Pheochromocytoma (PCC) is a catecholamine-secreting tumor originating from the adrenal gland. It is an emerging diagnosis in dogs, and currently, no medical treatment is available. This study will uncover the molecular signature of canine PCC and search for novel theranostic options. We will perform RNA sequencing to detect canine PCC transcriptomic profile(s) and the whole-genome sequencing to identify the mutation status. Both techniques will also be applied on organoids and tumoroids, cultured from canine normal adrenal medulla and PCC, respectively. These data will enable us to identify novel radioligands and radiotherapeutics that could lead to the future theranostic approach of canine PCC. Furthermore, the study will give the direction for potential medical treatment options. This project is an important step in veterinary medicine but will also provide a roadmap of translational research targets and will position the dog as an animal model of PCC in one medicine approach.

Pheochromocytoma (PCC) is a catecholamine-secreting tumor originating from the adrenal gland. It is an emerging diagnosis in dogs, and currently, no medical treatment is available. This study will uncover the molecular signature of canine PCC and search for novel theranostic options. We will perform RNA sequencing to detect canine PCC transcriptomic profile(s) and whole-genome sequencing to identify the mutational status of these tumors. Both techniques will also be applied on organoids and tumoroids, which will be cultured from canine normal adrenal medullas and PCCs, respectively. These data will enable us to identify novel radioligands and radiotherapeutics that could lead to the future theranostic approach of canine PCC. Furthermore, the study will give the direction for potential medical treatment options. This project is an important step in veterinary medicine but will also provide a roadmap of translational research targets and will position the dog as an animal model of PCC in one medicine approach.

• M.F. (Marit) van den Berg MSc

In this study, in vitro models of canine Cushing`s syndrome will be used to study new treatment options. In adrenocortical tumor tissue the investigators will assess the effectiveness of compounds, called SF-1 inverse agonists, on hormone production and tumor growth, which are the main characteristics of an adrenocortical tumor. Testing with these compounds holds promises for a new medical approach of even inoperable adrenocortical tumors. In normal adrenal glands, surrogating the pituitary-dependent hypercortisolism, the suppression of steroid production is sufficient. The investigators expect that compounds, called MC2 receptor antagonists, will be superior to the current treatment due to their selectivity.

Identifying novel medical options will improve current treatment of Cushing`s syndrome in dogs. 

• prof. dr. H.S. (Hans) Kooistra
• dr. ir. J.A. (Jan) Mol
• dr. Karin Sanders

• Enzo Lalli
• MD

Pheochromocytomas are catecholamine-secreting tumours derived from chromaffin cells in the adrenal medulla or extra-adrenal sympathetic nervous system. Symptoms and signs of pheochromocytoma result from secretion of excessive amounts of catecholamines or, infrequently from the space-occupying or invasive nature of the tumor. Clinical presentation of pheochromocytoma may be vague and in lack of reliable endocrine tests, it remains under diagnosed. Because pheochromocytoma are potentially malignant and their hormone secretion can lead to life-threatening situations and dead, there is need for a reliable endocrine test.
Measurement of catecholamine metabolites metanephrine (MN) and normetanephrine (NMN) in plasma and urine has been introduced in the diagnosis of pheochromocytoma in dogs. The major limitation of those studies is small sample size with regard to dogs with pheochromocytoma and healthy animals. In addition, acute stress can increase MN release, so exploring non invasive methods, like salivary MN and NMN measurements, is warranted.
The aims of this study are (1) to measure free MN and NMN in saliva (for the first time in veterinary medicine) (2) to establish the reference range for MN and NMN in saliva, plasma and urine and (3) to compare the concentrations of MN and NMS in urine and saliva collected at home with ones collected in-hospital.

In 50 healthy dogs voided urine samples will be collected by the owner in the morning one day before the visit to the hospital. In addition, saliva will be collected on the same day at home, written instructions will be provided. In the hospital, physical examination will be performed and thereafter blood, saliva and urine will be collected in this particular order. All samples will be stored at -20oC until analysis. In plasma and saliva, free MN and NMN will be measured and urinary deconjugated MN and NMN will be related to the urinary creatinine.

The results of urinary and salivary home–sampling will be compared to sampling in in-hospital environment and the influence of acute stress on MN and NMN values will be evaluated. Laboratory determinations and data analysis will take no more than 4 weeks and can be performed as soon as the material will be collected. The application to use healthy dogs in this study is in preparation.

