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Acinar Cell Carcinoma of Exocrine Pancreas in Two Horses
J. Comp. Path. 2014, Vol. 150, 388e392
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NEOPLASTIC DISEASE
Acinar Cell Carcinoma of Exocrine Pancreas in Two Horses S. de Brot*, H. Junge† and M. Hilbe* * Institute of Veterinary Pathology and † Clinic for Equine Internal Medicine, Equine Department, Vetsuisse Faculty, University of Zurich, Winterthurerstrasse 260, CH-8057 Zurich, Switzerland
Summary Two horses were presented with non-specific clinical signs of several weeks’ duration and were humanely destroyed due to a poor prognosis. At necropsy examination, both horses had multiple small, white nodules replacing pancreatic tissue and involving the serosal surface of the abdominal cavity, the liver and the lung. Microscopically, neoplastic cells were organized in acini and contained abundant (case 1) or sparse (horse 2) intracytoplasmic zymogen granules. Immunohistochemically, both tumours expressed amylase and pancytokeratin, but not insulin or neuron-specific enolase. In case 2, a low percentage of neoplastic cells expressed glucagon and synaptophysin. The presence of zymogen granules was confirmed in both cases by electron microscopy and occasional fibrillary or glucagon granules were observed in cases 1 and 2, respectively. A diagnosis of pancreatic acinar cell carcinoma was established in both horses. Ó 2014 Elsevier Ltd. All rights reserved. Keywords: electron microscopy; fibrillary granules; immunohistochemistry; pancreas
Neoplastic lesions of the pancreas are uncommon in domestic animals and originate more frequently from exocrine cells (Priester, 1974; Head et al., 2002; Charles, 2007). Most cases are reported in dogs (Rowlatt, 1967; Rabanal et al., 1992; Dennis et al., 2008). Such tumours are less common in cats (Banner et al., 1979; Seaman, 2004) and rare in horses (Church et al., 1987; Carrick et al., 1992; Rendle et al., 2006), cattle (Kelley et al., 1996; Lucena et al., 2011) and pigs (Rowlatt, 1967). This differs from man, where pancreatic neoplasia is among the five most common causes of cancerrelated mortality with an increasing rate of prevalence (Greenlee et al., 2001; Malvezzi et al., 2013). The diagnosis of pancreatic exocrine carcinoma by routine light microscopy may be difficult since it can be confused with other tumours, especially islet cell carcinoma (Ordon ~ez, 2001). In man, it has been demonstrated that both immunohistochemistry (IHC) and electron microscopy (EM) are useful in es-
tablishing the diagnosis of pancreatic cell carcinomas (Ordon ~ez, 2001; La Rosa et al., 2012). However, the few reports of equine pancreatic tumours are based on gross and light microscopical findings and do not include immunohistochemical or ultrastructural studies. The present study describes two horses with pancreatic acinar cell carcinoma (ACC). Case 1 was a 22-year-old Polish warmblood mare that was presented to the Clinic for Equine Internal Medicine, Vetsuisse Faculty, University of Zurich, with a 3-week history of apathy, anorexia, weight loss, polyuria and polydypsia. Case 2 was a 17-yearold Swiss warmblood gelding that was presented to the same institution with a several-week history of weight loss and anorexia. Laboratory investigations showed significant elevation of liver enzymes in both cases. Ultrasonography revealed the presence of multiple intrapulmonary nodules and a thickened colonic wall in case 1 and an irregular serosal surface of the small intestine in case 2. Exploratory laparotomy was performed in case 2, which revealed a thickened colonic wall, enlarged abdominal lymph nodes and a firm liver. Neoplasia was suspected in both cases and the
Correspondence to: S. de Brot (e-mail: [email protected]). 0021-9975/$ - see front matter http://dx.doi.org/10.1016/j.jcpa.2014.01.003
Ó 2014 Elsevier Ltd. All rights reserved.
