Plasma proteomic profiling identifies APOA4 as a downregulated biomarker of adrenocortical carcinoma: a multi-platform discovery and validation study

Abstract Objectives Adrenocortical carcinoma (ACC) is a rare, aggressive malignancy associated with heterogeneous prognosis. Preoperative differentiation from adrenocortical adenoma (ACA) remains challenging, and no serum tumor marker has been established. We aimed to identify circulating protein biomarkers that distinguish ACC from ACA using a stepwise, multi-platform proteomics strategy. Methods We assembled discovery (ACC=10, ACA=67) and verification (ACC=7, ACA=11) cohorts from a tertiary center and profiled fasting plasma using LC–MS/MS with data-independent acquisition. Differentially expressed proteins (DEPs) were defined by t-tests with P0.05 and |fold-change|1.2; DEPs common to both cohorts were prioritized. Targeted validation by parallel reaction monitoring (PRM) used an expanded, two-center cohort including additional cases from Asan Medical Center (ACC=31; ACA=78). Orthogonal validation employed the Olink Explore 384 Inflammation II panel in an independent set (ACC=15; ACA=24). Results The discovery cohort yielded 67 DEPs (22 up-regulated and 45 down-regulated in ACC), and the verification cohort identified 17 DEPs. Three proteins; CD44, PRG4, and APOA4 were common to both analyses and were under-expressed in ACC compared to ACA. In PRM, CD44 and APOA4 showed directionally concordant, significant decreases in ACC, prioritizing these markers for further evaluation. In the Olink analysis, 40 proteins differed between ACC and ACA after FDR correction; APOA4 remained significantly lower in ACC. Conclusion Across discovery, targeted, and orthogonal platforms, APOA4 consistently exhibited lower circulating levels in ACC, supporting its potential as a serum biomarker for the preoperative differentiation of ACC from ACA. External, multi-ethnic validation and clinically deployable assays, alone or within multi-marker panels are warranted.