Whole transcriptome analysis of canine pheochromocytoma and paraganglioma.

Pheochromocytomas and paragangliomas (PPGLs) are neuroendocrine tumors arising from the chromaffin cells in the adrenal medulla and extra-adrenal paraganglia, respectively. Local invasion, concurrent disorders, and metastases prevent surgical removal, which is the most effective treatment to date. Given the current lack of effective medical treatment, there is a need for novel therapeutic strategies. To identify druggable pathways driving PPGL development, we performed RNA sequencing on PPGLs (n = 19) and normal adrenal medullas (NAMs; n = 10) of dogs. Principal component analysis (PCA) revealed that PPGLs clearly clustered apart from NAMs. In total, 4,218 genes were differentially expressed between PPGLs and NAMs. Of these, 232 had a log2 fold change of >3 or < -3, of which 149 were upregulated in PPGLs, and 83 were downregulated. Compared with NAMs, PPGLs had increased expression of genes related to the cell cycle, tumor development, progression and metastasis, hypoxia and angiogenesis, and the Wnt signaling pathway, and decreased expression of genes related to adrenal steroidogenesis. Our data revealed several overexpressed genes that could provide targets for novel therapeutics, such as Ret Proto-Oncogene (RET), Dopamine Receptor D2 (DRD2), and Secreted Frizzled Related Protein 2 (SFRP2). Based on the PCA, PPGLs were classified into 2 groups, of which group 1 had significantly higher Ki67 scores (p = 0.035) and shorter survival times (p = 0.04) than group 2. Increased expression of 1 of the differentially expressed genes between group 1 and 2, pleiotrophin (PTN), appeared to correlate with a more aggressive tumor phenotype. This study has shed light on the transcriptomic profile of canine PPGL, yielding new insights into the pathogenesis of these tumors in dogs, and revealed potential novel targets for therapy. In addition, we identified 2 transcriptionally distinct groups of PPGLs that had significantly different survival times.

Laparoscopic vs. open adrenalectomy: perioperative data and survival analysis in 70 dogs with an adrenal tumor.

Adrenalectomy is the treatment of choice in case of functional adrenal tumors and malignant adrenal incidentalomas. Laparoscopic adrenalectomy (LA) in dogs has gained popularity in recent years, however, clinical studies on large patient populations are scarce. This retrospective study describes perioperative and recurrence data, survival, and prognostic factors in 70 dogs that underwent LA or open adrenalectomy (OA) in our hospital between 2008 and 2022. Diagnosis was based on history, clinical signs, endocrine function tests and advanced diagnostic imaging. Laparoscopic adrenalectomy was performed in 42 dogs (n = 27 naturally occurring hypercortisolism, n = 4 pheochromocytoma, n = 1 pheochromocytoma with concurrent hypercortisolism, n = 10 incidentaloma) and OA in 28 dogs (n = 22 hypercortisolism, n = 3 pheochromocytoma, n = 3 incidentaloma). Bilateral adrenalectomy was performed in 8/70 dogs. Surgical duration of LA and OA did not differ significantly in unilateral and bilateral procedures (P = 0.108 and P = 0.101, respectively). Systemic hypertension occurred in 7/41 and 1/28 dogs during LA and OA, respectively (P = 0.130). Hypotension occurred in 2/41 and 4/28 dogs during LA and OA, respectively (P = 0.214). A total of 40/42 dogs in the LA group and 27/28 in the OA group survived to discharge (P = 0.810). Mean hospital stay was significantly shorter (P = 0.006) after LA (1.5 days, range 1-3) than after OA (2.2 days, range 1-4). No significant differences were demonstrated between LA and OA groups in recurrence of adrenal-dependent endocrine disease (P = 0.332), disease-free period (P = 0.733) and survival time (P = 0.353). The disease-specific 1-, 2- and 3-year survival rates were 95, 89, and 89% after LA and 92, 88, and 81% after OA. Tumor size was significantly associated with the occurrence of a recurrence. In addition, tumor size had a negative effect on the disease-free period and survival time. This study shows a favorable outcome of both LA and OA in dogs. Based on low perioperative complication rate, short hospitalization time and long-term outcomes comparable to OA in selected cases, the less invasive laparoscopic approach is considered the preferred technique.

