Description of the use of contrastenhanced ultrasonography in four dogs with pancreatic tumours

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1 ttp:// CASE REPORT Description of the use of contrastenhanced ultrasonography in four dogs with pancreatic tumours K. Vanderperren*, H. Haers*, E. Van der Vekens*, E. Stock*, D. Paepe, S. Daminet and J. H. Saunders* *Department of Veterinary Medical Imaging and Small Animal Orthopaedics, Faculty of Veterinary Medicine, Ghent University, Salisburylaan 133, B-9820 Merelbeke, Belgium Department of Small Animal Medicine and Clinical Biology Medicine, Ghent University, Salisburylaan 133, B-9820 Merelbeke, Belgium Canine pancreatic tumours are rare compared to human medicine and the detection and differentiation of pancreatic neoplasia is challenging with B-mode ultrasonography, which often leads to late clinical diagnosis and poor prognosis. This case report describes the findings of contrast-enhanced ultrasonography in four dogs with pancreatic adenocarcinoma or insulinoma. B-mode ultrasonography of the pancreas revealed a hypoechoic nodule in three dogs and heterogenous tissue in one dog. Contrast-enhanced ultrasonography was able to differentiate between two tumour types: adenocarcinomas showed hypoechoic and hypovascular lesions, whereas insulinomas showed uniformly hypervascular lesions. Contrast-enhanced ultrasonography findings were confirmed by cytology and/or histopathology. The results demonstrated that contrast-enhanced ultrasonography was able to establish different enhancement patterns between exocrine (adenocarcinoma) and endocrine (insulinoma) tumours in dogs. Journal of Small Animal Practice (2013) 55, DOI: /jsap Accepted: 15 September 2013; Published online: 1 November 2013 INTRODUCTION Exocrine and endocrine pancreatic tumours are rare in veterinary practice compared to human medicine (Kircher & Nielsen 1976; Bailey & Page 2007). Exocrine pancreatic neoplasia mainly includes adenocarcinoma of ductular or acinar origin (Dennis et al. 2008), whereas insulinoma is the most common endocrine tumour (Jubb 1993). These tumours are diagnosed based on clinical signs and clinicopathological investigations, in combination with diagnostic imaging (Feldman & Nelson 2004). Several imaging techniques, including B-mode ultrasonography (US), Doppler US, computed tomography (CT), planar scintigraphy and single-photon emission CT, have been used to evaluate pancreatic tumours (Lamb et al. 1995; Robben et al. 2005; Iseri et al. 2007). A definitive diagnosis typically requires cytological or histopathological evaluation of the suspected neoplastic tissue (Steiner & Bryette 1996; Bennett et al. 2001). The sensitivity of abdominal US in detecting pancreatic tumours varies from 28 to 75% (Lamb et al. 1995; Robben et al. 2005; Goutal et al. 2012). With the availability of safe US contrast agents, contrastenhanced ultrasonography (CEUS) has become a reliable method for the differential diagnosis of pancreatic tumours in humans (D Onofrio et al. 2003; Rickes & Wermke 2004; Rickes et al. 2006). In dogs, CEUS of the pancreas has only been described for the normal pancreas and in dogs with pancreatitis (Gaschen et al. 2007; Johnson-Neitman et al. 2012). An ultrasound contrast agent is composed of tiny (<10 µ), gas-filled microbubbles, stabilised by an outer shell. They are injected into the systemic circulation, traverse the pulmonary capillary circulation, reach the organ vascular supply and remain at this level, unlike the contrast agents used in CT or magnetic resonance imaging (MRI). The gas content is gradually eliminated from the blood through the lungs, whereas the stabilising components are filtered by the kidneys and eliminated by the liver. The microbubbles are detected following vibration and bursting, which increases the signal-to-noise ratio. They are confined to the intravascular space and thus allow for optimal, detailed evaluation of tissue vascularisation and perfusion (Ohlerth & O Brien 2007; Haers & Saunders 2009). CEUS has been found to be a safe method in clinically ill dogs and cats. The incidence of adverse effects within 24 hours after CEUS was low, and the effects were transient and mild (vomiting or syncope) (Seiler et al. 2013). 164 Journal of Small Animal Practice Vol 55 March British Small Animal Veterinary Association

