Contrast Enhanced Harmonic Endoscopic Ultrasound (CEH-EUS) Used in the Differentiation of Focal Pancreatic Masses
Ultrasound contrast agents in conjunction with contrast specific imaging techniques are
increasingly accepted in clinical use for diagnostic imaging. The study of the pancreas is a
new and promising application of contrast-enhanced ultrasound (CE-US), including
contrast-enhanced endoscopic ultrasound (CE-EUS). The technique is not indicated to improve
the detection of pancreatic lesions, but to improve the delineation and differential
diagnosis of pancreatic lesions. One of the fluoro-gas-containing contrast agents used in
CE-US and CE-EUS is Sonovue®, which consists of phospholipids-stabilized bubbles of
sulfurhexafluoride (SF6). Sonovue® is isotonic, stable and resistant to pressure, with a
viscosity similar to blood. It does not diffuse into the extravascular compartment remaining
within the blood vessels until the gas dissolves and is eliminated in the expired air (blood
pool contrast agent). The safety profile of SonoVue showed a very low incidence of side
effects; it is not nephrotoxic and the incidence of severe hypersensitivity is similar to
other magnetic resonance imaging contrast agents. Moreover, Sono-Vue is approved for
clinical use in EU countries. The blood supply of the pancreas is entirely arterial, making
contrast-enhanced examinations feasible and readily available. Based on the European
Federation Societies of Ultrasound in Medicine and Biology guidelines and recommendations,
updated in 2008, two phases were defined for CE-US and CE-EUS of the pancreas: an
early/arterial phase (starting from 10 to 30 seconds) and a venous/late phase (from 30 to
Distinguishing pancreatic adenocarcinoma from other pancreatic masses remains challenging
with current imaging techniques. The specificity of the discrimination between benign and
malignant focal pancreatic lesions was found to be 93.3% using power Doppler
contrast-enhanced EUS (PD-CE-EUS) compared with 83.3% for conventional EUS. The hypovascular
aspect of lesions under PD-CE-EUS seemed highly sensitive and specific (higher than 90%) for
adenocarcinoma in several published studies. During PD-CE-EUS examinations the ultrasound
frequency returned to the transducer is the same with that transmitted, but the method is
associated with artifacts resulting from turbulent flow (flash and overpainting). At CE-EUS,
ductal adenocarcinoma is typically hypoenhanced compared to the adjacent pancreatic tissue
in all phases. Furthermore, the lesion size and margins are better visualized, as well as
the relationship with peripancreatic arteries and veins. Focal lesions in chronic
pancreatitis are reported to have similar or hyper enhancement features as compared to the
normal pancreatic parenchyma.
Dedicated contrast-enhanced harmonic EUS techniques (based on a low mechanical index) are
recently available in new EUS systems. The harmonic frequencies returned during CEH-EUS are
different from those transmitted by the transducer and are the result of non-linear
oscillations of the microbubbles. The image obtained is an addition of the signal created by
the distortion of the microbubbles and the tissue-derived signal. This can be optimized by
using low MI, which allows minimum bubble destruction and complete "subtraction" of the
tissue derived signal, obtaining a high resolution continuous real-time assessment of the
microvascularization during the contrast uptake period (real-time perfusion imaging).
CEH-EUS allows a more precise location of vascular structures within the parenchyma and
focal abnormalities, with better delineation of pancreatic lesions than EUS, especially in
the cases where air or fat causes artifacts and insufficient visualization of the pancreatic
parenchyma. An initial pilot study described an experimental technique of CEH-EUS based on a
linear prototype EUS scope, a low mechanical index (0.08 - 0.25) and a 2nd generation
contrast agent (Sono-Vue), which allowed the visualization of early arterial phase and late
parenchymal phase enhancement of the pancreas. Another pilot study demonstrated both
real-time continuous images of finely branching vessels of the pancreas and intermittent
homogenous parenchymal perfusion images, by using a radial prototype EUS scope, a low
mechanical index (0.4) and a 2nd generation contrast agent (Sono-Vue). Several other
research groups already reported the feasibility of CEH-EUS with low mechanical index. The
sensitivity, specificity and accuracy for diagnosing pancreatic adenocarcinoma were 88%,
89%, and 88.5% in one study and 80%, 91.7%, and 86% in the other study. However, the data is
still limited and a prospective, multicentric blinded study would certainly be necessary.
The study design is prospective, blinded and multi-center, comparing contrast-enhanced
harmonic endoscopic ultrasound (CEH-EUS) results for the detection and characterization of
focal pancreatic masses, in comparison with the gold standard represented by pathology. The
study will be performed with the approval of the institutional board review of each center.
The study protocol will be uploaded on ClinicalTrials.gov, the registry of federally and
privately supported clinical trials conducted in the United States and around the world.
