Contrast Enhanced Harmonic Endoscopic Ultrasound (CEH-EUS) in Focal Pancreatic Masses
Contrast Enhanced Harmonic Endoscopic Ultrasound (CEH-EUS) in Focal Pancreatic Masses
The aim of the study is to assess the accuracy of real-time perfusion imaging pattern of
 pancreatic focal lesions visualized by contrast-enhanced harmonic endoscopic ultrasound
 (CEH-EUS) for the differential diagnosis between chronic pseudotumoral pancreatitis and
 pancreatic cancer in a prospective multicenter design.

 The study will include patients with focal pancreatic masses evaluated by CEH-EUS and
 EUS-FNA. The diagnosis is usually unknown in the moment of the initial evaluation, the
 patients being included based on a suspicion of focal pancreatic masses after transabdominal
 ultrasound, CT or MR examinations. However, after a complete evaluation, a final diagnosis
 will be reached based on the combination of EUS-FNA cytology/pathology, surgical pathology
 and minimum 12 months follow-up.
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 120
 seconds).

 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
 pancreas examinations.

 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

 CEH-EUS procedure

 -  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
 pre-established presets.

 -  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.

 EUS-elastography procedures

 -  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
 contrast-enhanced EUS.
Inclusion Criteria:

 -  Patients with suspicion of solid pancreatic tumor masses by previous cross-sectional
 imaging techniques (US, CT, MR)

 -  Age 18 to 90 years old, men or women

 -  Signed informed consent for EUS with contrast-enhancement, elastography and EUS-FNA

 Exclusion Criteria:

 -  Prior surgical treatment with curative intent or chemo-radiotherapy

 -  Patients diagnosed with mucin producing tumors, pancreatic cystic tumors, etc.