2 Contrast-enhanced ultrasound pancreatic tumours Table 1. Signalment, clinical signs and clinicopathological results Case Signalment History and clinical signs Bloodwork 1 13 YO, FS, Toy poodle 2 10 YO, ME, golden retriever 3 9 YO, FS, golden retriever 4 8 YO, FE, French Braque Vomiting and diarrhoea (1 5 months), systolic heart murmur (grade 4 of 6), thin body condition Polyuria/polydipsia, excessive panting, exercise intolerance, weight loss despite good appetite, good body score condition, pale mucous membranes, uncomfortable abdomen (mainly cranially) Lethargy (1 year), weakness, tetraparesis. Physical examination: alert dog with weakness of the front limbs. Polyneuropathy was diagnosed on neurological examination, electromyography and magnetic nerve stimulation Mild seizures during exercise, spontaneous muscle twitching of the hind limbs FS Female spayed, FE Female entire, ME Male entire, US Ultrasonography, CEUS Contrast-enhanced ultrasonography, NA not available, YO Year old NA Haematocrit=31% (mild anaemia); increased alkaline phosphatase (242 U/L, reference interval: 20 to 200) and increased alkaline transferase (91 U/L; reference interval: 10 to 70) Hypoglycaemia (1 84 mmol/l; reference interval: 3 05 to 4 99 mmol/l); hyperinsulinaemia (28 mu/l; reference interval: 9 to 25 mu/l); high insulin/glucose ratio (80; reference value <30) Mild hypoproteinaemia (total protein 49 g/l; reference interval: 54 to 76); mild hypoglycaemia (2 83 mmol/l; reference interval: 3 05 to 4 99), fructosamine concentration=208 mol/l (reference interval: 200 to 300); low insulin concentration=2 4 mu/l (reference interval 9 to 25) To the authors knowledge, this is the first report describing the CEUS findings of dogs with insulinoma and pancreatic carcinoma. CASE HISTORIES Four cases with a final diagnosis of pancreatic neoplasia, which underwent CEUS, were included. Signalment, clinical findings and clinicopathological abnormalities are summarised in Table 1. The dogs were manually restrained in dorsal recumbency and scanned by B-mode US using a multi-frequency linear array transducer of 5 to 7 5 MHz. Subsequently, CEUS was performed with contrast-specific software using a 5-MHz linear transducer. A 20-gauge catheter was put in the left cephalic vein (operator is right-handed). The pancreas was centred on the screen and the contrast-specific imaging mode was turned on. Before injection, several imaging parameters were adjusted to decrease early destruction of the injected microbubbles by the US beam. Mechanical index (MI) was decreased (0 08 to 0 09), persistence was switched off and the focal spot was placed at the deepest level of the scan field. The image depth, gain and frame rate were optimised and held constant during the study. The contrast medium (Sonovue; Bracco Imaging) was injected intravenously (iv) as a 0 3 ml/10 kg bolus followed by 2 to 3 ml of sterile saline flush (NaCl 0 9%). A three-way stopcock was placed at the catheter to avoid any delay between the injection of the contrast and the saline. Two (only pancreas) or three (pancreas and liver in cases 1 and 2) injections were performed. The first injection was not used for interpretation. Between injections, to avoid attenuation artefacts, the microbubbles were destroyed by increasing acoustic power. The enhancement patterns were evaluated during the early arterial phase (20s) and the late parenchymal phase (28s) for the pancreas, and during the early arterial phase (15s) and the late portal-venous phase (45s) for the liver. The US and CEUS findings are summarised in Table 2. Native and contrast-enhanced (during parenchymal phase (28s)) MRI and CT of the abdomen was performed in case 4. The final diagnosis