• prof. dr. H.S. (Hans) Kooistra

• Ido Kema
• UMC Groningen

Subject: Surgical adrenalectomy is the most effective treatment for cortisol-secreting adrenocortical tumors (ATs) so far, unless metastases and/or local invasion are present at the time of diagnosis. However, adrenalectomy is a difficult procedure, requires an experienced surgeon and can be performed only in specialized institutions. An alternative is administration of the adrenocorticolytic drug mitotane (o,p`-DDD) or a competitive inhibitor of 3β-hydroxysteroid dehydrogenase (HSD3B), trilostane. Mitotane therapy is effective, but often associated with side effects and potential toxicity in both humans and animals. Recently, mitotane has been replaced widely by trilostane, which is well tolerated and effective in controlling the clinical signs of cortisol excess, but has no effect on tumor growth.
In this project, the therapeutic potential of steroidogenic factor 1 (SF-1) inverse agonist will be evaluated in vitro in a primary AT cell culture. The AT tissue will be obtained from dogs with hypercortisolism due to an AT.
Significance: SF-1 inverse agonist suppress steroid synthesis and adrenocortical cell growth and as such represent an ideal medical treatment of cortisol-secreting ATs.
Hypothesis/objective: SF-1 inverse agonist leads to decreased cortisol production by down-regulation of mRNA encoding steroidogenic enzymes and affects cell proliferation by induction of apoptosis. As such, it has a great potential becoming a new medical approach in treating canine cortisol-secreting ATs.
Study design: Primary cell cultures from at least 8 cortisol-secreting ATs will be incubated with 2 different concentrations of SF-1 inverse agonist for 72 hours. Its effect on steroidogenesis will be studied by mRNA expression levels of acute steroid regulatory protein (StAR), HSD3B and steroidogenic cytochrome P450 genes, which transcription is regulated by SF-1. At the end of incubation, the effect of SF-1 inverse agonist on cell growth will be assessed by measuring DNA concentration and the apoptosis will be evaluated by the amount of DNA fragmentation using a commercially available ELISA kit. The effect of SF-1 inverse agonist on steroidogenesis will be assessed by measuring the cortisol concentration in the cell culture media and will be determined by radioimmunoassay. All experiments will be performed in duplicate and a cell culture without any addition of SF-1 inverse agonist will serve as control. mRNA expression encoding SF-1 will be determined by QPCR in both primary cell cultures and ATs. Later, immunohistochemistry (IHC) on paraffin embedded AT tissue will be performed to judge the protein expression of SF-1. The primers for steroidogenic enzymes and SF-1 and the anti SF-1 antibody are known from our previous studies.
Expected results: Based on experiments with the human adrenocortical cell line H295R, dose-dependent suppression of steroidogenesis and tumor proliferation is expected in canine primary AT cell culture.
Anticipated outcomes: Recently, significant up-regulation of SF-1 in adrenocortical carcinomas and association between high SF-1 levels and poor outcome have been demonstrated by our research group. Based on these data and the biological role of SF-1 in canine AT, blocking its function would be beneficial. The present study will provide original information about the efficacy of the SF-1 inverse agonist in naturally occurring canine AT tissue.
Potential impact for animal health: SF-1 inverse agonist could become a new drug in treatment of canine cortisol-secreting ATs. As it affects both steroidogenesis and proliferation, it will be superior to trilostane therapy and could also be used in dogs in which surgery is not possible.

http://www.akcchf.org/news-events/multimedia/video/endo-hepatic.html

The comparative and translational oncology program is part of the focus areas Tissue Repair and Advances in Veterinary Medicine of the Faculty of Veterinary Sciences. 

In collaboration within the Utrecht University focus area "Growth and Differentiation" studies are oriented at the role of cancer stem cells in mammary, adrenal and insulin-producing carcinomas. 

• Mammary carcinoma
• Adrenocortical carcinoma
• Insulinoma

In the program Advances in Veterinary Medicine translational research is oriented at

• Soft tissue sarcoma
• Lymphoma
• Prostate carcinoma
• Molecular imaging 

• dr. ir. J.A. (Jan) Mol
• prof. dr. E. (Erik) Teske
• prof. dr. H.S. (Hans) Kooistra