Pancreatic Carcinoma in Two Horses
animals were humanely destroyed due to a poor prognosis. Necropsy examination was performed within 6 h of death in both cases. Samples of various organs were fixed in 10% neutral buffered formalin and processed routinely. Sections (3 mm) were stained with haematoxylin and eosin (HE), periodic acideSchiff (PAS), PAS after enzymatic digestion with diastase and Masson’s trichrome stain. IHC was performed using commercially available antibodies specific for amylase (anti-rabbit, 1 in 1,000 dilution; Catalogue number A8273; Sigma Chemical Company, St Louis, Missouri, USA) pan-cytokeratin (anti-mouse, 1 in 50 dilution; catalogue number M082101; Dako, Glostrup, Denmark), glucagon (anti-human, 1 in 100 dilution; catalogue number A0565, Dako), insulin (anti-guinea pig, 1 in 200 dilution; catalogue number A0564; Dako), neuron-specific enolase (NSE, anti-mouse, 1 in 150 dilution; catalogue number M087301, Dako) and synaptophysin (anti-mouse, 1 in 10 dilution; catalogue number M077601, Dako). The standard avidinebiotineperoxidase technique was used to demonstrate the binding of primary antibody. Normal horse pancreatic tissue was used as a positive control. Labelling was evaluated semiquantitatively by assessing the percentage of labelled neoplastic cells as follows: e, positive neoplastic cells not seen; +, <10% of neoplastic cells labelled; ++, 10e50% of neoplastic cells labelled; and +++, >50% of neoplastic cells labelled. For EM, selected tissues were fixed in 2.5% glutaraldehyde and 1% osmium tetroxide (OsO4), buffered with 0.1 M sodium phosphate (pH 7.4). After fixation, the tissues were dehydrated in an ethanol series, embedded in epon and ultrathin (90 nm) sections were stained with uranyl acetate and lead citrate. Necropsy examination of case 1 revealed the presence of 4 l of serosanguineous fluid in the thoracic cavity and 30 l of similar fluid in the abdominal cavity. The pancreas was markedly irregular and diffusely disrupted by the presence of innumerable small white to tan, firm nodules. Similar nodules (2e15 mm diameter), some of them umbilicated, were scattered over the mesentery and the serosal surface of the small intestine, colon, spleen and diaphragm. Approximately 50% of the hepatic parenchyma contained similar nodules with a multifocal to coalescing distribution. Mesenteric lymph nodes were markedly enlarged. In the thorax, approximately 10% of the pulmonary parenchyma was infiltrated by multiple tan to red, firm nodules (5e15 mm diameter) and the mediastinal lymph nodes were also markedly enlarged. Necropsy examination of case 2 revealed approximately 20 l of serosanguinous fluid in the abdominal
389
cavity. The pancreas was extensively replaced by a firm, white, multinodular mass with a smooth surface that adhered to the serosa covering the liver and diaphragm. The liver was firm and infiltrated by multifocal to coalescing pale foci (1e10 mm diameter), which occupied approximately 50% of the parenchyma. Hepatic and mesenteric lymph nodes were markedly enlarged, firm and cream in colour. Examination of the gastrointestinal tract showed a markedly oedematous ascending colon. In both cases, microscopical examination revealed the nodular masses to be comprised of a densely cellular, infiltrative and non-encapsulated tumour. No normal pancreatic tissue was observed in any of the sections. Neoplastic cells were organized mainly as acini and less frequently in a tubular or solid arrangement. The stroma was fibrous with a multifocal scirrhous reaction. Neoplastic cells were polyhedral, 15e25 mm in diameter, with mainly indistinct cell borders. In case 1, tumour cells showed a moderate amount of eosinophilic, homogeneous cytoplasm, often containing numerous intracytoplasmic deeply eosinophilic granules (Fig. 1). However, in case 2, only scant similar granules were seen within a low number of neoplastic cells. The nuclei of the neoplastic cells were round to oval, often basally located and contained finely stippled chromatin with 1e3 prominent eosinophilic nucleoli. Abundant mitotic figures were seen and were more numerous in areas of solid growth (up to 10 per 40 objective field). There was marked anisokaryosis and anisocytosis. Multifocally, there were extensive and abundant areas of necrosis, sometimes accompanied by haemorrhage. Often, the lumina of lymphatic and
Fig. 1. Pancreatic acinar cell carcinoma; horse 1. Neoplastic cells with basal nuclei and intracytoplasmic eosinophilic granules (zymogen granules) form acinar structures. HE. Bar, 50 mm.