of pancreatic adenocarcinoma or insulinoma was based on cytology (fine-needle aspiration, FNA) and/or histopathology. Imaging findings With B-mode US, pancreatic lesions were hypoechoic in cases 1, 3 and 4, and heterogenous in case 2 (Figs. 1 and 2). In cases 1 and 2, liver lesions were also detected. Compared with B-mode US, CEUS detected an increased number of nodules in the pancreas in case 1, and improved anatomical localisation and delineation of a lesion in case 2. In cases 1 and 2, the lesions were hypovascular during both arterial and parenchymal phases of contrast enhancement, whereas in cases 3 and 4 the pancreatic lesion had a hypervascular appearance with a rapid and intense wash-in of contrast during the arterial phase, and fast disappearance of contrast during the parenchymal phase of enhancement, respectively. In cases 1 and 2, CEUS of the liver showed a rapid and clear enhancement during the arterial phase, and hypovascular lesions during the portal-venous phase of enhancement (early wash-in, early wash-out). This enhancement pattern is indicative of malignancy (O Brien et al. 2004). In case 4, MRI and CT of the abdomen revealed a lesion of 1 2 cm in the right pancreatic lobe. On MRI, this lesion was hyperintense on T2 images, isointense on T1 and contrast enhancing after the injection of 0 3 ml/kg gadolinium iv. On CT, the lesion remained hypodense after contrast injection during the parenchymal phase (2 ml/kg Ultravist 300). Fine-needle aspiration and histopathology The final diagnosis of pancreatic adenocarcinoma (cases 1 and 2) with metastases in the liver was made by ultrasound-guided FNA of liver and pancreas. In case 3, FNA of the right pancreatic mass and left medial iliac lymph node revealed neuroendocrine tissue and a final diagnosis of insulinoma. In case 4, histopathological tissue evaluation confirmed the diagnosis of insulinoma (Fig 2). Journal of Small Animal Practice Vol 55 March British Small Animal Veterinary Association 165

3 K. Vanderperren et al. A B C D FIG 1. Pa ncreatic adenocarcinoma with metastasis to the liver (case 1). On B-mode ultrasound (US) images of pancreas (A) and liver (C), the liver had a mottled appearance. An ill-defined hypoechoic nodule (arrows) was present within the pancreatic parenchyma (A). Late phase contrast-enhanced US images of the pancreas (B) showed several hypovascular lesions (arrows). In the liver, multiple hypoechoic lesions (arrows) were visible during late phase of contrast enhancement (D), which is very suggestive for malignancy. *Acoustic reverberation artefacts Table 2. Ultrasound and contrast-enhanced ultrasound Case Ultrasound findings Contrast-enhanced ultrasound findings 1 Hypoechoic nodule of 1 cm in the right lobe of the pancreas ( surrounded by hyperechoic mesenteric fat); mottled appearance of the liver (more left lobes); calcification within left lateral liver lobe; enlarged and rounded cranial pole of the left adrenal gland (0 89 cm); enlarged slightly irregularly outlined and heterogeneous pancreaticoduodenal lymph node 2 Heterogenous tissue in the anatomic region of the pancreas; enlarged liver with rounded edges, containing isoechoic to mildly hyperechoic nodules of variable size (up to 2 cm diameter) with a hypoechoic rim 3 Ovoid hypoechoic mass lesion (2 2 cm) caudal to the right renal artery, representing enlarged mesenteric lymph node or pancreatic nodule, enlarged and rounded left medial iliac lymph node (1 2 cm) 4 Nodule (0 43 cm) in the right pancreatic lobe, prominent pancreaticoduodenal lymph nodes (but normal in size, shape and echogenicity) Pancreas: multiple hypovascular nodular lesions in whole pancreas during both arterial and parenchymal phase of contrast enhancement Liver: rapid and clear enhancement during arterial phase, and hypovascular lesions during portal-venous phase of enhancement (early wash-in, early wash-out) Pancreas: ill-defined hypovascular mass during both arterial and parenchymal phase of contrast enhancement. Liver: rapid and clear enhancement during arterial phase, and hypovascular lesions during portal-venous phase of enhancement (early wash-in, early wash-out) Pancreas: well-delineated hypervascular area in the pancreas with early arterial phase of contrast enhancement, and rapid disappearance of the contrast (early wash-in, early wash-out) Pancreas: hypervascular appearance of pancreatic nodule with a rapid and intense wash-in of contrast during the arterial phase, and fast disappearance of contrast during parenchymal phase of enhancement 166 Journal of Small Animal Practice Vol 55 March British Small Animal Veterinary Association

