Canine Insulinoma Research Articles

Advances in diagnosis and management of canine insulinoma: a review

Insulinomas are the most common pancreatic tumours in dogs. Diagnosis of insulinoma requires the concurrent presence of hypoglycaemia and serum insulin levels within or above the upper limit of the reference range. The diagnostic imaging modality of choice is triple-phase contrast enhanced tomography. Surgical resection is generally the most effective treatment option, leading to prolonged survival; however, medical management becomes necessary for many dogs at some point. After surgery, dogs can live for a few years without clinical signs of hypoglycaemia, but eventually clinical hypoglycaemia frequently recurs due to the outgrowth of micrometastases that were undetected at the time of surgery. This review consolidates and provides updated recommendations from the veterinary literature on the diagnosis and management of canine insulinomas.

Clinicopathological and immunohistochemical features of insulinoma in an adult mixed breed dog

Canine insulinoma is a common non-specific neoplasm with high metastasizing ability. Its epidemiology, diagnosis, and treatment are well described. However, its long-term prognosis is considered poor. We report a rare case of insulinoma in a small-size, mixed-breed adult dog, which presented with a circling walk, seizures, weakness, depressed mental state, and hypoglycemia. Glucose continuous rate infusion, diazepam, and phenobarbital were initiated to stabilize the glycemia and control the convulsion. Blood tests, fine needle aspiration cytology, ultrasound and radiological tests, and histology and immunohistochemical analysis confirmed the diagnosis of insulinoma, emphasizing the relevance of considering it as a differential diagnosis for small canine breeds and younger animals. Further, dogs can be regarded as an experimental animal model for human insulinoma research as they share characteristics.

Current Trends in Diagnosis, Treatment and Prognosis of Canine Insulinoma.

Simple SummaryDogs can develop a tumour of the pancreas that produces too much insulin, which is called an ‘insulinoma’. These tumours cause signs such as collapse at exercise and very low blood sugar. Insulinomas are difficult to cure because they spread to other tissues very commonly. The diagnosis of an insulinoma requires the demonstration of the simultaneous occurrence of low blood glucose levels and either normal or elevated blood insulin levels. The diagnostic imaging of insulinomas in dogs is preferably conducted by taking computed tomography scans. In general, the surgical treatment of insulinomas is the preferred therapy because it results in the best survival times, but many dogs also require medical treatment at some point. The prognosis of dogs with insulinomas is guarded, because clinical symptoms relating to low blood sugar almost always return due to tumour regrowth or spread. This review paper aims to provide a summary and update the current recommendations in the veterinary literature for the diagnosis, treatment and clinical outcome of insulinomas in dogs. The contents of this review are comprehensive and relevant to veterinarians that work at all levels and will inform and advance our understanding of insulinomas in dogs.The most common pancreatic tumour in dogs is the insulinoma. Canine insulinomas are considered to be malignant in more than 95% of the cases because they almost always tend to metastasise. The diagnosis of an insulinoma requires the demonstration of the simultaneous occurrence of hypoglycaemia and blood insulin levels that are within or above the high end of the reference interval. The staging of canine insulinomas is preferably conducted by contrast-enhanced computed tomography. In general, surgical treatment is the most effective because of it results in long survival times, but many dogs also require medical treatment at some point. The prognosis of canine insulinomas is guarded as clinical hypoglycaemia almost always returns due to metastasis or tumour regrowth. This review aims to provide a summary and update the current recommendations in the veterinary literature for the diagnosis, treatment and prognostication of canine insulinomas.

Toceranib phosphate in the management of canine insulinoma: A retrospective multicentre study of 30 cases (2009-2019).

BackgroundInsulinomas are the most common tumour of the endocrine pancreas in dogs. These malignant tumours have a high metastatic rate and limited chemotherapeutic options. The multi‐receptor tyrosine kinase inhibitor sunitinib malate has benefit in the treatment of metastatic insulinoma in people. Toceranib phosphate, an analogous veterinary agent, may provide benefit for dogs.MethodsA retrospective study describing the extent and duration of clinical outcomes and adverse events (AEs) in dogs diagnosed with insulinoma and receiving toceranib.ResultsRecords for 30 dogs diagnosed with insulinoma and having received toceranib were identified from a medical record search of five university and eight referral hospitals. The median progression‐free interval and overall survival time were 561 days (95% confidence interval (CI): [246, 727 days]) and 656 days (95% CI: [310, 1045 days]), respectively. Of the dogs for which the canine Response evaluation criteria for solid tumours tool could be applied, the majority (66.7%) showed either a complete response, partial response or stable disease. Time to clinical progression was associated with prior intervention and type of veterinary practice. Larger dogs were at increased risk for disease progression and death. No novel AEs were reported.ConclusionsMost dogs diagnosed with insulinoma and receiving toceranib appeared to have a clinical benefit. Randomised, prospective studies are needed to better elucidate and objectively quantify the potential effect and survival benefit of toceranib therapy for management of insulinoma in dogs.