S. de Brot et al.
390
blood vessels contained aggregates of neoplastic cells. Multifocally, areas of moderate lymphoplasmacytic infiltration were present. Overall, the observed features were compatible with pancreatic acinar carcinoma with metastasis to liver, lung, lymph nodes and peritoneal surfaces. Marked submucosal oedema of the ascending colon was observed in case 2, but no other significant abnormalities were seen in any other organ examined histologically in either case. In both cases, the intracytoplasmic granules within neoplastic cells were diastase resistant, PAS positive and clearly visible with Masson’s trichrome stain. Immunohistochemical results are summarized in Table 1. In both cases, diffuse, strong, intracytoplasmic, finely granular labelling for pan-cytokeratin was observed. Labelling for amylase demonstrated intense, granular, intracytoplasmic expression, often forming clumps, in approximately 30e40% of the neoplastic cells in case 2, while only occasional neoplastic cells reacted positively in case 1. Case 2 had scattered, rare, intracytoplasmic labelling for glucagon and synaptophysin, while sections from case 1 were negative. Neoplastic cells from both cases were negative for insulin and NSE. Ultrastructural examination of metastatic nodules showed neoplastic cells with obvious exocrine secretory characteristics that were more prominent in case 1. Neoplastic cells were polygonal, often organized in acini, with basally located nuclei and an apical small lumen. Frequently, neoplastic cells contained an abundant (case 1) or moderate (case 2) number of intracytoplasmic round to oval and electron dense granules, measuring 100e2,000 nm in diameter and surrounded by a limiting membrane, compatible with zymogen granules (Fig. 2). In addition, case 1 had some tumour cells that displayed another type of cytoplasmic granule, with an oval to rhomboid shape, measuring up to 600 nm. These inclusions showed a limiting membrane and contained
Fig. 2. Pancreatic acinar cell carcinoma; horse 1. Several neoplastic cells are organized in acini. They show basal nuclei, contain abundant intracytoplasmic round electron dense granules (zymogen granules) of up to 2,000 nm diameter and form a small lumen bordered by microvilli at their apical pole. Electron microscopy. Bar, 5 mm.
densely-packed fibrillary material (Fig. 3). No evidence of these granules was seen in case 2. Additionally, the tumour cells of case 2 revealed occasional round to oval cytoplasmic granules, 100e400 nm
Table 1 Immunohistochemical labelling in two equine pancreatic acinar cell carcinomas Antibody
Case 1
Case 2
Amylase Cytokeratin Glucagon Insulin NSE Synaptophysin
+ +++ e e e e
++ +++ + e e +
e, No labelled neoplastic cells; +, <10% of neoplastic cells labelled; ++, 10e50% of neoplastic cells labelled; +++, >50% of neoplastic cells labelled.
Fig. 3. Pancreatic acinar cell carcinoma; horse 1. Neoplastic cell with intracytoplasmic oval to rhomboid fibrillary granules. Electron microscopy. Bar, 0.2 mm.
Pancreatic Carcinoma in Two Horses
diameter, with a round to oval, eccentric, dense homogeneous core surrounded by a membranous envelope, compatible with neuroendocrine granules, more specifically, alpha granules. Similar granules were not seen in case 1. Based on these findings, a diagnosis of pancreatic ACC was made in both horses. Grossly, differential diagnoses in both horses included pancreatic islet cell carcinoma, exocrine pancreatic carcinoma, cholangiocellular carcinoma and mesothelioma. During necropsy examination, gastrointestinal carcinoma was also considered, but was ruled out as no neoplastic nodules affecting mucosa or intestinal wall were observed in any portion of the gastrointestinal tract. On histological examination, the observation of granules compatible with zymogen granules within neoplastic cells that were organized in acini (mainly in case 1) pointed towards a diagnosis of pancreatic exocrine carcinoma (Ord on ~ez, 2001; Head et al., 2002). According to the WHO Classification of Tumours of Domestic Animals, there are three histological types of epithelial tumours originating from the exocrine pancreas: adenomas, adenocarcinomas and non-differentiated carcinomas (Kircher and Nielsen, 1976). Adenocarcinomas are further divided into small tubular, large tubular and ACCs. In both of the present cases, the acinar pattern was dominant, but tubular and solid patterns of undifferentiated cells were also noted multifocally. Similarly, pancreatic ACCs in man frequently display variable histological features, with multiple appearances within a single tumour as well as between tumours (Ordon ~ez, 2001). Zymogen granules are characteristic of exocrine pancreatic carcinomas and, more specifically, of the ACC subtype, since tubular cells do not contain such granules (Ord on ~ez, 2001; La Rosa et al., 2012). However, zymogen granules may be unapparent or present in a low number of cells within the tumour (Ord on ~ez, 2001; Head et al., 2002), as was the case in horse 2. In these situations, the use of IHC to detect pancreatic hormones, neuroendocrine granules and pancreatic enzymes is useful in discriminating between endocrine and exocrine pancreatic carcinomas (Head et al., 2002). In the present cases, endocrine pancreatic neoplasia was ruled out in both horses, since negative results were obtained by IHC for insulin and NSE and only a low percentage of tumour cells expressed synaptophysin and glucagon in case 2. In contrast, expression of amylase was shown for both tumours. Surprisingly, amylase labelling was more intense in case 2, in which the zymogen granules were less frequently seen, while the labelling was observed in only a low percentage of cells in case 1. In man, pancreatic ACCs often have a