Challenges of transsphenoidal pituitary surgery in severe brachycephalic dogs.

Introduction: Transsphenoidal hypophysectomy is the standard surgical technique for the excision of pituitary neoplasms. Anatomy may be more obscured in brachycephalic skull types due to the crowding of soft tissue and osseous structures. We describe the unique challenges to approach the sphenoid bone and localize the correct burr hole site in severe brachycephalic dogs. Materials and methods: A single institution retrospective case series of brachycephalic dogs with pituitary-dependent hypercortisolism (PDH). Preoperative computed tomography enabled 3D-, and cross-sectional reconstruction to plan and dry-practice the position of the ideal burr hole in relation to the sella turcica, pterygoid hamular processes, and hard palate. Rostral burring of the caudal hard palate obscuring the direct sphenoid approach necessitated adaptations to the original transsphenoidal hypophysectomy procedure. Postoperative outcomes and complications with respect to those seen in mesocephalic dogs are described. Results: Ten brachycephalic dogs including French Bulldogs (n = 9) and a single Dogue de Bordeaux were included. All dogs were diagnosed with PDH and had preoperative advanced imaging performed on the skull. All but one dog had an enlarged pituitary gland, with a median pituitary/brain value of 0.5 (range 0.21-0.9). A total of 11 transsphenoidal hypophysectomy procedures were performed in these 10 dogs. Rostral extension of the soft palate incision into the hard palate was performed to access the burr hole site on the sphenoid bone. Major complications included aspiration pneumonia (n = 1), severe gastroesophageal reflux (n = 1), and central nervous signs (=1). All dogs survived until discharge, with a median time to follow-up of 618 days (range 79-1,669 days). Seven dogs experienced long-term remission of PDH. Conclusion: Brachycephalic dogs undergoing transsphenoid al hypophysectomy benefit from meticulous presurgical planning and extension of the approach into the caudal hard palate. Advanced surgical skills can render a good outcome in a technically challenging environment.

Plasma and urinary metanephrine and normetanephrine concentrations using liquid chromatography with tandem mass spectrometry in healthy cats and in a cat with pheochromocytoma.

BACKGROUND: Pheochromocytoma (PCC) is rare in cats and plasma (PL) and urinary (U) metanephrines (metanephrine [MN]; normetanephrine [NMN]) measurement is rarely described in cats. OBJECTIVES: We evaluated the utility of PL and U MNs measurement in 10 healthy cats and a cat with a confirmed diagnosis of pheochromocytoma (PheoCat), using liquid chromatography with tandem mass spectrometry (LC-MS-MS). METHODS: Urine and EDTA PL samples collected from each of the 10 cats and the PheoCat were promptly stored at -80°C and remained frozen until analysis. To evaluate U MNs stability, an additional urine sample collected from the healthy cats was refrigerated for 24 hours before freezing. Urinary creatinine concentration (Creat) was assessed using the same spot urine samples to calculate U MNs-to-creatinine ratios. RESULTS: The PL-MN and PL-NMN median concentrations of the healthy cats were 2.73 and 7.02 nmol/L, respectively. The median U-MN/Creat and U-NMN/Creat ratios were 70 and 139 µg/g, respectively. The PheoCat had a PL-MN of 3.68 nmol/L, PL-NMN of 66.27 nmol/L, U-MN/Creat of 179 µg/g, and U-NMN/Creat of 1262 µg/g. The PheoCat had markedly increased concentrations of both PL and U MNs when compared to the healthy cats. No significant difference was found between U MNs measured in urine samples that underwent 24 hours of refrigeration in comparison to those that were frozen immediately. CONCLUSIONS: We report preliminary reference intervals for PL and U MNs in cats using LC-MS-MS and the potential clinical applicability of these biomarkers for the diagnosis of PCC in cats.

Reference intervals for plasma, urinary, and salivary concentrations of free metanephrines in dogs: Relevance to the diagnosis of pheochromocytoma.