Fluorine‐18 fluorodeoxyglucose positron emission tomography‐computed tomography for staging of canine insulinoma: 3 cases (2019–2020)

Canine insulinomas are uncommon malignant functional pancreatic neuroendocrine tumours with a high metastatic rate. Diagnostic imaging aids with staging and surgical planning of these tumours; however, identification is unpredictable across modalities. High-grade human pancreatic neuroendocrine tumours display increased avidity on 18 F-fluorodeoxyglucose positron emission tomography-CT. Dogs with clinicopathologic findings consistent with pancreatic insulinoma were prospectively enrolled. Patients underwent 18 F-fluorodeoxyglucose positron emission tomography-CT and CT angiography, followed by exploratory laparotomy. Three patients met the inclusion criteria and had histologically confirmed insulinomas. Both metastatic lesions in patient 1 were mildly avid (SUVmax 2.79 and 3.01). In patient 2, the primary pancreatic insulinoma was minimally avid (SUVmax 2.16). The primary pancreatic lesion in patient 3 had similar avidity to normal pancreatic parenchyma (SUVmax 1.54) and was undetected on 18 F-fluorodeoxyglucose positron emission tomography-CT. Insulinomas demonstrated variable attenuation and contrast enhancement patterns on CT angiography and certain lesions were more conspicuous than on 18 F-fluorodeoxyglucose positron emission tomography-CT. Two metastatic lesions not visible on either imaging modality were discovered in patient 2 at surgery. Canine insulinomas were inconsistently avid on 18 F-fluorodeoxyglucose positron emission tomography-CT. This finding is likely attributable to the confounding clinicopathological features and multifaceted transformation of these tumours, in addition to the influence of variable tumour size, composition and vascularity. Unpredictable tumoural avidity limits the value of 18 F-fluorodeoxyglucose positron emission tomography-CT for staging canine insulinomas.

The appearance of canine insulinoma on dual phase computed tomographic angiography.

To further evaluate the appearance of insulinoma in dogs on dual-phase CT angiography, given the disparity of findings in recent publications. To establish whether CT angiographic localisation of insulinoma correlates with surgical findings. Single centre study of dogs with a final diagnosis of insulinoma which underwent abdominal CT angiography. Scans were retrospectively re-evaluated for specific features by two board-certified veterinary radiologists. These findings were also subsequently compared to surgical and histopathological reports to determine the accuracy of lesion localisation on CT. Thirty-five cases were included in final analysis, with pancreatic nodules identified in 33. Twenty-one were confirmed as insulinoma with histopathology. Jack Russell Terriers were over-represented. Twenty of 21 cases with confirmed insulinoma and 27 of 33 overall showed hyperattenuation in the arterial phase. The mean size of pancreatic insulinoma on CT was 15.1 mm, and 18.2% were larger than 20 mm. Eighteen of 21 confirmed and eight of 12 suspected insulinomas caused a deformation of the pancreatic shape, with two only identified as a result of this feature as these lesions were isoattenuating throughout the study. Pancreatic insulinoma location at surgery matched that described on the CT images in 17 of 19 cases where location was described in the surgical report. In contrast to recent publications, this study suggests hyperattenuation of insulinomas in the arterial phase is a predominant feature, and that hypoattenuation or isoattenuation are much less common. CT angiography is accurate in prediction of lesion location before surgery in most cases.

Clinical findings, neurological manifestations and survival of dogs withinsulinoma: 116 cases (2009-2020).

