ELSEVIER
DISEASE IN WILDLIFE OR EXOTIC SPECIES
Oncocytic Adrenocortical Carcinoma in a Putty- nosed Monkey (Cercopithecus nictitans) with Hyperadrenocorticism
E. Gruber-Dujardin*, K. Jurczynski1, F .- J. Kaup* and K. Matz-Rensing*
* Pathology Unit, German Primate Center, Göttingen and Veterinary Department, Duisburg Zoo, Germany
Summary
Oncocytic adrenocortical tumours are rare in man and have never been described in non-human primates. An oncocytic adrenocortical carcinoma was identified in an 18-year-old female putty-nosed monkey (Cercopithecus nictitans) with hyperadrenocorticism and invasive aspergillosis. Microscopically, the tumour consisted of large cells with abundant eosinophilic, granular cytoplasm containing numerous mitochondria as identified by elec- tron microscopy. Tumour cells had large nuclei with occasional intranuclear cytoplasmic pseudoinclusions. Immunohistochemically, tumour cells expressed vimentin, synaptophysin and neuron-specific enolase, while they were negative for cytokeratin, chromogranin-A, melan-A and S100.
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Keywords: hyperadrenocorticism; non-human primate; oncocytic tumour; pseudoinclusions
Oncocytic tumour cells are characterized by abun- dant intracytoplasmic mitochondria, causing a ‘swollen’ (oncocytic) appearance. Such tumours arise most commonly in the salivary, thyroid, or parathy- roid glands and kidney (Tallini, 1998). Oncocytic tu- mours of the adrenal cortex are rare in man and have never been described in non-human primates. Such tumours are usually benign, with only 18 reported malignant cases (El-Naggar et al., 1991; Alexander and Paulose, 1998; Kurek et al., 2001; Hoang et al., 2002; Seo et al., 2002; Bisceglia et al., 2004; Song et al., 2004; Tanaka et al., 2004; Ali and Raphael, 2007; Ohtake et al., 2010). The tumours are also generally non-functional. To our knowledge, only two human patients have presented with hyperadre- nocorticism together with oncocytic adrenocortical carcinoma (Alexander and Paulose, 1998; Ali and Raphael, 2007).
An 18-year-old, captive female putty-nosed mon- key (Cercopithecus nictitans) with a history of chronic infertility was submitted to the Pathology Unit of the German Primate Centre after being severely
injured by group members, with resultant pyothorax and invasive aspergillosis with pulmonary and cere- bral involvement. As described elsewhere, the animal also had signs of hyperadrenocorticism (Jurczynski et al., 2012). A well-circumscribed, oval mass (6.3 × 4.1 × 3.5 cm) was seen to replace the right ad- renal gland. The mass had multifocal areas of hae- morrhage and necrosis with a variegated, yellow, friable cut surface. The left adrenal gland was not de- tected despite thorough exploration.
The tumour was fixed in 10% neutral-buffered formalin, processed routinely and embedded in paraffin wax. Sections (3-4 um) were stained with haematoxylin and eosin (HE), periodic acid-Schiff (PAS) and phosphotungstic acid-haematoxylin (PTAH). Immunohistochemistry (IHC) was per- formed using the avidin-biotin-peroxidase method. Primary antibodies were specific for vimentin (V9, 1 in 100 dilution, monoclonal antibody; Dako, Glostrup, Denmark), cytokeratin (CK, MNF 116, broad-spectrum, 1 in 100 dilution, monoclonal anti- body; Dako), chromogranin-A (DAK-A3, 1 in 100 dilution, monoclonal antibody; Dako), neuron- specific enolase (NSE, BBS/NC/VI-H14, 1 in 400 dilu- tion, monoclonal antibody; Dako), synaptophysin
(anti-synaptophysin 1, 1 in 500 dilution, monoclonal antibody; Synaptic Systems GmbH, Göttingen, Germany), melan-A (A103, 1 in 50 dilution, mouse anti-human antibody; Dako), S100 (1 in 1,000 dilu- tion, polyclonal antibody; Dako) and Ki67 (MIB-1, 1 in 50 dilution, monoclonal antibody; Dako). 3, 3’diaminobenzidine (DAB) was used as the chromo- genic substrate and sections were counterstained with haematoxylin. For ultrastructural investigation with a Zeiss EM 10C electron microscope, tumour tis- sue was embedded in Epon 812 and contrasted with uranyl acetate and lead citrate.