4 Contrast-enhanced ultrasound pancreatic tumours A B C FIG 2. Pancreatic insulinoma (case 3). B-mode abdominal ultrasound image demonstrating an ovoid hypoechoic mass structure (2 2 cm) representing an enlarged mesenteric lymph node or a pancreatic nodule. Contrast-enhanced US images (B, C) during the early phase (13 seconds) of enhancement (B) showed a hypervascular lesion (arrows). After 24 seconds, the contrast medium rapidly disappeared (C). This early wash-in, early wash-out contrast behaviour of a pancreatic nodule is very suggestive of a well-vascularised lesion of neuroendocrine origin DISCUSSION In this report, CEUS demonstrated different enhancement patterns between two histologically different pancreatic tumours (adenocarcinoma and insulinoma). CEUS, compared to B-mode US, detected an increased number of nodules in the pancreas in case 1, and improved anatomical localisation and delineation of a lesion in case 2. This is mainly the result of the low contrast in the B-mode US images, which is caused by the similar acoustic impedances of different adjacent tissues within the pancreas (Cachard et al. 1997). With B-mode US, there was an overlap between imaging features of pancreatic adenocarcinoma and insulinoma, as they both presented on US as hypoechoic lesions. Because of the small size, its localisation and comparable echogenicity of the pancreas to surrounding mesentery, the evaluation of the pancreas is not always conclusive with US (Hecht & Henry 2007). Pancreatic diseases frequently investigated by means of US include pancreatitis, pseudocysts, abscesses, neoplastic lesions and nodular hyperplasia. Unfortunately, as in the cases presented here, US findings in various pancreatic disorders overlap. Frequently, conventional US cannot help to determine the aetiology of the process because of non-specific findings. Secondly, small and ill-defined malignant lesions or isoechoic lesions are often invisible with greyscale US. Moreover, pancreatic disorders may not cause changes in US appearance (Hecht & Henry 2007). Although US is the most widely used technique in the diagnosis of pancreatic tumours, it has a poor sensitivity for the diagnosis of insulinomas in dogs (Lamb et al. 1995; Robben et al. 2005). Contrast-enhanced US and contrast-enhanced CT (CECT) attempt to overcome these limitations. Compared to CECT, CEUS is lower in cost, does not require ionising radiation or anaesthesia, can be easily repeated, and eliminates the need for injection of iodinated contrast media, which can be contraindicated in critically ill patients (Thorelius 2004; Haers & Saunders 2009; Fan et al. 2013). With CEUS, significant differences have been observed in perfusion patterns between dogs with pancreatitis compared to normal dogs (Gaschen et al. 2007). In dogs with pancreatitis, non-vascularised regions could be easily visualised in contrast to the vascular surrounding tissues when using CEUS, findings that were not apparent on conventional US images. Also in canine pancreatic neoplasia, CEUS could have potential, as it provides information about perfusion and vascularisation of an organ or lesion (Haers & Saunders 2009). In humans, CEUS of the pancreas has been shown to improve the delineation and characterisation of pancreatic lesions compared to B-mode US (Badea et al. 2009). The results of a recent study revealed that, compared with B-mode US, the diagnostic confidence of CEUS for