To review the clinical findings and outcome in dogs diagnosed with insulinoma, and to assess which factors are predictors of overall survival. Additionally, to describe the neurological manifestations of this population and their correlation with survival. Retrospective multicentric study of canine insulinoma cases (2009 to 2020). Signalment, clinical history, neurological examination, diagnostic findings, treatment and outcome were obtained from clinical records. Univariate and multivariate analyses were used to compare the overall survival. One hundred and sixteen cases were included. Median duration of clinical signs before presentation was 1.5months. The most common presenting clinical signs were weakness (59.5%), epileptic seizures (33.6%) and changes in consciousness or behaviour (27.6%). Three dogs were suspected to have paroxysmal dyskinesia. Thirty-two dogs had an abnormal neurological examination, most commonly showing obtundation (28.1%), decreased withdrawal reflexes (21.9%) and absent menace response (18.8%). Overall survival for dogs undergoing surgery (20 months) was significantly longer than in medically treated (8months; adjusted hazard ratio: 0.33; 95% confidence interval: 0.18, 0.59). Presence of metastases was the only other variable associated with prognosis (adjusted hazard ratio 1.72; 95% confidence interval: 1.02, 2.91). Clinical signs of canine insulinoma are vague and non-specific. Weakness, epileptic seizures and changes in mentation or behaviour were the most commonly reported. Obtunded mentation and forebrain neurolocalisation were the main neurological manifestations. Dogs undergoing surgery had a longer overall survival compared to medically treated cases, and dogs with metastasis had a shorter overall survival regardless of treatment modality. Abnormalities in the neurological examination did not correlate with prognosis.

Evaluation of the expression of hexokinase 1, glucokinase, and insulin by canine insulinoma cells maintained in short-term culture.

To develop a technique for isolation and culture of canine insulinoma cells and assess expression of cellular hexokinases (glucokinase and hexokinase I) and expression and secretion of insulin from these cells in vitro. Pancreatic insulinomas and normal pancreatic tissue from 4 and 3 dogs, respectively. Tissues were collected by surgical excision or at necropsy. Insulinoma cells from 2 dogs were cultured for up to 10 weeks with standard techniques; insulin synthesis in vitro was confirmed by immunohistochemical analysis of freshly prepared slides of cultured cells, and insulin secretion was assessed by measurement of insulin concentrations in culture medium with an ultrasensitive mouse insulin ELISA. Expression of cellular hexokinases in insulinomas and adjacent normal (nontumor) pancreatic tissue from the same dog (n = 3) was examined by quantitative reverse transcriptase PCR assay. Insulinoma cells survived for up to 10 weeks but did not proliferate in culture. Insulin was detected in isolated cells and secreted into culture medium for up to 10 weeks. Both cellular hexokinases were expressed; glucokinase appeared to be overexpressed in insulinomas, compared with normal pancreatic tissue from the same dogs. Canine insulinomas expressed hexokinases responsible for glucose responsiveness. Insulinoma cells were successfully maintained in short-term culture; cultured cells remained functional for 10 weeks as evidenced by cellular insulin content and had detectable secretion of insulin into the culture medium for ≥ 5 weeks. Apparent glucokinase overexpression by insulinomas suggested a possible mechanism underlying excessive insulin release by these tumors.

Do Canine Pancreatic Neuroendocrine Neoplasms Resemble Human Pancreatic Neuroendocrine Tumours? A Comparative Morphological and Immunohistochemical Investigation

Although canine pancreatic neuroendocrine neoplasms (PanNENs) have been proposed as a model for the counterpart human neoplasms, the type or grade of human PanNEN that they resemble is unclear. PanNENs in animals are classified as adenoma or carcinoma, whereas in humans they are classified as pancreatic neuroendocrine tumour (PanNET) if well-differentiated, or as pancreatic neuroendocrine carcinoma (PanNEC) if poorly differentiated. We evaluated 16 canine primary PanNENs and two metastases histologically and immunohistochemically, and graded them using the animal and human grading systems. All neoplasms had local or vascular invasion and were classified as pancreatic islet cell carcinomas according to the current WHO classification. The Ki-67 index was low in all cases (0.01-1.50%). All had cytoplasmic expression of synaptophysin and insulin but were immunonegative for glucagon, confirming a functional diagnosis of canine insulinoma. Membranous expression of SSTR2A and nuclear expression of ATRX, but no p53 expression, was found in all neoplasms. One primary tumour was diagnosed as a mixed neuroendocrine-non-neuroendocrine neoplasm, which is the first report of this neoplasm in dogs. The other 15 primary tumours and both metastatic tumours were graded as PanNET G1, according to the human WHO classification. We conclude that canine PanNENs share well-differentiated histomorphology, SSTR2A expression and absence of nuclear p53 immunolabelling with human PanNETs G1. However, they differ in ATRX gene expression and functionality, and seem to have a worse prognosis than human PanNETs G1, although their generally low Ki-67 index precludes more precise assessment of prognosis. Membranous SSTR2A expression renders canine PanNENs potentially amenable to treatment with somatostatin analogues or SSTR targeted in-vivoimaging methods.

