Keratin 19 marks poor differentiation and a more aggressive behaviour in canine and human hepatocellular tumours

van Sprundel et al. Comparative Hepatology 2010, 9:4 http://www.comparative-hepatology.com/content/9/1/4 RESEARCH Open Access Keratin 19 marks poor differentiation and a more aggressive behaviour in canine and human hepatocellular tumours Renee GHM van Sprundel1, Ted SGAM van den Ingh2, Valeer J Desmet3, Azeam Katoonizadeh3, Louis C Penning1, Jan Rothuizen1, Tania Roskams3, Bart Spee1,3* Abstract Background: The expression of Keratin 19 (K19) was reported in a subset of hepatocellular carcinomas (HCCs). K19 positive HCCs are associated with an increased malignancy compared to K19 negative HCCs. No suitable mouse models exist for this subtype of HCC, nor is the incidence of K19 expression in hepatocellular neoplasia in model animals known. Therefore, we compared the occurrence and tumour behaviour of K19 positive hepatocellular neoplasias in dog and man. Results: The expression of hepatocellular differentiation (HepPar-1), biliary/progenitor cell (K7, K19), and malignancy (glypican-3) markers was semi-quantitatively assessed by immunohistochemistry. The histological grade of tumour differentiation was determined according to a modified classification of Edmondson and Steiner; the staging included intrahepatic, lymph node or distant metastases. Four of the 34 canine hepatocellular neoplasias showed K19 positivity (12%), of which two co-expressed K7. K19 positive tumours did not express HepPar-1, despite the histological evidence of a hepatocellular origin. Like in human HCC, all K19 positive hepatocellular neoplasias were glypican-3 positive and histologically poorly differentiated and revealed intra- or extrahepatic metastases whereas K19 negative hepatocellular neoplasias did not. Conclusions: K19 positive hepatocellular neoplasias are highly comparable to man and occur in 12% of canine hepatocellular tumours and are associated with a poorly differentiated histology and aggressive tumour behaviour. Background Hepatic progenitor cells (HPCs) are activated in the majority of liver diseases and are a potential cell of origin for hepatocellular carcinoma (HCC) [1,2]. HCC is a neoplasm of increasing incidence worldwide and is the fifth leading cause of death on a worldwide basis in man [3,4]. Although remarkable advances in surgical and imaging modalities have improved the prognosis of HCC patients [5], the high incidence of intrahepatic recurrence remains a major challenge in HCC therapy [6,7]. In man the only potentially curative modality for HCC is surgical resection (including whole organ transplantation), yet recurrence rates are high and the longterm survival is poor [8]. An additional dilemma is the * Correspondence: 1 Department of Clinical Sciences of Companion Animals, Faculty of Veterinary medicine, Utrecht University, Utrecht, The Netherlands limited availability of healthy donor livers. Thus, the ability to predict individual recurrence risk and subsequently prognosis would help guide surgical and chemotherapeutic treatment. As the understanding of hepatocarcinogenesis increases, the innumerable genetic and molecular events that drive the hepatocarcinogenic disease process, including angiogenesis, invasion and metastasis, are being unravelled in the human clinical situation. Keratin (K) 19 expression is normally found in hepatic progenitor cells (HPCs) and cholangiocytes but not hepatocytes [9-11]. However, several authors report the peculiar expression of K19 in HCC in man [12-15]. These K19 expressing HCCs had a higher rate of recurrence (hazard ratio 12.5) after transplantation [6]. Other studies also linked increased K19 expressions in HCC with a worse prognosis and faster recurrence after surgical treatment [14,16-18]. Others observed a significantly © 2010 van Sprundel et al; licensee BioMed Central Ltd. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. van Sprundel et al. Comparative Hepatology 2010, 9:4 http://www.comparative-hepatology.com/content/9/1/4 Page 2 of 11 shorter survival in patients with HCCs expressing K19 without any treatment [15]. Furthermore, one recent report showed that HCCs expressing K19 and K7 have a lower tumour free survival rate after curative resection [13]. Several studies show that a cut-off of five percent K19 positive cells already influences the outcome of the patient [12]. These studies in man validate K19 as a clinically meaningful and prognostically relevant marker for hepatocellular carcinoma. Other recently described markers include glypican-3 (GPC3) which is an extracellular proteoglycan that is inferred to play an important role in growth control in embryonic mesodermal tissues in which it is selectively expressed [19]. GPC3 is a member of the glypican family of glucosyl-phosphatidylinositol-anchored cellsurface heparin sulfate proteoglycans and is well established as a serologic and immunohistochemical diagnostic tool for hepatocellular carcinomas in man. The presence of GPC3 (mRNA and immuno-histochemistry) is much higher in hepatocellular carcinomas compared to cirrhotic tissue or small focal lesions, indicating that the transition from small premalignant lesions to hepatocellular carcinomas is associated with a sharp increase of GPC3 expression in the majority of cases [20,21]. In view of the similarities in cell biological mechanisms involved in regeneration and tumour development between human liver tumours and liver tumours in small domestic animals, it is conceivable that these acquisitions found in human hepatic tumour pathology may also be true for the canine liver tumours [22]. To this date, no mouse models exist which resemble K19 positive HCCs in man. Therefore clinicopathological prognostic markers including marker expression of K7, K19 (HPC and cholangiocytes), HepPar-1 (hepatocytes) and glypican-3 (malignant HCC) were examined in primary liver tumours of dogs and compared to man. Results indicate a high similarity in histopathology of primary liver tumours between man and dog, emphasizing the use of dogs as possible treatment models. Results Histological classification of canine primary liver tumours Liver material of 46 dogs was included in this study (male to female ratio: 0.7). Breeds represented included mixed breed, Flat coated retriever, Airedale terrier, German Sheppard, Alaskan malamute, Pit bull, Maltezer, Cocker spaniel, Appenzeller, Golden retriever and Yorkshire terrier. The age range was six to fourteen years. Microscopical examination (Table 1) classified the 46 primary liver tumours as: four nodular hyperplasia (9%) and 34 hepatocellular tumours (74%). Five hepatic carcinoids (11%) positive for one or more neuroendocrine differentiation markers (chromogranin-A, neuron-specific enolase, and synaptophysin) and three cholangiocellular carcinomas (7%) wer (...truncated)