Modeled Aqueous Humor Protein Concentrations to Enable Biomarker Development in Uveal Melanoma.

Uveal melanoma (UM) lacks minimally invasive and reproducible biomarkers to support clinical risk stratification, motivating the need for molecular profiling of aqueous humor (AH) as an alternative to fine-needle tumor aspiration (FNAB). This study aimed to generate a calibrated AH protein concentration map to identify tumor-associated signals present at clinically measurable levels and assess their associations with established molecular and clinical features. AH samples from 70 UM eyes were analyzed using next-generation sequencing-based proximity extension assays (PEAs), and leftover AH from 27 samples was further assessed using qPCR-based PEA to obtain reference concentration values. Regression models derived from overlapping proteins enabled extrapolation of calibrated pg/mL-level concentrations across the full cohort. Twenty-three proteins had median modeled concentrations above 5 pg/mL and were examined for clinical relevance and translational feasibility. Several proteins, including CXCL8, CXCL10, VEGFA, HGF, PDCD1, FLT1, FLT3LG, and CCL2, showed progressive increases from GEP1/PRAME- to GEP2/PRAME+ tumors and from AJCC Stage I/II to Stage III/IV, with Stage IV tumors demonstrating significant elevations in CXCL8, VEGFA, and PDCD1. Pathway analysis revealed activation of inflammatory and tumor microenvironment pathways, and upstream regulator analysis identified VEGFA and CCL2 as potential drivers. These findings demonstrate that calibrated AH proteomic profiling can identify clinically measurable protein changes associated with UM risk and stage, supporting its potential utility for biomarker development.