Quantitative proteomics of pediatric adrenocortical tumors provides insights into zone of origin and identifies overrepresented pathways

Andreas Metousis; Fincke Victoria E.; Wudy Stefan A.; Eva Jüttner; Marina Kunstreich; Johann Pascal D.; Antje Redlich; Lisa Schweizer; Rainer Claus; Matthias Mann; Michaela Kuhlen

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ESPE Abstracts (2024) 98 FC8.1

ESPE2024 Free Communications Adrenals and HPA Axis 2 (6 abstracts)

Quantitative proteomics of pediatric adrenocortical tumors provides insights into zone of origin and identifies overrepresented pathways

Andreas Metousis ¹ , Victoria E. Fincke ² , Stefan A. Wudy ³ , Eva Jüttner ⁴ , Marina Kunstreich ² , Pascal D. Johann ² , Antje Redlich ⁵ , Lisa Schweizer ¹ , Rainer Claus ⁶ , Matthias Mann ¹ & Michaela Kuhlen ²

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¹Department of Proteomics and Signal Transduction, Max Planck Institute of Biochemistry, Planegg, Germany. ²Pediatrics and Adolescent Medicine, Faculty of Medicine, University of Augsburg, Augsburg, Germany. ³Division of Pediatric Endocrinology & Diabetology, Laboratory for Translational Hormone Analysis, Steroid Research & Mass spectrometry Unit, Center of Child an Adolescent Medicine, Justus-Liebig- University, Giessen, Germany. ⁴Department of Pathology, University Hospital Schleswig-Holstein, Campus Kiel, Kiel, Germany. ⁵Pediatric Hematology/Oncology, Otto-von-Guericke-University, Magdeburg, Germany. ⁶Pathology, Faculty of Medicine, University of Augsburg, Augsburg, Germany

Introduction: Pediatric adrenocortical tumors (pACT), comprising highly malignant pediatric adrenocortical carcinomas (pACCs) and less aggressive adenomas (pACAs), present a rare yet clinically significant challenge. pACTs often pose a diagnostic and therapeutic dilemma due to their elusive differentiation and resistance to therapy. Originating from the adrenal cortex, pACTs are functional and, thus, characterized by a unique urinary steroid metabolome. This study provides for the first time valuable insights into the complex proteome of pACTs and may pave the way for identification of novel therapeutic targets.

Methods: Bulk proteomes of 76 pACTs from the German Malignant Endocrine Tumor studies and 36 matched healthy controls were analyzed using high sensitivity mass-spectrometry. Bulk pACT sections and corresponding healthy tissue were macro-dissected from H&E stained adrenal gland sections. The analytes were lysed and digested and consecutively measured on a Thermo Orbitrap Astral mass spectrometer, coupled to an Evosep One running a 60 samples per day liquid chromatography gradient.

Results: Our high-sensitivity mass-spectrometry workflow was able to quantitatively identify a total of 10714 proteins, with a median proteomic depth of more than 8100 proteins per sample. PCA clearly separated healthy controls from pACTs based on these proteomic data. Unsupervised clustering revealed several subgroups within the pACTs, some of which were clearly distinct, but no clear separation of the histological subgroups (ACTs, ACCs and ACXs). The t-test analysis identified more than 1300 proteins that were differentially regulated between tumours and healthy tissue. Specifically, 991 proteins were found to be downregulated and 353 upregulated in pACTs compared to healthy controls. Gene set enrichment analysis showed that several pathways, including those related to steroid metabolism and DNA synthesis, were significantly overrepresented in tumours. Of particular interest, key enzymes involved in steroid synthesis, such as 3β-hydroxysteroiddehydrogenase, were consistently downregulated in all pACTs, suggesting a common origin of ACTs, ACCs and ACXs in the zona reticularis of the adrenal gland.

Conclusion: We used a highly sensitive mass spectrometry workflow to analyse in depth the bulk proteomes of a large cohort of pACTs and healthy controls. Our results indicate the existence of relevant subgroups and contribute to a better understanding of the pathophysiology of pACTs, with the potential to suggest novel strategies for diagnosis and therapeutic intervention.