BACKGROUND: Measurement of free metanephrines is recommended for screening of pheochromocytoma (PCC) but requires appropriate reference intervals (RIs). HYPOTHESIS/OBJECTIVES: To report RIs for plasma, urinary and salivary concentrations of free metanephrines and to determine the diagnostic performance of plasma free normetanephrine (pNMN) and metanephrine (pMN) concentrations in dogs with PCC, hypercortisolism (HC), and nonadrenal illness (NAI). ANIMALS: Eighty healthy dogs, 11 PCC dogs, 25 HC dogs, 6 NAI dogs. METHODS: Plasma, urine, and saliva were collected prospectively from healthy dogs, and free metanephrine concentrations were determined by liquid chromatography-tandem mass spectrometry (LC-MS/MS). In addition, medical records of dogs that had plasma free metanephrine concentrations measured by LC-MS/MS between 2018-2021 were studied retrospectively. RESULTS: The RIs for free metanephrines in plasma, urine and saliva are reported. Dogs with PCC had significantly higher pNMN than dogs with HC (P < .001) and NAI (P = .002). The PCC dogs had significantly higher pMN than HC dogs (P < .001), but not higher than NAI dogs (P = .29). Using the upper reference limit, pNMN (>3.56 nmol/L) showed high sensitivity (100%, 95% confidence interval [CI]: 72-100) and specificity (94%, 95% CI: 79-99) for diagnosis of PCC, whereas pMN (>2.49 nmol/L) showed moderate sensitivity (73%, 95% CI: 39-94) and high specificity (94%, 95% CI: 79-99). CONCLUSIONS AND CLINICAL IMPORTANCE: With establishment of these RIs, biochemical testing for PCC in dogs can be substantially improved. Measurement of pNMN is superior to pMN in dogs with PCC.

Transcriptome sequencing reveals two subtypes of cortisol-secreting adrenocortical tumours in dogs and identifies CYP26B1 as a potential new therapeutic target.

Cushing's syndrome (CS) is a serious endocrine disorder that is relatively common in dogs, but rare in humans. In ~15%-20% of cases, CS is caused by a cortisol-secreting adrenocortical tumour (csACT). To identify differentially expressed genes that can improve prognostic predictions after surgery and represent novel treatment targets, we performed RNA sequencing on csACTs (n = 48) and normal adrenal cortices (NACs; n = 10) of dogs. A gene was declared differentially expressed when the adjusted p-value was <.05 and the log2 fold change was >2 or < -2. Between NACs and csACTs, 98 genes were differentially expressed. Based on the principal component analysis (PCA) the csACTs were separated in two groups, of which Group 1 had significantly better survival after adrenalectomy (p = .002) than Group 2. Between csACT Group G1 and Group 2, 77 genes were differentially expressed. One of these, cytochrome P450 26B1 (CYP26B1), was significantly associated with survival in both our canine csACTs and in a publicly available data set of 33 human cortisol-secreting adrenocortical carcinomas. In the validation cohort, CYP26B1 was also expressed significantly higher (p = .012) in canine csACTs compared with NACs. In future studies it would be interesting to determine whether CYP26B1 inhibitors could inhibit csACT growth in both dogs and humans.

Ultrasonographic Detected Adrenomegaly in Clinically Ill Cats: A Retrospective Study.

This retrospective study aimed to assess the prevalence of ultrasonographic detected adrenomegaly in clinically ill cats, evaluating the final established diagnosis, describe adrenal ultrasound findings and if the adrenomegaly was suspected or incidental. Abdominal ultrasonography reports of cats presenting to a veterinary teaching hospital between October 2018 and February 2021 were retrospectively reviewed. Cats showing adrenomegaly (one or both glands having a dorsoventral axis >4.8 mm) were selected and medical records respectively evaluated. Nine-hundred and eighty-three ultrasonographical reports were selected, of which, 68 (7%) disclosed adrenomegaly. European/Domestic Short-Hair (62/68; 91%) male (44/68; 65%) castrated (35/44; 80%) cats were overrepresented. Adrenomegaly was an incidental finding in 62/68 (91%) cats while in 6/68 (9%) it was identified in the context of investigating a potential adrenal disease. Concerning established diagnosis, chronic kidney disease was overrepresented (25/68; 37%), followed by endocrinopathies (20/68; 29%). Adrenomegaly was bilateral in 53% (36/68) of cases. In unilateral cases (32/68; 47%), it was more prevalent on the left side (23/32; 72%), with a normal-sized contralateral adrenal gland. Left adrenal demonstrated a larger size and a tendency to oval shape. This study assesses the prevalence of adrenomegaly in clinically ill cats, reinforcing it can be an incidental ultrasound finding.