pancreatic solid lesions was clearly improved with a higher diagnostic accuracy of CEUS compared to B-mode US (Fan et al. 2013). In the present cases, the enhancement pattern was different between the two tumour types. Following contrast agent administration, pancreatic adenocarcinomas were hypoechoic and hypovascular, whereas insulinomas were hypervascular. For adenocarcinomas, the overall enhancement remained low compared to the surrounding parenchyma, which was in agreement with human adenocarcinomas. This filling pattern results from poor vascularisation and fibrous content of the tumour, which hampers lesional blood flow (Dietrich et al. 2008). At histopathological examination, adenocarcinomas are characterised by marked desmoplasia (D Onofrio et al. 2010), causing the hard consistency of the tumour. Unlike adenocarcinomas, neuroendocrine tumours such as insulinomas have very rich vascularisation. Therefore, in cases 3 and 4 CEUS typically showed hypervascular lesions, characterised by rapid and marked enhancement during early phase of contrast enhancement, with a hyperechogenic pattern compared to the surrounding parenchyma. These findings are similar to those described for human insulinomas (D Onofrio et al. 2004). Other causes of nodules are pancreatic nodular hyperplasia, which is a common finding in old dogs, abscesses, haematomas, pancreatic pseudocysts and focal necrotic areas (Hecht & Henry 2007). Differentiation between focal necrosis, pseudocyst and abscess is usually not possible using conventional B-mode US Journal of Small Animal Practice Vol 55 March British Small Animal Veterinary Association 167

5 K. Vanderperren et al. images. Moreover, pancreatic neoplasms may mimic or be associated with abscessation, pancreatic necrosis or pancreatitis (Hecht & Henry 2007). Pseudocysts are seen as anechoic to slightly echoic cyst-like structures. The ultrasonographic aspect of pancreatic abscesses and necrotic masses can be variable in size, shape and echogenicity (Coleman & Robson 2005). In human medicine, CEUS of pseudocysts, as they are non-vascularised, reveal no signal and remain anechoic in all phases (Rickes & Wermke 2004). However, masses associated with pancreatitis may have different vascularisation patterns depending on the degree of inflammation and necrosis (Xu 2009). Although pancreatic nodular hyperplasia is a common finding in older dogs compared to humans, no information is available regarding CEUS contrast patterns in pancreatic hyperplastic lesions. However, in liver and spleen, CEUS of nodular hyperplasia showed a diffuse and homogeneous contrast pattern, which was iso- or hypoechogenic in the arterial phase and homogeneously isoechoic during the wash-out phase (Rossi et al. 2008; Wdowiak et al. 2010). In humans, dual-phase contrast-enhanced CT and MRI are the most reliable tools in the detection and staging of pancreatic tumours (Lu et al. 1996; Miura et al. 2006). Similarly as with the present observations using CEUS, pancreatic adenocarcinomas in humans are generally depicted as a hypoattenuating area on contrast-enhanced CT (Miura et al. 2006). Contrary, insulinomas are highly hyperattenuating at the arterial phase but not in the other phases in both humans and dogs (Lu et al. 1996; King et al. 1998; Mai & Cáreres 2008). Contrast-enhanced CT was available in case 4 which showed a hypovascularised lesion whereas this nodule had an arterial hypervascular appearance on CEUS. This was because only the late, parenchymal phase was available with CECT. Therefore, the arterial phase needs to be included for the distinction between pancreatic adenocarcinoma and insulinoma. Despite its benefit of higher detection of small pancreatic nodules, CT is not widely available and is of high cost. The most important disadvantage, however, is that general anaesthesia is required. The diagnosis of pancreatic adenocarcinoma (cases 1 and 2) was based on FNA. The diagnosis of insulinoma (case 3) was confirmed by the insulin and glucose concentrations and by the