Outcome after surgical management of canine insulinoma in 49 cases.

Canine insulinoma has historically been associated with a poor prognosis; however, prolonged survival times have recently been reported. Prognostic indicators that are available preoperatively are of limited predictive accuracy, and consensus on post-operative treatment recommendations is lacking. The objectives of this study were to describe outcomes in dogs with insulinoma treated surgically, and to assess whether selected potential risk factors are strongly associated with outcomes after surgery. Medical records of two institutions were searched for dogs with insulinoma that were treated surgically. Forty-nine dogs were included. Thirty-nine dogs (80%) had immediate resolution of hypoglycaemia and 10 (20%) remained persistently hypoglycaemic postoperatively. The median survival time (MST) for all dogs was 561 days. The MST for dogs that had resolution of hypoglycaemia was 746 days. The median of the overall euglycaemic time (times from surgery to first detection of hypoglycaemia at any time point after surgery) for all dogs was 424 days. Forty-four percent of those that had resolution of hypoglycaemia experienced recurrence of hypoglycaemia by 2 years postoperatively. Pathological stage was a predictor of persistent post-operative hypoglycaemia which, in turn, was a predictor of survival time. These results show that dogs with insulinoma can have prolonged survival, and that pathological stage is a predictor of outcome.

Transcriptomic analysis by RNA sequencing characterises malignant progression of canine insulinoma from normal tissue to metastatic disease

Insulinomas (INS) are the most common human and canine functioning pancreatic neuroendocrine tumours. The long-term prognosis for malignant INS is poor, because micrometastases are frequently missed during surgery. As human and canine malignant INS share clinical and histopathological features, dogs have been proposed as models for INS research. Using RNA-sequencing, we conducted a pilot study to better understand the underlying molecular mechanisms of canine INS. Normal canine pancreas and lymph node control tissues were compared with primary INS and INS-metastatic lymph nodes, revealing more than 3,000 genes differentially expressed in normal pancreas compared to primary INS. Only 164 genes were differentially expressed between primary INS and INS-metastatic lymph nodes. Hierarchical clustering analysis demonstrated similar genetic profiles in normal pancreas and early clinical stage primary INS, whereas late clinical stage primary INS resembled the genetic profile of INS-metastatic lymph nodes. These findings suggest that markers of malignant behaviour could be identified at the primary site of the disease. Finally, using the REACTOME pathways database, we revealed that an active collagen metabolism, extracellular matrix remodelling, beta-cell differentiation and non-beta-cell trans-differentiation might cause disease progression and hyperinsulinism in INS, identifying major pathways worthy of future research in this currently poorly controlled disease.

Utility of contrast-enhanced computed tomography in the evaluation of canine insulinoma location

Objectives: To determine 1) the sensitivity of contrast-enhanced CT (CECT) for detection of primary canine insulinomas and metastases 2) the sensitivity of CECT to locate canine insulinomas within the pancreas and 3) the CECT attenuation pattern of canine insulinomas and post-contrast phase in which insulinomas have the best visibility.Methods: A retrospective review was performed of the medical records of 27 canine insulinoma patients. Simultaneous occurrence of blood glucose < 3.5 mmol/L (reference interval: 4.2–5.8 mmol/L) and plasma insulin > 10 mIU/L (reference interval: 1.4–24.5 mIU/L) were considered diagnostic for insulinoma. The dogs had a mean age of 9.0 ± 1.7 (SD) years and comprised 11 males and 17 females.Results: Using CECT-scans, 26/27 insulinomas were successfully detected. However, CECT-scans predicted the correct location of insulinomas within the pancreas in only 14/27 dogs. In 9/13 inaccurately located insulinoma cases, the location error was major. There was no significant difference between triple, double and single-phase CECT-scans with location accuracies of 54%, 50% and 50%, respectively. Also, there was no specific post-contrast phase in which insulinomas could be visualised best. Detection of lymph node metastases with CECT-scans had a sensitivity of 67% (10/15 lymph node metastases). Detection of liver metastases had a sensitivity of 75% (6/8 liver metastases). This study highlights that major location errors mainly occurred if single- or double-phase CECT-scans were used (6/9 cases).Conclusion: It is suggested that triple-phase CECT-scans have superior outcome over single- or double-phase CECT-scans in pre-operative imaging of canine insulinomas.