CDC6: A novel canine tumour biomarker detected in circulating extracellular vesicles.

Circulating nucleic acids and extracellular vesicles (EV) represent novel biomarkers to diagnose cancer. The non-invasive nature of these so-called liquid biopsies provides an attractive alternative to tissue biopsy-based cancer diagnostics. This study aimed to investigate if circulating cell cycle-related E2F target transcripts can be used to diagnose tumours in canine tumour patients with different types of tumours. Furthermore, we assessed if these mRNAs are localised within circulating EV. We isolated total RNA from the plasma of 20 canine tumour patients and 20 healthy controls. Four E2F target genes (CDC6, DHFR, H2AFZ and ATAD2) were selected based on the analysis of published data of tumour samples available in public databases. We performed reverse transcription and quantitative real-time PCR to analyse the plasma levels of selected E2F target transcripts. All four E2F target transcripts were detectable in the plasma of canine tumour patients. CDC6 mRNA levels were significantly higher in the plasma of canine tumour patients compared to healthy controls. A subset of canine tumour patient and healthy control plasma samples (n = 7) were subjected to size exclusion chromatography in order to validate association of the E2F target transcripts to circulating EV. For CDC6, EV analysis enhanced their detectability compared to total plasma analysis. In conclusion, our study reveals circulating CDC6 as a promising non-invasive biomarker to diagnose canine tumours.

Circulating MicroRNAs as Non-invasive Biomarkers for Canine Cushing's Syndrome.

Canine Cushing's syndrome (hypercortisolism) can be caused by a pituitary tumor (pituitary-dependent hypercortisolism; PDH) or a cortisol-secreting adrenocortical tumor (csACT). For both cases, non-invasive biomarkers that could pre-operatively predict the risk of recurrence after surgery would greatly impact clinical decision making. The aim of this study was to determine whether circulating microRNAs (miRNAs) can be used as diagnostic (presence of PDH or csACT) and/or prognostic (disease recurrence, histological grade) non-invasive biomarkers for canine Cushing's syndrome. After a pilot study with 40 miRNAs in blood samples of healthy dogs (n = 3), dogs with PDH (n = 3) and dogs with a csACT (n = 4), we selected a total of 20 miRNAs for the definitive study. In the definitive study, these 20 miRNAs were analyzed in blood samples of healthy dogs (n = 6), dogs with PDH (n = 19, pre- and post-operative samples) and dogs with a csACT (n = 26, pre-operative samples). In dogs with PDH, six miRNAs (miR-122-5p, miR-126-5p, miR-141-3p, miR-222-3p, miR-375-3p and miR-483-3p) were differentially expressed compared to healthy dogs. Of one miRNA, miR-122-5p, the expression levels did not overlap between healthy dogs and dogs with PDH (p = 2.9x10-4), significantly decreased after hypophysectomy (p = 0.013), and were significantly higher (p = 0.017) in dogs with recurrence (n = 3) than in dogs without recurrence for at least one year after hypophysectomy (n = 7). In dogs with csACTs, two miRNAs (miR-483-3p and miR-223-3p) were differentially expressed compared to healthy dogs. Additionally, miR-141-3p was expressed significantly lower (p = 0.009) in dogs with csACTs that had a histopathological Utrecht score of ≥ 11 compared to those with a score of <11. These results indicate that circulating miRNAs have the potential to be non-invasive biomarkers in dogs with Cushing's syndrome that may contribute to clinical decision making.

Comparison of methods to monitor dogs with hypercortisolism treated with trilostane.