presence of neuroendocrine tissue on cytology. In case 4, the final diagnosis was confirmed by histopathological examination. Histopathology was not performed in all dogs. Nevertheless, FNA is a quick, cheap, low-invasive and effective method for collecting cells for cytology. Hence, endocrine tumours can be easily differentiated from pancreatic exocrine cells (Goutal et al. 2012). A limitation of this case report is the absence of a control group. Although quantitative analysis was not performed, different enhancement patterns were present between the exocrine and endocrine pancreatic tumours. Further research on the value of CEUS of different pancreatic lesions, including dogs with benign nodules and pancreatitis is needed. The value of CEUS in comparison with dual-phase CT angiography for the diagnosis of pancreatic tumours is warranted. In conclusion, B-mode US revealed hypoechoic and heterogeneous lesions in the pancreas with both adenocarcinoma and insulinoma. During CEUS, a differential uptake of contrast agent was seen between exocrine and endocrine pancreatic tumours in dogs, providing more diagnostic information. Adenocarcinomas remain hypovascular in all vascular phases, whereas insulinomas appear hypervascular. Acknowledgments K. Vanderperren is a post-doctoral fellow of the FWO vlaanderen. Conflict of interest None of the authors of this article has a financial or personal relationship with other people or organisations that could inappropriately influence or bias the content of the paper. References Badea, R., Seicean, A., Diaconu, B., et al. (2009) Contrast-enhanced ultrasound of the pancreas a method beyond its potential or a new diagnostic standard? Journal of Gastrointestinal and Liver Diseases 18, Bailey, D. B. & Page, R. L. (2007) Pancreatic beta-cell tumors (insulinomas). In: Small Animal Clinical Oncology. 4th edn. Eds S. J. Withrow and D. M. Vail. Saunders Elsevier, St. Louis, MO, USA. pp Bennett, P. F., Hahn, K. A., Toal, R. L., et al. (2001) Ultrasonographic and cytopathological diagnosis of exocrine pancreatic carcinoma in the dog and cat. Journal of the American Animal Hospital Association 37, Cachard, C., Finet, G., Bouakaz, A., et al. (1997) Ultrasound contrast agent in intravascular echography: an in vitro study. Ultrasound in Medicine and Biology 23, Coleman, M. & Robson, M. (2005) Pancreatic masses following pancreatitis: pancreatic pseudocysts, necrosis, and abscesses. Compendium on Continuing Education for the Practicing Veterinarian 27, Dennis, M. M., O Brien, T. D., Wayne, T., et al. (2008) Hyalinizing pancreatic adenocarcinomas in six dogs. Veterinary Pathology 45, Dietrich, C. F., Braden, B., Hocke, M., et al. (2008) Improved characterisation of solitary solid pancreatic tumours using contrast enhanced transabdominal ultrasound. Journal of Cancer Research and Clinical Oncology 134, D Onofrio, M., Mansueto, G., Vasori, S., et al. (2003) Contrast-enhanced ultrasonographic detection of small pancreatic insulinoma. Journal of Ultrasound in Medicine 22, D Onofrio, M., Mansueto, G., Falconi, M., et al. (2004) Neuroendocrine pancreatic tumor: value of contrast enhanced ultrasonography. Abdominal Imaging 29, D Onofrio, M., Gallotti, A., Principe, F., et al. (2010) Contrast-enhanced ultrasound of the pancreas. World Journal of Radiology 2, Fan, Z., Li, Y., Yan, K., et al. (2013) Application of contrast-enhanced ultrasound in the diagnosis of solid pancreatic lesions a comparison of conventional ultrasound and contrast-enhanced CT. European Journal of Radiology 82, Feldman, E. C. & Nelson, R. W. (2004) Beta cell neoplasia: insulinoma. In: Canine and Feline Endocrinology and Reproduction. 