Insulin-secreting β-cell neoplasia in a 7-year-old female dog, case report and review

AbstractA 7-year-old female dog presented with intermittent episodes of urinary incontinence, tremors, intermittent episodes of pelvic limb weakness and collapse, and lethargy were noticed over a period of two months. Hypoglycaemia, secondary to an insulin-secreting β-cell tumour, was suspected on the basis of clinical signs, ultrasound and serum biochemistry. A laparotomy was then performed to remove the tumour mass. Post-surgical pancreatitis was a major complication. Histology revealed a malignant neuroendocrine neoplasia of the β-cells of the pancreas. This case review is reported to keep clinicians abreast of the canine insulinoma disorder and the complications surrounding its diagnosis and treatment.

Contrast-Enhanced Ultrasonographic Detection and Dual-Phase Computed Tomographic Angiography in a 5-Year-Old Boxer with Pancreatic Insulinoma&amp;lt;br/&amp;gt;—Case Report

This case report describes the findings in a canine histopathologically confirmed pancreatic insulinoma using contrast-enhanced ultrasound (CEUS) and dual-phase computed tomographic angiography (CTA). The insulinoma was better demarcated in CEUS and CTA compared with conventional B-mode ultrasound. On the other hand, only one of two nodules visible in CTA was detected in CEUS. In this case, the insulinoma had an atypical non-contrast-enhancing appearance in both CEUS and CTA. Lack of enhancement in CEUS and CTA has previously been reported in human and canine studies, but this was the first report using both CEUS and CTA for detecting canine insulinoma.

Canine insulinoma: Part 2 – treatment

An earlier article discussed the diagnosis of canine insulinoma (Housley and Tappin (2014) 19(3): 138–42). This second article examines the treatment options available and the associated prognosis for these patients. In the acute situation, hypoglycaemic signs should be managed with the administration of glucose, either orally or by cautious intravenous injection. Medical management with frequent feeding of complex carbohydrates, prednisolone and diazoxide will control clinical signs in most patients. Surgery should be considered in all patients, even those with metastatic disease, as long-term prognosis is better after debulking surgery. Most patients will have a mass identified at surgery; if not, careful palpation, intra-operative ultrasound or new methylene blue administration may highlight the lesion. If a mass cannot be found, speculative hemipancreatectomy can be performed with little risk of either endocrine of exocrine insufficiency. Long-term prognosis depends on the presence or absence of metastasis, but disease-free intervals of more than two years have been reported, with survival times longer than this if medical management is commenced when signs recur.

Evaluation of prognostic indicators using validated canine insulinoma tissue microarrays

Tissue microarray (TMA) technology allows analysis of multiple tumour samples simultaneously on a single slide. The aim of the present study was to develop and assess a TMA containing 32 primary canine insulinomas and 13 insulinoma metastases. The results of histopathological and immunohistochemical analyses of triplicate core biopsies were compared with those of individual tissue sections using weighted κ statistics. Inter-observer agreement of TMA immunohistochemistry scores were assessed for chromogranin A (CgA), insulin, growth hormone (GH), growth hormone receptor (GHR) and Ki67 index, as well as the prognostic utility of clinicopathological, histopathological and immunohistochemical criteria.There was substantial agreement of scores for histopathological parameters (κ = 0.64–0.70) and a substantial to near-perfect agreement for homogenous immunohistochemical parameters (κ = 0.69–1.00). Except for GH, which demonstrated heterogeneous staining, there was good to excellent inter-observer agreement for all other immunohistochemical staining scores (intra-class correlation coefficients: 0.70–1.00). On univariate analysis, the presence of nuclear atypia was significantly predictive of disease-free intervals (DFIs) for canine insulinoma, while tumour size, TNM stage, necrosis and Ki67 index were significant in terms of prognosis, with respect to both DFI and survival time. On multivariate analysis, tumour size and Ki67 index retained predictive power for survival time, as did tumour size for DFI. This study confirms the applicability of TMA technology for evaluation of canine insulinoma.

