Germline targeted next-generation sequencing in patients with adrenal incidentalomas

Erika Messina¹Alessia Inglesi^1,2Soraya Puglisi^1*Anja Barač Nekić³Valentina Morelli⁴Ylenia Alessi⁵Karin Zibar Tomsic³Serena Palmieri⁶Pasquale Tomaiuolo²Enrico Grosso²Francesco Ferraù⁷Iacopo Chiodini⁸Darko Kastelan³Anna Pia⁹Maria Scatolini²Giuseppe Reimondo¹Massimo Terzolo¹

• ¹Department of Clinical and Biological Sciences, Internal Medicine, San Luigi Gonzaga Hospital, University of Turin, Orbassano, Italy
• ²Molecular Oncology Laboratory, Fondazione Edo ed Elvo Tempia, Ponderano (BI), Italy
• ³Department of Endocrinology, University Hospital Zagreb, Zagreb, Croatia
• ⁴Unit for Bone Metabolism Diseases and Diabetes and Lab of Endocrine and Metabolic Research, IRCCS, Istituto Auxologico Italiano, Milan, Italy
• ⁵Department of Biomedical, Dental and Morphological and Functional Imaging Sciences, University of Messina, Messina, Italy
• ⁶Endocrinology Unit, Fondazione IRCCS Ca' Granda-Ospedale Maggiore Policlinico, Milan, Italy
• ⁷Department of Human Pathology G. Barresi, Endocrine Unit, University Hospital G. Martino, University of Messina, Messina, Italy
• ⁸Department of Biotechnology and Translational Medicine, Unit of Endocrinology, Ospedale Niguarda Cà Granda, University of Milan, Milan, Italy
• ⁹Department of Endocrinology, Diabetes and Metabolism, Santa Croce and Carle Hospital, Cuneo, Italy

Objective: Adrenal incidentalomas are commonly detected in clinical practice. Despite growing interest in their molecular features, their germline genetic background remains largely unexplored. This study investigated the presence and potential pathogenic role of germline variants (GVs) in these patients using a targeted next-generation sequencing (NGS) approach, and explored possible genotype-phenotype correlations. Design: This multicenter retrospective study included 191 patients with incidentally discovered adrenal masses from four European reference centers. Patients with adrenocortical carcinoma, pheochromocytoma and primary aldosteronism were excluded Methods: Germline DNA was extracted from peripheral blood and analyzed using a custom next-generation sequencing (NGS) panel targeting 21 genes potentially involved in adrenal tumorigenesis. Bioinformatic analysis was followed by variant classification using the ClinVar and VarSome databases, in accordance with ACMG guidelines. Results: GVs were identified in 12 of 191 patients (6.3%), affecting 7 different genes: ZNRF3, ARMC5, APC, CACNA1H, SCNN1B, PDE11A, and KCNJ5. Most of the detected variants were classified as variants of uncertain significance (VUS). Genotype-phenotype analysis revealed that some patients with GVs had bilateral adrenal lesions and/or mild autonomous cortisol secretion (MACS). No variants were classified as clearly pathogenic. Conclusion: This study provides new insights into the germline genetic landscape of adrenal incidentalomas. Although GVs were identified in a minority of patients, their clinical significance remains unclear due to the predominance of VUS. These findings do not currently support widespread germline testing in routine clinical management of adrenal incidentalomas. Nevertheless, the detection of potentially pathogenic variants may inform future studies exploring their possible role in adrenal tumorigenesis.