391
low percentage of positive cells (La Rosa et al., 2012). In addition, among the few immunohistochemical data available for animals, Rabanal et al. (1992) suggested that IHC for both amylase and carboxypeptidase was of value in the diagnosis of pancreatic exocrine tumours in dogs, often showing abundant labelling within these tumours. These variations in amylase labelling might be attributed to factors such as the use of different antibodies, laboratory procedures as well as inter-species differences. Finally, the present tumours were strongly and diffusely positive for pan-cytokeratin, confirming their epithelial origin. Ultrastructurally, zymogen granules are generally larger than neuroendocrine granules. In man, zymogen granules in ACCs are reported to measure 125e1,000 nm in diameter, while neuroendocrine granules are usually <500 nm and often show a halo and granular contents (Ulich et al., 1982; Ordon ~ez, 2001). In both of the present cases, the dominant granules showed ultrastructural features characteristic of zymogen granules. In addition, in case 1, some neoplastic cells contained large irregular granules with a fine filamentous ultrastructure. Interestingly, these granules are similar to the so-called fibrillary granules, which are reported to be a frequent feature in human pancreatic ACC (Ordon ~ez, 2001) and have only been described occasionally in dogs (Banner et al., 1978) and a cat (Banner et al., 1979). Fibrillary granules are considered to be part of the spectrum of zymogen granules in neoplastic acinar cells and are considered to be an important diagnostic feature of pancreatic ACC in man (Ordon ~ez, 2001). Finally, in case 2, some neoplastic cells showed smaller round granules with a highly electronopaque core surrounded by a clear halo, which were compatible with glucagon granules. A low percentage of neoplastic cells in this horse also showed reactivity for both glucagon and synaptophysin. Taking into account that the EM technique was performed on tissue collected from metastatic nodules on the intestinal serosa, distant to the primary site of the tumour, it may be that the pancreatic ACC in case 2 showed scattered neuroendocrine differentiation associated with the neoplastic acini. Similarly, nearly 50% of primary and metastatic human pancreatic carcinomas show pluripotent capacity and express two or more cell lineage markers (Kim et al., 1990). Therefore, this finding suggests that in horses, as hypothesized in man, the original transformed cell type in some pancreatic exocrine carcinomas may retain the capability for differentiation along more than one cell lineage pathway (Kim et al., 1990). In man, a diagnosis of a mixed acinar-neuroendocrine
S. de Brot et al.
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carcinoma is established when >30% of neoplastic cells show endocrine differentiation (La Rosa et al., 2012). Since this was not so in these horses, pancreatic ACC was the final diagnosis in both cases.
Acknowledgements The authors would like to thank Prof. Dr. P. Wild and L. Nufer, University of Zurich, for their excellent assistance with EM and the Institute of Clinical Pathology, University Hospital Zurich, for performing IHC.
References Banner BF, Alroy J, Kipnis RM (1979) Acinar cell carcinoma of the pancreas in a cat. Veterinary Pathology, 16, 543e547. Banner BF, Alroy J, Pauli BU, Carpenter JL (1978) An ultrastructural study of acinic cell carcinomas of the canine pancreas. American Journal of Pathology, 93, 165e182. Carrick JB, Morris DD, Harmon BG, Fawzi M (1992) Hematuria and weight loss in a mare with pancreatic adenocarcinoma. Cornell Veterinarian, 82, 91e97. Charles JA (2007) Pancreas. In: Jubb, Kennedy and Palmer’s Pathology of Domestic Animals, 5th Edit., Vol. 2, MG Maxie, Ed., Saunders Elsevier, London, pp. 389e424. Church S, West HJ, Baker JR (1987) Two cases of pancreatic adenocarcinoma in horses. Equine Veterinary Journal, 19, 77e79. Dennis MM, O’Brien TD, Wayne T, Kiupel M, Williams M et al. (2008) Hyalinizing pancreatic adenocarcinoma in six dogs. Veterinary Pathology, 45, 475e483. Greenlee RT, Hill-Harmon MB, Murray T, Thun M (2001) Cancer statistics, 2001. Cancer Journal for Clinicians, 51, 15e36. Head KW, Else RW, Dubielzig RR (2002) Tumors of the alimentary tract. In: Tumors in Domestic Animals, 4th Edit., DJ Meuten, Ed., Iowa State Press, Ames, pp. 401e481. Kelley LC, Harmon BG, McCaskey PC (1996) A retrospective study of pancreatic tumors in slaughter cattle. Veterinary Pathology, 33, 398e406. Kim JH, Ho SB, Montgomery CK, Kim YS (1990) Cell lineage markers in human pancreatic cancer. Cancer, 66, 2134e2143.