3rd edn. Eds E. C. Feldman and R. W. Nelson. Saunders Elsevier, St Louis, MO, USA. pp Gaschen, L., Schur, D. & Kearney, M. (2007) Contrast harmonic ultrasound imaging of the normal pancreas and pancreatitis in dogs. Proceedings of the American College of Veterinary Radiologists Annual Scientific Meeting. ACVR Meeting, Chicago, November 27-December 1, 21 Goutal, C. M., Brugmann, B. L. & Ryan, K. A. (2012) Insulinoma in dogs: a review. Journal of the American Animal Hospital Association 48, Haers, H. & Saunders, J. H. (2009) Review of clinical characteristics and applications of contrast-enhanced ultrasonography in dogs. Journal of the American Veterinary Medical association 234, Hecht, S. & Henry, G. (2007) Sonographic evaluation of the normal and abnormal pancreas. Clinical Techniques in Small Animal Practice 22, Iseri, T., Yamada, K., Chijiwa, K., et al. (2007) Dynamic computed tomography of the pancreas in normal dogs and in a dog with pancreatic insulinoma. Veterinary Radiology and Ultrasound 48, Johnson-Neitman, J. L., O Brien, R. T. & Wallace, J. D. (2012) Quantitative perfusion analysis of the pancreas and duodenum in healthy dogs by use of contrast-enhanced ultrasonography. American Journal of Veterinary Research 73, Jubb, K. (1993) The pancreas. In: Pathology of Domestic Animals. 4th edn. vol. 2. Eds K. Jubb, P. Kennedy and N. Palmer. Academic Press, San Diego, CA, USA. pp Kircher, C. H. & Nielsen, S. W Tumours of the pancreas. Bulletin of the World Health Organization 53, King, A. D., Ko, G. T., Yeung, V. T., et al. (1998) Dual phase spiral CT in the detection of small insulinomas of the pancreas. British Journal of Radiology 71, Lamb, C. R., Simpson, K. W., Boswood, A., et al. (1995) Ultrasonography of pancreatic neoplasia in the dog: a retrospective review of 16 cases. Veterinary Record 137, Journal of Small Animal Practice Vol 55 March British Small Animal Veterinary Association

6 Contrast-enhanced ultrasound pancreatic tumours Lu, D. S., Vedantham, S., Krasny, R. M., et al. (1996) Two-phase helical CT for pancreatic tumors: pancreatic versus hepatic phase enhancement of tumor, pancreas, and vascular structures. Radiology 199, Mai, W. & Cáceres, A. V. (2008) Dual-phase computed tomographic angiography in three dogs with pancreatic insulinoma. Veterinary Radiology and Ultrasound 49, Miura, F., Takada, T., Amano, H., et al. (2006) Diagnosis of pancreatic cancer. Official Journal of the International Hepato Pancreato Biliary Association 8, O Brien, R. T., Iani, M., Matheson, J., et al (2004) Contrast harmonic ultrasound of spontaneous liver nodules in 32 dogs. Veterinary Radiology and Ultrasound 45, Ohlerth, S. & O Brien, R. T. (2007) Contrast ultrasound: General principles and veterinary clinical applications. The Veterinary Journal 174, Rickes, S. & Wermke, W. (2004) Differentiation between cystic pancreatic neoplasms and pseudocysts by conventional and echo-enhanced ultrasound. Journal of Gastroenterology and Hepatology 19, Rickes, S., Mönkemüller, K. & Malfertheiner, P. (2006) Contrast-enhanced ultrasound in the diagnosis of pancreatic tumors. Journal of the Pancreas 7, Robben, J. H., Pollak, Y. W., Kirpensteijn, J., et al. (2005) Comparison of ultrasonography, computed tomography, and single-photon emission computed tomography for the detection and localization of canine insulinoma. Journal of Veterinary Internal Medicine 19, Rossi, F., Leone, V. F., Vignoli, M., et al. (2008) Use of contrast-enhanced ultrasound for characterization of focal splenic lesions. Veterinary Radiology and Ultrasound 49, Seiler, G. S., Brown, J. C., Reetz, J. A., et al. (2013) Safety of contrast-enhanced ultrasonography in dogs and cats: 488 cases ( ). Journal of the American Veterinary Medical association 242, Steiner, J. M. & Bruyette, D. S. (1996) Canine insulinoma. Compendium on Continuing Education for the Practicing Veterinarian 18, Thorelius, L. (2004) Contrast-enhanced ultrasound for extrahepatic lesions: preliminary experience. European Journal of Radiology 51, S31-S38 Wdowiak, M., Rychlik, A., Nieradka, R., et al. (2010) Contrast-enhanced ultrasonography (CEUS) in canine liver examination. Polish Journal of Veterinary Sciences 13, Xu, H. X. (2009) Contrast-enhanced ultrasound: the evolving applications. World Journal of Radiology 1, Journal of Small Animal Practice Vol 55 March British Small Animal Veterinary Association 169