Unraveling canine pheochromocytoma: From diagnosis to multi-omics, prognosis, and translational modeling

Abstract

Pheochromocytomas (PCCs) are neuroendocrine tumors of the adrenal medulla that occur in both dogs and humans. They often produce excess catecholamines, potentially causing serious cardiovascular complications such as arrhythmias and hypertensive crisis. Diagnosis is challenging because clinical signs are often nonspecific, and reference intervals for diagnostic tests have so far been lacking. How these tumors arise in dogs remains largely unknown, and apart from surgery no effective treatments are available. In addition, some dogs develop recurrence or metastases after surgery, making the prognosis uncertain. This thesis set out to improve the biochemical diagnosis of canine PCC, characterize the molecular and metabolomic landscape, develop a histopathology-based prognostic tool after adrenalectomy, and establish a preclinical in vitro model for drug screening. The first part focuses on biochemical testing. We established reference intervals for metanephrines in plasma, urine, and saliva using LC-MS/MS. Plasma normetanephrine clearly outperformed plasma metanephrine, showing 100% sensitivity and 94% specificity for diagnosing PCC, versus 73% sensitivity and 94% specificity for plasma metanephrine. The second part addresses tumor biology. Transcriptomic profiling of canine PCCs versus normal adrenal medullas (NAMs) identified 4,218 differentially expressed genes with upregulated genes involved in the cell cycle, tumor development, progression and metastasis, hypoxia/angiogenesis, and the Wnt-signaling pathway. High pleiotrophin expression correlated with a more aggressive phenotype. Two genes, Ret Proto-Oncogen and Dopamine Receptor D2, emerged as promising therapeutic targets, consistent with human PCCs where drugs against these targets already exist. Metabolomic profiling revealed a distinct tumor catecholamine phenotype characterized by high norepinephrine and low epinephrine content. Moreover, an aberrant succinate-to-fumarate ratio in one case suggested an underlying succinate dehydrogenase (SDHx) mutation. The third part focuses on prognosis after surgery. Ten histopathological parameters and the Ki67 proliferation index (PI) were evaluated for reproducibility and prognostic value. Necrosis, tumor cell spindling, and extension into adipose tissue achieved acceptable inter- and intraobserver agreement, while the Ki67 PI showed excellent reproducibility. Dogs that survived the immediate perioperative period generally had a favorable outlook with a mean overall survival of 2,456 days, although a subset (9%) developed late recurrence or metastasis. A Ki67 PI ≥18% was associated with shorter survival after adrenalectomy. The final part establishes new preclinical tools. We generated the first canine organoid lines from NAM and PCC. These organoids self-renewed and expressed progenitor markers (NES, SOX10). Early passages retained chromaffin features (metanephrine production, expression of CHGA, SYP, PNMT), but this declined over time. Expansion was limited in PCC organoids, underscoring the need for culture optimization to sustain both proliferation and differentiation and to enable scalable drug screening. In summary, this thesis establishes metanephrine reference intervals (demonstrating plasma normetanephrine as a key marker), elucidates the molecular and metabolomic characteristics of canine PCC, identifies promising therapeutic targets, shows the feasibility of SDHx screening via targeted metabolomics, validates Ki67≥18% for risk stratification post-adrenalectomy, and introduces the first canine PCC organoids. Together, these advances improve the diagnosis of canine PCC, move the field closer to targeted therapy, and may accelerate bench-to-bedside translation in PCC management for both veterinary and human medicine.