MON-517 - Quantitative Proteomic Profiling of Adrenal Tumors Identifies Key Drivers of Malignancy

Background: Adrenocortical carcinoma (ACC) is a rare but aggressive neoplasm, for which early diagnosis and differentiation from adrenocortical adenoma (ACA) is crucial for subsequent treatment and prognosis.The aim of the study was to identify proteomic markers that differentiate ACC, ACA, and normal adrenal tissue based on analysis of archival adrenal tissue sections preserved as formalin-fixed paraffin-embedded (FFPE) samples.

Methods: A total of 24 FFPE tissues were included from 9 patients with ACC (median age of 55.5 years), 9 patients with ACA (median age of 60.5 years), and 6 patients with normal adrenal glands (controls), based on histological evaluation. All ACA cases (n = 9), as well as 4/9 ACC tumors, were secretory. The majority of ACC cases (n=7/9) were stage III/IV according to ENSAT, with a median Ki-67 proliferation index of 29%. Tissue was collected by scraping from microscope slides, and proteins were extracted using repeated cycles of heating and sonication in a 4% SDS lysis buffer. Digestion was performed using the SP3 method, and quantification was carried out by LC–MS/MS using Data-Independent Acquisition. Data processing was performed using DIA-NN (library-free), followed by statistical analysis and pathway enrichment.

Results: More than 6000 proteins were quantified from FFPE material. One hundred eighty-six of them presented extremely statistically significant differences between ACC and ACA groups (q value=0). Among them, ACC samples showed increased expression of the Endoplasmic Reticulum-Golgi Intermediate Compartment 2 (ERGIC2) and of the insulin-like growth factor 2 (IGF2) and reduced expression of mitochondrial membrane and steroidogenesis-related proteins ( Ferredoxin Reductase (FDXR), 11-beta-hydroxylase (CYP11B1), and Glycosylphosphatidylinositol Specific Phospholipase D1 (GPLD1)). Secretogranin-1 and Chromogranin-A, proteins associated with hormone storage and secretion, were also decreased in ACC. In addition, enrichment of the Nucleotide Excision Repair pathway was observed, consistent with increased cellular proliferation.

Conclusions: DIA-based proteomic analysis of FFPE tissues reveals distinct molecular signatures that differentiate ACC from ACA and normal adrenal tissue. Alterations in the IGF2,, steroidogenesis, and DNA repair mechanisms may serve as useful diagnostic and biological markers for understanding adrenal tumorigenesis

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