The Immunohistochemical Confirmation of Insulinoma After the Curative Treatment by Complete Resection of the Ectopic Nodule
D.A.P.C. Zuccari; R. Castro; C.L. da Silva; A.F.F. Sabbag; C.U. Cunha; C.C. Ferrari; C. Meneghetti
Department of Molecular Biology, Faculty of Medicine of São José do Rio Preto, Vila São Pedro, São José do Rio Preto, SP, Brazil
Insulinoma is a tumor of pancreatic islets cells that produces an excessive amount of insulin. The excessive secretion leads to capture and excessive use of glucose by insulin sensitive tissues and a reduction in liver production of glucose causing hypoglycemia and its associated clinical signs. The cell tumors are uncommon in dogs and rare in cats. Their potential for malignancy is usually underestimated. The clinical signs include seizures, weakness, collapse, ataxia, muscle fasciculation and bizarre behavior (Nelson & Couto 2001, Tilley & Smith 2003, Withrow & Vail 2007). The significant progress made in recent years has brought about great improvement in the identification and differentiation of these neoplasms. Although uncommon in dogs, the only curative treatment for islet cell tumors in humans is complete surgical resection (Krysiak 2008). An adult female dog has received a diagnosis of insulinoma after syncope, weakness and ataxia confirmed by biochemical tests with the values of PIGC (Proportion of insulin: glucose) above normal and compatible with the disease. The aim of this study was to confirm by immunohistochemistry the histogenesis of the nodule cells found in the curative surgery of this tumor.
Materials and Methods
The dog was submitted to exploratory laparotomy for excision of the ectopic nodules, a controversial procedure in literature. The nodules were identified by macroscopic similarity with the pancreatic tissue. Surgery and post surgical delay: The procedure was a median laparotomy. At the inspection of the cavity, the organs had a normal appearance and there was no evidence of liver metastases. The approach of the pancreas was through gastrocolic omentum. A small node of about 1 cm in the lower edge of the body of the pancreas was found and surgically removed with cotton 2-0. Adjacent to it, a lymph node was compromised at the root of the transverse mesocolon and it was resected at the same way. After all, the cavity was drained with a fenestrated tubular drain sutured with a Vicryl 0. In the immediate postoperative, the diet was only water for three days and released light diet after this period if: there was no significant flow in the drain; normal amylosuria; normal amylase at the drain. Clinically well and with no fluid in the drain, this was removed on the fifth day after the surgery. Insulin immunostaining: The monoclonal antibody used in this study was Polyclonal Guinea Pig Anti-insulin (code A0564, DakoCytomation) diluted 1:50 in Bovine Serum Albumin (BSA, Sigma-aldrich), until then not mentioned in the literature for use in dogs. Sections 3 μm thick were cut from at least one representative block this case and collected onto salinazid slides for adhesion of tissue sections. After this, the slides were deparaffined, rehydrated in graded alcohols, and incubated with 3% hydrogen peroxidase for 30 minutes to block endogenous peroxidase activity. Induced antigen retrieval in Pan Steam at 95°C with citric acid (pH 6.0, 35 minutes) was used. After cooling down, the slides were covered with Bovine Serum Albumin (BSA, Sigma-aldrich) for 30 minutes before incubation with the primary antibody Polyclonal Guinea Pig Anti-insulin (code A0564, DakoCytomation) for 2 hours at room temperature, and the avidin-biotin-peroxidase complex (ABC, Erviegas) for 1 hour. The chromogen, 3,3' diaminobenzidine tetrahydrochloride 0.5% (Signet®) diluted in Phosphate-buffered Saline (PBS), was applied to the slides for 2-5 minutes at 20-22°C. Slides were counterstained with Harris's Hematoxylin. Negative controls were obtained by omitting the primary antibody, whereas normal pancreatic tissue served as an internal positive control in every assay. Expression of the marker was verified in accordance with graduation of expression proposed by Allred et al. (1998).
Results and Discussion
Insulinomas are rare tumors of the pancreas. Nevertheless, it is the most common endocrine tumor of this organ (Vasques 2007). The distinction between benign and malignant insulinomas is based on the presence or absence of metastases and clinical course of disease. The management of insulinoma involves the diagnosis, localization of the tumor and treatment. Meleo et al. (1990) considered that insulinomas in dogs have been described frequently and surgical exploration of the pancreas and histologic evaluation is required for definitive diagnosis of insulinoma. Also they consider that whenever is possible, surgical excision of the primary lesion and associated metastases should be performed. For Krysiak (2007) the only curative treatment for insulinoma is complete resection of the tumor. The confirmation of the diagnosis can only happen after marking the cells by the specific antibody. For this, multiple fields of each slide were examined and demarcation was indicated by the presence of distinct brown cytoplasmatic staining. This antibody cross-reacts with insulin from several mammalian species. Insulin producing islet cell tumors, hyperplastic islet cells and islet cells originating in pancreatic ductules, but in the mesenchymal tumours are not reactive. We observed in this study a really strong cytoplasmatic staining of the cells. There are no reports in the literature of the effectiveness of surgical treatment in dogs and the immunohistochemical confirmation of the diagnosis. Six months after surgery the female dog shows no clinical signs and the values of plasma insulin and glucose are within the norm. Both procedures were considered satisfactory for diagnostic confirmation and treatment of insulinoma.
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