BACKGROUND: The use of adrenocorticotropic hormone stimulation test as method to monitor efficacy of trilostane treatment of hypercortisolism (HC) in dogs has been questioned. OBJECTIVES: To evaluate and compare 12 methods with which to monitor efficacy of trilostane treatment in dogs with HC. ANIMALS: Forty-five client-owned dogs with HC treated with trilostane q12h. METHODS: Prospective cross-sectional observational study. The dogs were categorized as well-controlled, undercontrolled, and unwell through a clinical score obtained from an owner questionnaire. The ability to correctly identify trilostane-treatment control of dogs with HC with the following variables was evaluated: before trilostane serum cortisol (prepill), before-ACTH serum cortisol, post-ACTH serum cortisol, plasma endogenous ACTH concentrations, prepill/eACTH ratio, serum haptoglobin (Hp) concentration, serum alanine aminotransferase (ALT), gamma-glutamyl transferase (γGT) and alkaline phosphatase activity, urine specific gravity, and urinary cortisol : creatinine ratio. RESULTS: Ninety-four re-evaluations of 44 dogs were included; 5 re-evaluations of 5 unwell dogs were excluded. Haptoglobin was significantly associated with the clinical score (P < .001) and in the receiver operating characteristic analysis, Hp cutoff of 151 mg/dL correctly identified 90.0% of well-controlled dogs (specificity) and 65.6% of undercontrolled dogs (sensitivity). Alanine aminotransferase (P = .01) and γGT (P = .009) were significantly higher in undercontrolled dogs. Cutoff of ALT and γGT greater than or equal to 86 U/L and 5.8 U/L, respectively, were significantly associated with poor control of HC by trilostane. CONCLUSIONS AND CLINICAL IMPORTANCE: Of all the 12 variables, Hp, and to a lesser degree ALT and γGT, could be considered additional tools to the clinical picture to identify well-controlled and undercontrolled trilostane-treated dogs.

Evaluation of hypophysectomy for treatment of hypersomatotropism in 25 cats.

BACKGROUND: Successful treatment of cats with hypersomatotropism by transsphenoidal hypophysectomy is described in small numbers of cats. OBJECTIVES: To describe the endocrine profile, survival, and remission rates of hypersomatotropism and diabetes mellitus in a cohort of cats with hypersomatotropism that underwent hypophysectomy between 2008 and 2020. ANIMALS: Twenty-five client-owned cats with spontaneous hypersomatotropism. METHODS: Retrospective study. Diagnosis of hypersomatotropism was based on clinical signs, plasma insulin-like growth factor-1 (IGF-1) concentration, and imaging of the pituitary gland. Growth hormone (GH) and IGF-1 concentrations were measured repeatedly after surgery. Survival times were calculated based on follow-up information from owners and referring veterinarians. RESULTS: Median postoperative hospital stay was 7 days (range, 3-18 days). One cat died within 4 weeks of surgery. Median plasma GH concentration decreased significantly from 51.0 ng/mL (range, 5.0-101.0 ng/mL) before surgery to 3.8 ng/mL (range, 0.6-13.0 ng/mL) at 5 hours after surgery. Remission of hypersomatotropism, defined as normalization of plasma IGF-1 concentration, occurred in 23/24 cats (median, 34 ng/mL; range, 14-240 ng/mL) and 22/24 cats entered diabetic remission. Median survival time was 1347 days (95% confidence interval, 900-1794 days; range, 11-3180 days) and the overall 1-, 2-, and 3-year all-cause survival rates were 76%, 76%, and 52%, respectively. CONCLUSIONS AND CLINICAL IMPORTANCE: This study shows the beneficial outcome of hypophysectomy in cats with hypersomatotropism, marked by low death rate and a high percentage of diabetic remission and definitive cure.

Feline plasma adrenocorticotropic hormone: validation of a chemiluminescent assay and concentrations in cats with hypercortisolism, primary hypoadrenocorticism and other diseases.

OBJECTIVES: The aims of this study were to validate a commercially available chemiluminescent assay for measurement of feline plasma adrenocorticotropic hormone concentration (ACTH), to determine the normal reference interval (RI) of plasma ACTH in healthy cats, to assess plasma ACTH in cats with naturally occurring hypercortisolism (HC), primary hypoadrenocorticism (PH) and other diseases (OD), and to evaluate the effect of aprotinin on plasma ACTH degradation. METHODS: Forty healthy cats, 10 with HC, 11 with PH and 30 with OD, were included. The chemiluminescent enzyme immunometric assay was evaluated by measurement of intra-assay precision, interassay precision and linearity. The RI for plasma ACTH in healthy cats was established using robust methods. Plasma ACTH of samples collected with and without aprotinin, stored at 4°C and assayed over a 6-day period, was measured. RESULTS: The intra-assay coefficients of variance (CVs) ranged from 2.7% to 4.3% and interassay CVs from 3.3% to 10.7%. Dilution studies showed excellent accuracy (R2 >0.99). The RI for plasma ACTH in healthy cats was 32-370 pg/ml. Plasma ACTH was not significantly different between healthy cats and the OD group. Cats with pituitary-dependent hypercortisolism (PDH) and PH had significantly higher plasma ACTH than the other groups. Plasma ACTH did not show significant differences when samples collected with and without aprotinin were compared. CONCLUSIONS AND RELEVANCE: The Immulite chemiluminescent assay is a valid technique for measuring plasma ACTH in cats and the RI of plasma ACTH is quite wide. Owing to the low overlap between healthy or OD cats and cats with HC or PH, the measurement of plasma ACTH appears to be useful and should be included in the diagnostic work-up when HC or PH are suspected. Furthermore, the measurement of plasma ACTH may be an accurate test for differentiating PDH from adrenal-dependent hypercortisolism.