Kircher CH, Nielsen SW (1976) Tumours of the pancreas. Bulletin of the World Health Organization, 53, 195e202. La Rosa S, Adsay V, Albarello L, Asioli S, Casnedi S et al. (2012) Clinicopathologic study of 62 acinar cell carcinomas of the pancreas: insights into the morphology and immunophenotype and search for prognostic markers. American Journal of Surgical Pathology, 36, 1782e1795. Lucena RB, Rissi DR, Kommers GD, Pierezan F, OliveiraFilho JC et al. (2011) A retrospective study of 586 tumours in Brazilian cattle. Journal of Comparative Pathology, 145, 20e24. Malvezzi M, Bertuccio P, Levi F, La Vecchia C, Negri E (2013) European cancer mortality predictions for the year 2013. Annals of Oncology, 24, 792e800. Ord on ~ez NG (2001) Pancreatic acinar cell carcinoma. Advances in Anatomic Pathology, 8, 144e159. Priester WA (1974) Data from eleven United States and Canadian colleges of veterinary medicine on pancreatic carcinoma in domestic animals. Cancer Research, 34, 1372e1375. Rabanal R, Fondevila D, Vargas A, Ramis A, Badiola J et al. (1992) Immunocytochemical detection of amylase, carboxypeptidase A, carcinoembryonic antigen and alpha 1-antitrypsin in carcinomas of the exocrine pancreas of the dog. Research in Veterinary Science, 52, 217e223. Rendle DI, Hewetson M, Barron R, Baily JE (2006) Tachypnoea and pleural effusion in a mare with metastatic pancreatic adenocarcinoma. Veterinary Record, 159, 356e359. Rowlatt U (1967) Spontaneous epithelial tumours of the pancreas of mammals. British Journal of Cancer, 21, 82e107. Seaman RL (2004) Exocrine pancreatic neoplasia in the cat: a case series. Journal of the American Animal Hospital Association, 40, 238e245. Ulich T, Cheng L, Lewin KJ (1982) Acinar-endocrine cell tumor of the pancreas. Report of a pancreatic tumor containing both zymogen and neuroendocrine granules. Cancer, 50, 2099e2105.
August 15th, 2013 ½ Received, Accepted, January 4th, 2014
Available online at www.sciencedirect.com
ScienceDirect www.elsevier.com/locate/jcpa
NEOPLASTIC DISEASE
Acinar Cell Carcinoma of Exocrine Pancreas in Two Horses S. de Brot*, H. Junge† and M. Hilbe* * Institute of Veterinary Pathology and † Clinic for Equine Internal Medicine, Equine Department, Vetsuisse Faculty, University of Zurich, Winterthurerstrasse 260, CH-8057 Zurich, Switzerland
Summary Two horses were presented with non-specific clinical signs of several weeks’ duration and were humanely destroyed due to a poor prognosis. At necropsy examination, both horses had multiple small, white nodules replacing pancreatic tissue and involving the serosal surface of the abdominal cavity, the liver and the lung. Microscopically, neoplastic cells were organized in acini and contained abundant (case 1) or sparse (horse 2) intracytoplasmic zymogen granules. Immunohistochemically, both tumours expressed amylase and pancytokeratin, but not insulin or neuron-specific enolase. In case 2, a low percentage of neoplastic cells expressed glucagon and synaptophysin. The presence of zymogen granules was confirmed in both cases by electron microscopy and occasional fibrillary or glucagon granules were observed in cases 1 and 2, respectively. A diagnosis of pancreatic acinar cell carcinoma was established in both horses. Ó 2014 Elsevier Ltd. All rights reserved. Keywords: electron microscopy; fibrillary granules; immunohistochemistry; pancreas
Neoplastic lesions of the pancreas are uncommon in domestic animals and originate more frequently from exocrine cells (Priester, 1974; Head et al., 2002; Charles, 2007). Most cases are reported in dogs (Rowlatt, 1967; Rabanal et al., 1992; Dennis et al., 2008). Such tumours are less common in cats (Banner et al., 1979; Seaman, 2004) and rare in horses (Church et al., 1987; Carrick et al., 1992; Rendle et al., 2006), cattle (Kelley et al., 1996; Lucena et al., 2011) and pigs (Rowlatt, 1967). This differs from man, where pancreatic neoplasia is among the five most common causes of cancerrelated mortality with an increasing rate of prevalence (Greenlee et al., 2001; Malvezzi et al., 2013). The diagnosis of pancreatic exocrine carcinoma by routine light microscopy may be difficult since it can be confused with other tumours, especially islet cell carcinoma (Ordon ~ez, 2001). In man, it has been demonstrated that both immunohistochemistry (IHC) and electron microscopy (EM) are useful in es-
tablishing the diagnosis of pancreatic cell carcinomas (Ordon ~ez, 2001; La Rosa et al., 2012). However, the few reports of equine pancreatic tumours are based on gross and light microscopical findings and do not include immunohistochemical or ultrastructural studies. The present study describes two horses with pancreatic acinar cell carcinoma (ACC). Case 1 was a 22-year-old Polish warmblood mare that was presented to the Clinic for Equine Internal Medicine, Vetsuisse Faculty, University of Zurich, with a 3-week history of apathy, anorexia, weight loss, polyuria and polydypsia. Case 2 was a 17-yearold Swiss warmblood gelding that was presented to the same institution with a several-week history of weight loss and anorexia. Laboratory investigations showed significant elevation of liver enzymes in both cases. Ultrasonography revealed the presence of multiple intrapulmonary nodules and a thickened colonic wall in case 1 and an irregular serosal surface of the small intestine in case 2. Exploratory laparotomy was performed in case 2, which revealed a thickened colonic wall, enlarged abdominal lymph nodes and a firm liver. Neoplasia was suspected in both cases and the
Correspondence to: S. de Brot (e-mail: [email protected]). 0021-9975/$ - see front matter http://dx.doi.org/10.1016/j.jcpa.2014.01.003
Ó 2014 Elsevier Ltd. All rights reserved.
