Canine adrenocorticotropic hormone-producing sinusoidal neuroendocrine tumor associated with Cushing's disease.

An 18-year-old male Yorkshire Terrier was admitted with a history of neurological signs including dullness and progressive tetraparesis. Physical examination revealed bilaterally symmetrical alopecia and pot-bellied abdomen. Computed tomography and necropsy examination showed a mass across the frontal sinus and cerebral frontal lobe, bilateral adrenocortical hyperplasia, and hepatomegaly. Histopathologically, the tumor lesions consisted of sheets, nests, or cords of small- to medium-sized round-to-polyhedral cells. Adrenal cortex showed bilateral diffuse cellular proliferation, and some hepatocytes showed intracytoplasmic glycogen accumulation. Immunohistochemically, the tumor cells were positive for pancytokeratin, chromogranin-A, neuron-specific enolase, S100, synaptophysin, and thyroid transcription factor-1 but negative for microtubule-associated proein-2 and neurofilament, leading to the diagnosis of neuroendocrine tumor. These tumor cells were also positive for adrenocorticotropic hormone.

Neuroendocrine tumors (NETs) are rare tumors in domestic animals. In dogs and cats, these tumors occur in the intestines, esophagus, lungs, naso-oral cavity and skin [13,14,15,16, 18, 23]. In humans, the most common sites are the gastrointestinal and respiratory tracts [2, 6, 10, 22]. Most of these tumors are malignant and aggressive [13, 18, 20, 23]. Identification of NETs primarily relies on histopathological, immunohistochemical, and ultrastructural findings [8, 9, 13,14,15,16, 19, 21]. As the clinical, histopathological, and immunohistological characteristics of these tumors, particularly in the nasal cavities and brain, are similar to other tumors, such as olfactory neuroblastomas, differential diagnosis is usually required [24]. NETs are rarely functional in animals to secrete hormones, including glucagon and serotonin, leading to hormone-associated diseases [1, 3, 17, 20]. As far as we known, adrenocorticotropic hormone (ACTH)-producing NET has been only reported in one dog [3]. The present report describes the computed tomographical, histopathological, and immunohistochemical findings of ACTH-producing NET found in the sinusoidal cavity and brain in an 18-year-old dog in association with Cushing’s disease.

An 18-year-old, male, Yorkshire Terrier was admitted to the Chonnam National University Veterinary Teaching Hospital with a history of neurological signs including dullness and progressive tetraparesis for 3 months, and a perianal tumor. Physical examination revealed severe, bilaterally symmetric alopecia with pot-bellied distended abdomen. Blood test revealed stress leukogram such as neutrophilia (12.56 K/µl over the reference range, 2.95–11.64 K/µl), monocytosis (1.61 K/µl over the reference range, 0.16–1.12 K/µl) and eosinopenia (0.02 K/µl below the reference range, 0.06–1.23 K/µl) and elevated alkaline phosphatase (714 U/l over the reference range, 23–212 U/l). Enlargement of bilateral adrenal glands without change in shape was detected on ultrasonography. Further imaging via computed tomography (CT) showed multiple masses in the frontal sinus, brain, stomach wall, and testes. In the reformatted sagittal plane (Fig. 1A), the sinusoidal tumor destroyed the cribriform plate and frontal bone, invading into the left cerebral frontal lobe and forming a hyperattenuated mass (1.5 × 2 × 3 cm). Serial transverse images of the sinonasal region showed a hyperattenuated mass, measuring 1.5 × 2 × 3 cm, in the left frontal sinus (Fig. 1B) and left cerebral frontal lobe (Fig. 1C). The mass showed strong homogeneous contrast enhancement on post-contrast imaging. Midline shift and displacement of the left ventricle to the right side were observed. There was no abnormal finding of the pituitary gland in size, shape, and density on the pre- and post-contrast CT images.

Fig. 1.