Expression of insulin-like growth factor-1 by canine insulinomas and their metastases

The long-term prognosis after surgical resection of canine insulinoma is poor. Signs of hypoglycaemia often recur soon after surgery because tumour tissue has only been resected partially and/or functional (micro-)metastases were present. Using quantitative real-time PCR, the expression of 16 target genes was compared between primary canine insulinomas and their corresponding metastases. There was significantly higher expression of genes encoding for growth hormone (GH) and insulin-like growth factor-1 (IGF-1) in metastases compared to their primary tumours. Immunohistochemical examination of proteins of the GH-IGF-1 axis revealed expression of GH, IGF-1 and GH receptor (GHR) in both primary insulinomas and metastases. Immunohistochemical staining for IGF-1 was significantly higher in metastases compared to primary tumours.

Use of a bipolar vessel‐sealing device in resection of canine insulinoma

To describe partial pancreatectomy using a bipolar vessel-sealing device (BVSD) and compare this novel technique to the conventional suture-fracture (SF) method for canine insulinoma. Pre-, intra- and postoperative data of eight dogs with insulinoma, which underwent resection using the BVSD (LigaSure V), were prospectively collected and compared with those of eight randomly selected case-matched patients that underwent resection using the conventional SF technique. Mean surgical time was significantly (P=0·022) shorter in the BVSD (107 ± 9 minutes) than in the SF (135 ± 22 minutes) group. The BVSD technique was negatively associated with surgical time and duration of the hospitalisation period. Neither technique caused intraoperative complications, such as bleeding, collateral damage to adjacent tissues or problems with sealing or suturing the pancreatic tissue. Three dogs in the SF group and none in the BVSD group developed postoperative clinical signs associated with pancreatitis. BVSD is a safe and viable alternative to conventional methods of pancreatectomy for canine insulinoma. It provides the possibility to remove insulinomas in the pancreatic limbs and corpus with relative ease. BVSD pancreatectomy in dogs with insulinoma significantly decreases operative and hospitalisation times and is not associated with more clinical complications than SF pancreatectomy.

Evaluation of clinico-pathological criteria and the Ki67 index as prognostic indicators in canine insulinoma

The establishment of reliable prognostic biomarkers for canine insulinoma are warranted to facilitate optimal patient management. Using univariate and multivariate analyses, the present study evaluated the prognostic power of several clinical and histopathological criteria, as well as the Ki67 index, in 26 dogs with insulinoma. On univariate analysis, stromal fibrosis within these tumours was found to be significantly predictive for survival, while tumour size, the Ki67 index, and TNM (Tumour, Node, Metastasis) stage were significant in the prognosis of both disease-free interval and survival time. On multivariate analysis, tumour size retained predictive power for disease-free intervals, while the Ki67 index proved prognostically significant for both the disease-free interval and overall survival time. This study demonstrates that, in addition to known factors such as tumour size and stage, Ki67 can act as a biomarker of insulinoma that can be used to predict clinical outcome.

Autoantibodies to GAD65 and IA-2 in canine diabetes mellitus

Diabetes mellitus in dogs shares many characteristics with the human type 1 disease and virtually all diabetic dogs require insulin therapy to control hyperglycaemia. Insulin deficiency is suspected to result from immune-mediated destruction of pancreatic beta cells in some cases. Human patients suffering from Type 1A (immune-mediated) diabetes or latent autoimmune diabetes of the adult (LADA) demonstrate circulating autoantibodies against the 65 kDa isoform of glutamic acid decarboxylase (GAD65) and/or insulinoma antigen-2 (IA-2). The aims of the current study were to develop radio-immunoassays to detect serum antibodies against recombinant canine GAD65 and IA-2 and to identify diabetic dogs showing serological evidence of autoreactivity to these pancreatic beta cell antigens. Canine GAD65 and the 3′ end of IA-2 (coding for amino acids 771–979 of the intracellular domain) were amplified by PCR from cDNA prepared from canine insulinoma tissue and cloned into the pCRII vector. The canine sequences were later confirmed by identifying GAD2 and PTPRN genes from the dog genome assembly. Recombinant 35S-methionine-radiolabelled canine GAD65 and IA-2 (771–979) proteins were used in radio-immunoprecipitation assays to screen sera from 30 newly diagnosed diabetic dogs and 30 control dogs. Four of 30 canine diabetic patients had significant GAD65 autoreactivity ( p < 0.01) compared to controls and 3 dogs were positive for autoantibodies to IA-2 (771–979). Two diabetic dogs showed dual autoantigen reactivity. These preliminary data indicate that serological reactivity to GAD65 and IA-2 is present in a proportion of diabetic dogs and suggests that, in some cases, canine diabetes is associated with an autoimmune response to these antigens.