Microscopically, the mass was densely cellular and completely effaced the right adrenal gland. It was partially encapsulated and multilobular, composed of large round to polygonal cells that were arranged diffusely in anastomosing nests or trabeculae and sup- ported by a delicate fibrovascular stroma. In some areas, neoplastic cells formed small sinusoids, lined by flattened endothelium (Fig. 1). Tumour cells had indistinct borders and moderate to abundant brightly eosinophilic, finely granular or sometimes vacuolated cytoplasm. The large round to oval nuclei showed marginated or finely stippled chromatin, often with one distinct large magenta nucleolus and occasional striking round to oval, eosinophilic, PAS-negative nu- clear inclusions of varying size (Fig. 2). Numerous neoplastic cells displayed karyomegaly or were multi- nucleated and sometimes invaded the fibrous capsule as well as some sinusoids (Fig. 1). PTAH staining re- vealed a high cytoplasmic density of mitochondria, indicated by dark blue, fine cytoplasmic granulation (Fig. 3). There was moderate anisocytosis and aniso- karyosis with an average of two mitoses per 10 high- power fields (HPFs, x40 objective), determined in
non-necrotic areas with high mitotic activity, in addi- tion to sporadic atypical mitoses. The proliferation in- dex (PI), determined by the ratio of Ki67+ cells, was 18% of 850 neoplastic cells counted. Throughout the tumour there were multiple areas of necrosis, haemor- rhage and mineral deposition.
IHC revealed diffuse expression of vimentin, while 40% of the neoplastic cells expressed synaptophysin and 25% expressed NSE. There was no expression of CK, chromogranin-A, melan-A or S100.
Electron microscopy revealed large tumour cells with a round to oval nucleus that sometimes showed marked undulation or invagination of the nuclear enve- lope, forming intranuclear pseudoinclusions that incor- porated numerous cytoplasmic organelles, mainly mitochondria and some stacks of rough endoplasmic
reticulum (ER) (Figs. 4a-e). There was clumping of heterochromatin along the nuclear envelope and a prominent, round nucleolus was located at the nuclear periphery. The cytoplasm was packed with numerous enlarged globular mitochondria, showing atypical morphological variation such as stacks of lamelliform, tubular to flat cristae or complete loss of these structures (Fig. 4d). Additionally, round to polygonal, lysosomal, electron-dense inclusions as well as lamellar body-like structures with a furled double membrane (Fig. 4e) and a granular or electron-dense core were noted occa- sionally. Other cytoplasmic organelles were sparse, except for focal prominence of smooth ER or randomly scattered parallel stacks of rough ER. A few tumour cells contained lipid droplets (Fig. 4b). On the basis of the histopathological, ultrastructural and immuno- histochemical characteristics, a diagnosis of oncocytic adrenocortical carcinoma was made.
Oncocytic tumours have distinct morphological features including abundant eosinophilic granular cytoplasm composed of numerous aberrant mito- chondria (Gasparre et al., 2011). Little is known about the mechanism leading to oncocytosis in general. Mitochondrial proliferation as a compensatory mech- anism due to dysfunction or differentiation into cells with high energy output is suggested. Another hy- pothesis is that oncocytomas are tumours of mito- chondria because they have their own DNA (Gasparre et al., 2011). However, all of these hypoth- eses suppose that the observed structural changes are associated with functional alteration. Mitochondrial biogenesis is induced by retrograde signalling from or- ganelles to the nucleus in cases of metabolic stress and/ or loss of mitochondrial function (Butow and Avadhani, 2004). Generally, mitochondrial stress re- sults from decreased oxygen tension (e.g. in tumour
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cells at initial stages before neovascularization). Decreased oxygen tension leads to increased levels of reactive oxygen species (ROS), which may represent the trigger for retrograde signalling to the nucleus for oncocytic modification, either via induction of mito- chondrial DNA damage or another, yet unknown, proliferative stimulus (Gasparre et al., 2011). Accord- ingly, defective mitochondrial ATP synthesis, caused by oxidative phosphorylation coupling defects or overexpression of uncoupling mitochondrial proteins, are discussed as possible stimuli for mitochondrial hy- perplasia in oxyphilic thyroid tumours (Savagner et al., 2001). In the present case, the cause of the mito- chondrial proliferation and morphological alteration remains speculative. One possible trigger could have been a markedly decreased oxygen tension within the tumour, resulting from fast tumour growth. More- over, systemic hypoxia due to the severe pulmonary aspergillosis and tissue infarction in this animal might also have contributed to low cellular oxygen levels.
Another indication for increased ROS within the tumour cells might have been the presence of lamellar body-like structures and lysosomal electron-dense in- clusions, which are also described in cases of adreno- cortical phospholipidosis (e.g. after toxin-induced activation of cytochrome P-450 enzymes; Rosol et al., 2001).