Planar and single-photon emission computed tomography imaging in dogs with thyroid tumors: 68 cases.

BACKGROUND: Information on scintigraphy findings in dogs with thyroid neoplasia is scarce. The use of single-photon emission computed tomography (SPECT) could improve detection of metastatic disease. HYPOTHESIS/OBJECTIVES: To describe planar and SPECT imaging findings in dogs with thyroid tumors, and to compare SPECT and thoracic radiography for metastasis detection. ANIMALS: Sixty-eight dogs with thyroid neoplasia. METHODS: Retrospective study, search of medical records for dogs with thyroid neoplasia (2008-2018). RESULTS: Thyroid scintigraphy was available from 68 dogs, of which 6 presented after surgical resection. Radionuclide uptake was increased in 56% of dogs, decreased in 24%, and comparable to that of the salivary glands in 13%. The remainder had multiple masses with variable uptake. A homogeneous uptake pattern was present in 16% and a heterogeneous uptake pattern in 73%. In 11% (all dogs with multiple masses), various uptake patterns were present. Thyroid tumors were well delineated in 55%. There was a significant association between hormone status and uptake pattern (P = .009), with a heterogeneous uptake pattern in the majority of euthyroid dogs, and hormone status and tumor circumscription (P = .003), with well-circumscribed margins in the majority of hypothyroid and hyperthyroid dogs. Thoracic SPECT imaging was available in 39 dogs and identified metastatic lesions in 15 dogs. Thoracic radiographs were performed in 14 of these dogs, and detected metastases in 3 dogs. CONCLUSIONS AND CLINICAL IMPORTANCE: SPECT imaging is a viable imaging technique to screen for thoracic metastasis and wider use of SPECT imaging is recommended in dogs with thyroid neoplasia.

Molecular markers of prognosis in canine cortisol-secreting adrenocortical tumours.

Hypercortisolism is caused by a cortisol-secreting adrenocortical tumour (ACT) in approximately 15%-20% of cases in dogs. Little is known about which molecular markers are associated with malignant behaviour of canine ACTs. The objective of this study was to identify molecular markers of prognosis, which could be useful to refine prognostic prediction and to identify potential treatment targets. Cortisol-secreting ACTs were included from 40 dogs, of which follow-up information was available. The ACTs were classified as low risk of recurrence tumours (LRT; n = 14) or moderate-high risk of recurrence tumours (MHRT; n = 26), based on the novel histopathological Utrecht score. Normal adrenals (NAs) were included from 11 healthy dogs as reference material. The mRNA expression of 14 candidate genes was analysed in the 40 ACTs and in 11 NAs with quantitative RT-PCR. The genes' expression levels were statistically compared between NAs, LRTs and MHRTs. Univariate and multivariate analyses were performed to determine the association of the genes' expression levels with survival. Seven genes were differentially expressed between NAs and ACTs, of which pituitary tumour-transforming gene-1 (PTTG1) and topoisomerase II alpha (TOP2A) were also differentially expressed between LRTs and MHRTs. In survival analyses, high expression levels of Steroidogenic factor-1 (SF-1), PTTG1 and TOP2A were significantly associated with poor survival. In conclusion, we have identified several genes that are part of the molecular signature of malignancy in canine ACTs. These findings can be used to refine prognostic prediction, but also offer insights for future studies on druggable targets.

Molecular Alterations in Dog Pheochromocytomas and Paragangliomas.

