A 7-year-old, intact female, Gordon Setter was examined for a 6-month history of progressive weakness and ataxia without loss of appetite or change in weight. The owner reported a slight improvement in the signs after food consumption. The dog was kept indoors, regularly vaccinated, and fed a commercial maintenance diet.
Physical examination revealed weakness, difficulty in holding quadrupedal posture, and mild muscle hypotrophy. No abnormalities were detected in the CBC, morphologic evaluation of the smear, and coagulation profile. Biochemical profile showed moderate hypoglycemia on fasting (57 mg/dL; range 80–120 mg/dL). Abdominal ultrasound showed an hypoechoic pancreatic lesion of 23 mm in diameter, with indistinct margins and poorly contrasting with adjoining structures. Eco-guided fine needle biopsy of the lesion was performed. The cytologic specimen contained a large number of naked nuclei on a cytoplasmic background with indistinct margins, occasionally acinar structures with moderate anisokaryosis. The cytologic pattern, together with clinical signs, suggested neuroendocrine tumor. Abnormalities were not detected on chest X-ray in 3 standard projections. Serum concentration of insulin was 0.5 mIU/mL (range 4–16 mIU/mL). Serum concentration of insulin-like growth factor type II (IGF-II) was evaluated by immunoradiometric assay (IRMA) after chromatographic separation. Five hundred microliters of serum were obtained from the dog and gel-filtered by fast protein liquid chromatography on HyPrep Sephacryl S-200 High Resolution column (GE Healthcare, Amersham Place, Little Chalfont, Buckinghamshire, UK) in a buffer containing 50 mM NaH2PO4, 0.15 M NaCl, 0.02% NaN3, pH 7.2. Samples were eluted at 0.8 mL/min and collected at 3-minute intervals.1 The 44 fractions collected were pooled and tested for IGF-II immunoreactivity as follows: fractions 8–11 corresponding to the 150 kDa ternary complex, fractions 12–16 corresponding to the 45 kDa binary complex, and fractions 24–27 corresponding to the free form of IGF-II. The fraction of IGF-II bound to insulin-like growth factor binding protein (IGFBP)-3 and acid labile subunit (ALS) forming a 150-kDa complex was 0.8 ng/mL. Similarly to normal dogs, IGF-II was only measurable in the 150 kDa region and undetectable in the others. IGF-II was measured by IRMA with reagents kit provided by DSL,a on acid ethanol pretreated samples.2 The sensitivity of the assay was 0.13 ng/mL; the intra-assay and interassay coefficients of variation were 5.3 and 8.7%, respectively. No detectable cross-reactivity was found against IGF-I, up to 480,000 ng/mL, proinsulin, up to 2 μg/mL, and insulin, up to 4.3 μg/mL.
In native plasma total IGF-II was measured after an extraction step in which IGF-II was separated from the IGFBPs. Circulating IGF-II reference values (55–70 ng/mL) were established in a group of 10 healthy adult dogs, matched for age and sex. Plasma IGF-II concentration was 94.5 ng/mL in the affected dog.
Prednisone (0.25 mg/kg PO q12 h) was administered with frequent small meals of high-protein food and honey to control hypoglycemia. Explorative laparotomy and partial pancreatectomy were performed. Using a Tumor Node Metastasis (TNM) staging system based on insulinoma, the tumor was classified as stage 1 (T1N0M0). Blood glucose concentration was 170 mg/dL 60 minutes after surgery.
The surgical specimen was fixed in 10% formalin before being processed in paraffin. Representative sections of the lesion were selected for immunohistochemical analysis. All sections were deparaffinized in Bio-Clearb and hydrated with grade ethanol concentration until distilled water. Antigen retrieval was performed by calibrated water bath capable of maintaining the epitope retrieval solution in 10mM sodium citrate buffer (pH 6.0) at 97°C for 30 minutes. The sections were allowed to cool down to room temperature for 20 minutes. To block endogenous peroxidase activity, slides were treated with 3.0% hydrogen peroxide in distilled water for 10 minutes and, after washing with phosphate-buffered saline (PBS)c two or three times, the primary antibodies anti-CD56 (MoAb clone 123C3.D5),d antisynaptophysin,e antichromogranin A (MoAb clone LK2H10),f anti-insulin,g antiglucagon,h antisomatostatin,i anti-cytokeratin 7 (CK7) (MoAb clone OV-TL12/30),j antipancytokeratin (MoAb clone AE1/AE3/PCK26)k and anti-pancreatic polypeptide (PP)l were incubated for 1 hour at room temperature. Immunohistochemical analysis was performed using the streptavidin-biotin peroxidase complex (UltraVision)m and 3,3′-diaminobenzidine (DAB)n as the chromogen. Positive controls comprised pancreas for synaptophysin, chromogranin, insulin, glucagon, and somatostatin, intestine for pancytokeratin, neuroblastoma for CD56, and salivary gland for CK7.
