

• Elastography and CEUS allowed diagnoses with

high accuracy in this experimental rat model of

NASH.
• Further studies are needed in order to evaluate

the effectiveness of this method for diagnosis of

HCC in NASH in other species.
 References
1. LIMA, V.M.R.; OLIVEIRA, C.P.M.S.; ALVES, V.A.F.; CHAMMAS, M.C.; OLIVEIRA, E.P.; STEFANO,
J.T.; MELLO, E.S.; CERRI, G.G.; CARRILHO, F.J.; CALDWELL, S.H. A rodent model of NASH with
cirrhosis, oval cell proliferation and hepatocellular carcinoma. Journal of Hepatology. v.49, n.6,
p.1051-61, 2008.
2. WANG, Z.; TANG, J.; WESKOTT, H.P.; LI, J.; WANG, W.; LUO, Y.; AN, L.; XU J. Undetermined focal
liver lesions on gray-scale ultrasound in pattients with fatty liver: characterization with contrast-
enhanced ultrasound. Journal of Gastroenterology and Hepatology. V.23, 1511-1519, 2006
3. KONOFAGOU, E.E.; OPHIR, J.; KROUSKOP, T.A.; GARRA, B.S., 2003 Summer Bioenfineering
Conference, June-25-29, Sonesta Beach Resort in Key Biscayne, Florida.
4. MORIYASU, f.; ITOH, K. Efficacy of perflubutane microbubble-enhanced ultrasound in the
characterization and detection of focal liver lesions: phase3 multicenter clinical trial American Journal
of Radiology; 193:86-95, 2009.
5. Kleiner DE,Brunt EM, Van Natta M, Behling C, Contos MJ, Cummings OW et al. Nonalcoholic
steatohepatitisclinical research network. Design and validation of a histological scoringsystem for
nonalcoholic fatty liver disease. Hepatology. 41:1313-1321, 2005.
6. EdmondonH, Steiner PE. Primary carcinoma of the liver: a study of 100 cases among 48,900
necropsies. Cancer. 7:462-503, 1954.
7. KojiroM. Advanced hepatocellular carcinoma. In: Pathology of hepatocellularcarcinoma. Malden, USA:
Blackwell Publishing.p.77-96, Chapter6, 2006.
8. Takahashi H, Ono N, Egushi Y, Egushi T, et al. Evaluation of acoustic radiation force impulse
elastography for fibrosis staging of chrronic liver disease: a pilot study. Liver International. 30:538-545,
2010.
9. Wilson S, Greenbaum LD, Goldberg B. Contrast-Enhanced Ultrasound: What Is the Evidence and
WhatAre the Obstacles? American Journal of Radiology. 193:55–60, 2009.
10. O'Brien, R., Iani, M., Matheson, J., Delaney, F. and Young, K. Contrast harmonic ultrasound of
spontaneous liver nodules in 32 dogs. Veterinary Radiology & Ultrasound. 45: 547–553, 2004.
Thank you for your attention!