8658860258318000Recently, genetic alterations in the genes encoding succinate dehydrogenase subunit B and D (SDHB and SDHD) were identified in pet dogs that presented with spontaneously arising pheochromocytomas (PCC) and paragangliomas (PGL; together PPGL), suggesting dogs might be an interesting comparative model for the study of human PPGL. To study whether canine PPGL resembled human PPGL, we investigated a series of 50 canine PPGLs by immunohistochemistry to determine the expression of synaptophysin (SYP), tyrosine hydroxylase (TH) and succinate dehydrogenase subunit A (SDHA) and B (SDHB). In parallel, 25 canine PPGLs were screened for mutations in SDHB and SDHD by Sanger sequencing. To detect large chromosomal alterations, single nucleotide polymorphism (SNP) arrays were performed for 11 PPGLs, including cases for which fresh frozen tissue was available. The immunohistochemical markers stained positive in the majority of canine PPGLs. Genetic screening of the canine tumors revealed the previously described variants in four cases; SDHB p.Arg38Gln (n = 1) and SDHD p.Lys122Arg (n = 3). Furthermore, the SNP arrays revealed large chromosomal alterations of which the loss of chromosome 5, partly homologous to human chromosome 1p and chromosome 11, was the most frequent finding (100% of the six cases with chromosomal alterations). In conclusion, canine and human PPGLs show similar genomic alterations, suggestive of common interspecies PPGL-related pathways.

The Utrecht Score: A novel histopathological scoring system to assess the prognosis of dogs with cortisol-secreting adrenocortical tumours.

A cortisol-secreting adrenocortical tumour (ACT) is the cause of naturally occurring canine hypercortisolism in approximately 15% to 20% of cases. The differentiation between an adrenocortical adenoma and carcinoma is usually based on histopathology. However, histopathological parameters have never been linked to the dogs' survival. Moreover, in human medicine the inter-observer variability of some histopathological parameters that are used for ACTs is high. The objective of this study was to establish a reliable and easy-to-use histopathological scoring system for cortisol-secreting ACTs that can assess the prognosis of dogs after adrenalectomy. Cortisol-secreting ACTs of 50 dogs, collected between 2002 and 2015, were included in this study. Twenty histopathological features were assessed by one veterinary pathologist and one resident in veterinary pathology. In addition, the Ki67 proliferation index was assessed by two observers. Only parameters with intra- and inter-observer agreement scores (intra-class correlation or Cohen's kappa coefficient) of ≥0.40 were included in survival analyses. Use of multivariate forward stepwise regression analysis with associated hazard ratios led us to a scoring system which we call the Utrecht score: the Ki67 proliferation index, +4 if more than 33% of the tumour cells have clear/vacuolated cytoplasm and + 3 if necrosis is present. Using cut-off values of 6 and 11, we could distinguish three groups that had significantly shorter survival times with increasing Utrecht scores. We conclude that the Utrecht score can be used to assess the prognosis of dogs with cortisol-secreting ACTs after adrenalectomy, which can help to select high-risk dogs that might benefit from adjuvant treatment or additional monitoring.

Concurrent pituitary and adrenocortical lesions on computed tomography imaging in dogs with spontaneous hypercortisolism.

BACKGROUND: Spontaneous hypercortisolism or Cushing's syndrome in dogs is either pituitary or adrenal dependent, but concurrent pituitary and adrenal hypercortisolism also has been reported. OBJECTIVE: To determine how often concurrent pituitary and adrenal lesions are present in dogs with spontaneous hypercortisolism. ANIMALS: Two hundred one client-owned dogs with spontaneous hypercortisolism. METHODS: Retrospective study. Pre- and post-contrast computed tomography (CT) scans of the pituitary and adrenal glands were performed in dogs with confirmed hypercortisolism. RESULTS: In dogs with dexamethasone-suppressible hypercortisolism (122/201), 78 dogs (64%) had an enlarged pituitary gland (median pituitary height/brain area [P/B], 0.43 × 10-2 mm-1 ; range, 0.32-1.21 × 10-2 mm-1 ). Two of these 78 dogs had concurrent adrenal lesions. In the remaining dogs (44/122; 36%), the pituitary gland was not enlarged. In the dexamethasone-resistant group (79/201), the pituitary gland was enlarged in 47 dogs (59%; median P/B, 0.57 × 10-2 ; range, 0.32-1.50 × 10-2 mm-1 ). Eight of these 47 dogs (17%) had concurrent adrenal lesions. In the remaining 32 dexamethasone-resistant dogs (41%), the pituitary gland was not enlarged. Among them, 27 dogs had adrenal lesions and suppressed ACTH concentrations consistent with adrenal-dependent hypercortisolism and 5 dogs were diagnosed with pituitary-dependent hypercortisolism. CONCLUSIONS AND CLINICAL IMPORTANCE: Concurrent pituitary and adrenal lesions were present in 5% of all dogs with hypercortisolism and in 10% of the dexamethasone-resistant dogs. Diagnostic imaging of both pituitary and adrenal glands should be included in the diagnostic evaluation of every dog with spontaneous hypercortisolism to obtain information needed for estimation of prognosis and choosing the optimal treatment.

