Clinical Impact of EUS Elastography Mean Strain Histograms (SH) and Contrast Peak-enhancement in Focal Pancreatic Masses and Lymph Nodes
Condition: Pancreatic Cancer; Secondary Malignant Neoplasm of Lymph Node; Benign Neoplasm of Lymph Nodes; Benign Pancreatic Tumors
MeSH terms: Neoplasms; Pancreatic Neoplasms
Study type: Observational

Summary: 
      The study protocol is based on a multi-center semi-quantitative approach of EUS elastography
      data in combination with contrast-enhanced EUS, consisting of measuring SR and SH for focal
      pancreatic masses and lymph nodes, as well as several parameters of CE-EUS based on
      time-intensity-curve (TIC) analysis. A number of parameters must be taken into consideration,
      as the ROIs are still manually selected by the user. The aim of the study is to establish an
      EUS based diagnostic algorithm in patients with pancreatic masses and lymph nodes, with
      negative or inconclusive cytopathology after EUS-FNA, based on previously published results
      and cut-offs of elastography and contrast-enhancement. The proposed algorithm of sequential
      use of real-time elastography, followed by contrast-enhanced EUS could be a good clinical
      tool to help select the patients with possible pancreatic adenocarcinoma or malignant lymph
      nodes, in the setting of patients with negative EUS-FNA results.
    

Detailed description: 
      1. Background Endoscopic ultrasound (EUS) is a technique with a major clinical impact in
      digestive diseases, determining a change in the diagnosis and management of more than half of
      examined patients [1]. Recent advances in EUS-FNA techniques, but also the development of
      real-time EUS elastography and contrast-enhancement, allowed a better characterization of
      focal pancreatic masses, with possible implications in the management of patients with
      negative EUS-FNA and a strong suspicion of malignancy.

      1.a Elastography Elastography is a recent ultrasound method used for the reconstruction of
      tissue elasticity distribution in real-time [2]. The method allows the calculation of the
      elasticity modulus, consequently showing differences in tissue hardness patterns that are
      determined by diseases. The main intended use is to differentiate between benign and
      malignant focal lesions based on the significantly smaller strain of the latter [3]. Second
      generation elastography introduces strain ratio (SR) and strain histogram (SH) as
      reproducible parametric measurements that retrieve numerical values in real time, adding
      quantification possibilities to the technique [4]. Elastography typically estimates the axial
      strain (along the direction of insonification / compression) by analyzing ultrasonic signals
      obtained with standard ultrasonographic systems - the RF signals returned from tissue
      structures before and after slight compression (about 1%) are compared [5]. Tissue
      elastography can be easily performed with conventional probes, including the linear EUS
      probes used for the examination of the pancreas and/or lymph nodes. The calculation of tissue
      elasticity distribution is performed in real-time under freehand compression and the
      examination results are represented as transparent overlay colour images overimposed on the
      conventional gray-scale B-mode images [6]. Thus, this method allows the characterization of
      many tumors, because they are stiffer than normal tissues. Ultrasound elastography was
      previously used for the diagnosis of non-digestive as well as digestive tumors: breast
      lesions [7], prostate cancer [8], thyroid nodules [9], rectal tumors [10]. Regarding the
      diagnosis of pancreatic focal masses, some authors could not differentiate between malignancy
      and benign tumors or chronic pancreatitis [11], while others have obtained good results,
      especially when using computer assisted means of evaluation like hue histogram analysis [12]
      and artificial neural networks [13]. More recently, lymph node involvement of several tumors
      has been succesfully determined using this method: esophagus [14], oral squamous cell
      carcinoma [15], breast cancer [16].

      Since elastography images and movies represent a qualitative type output that entails a
      subjective interpretation by the examiner, human bias is always susceptible to interfere with
      the results and diagnoses, due to color perception errors, moving artifacts, or possible
      selection bias induced by the analysis of still images. More objective, computer-assisted
      semi-quantitative means of interpreting the results were developed, but these have the
      disadvantage of being labor-intensive and using third-party software that cannot be used in
      real time [17]. Second generation elastography introduces strain ratio (SR) and strain
      histogram (SH) as two reproducible measurements that retrieve numerical values in real time,
      thus greatly reducing the human bias without the need for third-party software [4]. SR
      calculates the relative strain between two regions of interest (ROI) (normal and
      pathological). SH measures the strain values of elemental areas inside a ROI and divides the
      measurement range into intervals; if the strain value of an element falls into an interval,
      its initial area normalized by the initial total surface area is added to the running total
      of that interval; the total values of each interval are used to produce a graph and an
      average value. Both SR and SH have already been used in vivo for pancreatic masses or lymph
      nodes, with promising results [18].

