Real-time Semi-quantification of Endoscopic Ultrasound Elastography and Contrast-enhancement Using Strain Histograms (SH) and Contrast-enhancement (CE) for the Differentiation of Focal Pancreatic Masses and the Assessment of Lymph Node Involvement

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].