Head, dog, computed tomography. (A) Reformatted sagittal image of the head shows a mass in the cerebral frontal lobe (asterisk) invading the subcutaneous tissue through the frontal bone (arrow). (B) A transverse image of the sinonasal region reveals a hyperattenuated mass (asterisk) in the left frontal sinus. (C) A well-outlined mass in the cerebral frontal lobe (asterisk).

The animal finally fell into a coma and was euthanized to relieve from this terminal illness by intravenous injection of potassium chloride after anesthesia by an intramuscular injection of a combination of 1.5 mg/kg zolazepam hydrochloride-tiletamine hydrochloride (Zoletil, Virbac, Carros, France) and 0.03 mg/kg medetomidine (Domitor, Oriion Corp., Espoo, Finland). Grossly, in consistent with the CT results, a white-to-greyish tumor mass was observed in the left cerebral frontal lobe over the cribriform plate starting from the left frontal sinus (Fig. 2A). The left testis contained a sharply delineated, soft, bulging yellow mass of 2.4 × 2 cm in diameter. The adrenal glands showed severe diffuse bilateral cortical hyperplasia, resulting in an increased cortico-medullary ratio of 3:1. Gross abnormality in the pituitary gland was not observed.

Fig. 2.

Gross and histopathological findings. (A) A cross-section through the frontal lobe. The tumor (asterisk) occupies the lower right frontal lobe, compressing the left frontal lobe. Bar=1 cm. (B) The tumor mass consists of nests of small-to-medium-sized cells separated by a neuropil stroma; some tumor nests undergo central necrosis (asterisk), characterized by necrotic debris, macrophages, and neutrophils. H&E. Bar=200 µm. (C) Tumor cells are round to polyhedral, containing round-to-polyhedral, centrally located nuclei and distinct granular, hyperchromatic cytoplasm. H&E. Bar=30 µm. (D) Homer-Wright rosette formation characterized by central neurophil and peripheral tumor cells (arrow). The tumor nest has central necrosis (asterisk). H&E. Bar=40 µm. (E) Tumor cells contain many intracytoplasmic endocrine granules. Grimelius stain. Bar=25 µm. (F) The liver has severe, multiple, locally extensive swollen hepatocytes, which show intracytoplasmic glycogen accumulation identified by Periodic acid-Schiff reaction (inset). H&E and Periodic acid-Schiff reaction. Bars=40 µm.

For histopathological examination, the tumor masses and major organs were fixed in 10% neutral buffered formalin, embedded in paraffin-wax, sectioned (3 µm), and routinely stained with hematoxylin and eosin (H&E). The selected sections were also subjected to immunohistochemistry (IHC) and Grimelius stain. To identify the nature of swollen hepatocytes, formalin-fixed, frozen liver sections were stained with Periodic acid-Schiff (PAS) reaction. Microscopically, the tumor masses in the left frontal sinus and left cerebral frontal lobe were composed of small-to-medium-sized cells arranged in nests separated by a delicate fibrovascular stroma in the sinus and a neuropil stroma, giving an endocrine-type packaging (Fig. 2B). The tumor cells were round to polyhedral, containing round-to-polyhedral nuclei with distinct nucleolus and a distinct granular eosinophilic cytoplasm (Fig. 2C). Large clear neoplastic cells were mixed with round-to-polyhedral cells in some tumor islands. The peripheral tumor cells in the tumor nests and islands showed a palisading appearance. Homer-Wright rosette formation was rarely found in the tumor lesions (Fig. 2D). Some tumor nests underwent central necrosis containing necrotic debris, macrophages, and neutrophils (Fig. 2B). By Grimelius stain, the tumor cells contained intracytoplasmic endocrine granules (Fig. 2E). The pituitary gland showed no abnormality. The adrenal cortex in both glands showed diffuse proliferation of cells, with alveolar and trabecular patterns. The liver had severe, multiple, locally extensive swollen hepatocytes, which were due to glycogen accumulation confirmed by PAS reaction (Fig. 2F). These findings suggested severe bilateral adrenocortical hyperplasia and resulting glucocorticoid-induced hepatopathy. In other organs, leiomyoma (stomach), Leydig cell tumor (testis) and hepatoid gland adenoma (perianal gland) were observed, respectively.