        1. b Contrast-enhancement Ultrasound contrast agents in conjunction with contrast specific
           imaging techniques are increasingly accepted in clinical use for diagnostic imaging
           [19]. 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 [20-23]. 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) [24]. 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)
           [25]. 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) [19].

           Distinguishing pancreatic adenocarcinoma from other pancreatic masses remains
           challenging with current imaging techniques [22-27]. 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 [26]. The hypovascular aspect of lesions under PD-CE-EUS seemed highly
           sensitive and specific (higher than 90%) for adenocarcinoma in several published studies
           [22-27]. 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) [28]. At CE-EUS, ductal
           adenocarcinoma is typically hypoenhanced compared to the adjacent pancreatic tissue in
           all phases [19]. 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 [19].

           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 [24]. 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) [29-31]. 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 [32]. 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) [33]. Several
           other research groups already reported the feasibility of CEH-EUS with low mechanical
           index [34-36]. The sensitivity, specificity and accuracy for diagnosing pancreatic
           adenocarcinoma were 88%, 89%, and 88.5% in one study [34] and 80%, 91.7%, and 86% in the
           other study [33]. However, the data is still limited and a prospective, multicentric
           blinded study would certainly be necessary.

           The study protocol is based on a multi-center semi-quantitative approach of EUS
           elastography data in combination with contrast-enhanced EUS, consisting of measuring SR
           and SH for focal pancreatic masses and lymph nodes, as well as several parameters of
           CE-EUS based on time-intensity-curve (TIC) analysis. A number of parameters must be
           taken into consideration, as the ROIs are still manually selected by the user. The aim
           of the study is to establish an EUS based diagnostic algorithm in patients with
           pancreatic masses and lymph nodes, with negative or inconclusive cytopathology after
           EUS-FNA, based on previously published results and cut-offs of elastography and
           contrast-enhancement. The proposed algorithm of sequential use of real-time
           elastography, followed by contrast-enhanced EUS could be a good clinical tool to help
           select the patients with possible pancreatic adenocarcinoma or malignant lymph nodes, in
           the setting of patients with negative EUS-FNA results.

        2. Aims of the study The aim of the study is to assess quantitative elastography and
           contrast-enhancement parameters during EUS examinations of focal pancreatic masses and
           lymph nodes, to standardize an algorithm for their use and to consequently differentiate
           benign vs malignant pancreatic masses and evaluate lymph node involvement in a
           prospective multicenter design.

        3. Patients and methods The study design is prospective, blinded and multi-center,
           comparing endoscopic ultrasound elastography (EG-EUS) and contrast-enhnecement (CE-EUS)
           results for the characterization of focal pancreatic masses and lymph nodes by using
           parametric measurements, in comparison with the gold standard represented by pathology.

           The study will be performed with the approval of the institutional board (ethical
           committee) review of each center. The complete study protocol and particpating centers
           will be uploaded on ClinicalTrials.gov, the registry of federally and privately
           supported clinical trials conducted in the United States and around the world.

           Inclusion criteria

             -  Patients diagnosed with solid pancreatic tumor masses, with cytological /
                histo-logical confirmation

             -  Patients with or without suspected lymph node involvement are eligible

             -  Age 18 to 90 years old, men or women

             -  Signed informed consent for EG-EUS, CE-EUS and FNA biopsy Exclusion criteria

             -  Prior surgical treatment with curative intent or chemo-radiotherapy

             -  Patients diagnosed with mucin producing tumors, pancreatic cystic tumors, etc.

        4. Data collection

           • Personal data (name, surname, age, admission date, SSN, diagnosis at admission)

        5. Imaging tests

             -  All patients with a suspicion of pancreatic masses or lymph nodes should undergo
                EUS, with sequential EG-EUS and CE-EUS

             -  EUS with EUS-guided FNA and elastography

                  -  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

             -  EG-EUS procedure:

                  -  EUS-EG will be performed during usual EUS examinations, with two movies of 10
                     seconds recorded on the embedded HDD in order to minimize variability and to
                     increase repeatability of acquisition.

                  -  A two panel image with the usual conventional gray-scale B-mode EUS image on
                     the right side and with the elastography image on the left side will be used.
                     The same parameters will be set-up in all systems used: e-dynamic range 2,
                     persistence 3, etc.

