Pancreatic Islet Cell Tumor Secreting Insulin-Like Growth Factor Type-II in a Dog

A7-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 23mm 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 0.5mIU/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 50mM NaH2PO4, 0.15M 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 150kDa 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 150kDa 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 mg/mL, and insulin, up to 4.3 mg/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.A7-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 23mm 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 0.5mIU/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 50mM NaH2PO4, 0.15M 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 150kDa 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 150kDa 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 mg/mL, and insulin, up to 4.3 mg/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.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.