Clinicopathological data and the role of miRNA expression in patients with Pheochromocytomas/Paragangliomas

Foteini Thanasoula^1,2Maria Giavropoulou³Georgios Kyriakopoulos⁴Athanasios Papatheodorou⁵Chrysostomos Maltezos⁶Konstantinos Maltezos⁶Vasiliki Vasileiou²Eva Kassi^7,8Gregory Kaltsas⁷Anna Angelousi^1*

• ¹First Department of Internal Medicine, European Reference Network on Rare Endocrine Conditions (ENDO-ERN), Laikon General Hospital, Medical School, National and Kapodistrian University, Athens, Greece
• ²Endocrinology Department and Diabetological Center, Alexandra General Hospital, Athens, Greece
• ³Department of Propaedeutic and Internal Medicine, European Reference Network on Rare Endocrine Conditions (ENDO-ERN), Laikon General Hospital, National and Kapodistrian University of Athens, Athens, Greece
• ⁴Department of Pathology, Evangelismos Hospital, Athens, Greece
• ⁵Department of Medical Research,251 Hellenic Air Force & VA General Hospital, Athens, Greece
• ⁶Vascular Surgery Department, General Hospital of Athens, “K.A.T”, Athens, Greece
• ⁷Department of Propaedeutic and Internal Medicine, European Reference Network on Rare Endocrine Conditions (ENDO-ERN), Laikon General Hospital, National and Kapodistrian University, Athens, Greece
• ⁸Department of Biological Chemistry, Medical School, National and Kapodistrian University, Athens, Greece

Background: Pheochromocytomas and paragangliomas (PPGLs) are rare neuroendocrine tumors with variable behavior and metastatic potential. Reliable prognostic biomarkers are needed to improve risk stratification and long-term management. Emerging data suggest a role for microRNAs (miRNAs) and immune checkpoint pathways (or inhibitors) in tumor aggressiveness. Objective: To evaluate the expression of five candidate miRNAs (miR-15a, miR-16, miR-101, miR-183, and miR-483-5p) and programmed death ligand-1 (PD-L1) in PPGL tissues, and assess their associations with clinicopathological features, genetic status and outcomes. Methods: A retrospective cohort of 130 patients with PPGLs was analyzed, including 53 PPGL tumor and 20 normal adrenal medulla tissues (controls). MiRNA expression was assessed by RT-qPCR in 53 formalin-fixed paraffin-embedded samples (FFPE). PD-L1 and microsatellite instability (MSI) were evaluated by immunohistochemistry in FFPE samples. Associations with tumor type and size, functionality, Ki-67 index, grading scores (PASS, GAPP), metastatic status, localization, and genotype were examined. Results: Overall, 21.5% (28/130) of patients developed metastases during a median period of follow-up of 45 months and 35 out of the 61 tested (57.4%), harbored pathogenic germline mutations. In tumor samples (n=53), PD-L1 expression was observed in 18.9% (10/53) whereas no MSI expression was detected. MiR-483-5p was the most consistently upregulated marker in biochemically negative and in high–Ki-67 tumors along with significant up regulation in metastatic PPGL, supporting its role in cellular proliferation and metastatic potential. MiR-183 and miR-101 were overexpressed in pheochromocytomas (PHEOs) with high PASS and Ki-67 indices, while miR-15a and miR-16 displayed higher levels in non-metastatic tumors. Conclusion: MiR-483-5p emerges as a promising biomarker of aggressive PPGL behavior, while other miRNAs reflect distinct biological behaviors. PD-L1 expression in a subset of cases highlights immune checkpoint inhibition as a potential therapeutic strategy. Prospective validation is warranted.