The study is already approved by the ethical committee of the University of Medicine and
Pharmacy Craiova, Romania (attached). According to EFSUMB (European Federation Societies for
Ultrasound in Medicine and Biology) guidelines published in 2008, second-generation contrast
agents are also approved in the E.U. for ultrasound examinations, including liver and
All patients with a suspicion (clinical, US, CT/MR) of pancreatic masses should undergo EUS,
contrast-enhanced harmonic EUS and EUS-elastography, as well as EUS-FNA
EUS with EUS-guided FNA
- Protocol of EUS with EUS-FNA should include linear EUS instruments with complete
examinations of the pancreas.
- Tumor characteristics (echogenicity, echostructure, size) will be described as well as
presence / absence of power Doppler signals.
- EUS-FNA will be performed in all pancreatic masses with at least three passes
- All examiners should be blinded for the results of pathology
- A two panel image with the usual conventional gray-scale B-mode EUS image on the right
side and with the contrast harmonic image on the left side will be used, according to
- A low mechanical index procedure (dynamic wide-band contrast harmonic imaging mode)
will be used, with a mechanical index of 0.08-0.25 and corresponding powers.
- The starting point of the timer will be considered the moment of intravenous contrast
injection (Sonovue 4.8 mL).
- CEH-EUS will be performed during usual EUS examinations, with the whole movie
(T0-T120s) recorded on the embedded HDD of the ultrasound system, for later analysis.
- In order to minimize human bias, all post-processing and computer analysis of digital
movies will be performed within the coordinating IT Center, with all programmers and
statisticians being blinded to the clinical, imaging and pathological data.
- An additional EUS elastography movie of 30 seconds should be saved on the embedded HDD.
- The following settings will be used for EUS elastography: examination frequency is
usually set at 7.5 MHz, while preinstalled system settings are used in all patients:
reject (2), E-smoothing (2), persistence (3), and E-dynamic range (4).
Final diagnosis The diagnosis of chronic pancreatitis will be based on the clinical
information (history of alcohol abuse, previous diagnosis of chronic pancreatitis or
diabetes mellitus), as well as a combination of imaging methods (ultrasound, CT and EUS). At
least four criteria of chronic pancreatitis during EUS will be considered for the positive
diagnosis. The diagnosis of chronic pseudotumoral pancreatitis will always be confirmed by
surgery or by a follow-up of at least six months used to exclude malignancy in the patients
that will not be operated on.
A positive cytological diagnosis will be taken as a final proof of malignancy of the
pancreas mass. The diagnoses obtained by EUS-FNA will be further verified either by surgery
or during a clinical follow-up of at least 6 months.
Pathology samples obtained from duodeno-pancreatectomies or caudal pancreatectomies done
with curative intent, as well as microhistological fragments obtained through EUS-FNA biopsy
will be processed by paraffin embedding with usual colorations (haematoxylin-eosin), with
subsequent immune-histochemistry at the discretion of the participating centers pathologists
in order to exclude neuroendocrine tumors / pancreatic metastases.
The patients will be followed-up for at least six months through clinical examination,
biological exams and transabdominal ultrasound, eventually with a repeat spiral CT / EUS
after six months.
Statistical analysis All results will be expressed as mean ± standard deviation (SD).
Differences between the patients with pancreatic cancer and chronic pancreatitis will
performed by the two-sample t-test (two independent samples). Since this parametric method
makes assumptions about normality and similar variances, we will initially perform both the
Kolmogorov-Smirnov and Shapiro-Wilk W normality tests and verify the equality of variances
assumption with the F test. In the case of the two-sample t-test, we will also perform the
non-parametric alternative given by the Mann-Whitney U test, since in some instances it may
even offer greater power to reject the null hypothesis than the t-test.
Since with more than two groups of observations it is far better to use a single analysis
that enables us to look at all the data in the same time, we will also perform the one-way
analysis of variance (ANOVA) method with the same baseline assumptions. A p-value less than
0.05 will be considered as statistically significant.
Sensitivity, specificity, positive predictive value, negative predictive value and accuracy
of CEH-EUS +/- will be determined in comparison with the final diagnosis.
The estimated number of patients included is at least 210, based on at least 10 centers with
at least 20 patients each, which will be enrolled in an 18 months period. The power analysis
was based on the following assumption: in order to use the powerful t-test for independent
samples, a sample size equaling 105 patients in each group is sufficient to provide 95%
statistical power to detect a difference of 5% in mean, for a type I error alpha = 0.05, and
the population standard deviation = 10%. The difference in mean was based on previously
published data which report an accuracy of approximately 80-85% for EUS-FNA, and 90% for
Observational Model: Case Control, Time Perspective: Prospective
Contrast enhanced harmonic endoscopic ultrasound
Value of contrast enhanced harmonic endoscopic ultrasound for the differential diagnoses of pancreatic masses.
Every six months
Adrian Săftoiu, Professor
University of Medicine and Pharmacy Craiova, Romania
Romania: Ministry of Education, Research, Youth and Sport