Pancreatic Carcinoma in Two Horses
animals were humanely destroyed due to a poor prognosis. Necropsy examination was performed within 6 h of death in both cases. Samples of various organs were fixed in 10% neutral buffered formalin and processed routinely. Sections (3 mm) were stained with haematoxylin and eosin (HE), periodic acideSchiff (PAS), PAS after enzymatic digestion with diastase and Masson’s trichrome stain. IHC was performed using commercially available antibodies specific for amylase (anti-rabbit, 1 in 1,000 dilution; Catalogue number A8273; Sigma Chemical Company, St Louis, Missouri, USA) pan-cytokeratin (anti-mouse, 1 in 50 dilution; catalogue number M082101; Dako, Glostrup, Denmark), glucagon (anti-human, 1 in 100 dilution; catalogue number A0565, Dako), insulin (anti-guinea pig, 1 in 200 dilution; catalogue number A0564; Dako), neuron-specific enolase (NSE, anti-mouse, 1 in 150 dilution; catalogue number M087301, Dako) and synaptophysin (anti-mouse, 1 in 10 dilution; catalogue number M077601, Dako). The standard avidinebiotineperoxidase technique was used to demonstrate the binding of primary antibody. Normal horse pancreatic tissue was used as a positive control. Labelling was evaluated semiquantitatively by assessing the percentage of labelled neoplastic cells as follows: e, positive neoplastic cells not seen; +, <10% of neoplastic cells labelled; ++, 10e50% of neoplastic cells labelled; and +++, >50% of neoplastic cells labelled. For EM, selected tissues were fixed in 2.5% glutaraldehyde and 1% osmium tetroxide (OsO4), buffered with 0.1 M sodium phosphate (pH 7.4). After fixation, the tissues were dehydrated in an ethanol series, embedded in epon and ultrathin (90 nm) sections were stained with uranyl acetate and lead citrate. Necropsy examination of case 1 revealed the presence of 4 l of serosanguineous fluid in the thoracic cavity and 30 l of similar fluid in the abdominal cavity. The pancreas was markedly irregular and diffusely disrupted by the presence of innumerable small white to tan, firm nodules. Similar nodules (2e15 mm diameter), some of them umbilicated, were scattered over the mesentery and the serosal surface of the small intestine, colon, spleen and diaphragm. Approximately 50% of the hepatic parenchyma contained similar nodules with a multifocal to coalescing distribution. Mesenteric lymph nodes were markedly enlarged. In the thorax, approximately 10% of the pulmonary parenchyma was infiltrated by multiple tan to red, firm nodules (5e15 mm diameter) and the mediastinal lymph nodes were also markedly enlarged. Necropsy examination of case 2 revealed approximately 20 l of serosanguinous fluid in the abdominal
389
cavity. The pancreas was extensively replaced by a firm, white, multinodular mass with a smooth surface that adhered to the serosa covering the liver and diaphragm. The liver was firm and infiltrated by multifocal to coalescing pale foci (1e10 mm diameter), which occupied approximately 50% of the parenchyma. Hepatic and mesenteric lymph nodes were markedly enlarged, firm and cream in colour. Examination of the gastrointestinal tract showed a markedly oedematous ascending colon. In both cases, microscopical examination revealed the nodular masses to be comprised of a densely cellular, infiltrative and non-encapsulated tumour. No normal pancreatic tissue was observed in any of the sections. Neoplastic cells were organized mainly as acini and less frequently in a tubular or solid arrangement. The stroma was fibrous with a multifocal scirrhous reaction. Neoplastic cells were polyhedral, 15e25 mm in diameter, with mainly indistinct cell borders. In case 1, tumour cells showed a moderate amount of eosinophilic, homogeneous cytoplasm, often containing numerous intracytoplasmic deeply eosinophilic granules (Fig. 1). However, in case 2, only scant similar granules were seen within a low number of neoplastic cells. The nuclei of the neoplastic cells were round to oval, often basally located and contained finely stippled chromatin with 1e3 prominent eosinophilic nucleoli. Abundant mitotic figures were seen and were more numerous in areas of solid growth (up to 10 per 40 objective field). There was marked anisokaryosis and anisocytosis. Multifocally, there were extensive and abundant areas of necrosis, sometimes accompanied by haemorrhage. Often, the lumina of lymphatic and
Fig. 1. Pancreatic acinar cell carcinoma; horse 1. Neoplastic cells with basal nuclei and intracytoplasmic eosinophilic granules (zymogen granules) form acinar structures. HE. Bar, 50 mm.