Subsequently, we tested antibody to IGF-II.o Antigen retrieval was performed in thermostat bath at 98°C with citrate buffer 10mM pH 6 for 10 minutes. The tumor section was incubated with the primary antibody at 1 : 200 dilution for 1 hour at room temperature. The bound antibody was evaluated by peroxidase conjugated polymer (EnVision Detection KIT)p for 30 minutes and DABp as the chromogen. Normal colon was used as positive control.
Negative controls were performed by substituting the primary antibodies with nonimmune serum at the same concentration. Control sections were treated at the same time as sample sections. Nuclei of all sections were counterstained with Mayer's hematoxylin.
Microscopic examination of the lesion showed polygonal cells with a round central nucleus and finely granular cytoplasm, arranged in trabeculae or cords (Fig 1). Mitotic figures were rare (<1/10 HPF). There was no evidence of necrosis or vascular invasion. The surgical resection margins resulted free from tumor.
With the aim of clarifying the exact nature of the neoplasia, an immunohistochemical analysis was carried out, using a panel of antibodies against CK7, pancytokeratin AE1/AE3/PCK26, chromogranin A, synaptophysin, CD56, glucagon, somatostatin, PP, insulin, and IGF-II. Neoplastic cells were immunoreactive for pancytokeratin AE1/AE3/PCK26, chromogranin A, CD56, and synaptophysin and negative for CK7, suggesting the diagnosis of well-differentiated neuroendocrine tumor. The positive staining for IGF-II and negative for insulin and the other pancreatic hormones indicated the tumor producing IGF-II (Fig 2).
One month after surgery clinical signs had improved; insulin normalized from nearly undetectable levels to a concentration of 7 mIU/mL (range 4–16 mIU/mL), glucose concentration was 87 mg/dL (range 80–120 mg/dL), IGF-II concentration was 62,5 ng/mL (range 55–70 ng/mL) and chromatographic analysis showed an increase of the 150 kDa complex (ALS-IGFB3-IGF) from 0.8 to 2.2 ng/mL.
Ten months after surgery, the dog was in a good health, serum biochemical values continued to be within the normal limits (insulin concentration 6 mIU/mL; reference range, 4–16 mIU/mL; glucose concentration 88 mg/dl; reference range, 80–120 mg/dL; IGF-II concentration 68,1 ng/mL; reference range, 55–70 ng/mL) and showed no sign of local relapse or distant metastasis.
The main causes of hypoglycemia are liver insufficiency or failure, hypoadrenocorticism, hypopituituarism, sepsis, hormonal imbalance, cancer or, in dogs with diabetes mellitus, the incorrect administration of oral hypoglycemic drugs, insulin, or both. In some instances hypoglycemia might be because of poor food intake or the excessive ingestion of glucose after intense physical exercise.3
Hypoglycemia associated with a neoplasia might be the direct consequence of insulin hypersecretion by the tumor (hyperinsulinemia in insulinomas) or of excessive glucose utilization, reduced gluconeogenesis because of liver destruction by tumor mass, suppression of insulin counteractive hormones or overproduction of IGFs (IGF-I, IGF-II). In the latter cases, blood insulin concentration is normal or reduced.4,5
IGF-I and IGF-II are 7.5-kDa single-chain proteins, homologs of proinsulin, which promote cell growth, survival, migration, and differentiation, and have insulin-like activity through interaction with the cell membrane insulin receptor. IGF-I and IGF-II are synthesized and secreted by many tissues and can act both locally as autocrine or paracrine factors, or at distant sites as endocrine growth factors.6
In some cases of nonislet cell tumor-induced hypoglycemia, hypoglycemia is promoted by excessive neoplastic overproduction of IGF-II which is frequently in a variant form unable to constitute the ternary 150-kDa complex.6,7 In these case, low levels of insulin and IGF-I and high levels of IGF-II are found in the circulation.
Only a few cases of extrapancreatic tumors with paraneoplastic hypoglycemia, hypo- or normo-insulinemia and raised levels of IGF-II have been reported in animals.8,9 Literature about IGF-II pancreatic expression in dogs is very scanty. In our study perilesional normal tissue showed a low IGF-II immunohistochemistry positivity in 30% of islet cells as well as in a pancreatic tissue obtained from an adult normal dog. On the contrary neoplastic tissue showed a high IGF-II positivity in 80% of tumor cells.
In our dog, hypoinsulinemia excluded insulinoma. The serum levels of IGF-II were only slighty increased, although immunohistochemistry showed the production of IGF-II by the tumor.
Surgery produced immediate improvement of clinical signs so demonstrating the causative effect of the tumor and the comparison between before and after surgery chromatographic analysis showed an increase of 150-kDa heterotrimeric complexes confirming the hypothesis of IGF-II pathway normalization.
In human medicine, only one such case has been reported.10
At the time of writing, 14 months after surgery, the subject was in good health and its survival time overlapped with an analog case of insulinoma according to TNM staging system.11