To differentiate the tumors in the frontal sinus and cerebral frontal lobe, IHC was performed with 3-µm-thick selected sections using the Dako REALTM EnVisionTM Detection System (DakoCytomation, Denmark), following the manufacturer’s instructions. Tissue sections were incubated overnight in a moist chamber at 4°C with antibodies (Table 1). For negative control, the primary antibodies were replaced with phosphate-buffered saline (PBS, pH 7.4). Table 1 shows various immunolabelling reactivities of the tumor cells with various antibodies. The tumor cells were positive for pancytokeratin, chromogranin-A, neuron-specific enolase (NSE), S100, and synaptophysin, which are makers for NETs and olfactory neuroblastomas (Fig. 3A–E). However, tumor cells were negative for microtubule-associated protein 2 (MAP2) and neurofilament, as it could differentiate from olfactory neuroblastomas. In addition, tumor cells were negative for neuronal and glial cell markers (glial fibrillary acidic protein [GFAP], and 2ʹ,3ʹ-cyclic nucleotide-3ʹ-phosphodiesterase [CNPase], ruling out astrocytoma, oligodendroglioma, ependymoma, and gangliocytoma. Antibody against CD56 antigen (also known as neural cell adhesion molecule), which is detected in human NETs as well as non-Hodgkin lymphoma-natural killer cell type and other hematological malignancies [4], identified some tumor cells in the present case (Fig. 3F). Antibody against thyroid transcription factor-1 (TTF-1), which is used for identifying human respiratory NETs [6, 10], labeled the tumor cells (Fig. 3G) by cross reactivity of this antibody to dog cells. The CDX-2 antibody, which is used for determining human intestinal NETs [6, 10], did not react in the tumor cells. Interestingly, antibody against ACTH detected the tumor cells (Fig. 3H).

Table 1.

Primary antibodies used in immunohistochemistry

Antigen	Type	Dilution	Antigen retrieval	Source	Reactivity
Pancytokeratin	Mouse Mab	1:50	HIARa)	DakoCytomation, Glostrup, Denmark	Positive
Chromogranin-A	Rabbit PCab	1:400	HIARa)	Novus Biotech, Centennial, CO, U.S.A.	Positive
NSE	Mouse Mab	1:100	HIARa)	Millipore, Burlington, MA, U.S.A.	Positive
S100	Rabbit PCab	1:400	HIARb)	DakoCytomation, Glostrup, Denmark	Positive
Synaptophysin	Mouse Mab	1:400	HIARa)	DakoCytomation, Glostrup, Denmark	Positive
CD56	Mouse Mab	1:200	HIARa)	DakoCytomation, Glostrup, Denmark	Positive
TTF-1	Mouse Mab	1:200	HIARa)	DakoCytomation, Glostrup, Denmark	Positive
ACTH	Mouse Mab	1:50	HIARa)	Santa Cruz Biotech, Dallas, TX, U.S.A.	Positive
MAP2	Mouse Mab	1:500	HIARc)	Sigma-aldrich, St. Louis, MO, U.S.A.	Negative
Neurofilament	Mouse Mab	1:400	HIARa)	Sigma-aldrich, St. Louis, MO, U.S.A.	Negative
CNPase	Mouse Mab	1:100	HIARc)	Merck, Darmstadt, Germany	Negative
GFAP	Rabbit PCab	1:1,000	HIARc)	DakoCytomation, Glostrup, Denmark	Negative
CDX-2	Mouse Mab	1:50	HIARa)	DakoCytomation, Glostrup, Denmark	Negative

NSE, neuron-specific enolase; TTF-1, thyroid transcription factor 1; ACTH, adrenocorticotropic hormone; MAP2, microtubule-associated protein 2; CNPase, 2ʹ,3ʹ-cyclic nucleotide-3ʹ-phosphodiesterase; GFAP, glial fibrillary acidic protein; Mab, mouse monoclonal antibody; PCab, rabbit polyclonal antibody; HIAR, heat-induced antigen retrieval (a) microwave: 100°C/10 mM Tris buffer for 20 min; b) microwave: 100°C/10 mM citrate buffer for 20 min; c) autoclaving at 121°C in 10 mM citrate buffer for 10 min).