                  -  The region of interest for EUS-EG will be preferably larger than the focal
                     mass (approximately 50%-50%), in order to include the surrounding structures.
                     If the focal mass is larger than 3 cm, part of the mass will be included in
                     the ROI, as well as the surrounding structures (preferably avoiding large
                     vessels). Very large ROI for the elastography calculations will be avoided due
                     to the appearance of side artifacts.

                  -  The following pre-settings will be used in all centers: elastography colour
                     map 1, frame rejection 2, noise rejection 2, persistence 3, dynamic rage 4,
                     smoothing 2, blend 50%.

                  -  SR and SH will be measured; with three measurements made and recorded on the
                     embedded HDD. For SR, the reference area should be placed at the same level
                     with the lesion, if possible.

             -  CE-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.

                  -  The starting point of the timer will be considered the moment of intravenous
                     contrast injection (Sonovue 4.8 mL).

                  -  CE-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.

                  -  A low mechanical index procedure (dynamic wide-band contrast harmo¬nic imaging
                     mode) will be used, with a mechanical index of 0.2 and corresponding powers.

                  -  The following pre-settings will be used in all centers: contrast mode dCHI-W,
                     WPI-R/P (resolution / penetration for superficial vs deep structures),
                     mechanical index (variable starting with 0.1, preferred 0.2), MI gray-scale
                     (0.03), grey map 4, AGC 0, R-filter C, persistence 2, dynamic range 50,
                     B-colour 21, smoothing 3, gamma curve linear.

                  -  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.

                  -  Off-line analysis of time-intensity curves will be performed using Vue-Box,
                     which yields the following quantitative parameters: Peak Enhancement (PE),
                     Wash-in Area Under the Curve (Wi-AUC), Rise Time (RT), mean Transit Time
                     (mTT), Time To Peak (TTP), Wash-in Rate (WiR) and Wash-in Perfusion Index
                     (WiPI). The software also provides referenced values (expressed in
                     percentages), aligning the set of values for the tumor ROI to the parenchymal
                     ones.

        6. Final diagnosis

             -  A positive cytological diagnosis will be taken as a final proof of malignancy of
                the pancreas mass or lymph node. The diagnoses obtained by EUS-FNA will be further
                verified either by surgery or during a clinical follow-up of at least 6 months.

             -  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 or benign
                lymph nodes 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.

             -  Pathology samples obtained from duodeno-pancreatectomies or caudal
                pancreatecto-mies done with curative intent, as well as microhistological fragments
                obtained through EUS-FNA biopsy will be processed by paraffin embedding with usual
                stainings (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.

        7. Statistical analysis

             -  Descriptive statistics

                  -  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 EG-EUS and CE-EUS will be determined in comparison with the final
                diagnosis. Also, the sensitivity, specificity, positive predictive value, negative
                predictive value and accuracy for the subgroup of patients with negative EUS-FNA
                and a positive diagnosisi of malignancy during ensuing follow-up will be calculated
                separately.

        8. Power analysis

             -  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 a 12 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 EG-EUS and/or CE-EUS.
    

Intervention name(s): Elastography, contrast enhancement
Intervention type(s): Other
Intervention description(s): Elastography is an ultrasound method used for the reconstruction of tissue elasticity distribution in real-time. The main intended use is to differentiate between benign and malignant focal lesions based on the significantly smaller strain of the latter real time. Tissue elastography can be easily performed with conventional probes, including the linear EUS probes used for the examination of the pancreas and/or lymph nodes.
Contrast-enhancement Ultrasound contrast agents in conjunction with contrast specific imaging techniques are increasingly accepted in clinical use for diagnostic imaging. Pancreatic adenocarcinomas are usually hypovascular tumors.
Observational model: Cohort
Time perspective: Prospective
Sampling method: Non-Probability Sample
Study population: 
        All patients with a suspicion of pancreatic masses or lymph nodes should undergo EUS, with
        sequential EG-EUS and CE-EUS. Personal data (name, surname, age, admission date, SSN,
        diagnosis at admission) will be registered in the study.

        EUS and EUS-guided fine needle aspiration are methods used for pancreatic masses and
        lymphnodes assessment in daily clinical practice. Contrast enhanced-EUS (CE-EUS) is aproved
        in european countries, and this mehode has been accepted for EUS procedures in daily
        practice, as well as elastography (EG-EUS). Anyway, signed informed consent for EG-EUS,
        CE-EUS and FNA biopsy is required for participation in the study.
      
Biospecimen description: 
      Pancreas and lymphnode samples (EUS-guided fine needle aspiration)