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

A7-year-old, intact female, Gordon Setter was examinedfor a 6-month history of progressive weaknessand ataxia without loss of appetite or change in weight.The owner reported a slight improvement in the signs afterfood consumption. The dog was kept indoors,regularly vaccinated, and fed a commercial maintenancediet.Physical examination revealed weakness, difficulty inholding quadrupedal posture, and mild muscle hypotrophy.No abnormalities were detected in the CBC, morphologicevaluation of the smear, and coagulation profile.Biochemical profile showed moderate hypoglycemia onfasting (57 mg/dL; range 80–120 mg/dL). Abdominal ultrasoundshowed an hypoechoic pancreatic lesion of 23mm indiameter, with indistinct margins and poorly contrastingwith adjoining structures. Eco-guided fine needle biopsy ofthe lesion was performed. The cytologic specimen containeda large number of naked nuclei on a cytoplasmicbackground with indistinct margins, occasionally acinarstructures with moderate anisokaryosis. The cytologic pattern,together with clinical signs, suggested neuroendocrinetumor. Abnormalities were not detected on chest X-ray in 3standard projections. Serum concentration of insulin was0.5mIU/mL (range 4–16 mIU/mL). Serum concentrationof insulin-like growth factor type II (IGF-II) was evaluatedby immunoradiometric assay (IRMA) after chromatographicseparation. Five hundred microliters of serumwere obtained from the dog and gel-filtered by fast proteinliquid chromatography on HyPrep Sephacryl S-200 HighResolution column (GE Healthcare, Amersham Place,Little Chalfont, Buckinghamshire, UK) in a buffer containing50mM NaH2PO4, 0.15M NaCl, 0.02% NaN3, pH7.2. Samples were eluted at 0.8 mL/min and collected at 3-minute intervals.1 The 44 fractions collected were pooledand tested for IGF-II immunoreactivity as follows: fractions8–11 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 ofIGF-II. The fraction of IGF-II bound to insulin-likegrowth factor binding protein (IGFBP)-3 and acid labilesubunit (ALS) forming a 150-kDa complex was 0.8 ng/mL.Similarly to normal dogs, IGF-II was only measurable inthe 150kDa region and undetectable in the others. IGF-IIwas measured by IRMA with reagents kit provided byDSL,a on acid ethanol pretreated samples.2 The sensitivityof the assay was 0.13 ng/mL; the intra-assay and interassaycoefficients of variation were 5.3 and 8.7%, respectively.No detectable cross-reactivity was found against IGF-I, upto 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 anextraction step in which IGF-II was separated from theIGFBPs. 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 concentrationwas 94.5 ng/mL in the affected dog.A7-year-old, intact female, Gordon Setter was examinedfor a 6-month history of progressive weaknessand ataxia without loss of appetite or change in weight.The owner reported a slight improvement in the signs afterfood consumption. The dog was kept indoors,regularly vaccinated, and fed a commercial maintenancediet.Physical examination revealed weakness, difficulty inholding quadrupedal posture, and mild muscle hypotrophy.No abnormalities were detected in the CBC, morphologicevaluation of the smear, and coagulation profile.Biochemical profile showed moderate hypoglycemia onfasting (57 mg/dL; range 80–120 mg/dL). Abdominal ultrasoundshowed an hypoechoic pancreatic lesion of 23mm indiameter, with indistinct margins and poorly contrastingwith adjoining structures. Eco-guided fine needle biopsy ofthe lesion was performed. The cytologic specimen containeda large number of naked nuclei on a cytoplasmicbackground with indistinct margins, occasionally acinarstructures with moderate anisokaryosis. The cytologic pattern,together with clinical signs, suggested neuroendocrinetumor. Abnormalities were not detected on chest X-ray in 3standard projections. Serum concentration of insulin was0.5mIU/mL (range 4–16 mIU/mL). Serum concentrationof insulin-like growth factor type II (IGF-II) was evaluatedby immunoradiometric assay (IRMA) after chromatographicseparation. Five hundred microliters of serumwere obtained from the dog and gel-filtered by fast proteinliquid chromatography on HyPrep Sephacryl S-200 HighResolution column (GE Healthcare, Amersham Place,Little Chalfont, Buckinghamshire, UK) in a buffer containing50mM NaH2PO4, 0.15M NaCl, 0.02% NaN3, pH7.2. Samples were eluted at 0.8 mL/min and collected at 3-minute intervals.1 The 44 fractions collected were pooledand tested for IGF-II immunoreactivity as follows: fractions8–11 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 ofIGF-II. The fraction of IGF-II bound to insulin-likegrowth factor binding protein (IGFBP)-3 and acid labilesubunit (ALS) forming a 150-kDa complex was 0.8 ng/mL.Similarly to normal dogs, IGF-II was only measurable inthe 150kDa region and undetectable in the others. IGF-IIwas measured by IRMA with reagents kit provided byDSL,a on acid ethanol pretreated samples.2 The sensitivityof the assay was 0.13 ng/mL; the intra-assay and interassaycoefficients of variation were 5.3 and 8.7%, respectively.No detectable cross-reactivity was found against IGF-I, upto 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 anextraction step in which IGF-II was separated from theIGFBPs. 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 concentrationwas 94.5 ng/mL in the affected dog.With the aim of clarifying the exact nature of the neoplasia,an immunohistochemical analysis was carriedout, using a panel of antibodies against CK7, pancytokeratinAE1/AE3/PCK26, chromogranin A, synaptophysin,CD56, glucagon, somatostatin, PP, insulin, andIGF-II.Neoplastic cells were immunoreactive for pancytokeratinAE1/AE3/PCK26, chromogranin A, CD56,and synaptophysin and negative for CK7, suggesting thediagnosis of well-differentiated neuroendocrine tumor.The positive staining for IGF-II and negative for insulinand the other pancreatic hormones indicated the tumorproducing IGF-II (Fig 2).One month after surgery clinical signs had improved;insulin normalized from nearly undetectable levels to aconcentration of 7 mIU/mL (range 4–16 mIU/mL), glucoseconcentration 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 increaseof the 150 kDa complex (ALS-IGFB3-IGF) from 0.8 to2.2 ng/mL.Ten months after surgery, the dog was in a goodhealth, serum biochemical values continued to be withinthe normal limits (insulin concentration 6 mIU/mL; referencerange, 4–16 mIU/mL; glucose concentration88 mg/dl; reference range, 80–120 mg/dL; IGF-II concentration68,1 ng/mL; reference range, 55–70 ng/mL)and showed no sign of local relapse or distant metastasis