An 11-year-old male castrated mixed-breed dog was presented for exercise intolerance, tetraparesis, and persistent hypoglycemia. Abdominal ultrasound examination revealed 2 nodules within the right limb of the pancreas. Cytology from one nodule was consistent with a carcinoma of neuroendocrine origin, with a primary differential diagnosis of insulinoma. Histologic evaluation and immunohistochemistry for synaptophysin and insulin confirmed the diagnosis of insulinoma. Additionally, there was a solitary nodule of mineralized compact bone composing approximately 60% of the mass. To the authors' knowledge, this is the first report of osseous metaplasia within an insulinoma (islet cell carcinoma).
An 11-year-old male castrated mixed-breed dog was presented to the Ohio State University Veterinary Medical Center (OSUVMC) for evaluation of exercise intolerance and tetraparesis for several weeks' duration, as well as persistent hypoglycemia. Serum chemistry performed at OSUVMC showed sustained marked hypoglycemia (36 mg/dL; reference interval [RI] 77–126 mg/dL), mild hypokalemia (3.92 mEq/L; RI 4.2–5.4 mEq/L), and mildly elevated total serum ALP activity (148 IU/L; RI 15–120 IU/L]) with significantly increased corticosteroid-induced ALP isoenzyme activity (138 IU/L; RI 0–6 IU/L). An abdominal ultrasound examination identified 2 hypoechoic nodular structures measuring 2.9 × 1.9 cm (cranial nodule) and 3.3 × 1.4 cm (caudal nodule) in the right limb of the pancreas. The echogenicity of these nodules was heterogenous with focal hyperechoic shadowing regions. A fine-needle aspirate of one pancreatic nodule revealed many variably sized clusters of large, round to polygonal shaped cells within a background of blood and numerous bare nuclei (Figure 1). Neoplastic cells displayed mild-to-moderate anisocytosis and anisokaryosis. Nuclei were round to oval, centrally placed, and had a fine chromatin structure with 1–3 prominent round to ovoid nucleoli. These cells had moderate amounts of basophilic cytoplasm that frequently contained multiple small punctate vacuoles. Occasional cell clusters contained distinct intercellular junctions and/or piling of cells, as well as multiple ruptured cells. Rare binucleated cells, nuclear molding, and mitotic figures were observed. These findings were compatible with a carcinoma of neuroendocrine origin. The primary differential diagnosis was an insulinoma.
Thoracic radiographs were acquired prior to surgery and revealed no evidence of metastasis. A partial pancreatectomy was performed of the right limb along with surgical excision of 2 regional lymph nodes, and all tissues were submitted for routine histopathologic examination. Gross examination of the surgical specimen revealed a solitary, hard, roughly round, white to tan mass about 9 mm in diameter within the right limb of the pancreas. The second nodule identified via ultrasound was not found on gross examination of the surgical specimen despite complete sectioning of the tissue. Microscopically, approximately 50% of the mass was composed of densely arranged packets of monomorphic polygonal cells interspersed by a delicate fibrovascular network. The mass was partially delineated by a thick fibrous capsule, with nodular aggregates of neoplastic cells that infiltrated the capsule and the surrounding parenchyma. The architecture of the lymph node had been entirely effaced by similar cells. They had ample, finely granular eosinophilic cytoplasm, with basilar to centrally located round nuclei, which had clumped chromatin, and a single centrally located nucleolus. There was mild-to-moderate anisocytosis and anisokaryosis with occasional binucleated cells. Mitotic figures were rare (< 1 per 10 high-power fields). Embedded within the neoplasm and composing the remainder of the mass, there was a single nodule of broad and irregularly arranged, anastomosing trabeculae of mineralized compact bone. Bony trabeculae were lined by a single layer of osteoblasts, and the matrix contained loosely arranged osteoblasts within lacunae (Figures 2 and 3). Numerous neoplastic cells and pigment-laden macrophages (hemosiderophages) were scattered between the trabeculae.
Immunohistochemistry of the neoplasm was performed by the avidin-biotin-peroxidase method (ABC elite reagent; Vector Laboratories, Burlingame, CA, USA). Primary antibodies included synaptophysin (1:100; Dako, Glostrup, Denmark), chromogranin A (1:400; Dako), and insulin (1:50; Invitrogen, Grand Island, NY, USA). Diaminobenzidine was used as the chromogen; slides were counterstained with Harris' hematoxylin. Neoplastic cells showed strong intracellular staining with synaptophysin immunohistochemistry (not shown), supportive of the diagnosis of neuroendocrine carcinoma. Subsequent staining of the neoplastic cells with insulin was also strongly positive, compatible with a diagnosis of an insulinoma (Figure 4). The chromogranin A immunohistochemistry was not interpretable due to excessive background staining.