S. de Brot et al.
390
blood vessels contained aggregates of neoplastic cells. Multifocally, areas of moderate lymphoplasmacytic infiltration were present. Overall, the observed features were compatible with pancreatic acinar carcinoma with metastasis to liver, lung, lymph nodes and peritoneal surfaces. Marked submucosal oedema of the ascending colon was observed in case 2, but no other significant abnormalities were seen in any other organ examined histologically in either case. In both cases, the intracytoplasmic granules within neoplastic cells were diastase resistant, PAS positive and clearly visible with Masson’s trichrome stain. Immunohistochemical results are summarized in Table 1. In both cases, diffuse, strong, intracytoplasmic, finely granular labelling for pan-cytokeratin was observed. Labelling for amylase demonstrated intense, granular, intracytoplasmic expression, often forming clumps, in approximately 30e40% of the neoplastic cells in case 2, while only occasional neoplastic cells reacted positively in case 1. Case 2 had scattered, rare, intracytoplasmic labelling for glucagon and synaptophysin, while sections from case 1 were negative. Neoplastic cells from both cases were negative for insulin and NSE. Ultrastructural examination of metastatic nodules showed neoplastic cells with obvious exocrine secretory characteristics that were more prominent in case 1. Neoplastic cells were polygonal, often organized in acini, with basally located nuclei and an apical small lumen. Frequently, neoplastic cells contained an abundant (case 1) or moderate (case 2) number of intracytoplasmic round to oval and electron dense granules, measuring 100e2,000 nm in diameter and surrounded by a limiting membrane, compatible with zymogen granules (Fig. 2). In addition, case 1 had some tumour cells that displayed another type of cytoplasmic granule, with an oval to rhomboid shape, measuring up to 600 nm. These inclusions showed a limiting membrane and contained
Fig. 2. Pancreatic acinar cell carcinoma; horse 1. Several neoplastic cells are organized in acini. They show basal nuclei, contain abundant intracytoplasmic round electron dense granules (zymogen granules) of up to 2,000 nm diameter and form a small lumen bordered by microvilli at their apical pole. Electron microscopy. Bar, 5 mm.
densely-packed fibrillary material (Fig. 3). No evidence of these granules was seen in case 2. Additionally, the tumour cells of case 2 revealed occasional round to oval cytoplasmic granules, 100e400 nm
Table 1 Immunohistochemical labelling in two equine pancreatic acinar cell carcinomas Antibody
Case 1
Case 2
Amylase Cytokeratin Glucagon Insulin NSE Synaptophysin
+ +++ e e e e
++ +++ + e e +
e, No labelled neoplastic cells; +, <10% of neoplastic cells labelled; ++, 10e50% of neoplastic cells labelled; +++, >50% of neoplastic cells labelled.
Fig. 3. Pancreatic acinar cell carcinoma; horse 1. Neoplastic cell with intracytoplasmic oval to rhomboid fibrillary granules. Electron microscopy. Bar, 0.2 mm.