TRH-induced secretion of adrenocorticotropin and cortisol in dogs with pituitary-dependent hypercortisolism.

BACKGROUND: In dogs, spontaneous Cushing's syndrome is most often pituitary-dependent and caused by hypersecretion of adrenocorticotropic hormone (ACTH), resulting in increased adrenocortical glucocorticoid secretion similar to horses. In horses with Cushing's syndrome (or pituitary pars intermedia dysfunction [PPID]) a thyrotropin-releasing hormone (TRH) stimulation test can be used for diagnosis, as TRH administration results in increased circulating ACTH and cortisol concentrations in affected horses. OBJECTIVE: The aim of this study was to investigate the effect of TRH administration on the circulating ACTH and cortisol concentrations in dogs with pituitary-dependent hypercortisolism (PDH). METHODS: Ten clinically normal control dogs and 10 dogs with PDH, all client owned, underwent a TRH stimulation test with measurement of plasma concentrations of ACTH and cortisol, before and after intravenous administration of 10 µg TRH/kg bodyweight. RESULTS: Plasma ACTH concentration did not rise significantly after TRH stimulation, neither in PDH dogs nor in clinically normal dogs. In contrast, the plasma cortisol concentration did increase significantly after TRH stimulation in both groups (p = .003 in PDH and p < .001 in control). Immunohistochemistry of normal adrenal glands demonstrated the presence of TRH receptors in the whole adrenal cortex. CONCLUSIONS: The results of this study demonstrate that the TRH stimulation test should be rejected as a tool to diagnose PDH in dogs. The observed TRH-induced increase in plasma cortisol concentration without a significant rise in plasma ACTH concentration may be explained by a direct effect of TRH on adrenocortical cells mediated by adrenocortical TRH receptors.

Abiraterone Acetate for Cushing Syndrome: Study in a Canine Primary Adrenocortical Cell Culture Model.

Abiraterone acetate (AA) is a potent inhibitor of steroidogenic enzyme 17α-hydroxylase/17,20-lyase (CYP17A1). AA is approved for the treatment of prostate cancer but could also be used to treat patients with Cushing syndrome (CS). Similar to humans, canine glucocorticoid synthesis requires CYP17A1, providing a useful animal model. The objective of this study was to preclinically investigate the effect of AA on adrenocortical hormone production, cell viability, and mRNA expression of steroidogenic enzymes in canine primary adrenocortical cell cultures (n = 9) from the adrenal glands of nine healthy dogs. The cells were incubated with AA (0.125 nM to 10 µM) for 72 hours under basal conditions and with 100 nM ACTH(1-24). Adrenocortical hormone concentrations were measured in culture medium using liquid chromatography-mass spectrometry, RNA was isolated from cells for subsequent real-time quantitative PCR analysis, and cell viability was assessed with an alamarBlue™ assay. AA reduced cortisol (IC50, 21.4 ± 4.6 nM) without affecting aldosterone under basal and ACTH-stimulated conditions. AA increased progesterone under basal and ACTH-stimulated conditions but reduced corticosterone under basal conditions, suggesting concurrent inhibition of 21-hydroxylation. AA did not affect the mRNA expression of steroidogenic enzymes and did not inhibit cell viability. In summary, primary canine adrenocortical cell culture is a useful model system for drug testing. For the treatment of CS, AA may to be superior to other steroidogenesis inhibitors due to its low toxicity. For future in vivo studies, dogs with endogenous CS may provide a useful animal model.