Normal adrenocortical cells of all three zones have prominent mitochondria that can be distinguished ul- trastructurally by the shape of their cristae. Further- more, they contain lipid droplets, Golgi apparatus, smooth ER and lysosomes in varying composition (Rosol et al., 2001). All of these organelles were also identified in the present tumour cells. In contrast, chromaffin cells of the adrenal medulla are character- ized ultrastructurally by membrane-bound dense core granules, which were not detected in this case. Additionally, IHC for chromogranin-A was diffusely negative in all neoplastic cells, so that this tumour was unlikely to have been of adrenal medullary origin.
Further differential diagnoses for adrenocortical carcinomas include other primary tumours, such as renal or hepatocellular carcinoma, or metastatic tu- mours such as malignant melanoma carcinoma of the ovary and uterus, or lung (Sasano et al., 2006). In the present case, IHC for CK was negative, while neoplastic cells diffusely expressed vimentin. Although oncocytic metaplasia is often described in conjunction with epithelial cells (Doughty et al., 2006), there are many cases of human adrenocortical oncocytomas that are immunoreactive for vimentin with variable CK positivity (Wong et al., 2011). However, the immunoreactivity profile can be unusual in non- human primates, especially in exotic species, although the CK antibody used here showed positive labelling
of other epithelial tissue in the same animal. The lack of CK expression and absence of glycogen accu- mulation within tumour cells in the present case makes a primary hepatocellular or renal origin of the tumour unlikely. Moreover, the tumour cells did not show any immunoreactivity for S100 or melan-A and the lungs, ovaries and uterus of the monkey had no neoplastic changes. Therefore, the histological, immunohisto- chemical and ultrastructural cell characteristics were consistent with an adrenocortical tumour.
Oncocytic adrenocortical tumours exhibit a diffuse architecture with few vacuolated cells and frequent nuclear atypia, even when they lack malignant behaviour (Lin et al., 1998; Bisceglia et al., 2004). Therefore, it is appropriate to apply different criteria of malignancy than are used usually for adrenocor- tical tumours (Weiss, 1984). Following recent publi- cations (Bisceglia et al., 2004; Ohtake et al., 2010) a human oncocytic tumour is considered malignant if it exhibits more than one of the following features: >5 mitoses per 50 HPFs, PI >10%, any atypical mi- toses, distant metastasis, venous invasion or direct in- vasion of adjacent organs. A tumour with uncertain malignant potential is defined by: large tumour size (>10 cm and/or >200 g), necrosis, capsular or sinu- soidal invasion. If all these features are absent, the tumour is considered benign (Ohtake et al., 2010). Since an oncocytic adrenocortical tumour has not been described previously in a non-human primate, the human criteria of malignancy were used in the present case. The present tumour showed capsular and sinusoidal invasion, atypical mitoses and multi- focal necrosis. The mitotic index was 2 per 10 HPFs and the PI was 18%. Therefore, this oncocytic adre- nocortical tumour was classified as malignant.
A striking feature of the neoplastic cells was the pres- ence of scattered intranuclear pseudoinclusions, result- ing from invagination of the nuclear envelope with incorporation of cytoplasmic organelles. Similar inclu- sions have been described in papillary thyroid carci- nomas, salivary gland tumours, hepatocellular carcinomas, renal clear cell carcinomas, pheochromo- cytomas (Arora and Dey, 2012) and tumours of the pi- tuitary (Yang et al., 2003). However, mechanisms leading to the formation of these nuclear cytoplasmic inclusions are unknown (Arora and Dey, 2012). Some authors have found inclusions predominantly in cells with abundant cytoplasm (e.g. cytomegalic ad- renal cells; Nakamura et al., 1981), considering that in- vaginations of the nuclear envelope result from extensive cytoplasm extruding into the nucleus. In the present tumour cells, the increased cytoplasmic volume might have impressed on the nuclear envelope. In another report, intranuclear cytoplasmic inclusions in the pituitary gland of hamsters were related to
Adrenocortical Carcinoma in a Putty-nosed Monkey
ageing, probably associated with a sex hormone defi- ciency (Serber, 1961). This observation would also fit with the present monkey.
In general, adrenocortical tumours with oncocytic differentiation are non-functional. Only two human cases of hormone-producing oncocytic adrenocortical carcinoma associated with hyperadrenocorticism are reported (Alexander and Paulose, 1998; Ali and Raphael, 2007). However, the oncocytic adrenocor- tical carcinoma in the present case was associated with signs of hyperadrenocorticism, including elev- ated blood cortisol concentration, long-term infer- tility, visceral obesity, alopecia with atrophic skin changes, hyperglycemia, elevated liver enzymes and inactivity of the immune system, which had facil- itated the development of invasive aspergillosis (Jurczynski et al., 2012).
Supplementary data
Supplementary data related to this article can be found at http://dx.doi.org/10.1016/j.jcpa.2013.04.002.
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[ Received, June 21st, 2012 Accepted, April 6th, 2013
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