Pancreatic Carcinoma in Two Horses
diameter, with a round to oval, eccentric, dense homogeneous core surrounded by a membranous envelope, compatible with neuroendocrine granules, more specifically, alpha granules. Similar granules were not seen in case 1. Based on these findings, a diagnosis of pancreatic ACC was made in both horses. Grossly, differential diagnoses in both horses included pancreatic islet cell carcinoma, exocrine pancreatic carcinoma, cholangiocellular carcinoma and mesothelioma. During necropsy examination, gastrointestinal carcinoma was also considered, but was ruled out as no neoplastic nodules affecting mucosa or intestinal wall were observed in any portion of the gastrointestinal tract. On histological examination, the observation of granules compatible with zymogen granules within neoplastic cells that were organized in acini (mainly in case 1) pointed towards a diagnosis of pancreatic exocrine carcinoma (Ord on ~ez, 2001; Head et al., 2002). According to the WHO Classification of Tumours of Domestic Animals, there are three histological types of epithelial tumours originating from the exocrine pancreas: adenomas, adenocarcinomas and non-differentiated carcinomas (Kircher and Nielsen, 1976). Adenocarcinomas are further divided into small tubular, large tubular and ACCs. In both of the present cases, the acinar pattern was dominant, but tubular and solid patterns of undifferentiated cells were also noted multifocally. Similarly, pancreatic ACCs in man frequently display variable histological features, with multiple appearances within a single tumour as well as between tumours (Ordon ~ez, 2001). Zymogen granules are characteristic of exocrine pancreatic carcinomas and, more specifically, of the ACC subtype, since tubular cells do not contain such granules (Ord on ~ez, 2001; La Rosa et al., 2012). However, zymogen granules may be unapparent or present in a low number of cells within the tumour (Ord on ~ez, 2001; Head et al., 2002), as was the case in horse 2. In these situations, the use of IHC to detect pancreatic hormones, neuroendocrine granules and pancreatic enzymes is useful in discriminating between endocrine and exocrine pancreatic carcinomas (Head et al., 2002). In the present cases, endocrine pancreatic neoplasia was ruled out in both horses, since negative results were obtained by IHC for insulin and NSE and only a low percentage of tumour cells expressed synaptophysin and glucagon in case 2. In contrast, expression of amylase was shown for both tumours. Surprisingly, amylase labelling was more intense in case 2, in which the zymogen granules were less frequently seen, while the labelling was observed in only a low percentage of cells in case 1. In man, pancreatic ACCs often have a
391
low percentage of positive cells (La Rosa et al., 2012). In addition, among the few immunohistochemical data available for animals, Rabanal et al. (1992) suggested that IHC for both amylase and carboxypeptidase was of value in the diagnosis of pancreatic exocrine tumours in dogs, often showing abundant labelling within these tumours. These variations in amylase labelling might be attributed to factors such as the use of different antibodies, laboratory procedures as well as inter-species differences. Finally, the present tumours were strongly and diffusely positive for pan-cytokeratin, confirming their epithelial origin. Ultrastructurally, zymogen granules are generally larger than neuroendocrine granules. In man, zymogen granules in ACCs are reported to measure 125e1,000 nm in diameter, while neuroendocrine granules are usually <500 nm and often show a halo and granular contents (Ulich et al., 1982; Ordon ~ez, 2001). In both of the present cases, the dominant granules showed ultrastructural features characteristic of zymogen granules. In addition, in case 1, some neoplastic cells contained large irregular granules with a fine filamentous ultrastructure. Interestingly, these granules are similar to the so-called fibrillary granules, which are reported to be a frequent feature in human pancreatic ACC (Ordon ~ez, 2001) and have only been described occasionally in dogs (Banner et al., 1978) and a cat (Banner et al., 1979). Fibrillary granules are considered to be part of the spectrum of zymogen granules in neoplastic acinar cells and are considered to be an important diagnostic feature of pancreatic ACC in man (Ordon ~ez, 2001). Finally, in case 2, some neoplastic cells showed smaller round granules with a highly electronopaque core surrounded by a clear halo, which were compatible with glucagon granules. A low percentage of neoplastic cells in this horse also showed reactivity for both glucagon and synaptophysin. Taking into account that the EM technique was performed on tissue collected from metastatic nodules on the intestinal serosa, distant to the primary site of the tumour, it may be that the pancreatic ACC in case 2 showed scattered neuroendocrine differentiation associated with the neoplastic acini. Similarly, nearly 50% of primary and metastatic human pancreatic carcinomas show pluripotent capacity and express two or more cell lineage markers (Kim et al., 1990). Therefore, this finding suggests that in horses, as hypothesized in man, the original transformed cell type in some pancreatic exocrine carcinomas may retain the capability for differentiation along more than one cell lineage pathway (Kim et al., 1990). In man, a diagnosis of a mixed acinar-neuroendocrine
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carcinoma is established when >30% of neoplastic cells show endocrine differentiation (La Rosa et al., 2012). Since this was not so in these horses, pancreatic ACC was the final diagnosis in both cases.
Acknowledgements The authors would like to thank Prof. Dr. P. Wild and L. Nufer, University of Zurich, for their excellent assistance with EM and the Institute of Clinical Pathology, University Hospital Zurich, for performing IHC.
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August 15th, 2013 ½ Received, Accepted, January 4th, 2014