The dog recovered well following surgery. At the time of discharge, the tetraparesis had significantly improved and blood glucose was stable. Within one month of discharge postoperatively, the dog was re-presented for increased liver enzymes, tetraparesis, and respiratory distress. Fluid was noted in the abdomen, and cytology of an abdominal fluid sample showed neutrophilic inflammation with clusters of malignant neuroendocrine epithelial cells consistent with an exudate accompanied by a neoplastic effusion. Based on cell morphology, the neuroendocrine cells were most consistent with the previously diagnosed islet cell carcinoma. However, per owner request, no further testing was performed to evaluate the extent of metastasis. Due to the grave prognosis, the dog was humanely euthanized. A necropsy was declined.
Insulin-secreting beta-cell neoplasia (insulinoma) is the most common tumor arising from pancreatic islets in the dog. The majority of canine insulinomas are malignant solitary masses, predominantly identified in the duodenal (right) limb of the pancreas.[2-4] Beta-cell adenomas occur less frequently than carcinomas, but both tumors may cause significant endocrine disturbances and clinical signs through oversecretion of insulin.[5-7] Insulinomas have been found in dogs from 3–15 years of age, with a mean of 9 years. There is no apparent sex predilection and no breed predisposition has been reported; however, most cases involve medium- to large-breed dogs. Islet cell adenomas typically occur as small (1–3 cm), single or less often multiple nodules that are sharply delineated and surrounded by a partial or complete thin, fibrous capsule. Islet cell carcinomas are usually larger than adenomas, are multilobular, invade adjacent pancreatic parenchyma, and may metastasize. Histologic evidence of capsular invasion is considered a critical diagnostic feature of malignancy.
The clinical signs of tetraparesis and clinicopathologic findings of persistent hypoglycemia, mild hypokalemia, and mildly increased serum ALP activity in the current case are consistent with previous reports of dogs with insulin-secreting islet cell tumors.[2-4] In healthy dogs, hypoglycemia of less than 80 mg/dL is corrected through complete inhibition of insulin secretion, while counterregulatory hormones such as glucagon, catecholamines, growth hormone, and glucocorticoids are increased. However, neoplastic beta cells secrete insulin independent of blood glucose concentration, resulting in persistent and unregulated hypoglycemia accompanied by normal or increased blood insulin levels in most cases. As a consequence, hypoglycemia of the central nervous system or neuroglyocopenia leads to the clinical signs of tetraparesis. It is well-recognized that hyperinsulinism induces hypokalemia via intracellular potassium shifting, independent of glucose transport. Although serum insulin was not measured in the current case, the mild hypokalemia is likely the result of hyperinsulinemia due to unregulated insulin secretion from tumor cells. The mechanism of increased ALP in dogs with insulinomas has not been clearly defined, but may be associated with increased secretion of glucocorticoids in response to the hypoglycemia, and/or chronic stress. This explanation is supported in the current case because the majority of increased serum ALP activity was attributable to the corticosteroid-induced ALP isoenzyme. Although bone ALP has been linked to increased osteoblastic activity in tumors such as osteosarcoma, the amount of bone formation in the current case was likely insufficient to appreciably contribute to total ALP activity. In a study comparing dogs with mammary tumors with and without the presence of osseous transformation, there was no significant difference in bone ALP activity between the 2 groups. Furthermore, when the 2 groups were independently compared to a control population, bone ALP activity was significantly increased only in the group with mammary tumors lacking bone, suggesting that small amounts of bone formation in tumors may not significantly affect measured serum activity of bone ALP.
Intratumoral bone can be found in a variety of epithelial and mesenchymal neoplasms other than those primarily derived from osteoblasts. In people, mature bone has been reported in a number of extra-osseous neoplasms. In veterinary species, bone is commonly identified in fibromas derived from the periodontal ligament, maxillary fibromas of young horses, canine mixed mammary tumors, and in some chondrosarcomas.[13-16] Less frequently, bone has been described within canine mixed thyroid tumors, mixed apocrine sweat gland tumors, gastrointestinal adenocarcinomas, oral malignant melanomas, and a pancreatic exocrine adenocarcinoma in a ferret.[17-22] The current case represents the first report of bone formation within an insulinoma (islet cell carcinoma) in animals or people.
Intratumoral bone formation can originate from 2 basic mechanisms, either osseous metaplasia or endochondral ossification. In osseous metaplasia, nonosteoblastic cells are directly transformed into osteoblasts. However, identifying the cell of origin responsible for metaplastic bone can be challenging. In canine mammary tumors, for example, it is still unclear whether bone forms from metaplasia of epithelial cells, stromal connective tissue, or basal/myoepithelial cells.[23, 24] In endochondral ossification, ground substance is secreted and converted into hyaline cartilage, which subsequently undergoes endochondral ossification. For instance, myoepithelial cells in benign or malignant mammary tumors can secrete a mucinous ground substance that converts to cartilage, and then undergoes endochondral ossification. As there was no evidence of ground substance or cartilage in the current case, it was concluded that the bone formed via osseous metaplasia.
Special thanks to Drs. Bridget Urie and Andrew Mercurio for their clinical management and follow-up with the case. The authors also thank Marc Hardman for providing technical assistance with the images.