Fig. 3.

Immunohistochemical findings. (A–N) The tumor cells are found to be positive for pancytokeratin (A), chromogranin-A (B), neuron-specific enolase (C), S100 (D), synaptophysin (E), CD56 (F), thyroid transcription factor-1 (G), and adrenocorticotropic hormone (H). Immunostaining with hematoxylin counter stain. Bars=50 µm.

The characteristic morphological and immunohistochemical findings suggested sinusoidal NET in the present case. However, NET and olfactory neuroblastomas have some common histopathological and immunohistochemical characteristics, such as microscopic rosette formation and positive immunoreactivity for some neural and neuroendocrine markers (NSE, synaptophysin, S100, and chromogranin-A), making it difficult to distinguish these tumors [24]. Some neuronal tumor makers including MAP2, neurofilament, and class III beta-tubulin isotype can differentiate these tumors, all of which are positive for neuroblastomas but negative for NETs [9, 20, 24]. The tumor cells in the present case were negative for MAP2 and neurofilament, which can differentiate this tumor from olfactory neuroblastomas. In addition, negative IHC reactions of this tumor with MAP2, GFAP, CNPase, and Iba1 allowed differentiation from neurogenic tumors, such as astrocytoma, oligodendroglioma, ependymoma, and gangliocytoma [7].

In animals, hematogenous dissemination plays a major role in tumor metastasis to the central nervous system, while the remainder is caused by local direct invasion [7]. Secondary intracranial tumors with hematogenous origin were usually multiple, well-circumscribed, round masses. In veterinary literature, direct invasion of nasal NETs have rarely been reported [5, 7]. In the present case, NET was found across the frontal sinus and cerebral frontal lobe. Moreover, NETs were not found in other organs or tissues on CT or gross or histopathological examination, indicating that the tumor was not metastasized from any other organ. Therefore, invasion of the frontal sinusoidal NET through the cribriform plate in the olfactory bulbs and frontal lobes is plausible [7]. As primary NETs in the brain have been reported in humans [11, 12, 22], direct invasion of the olfactory bulb or frontal lobe by NETs through the cribriform plate into the frontal sinus could not be ruled out. In human pathology, TTF-1 and CDX-2 are used for the differentiation between pulmonary and intestinal NETs [10]. In the present study, tumor cells in the frontal sinus and brain were positive for TTF-1 but not for CDX-2, suggesting nasal origin of this tumor. To confirm this hypothesis, more intensified studies using many pulmonary, sinonasal, and intestinal NETs of dogs are needed.

Hormone hypersecretory syndromes caused by NETs have rarely been reported in animals compared to humans. For example, glucagonomas in the pancreas and liver can induce superficial necrolytic dermatitis through excessive glucagon production by tumor cells [1], whereas intestinal NETs can cause diarrhea through hypersecretion of serotonin [20]. In this case, physical examination revealed bilaterally symmetric alopecia and pot-bellied distended abdomen. Moreover, this case had bilateral adrenocortical hyperplasia and was suspected with glucocorticoid-induced hepatopathy. All these findings indicated that this case was of Cushing’s disease. As there was no history of corticosteroid medication or gross or histopathological abnormality in the pituitary gland, Cushing’s disease due to iatrogenic corticosteroid medication and functional pituitary gland tumor could be excluded [17]. Interestingly, tumor cells in the present case were positive for ACTH. This indicated that hypersecretion of ACTH from NET caused bilateral adrenocortical hyperplasia, and subsequently, excessive production of glucocorticoid hormone, which induced Cushing’s syndrome.