Age-Stratified Transcriptomic Profiling Reveals Biologically Distinct Molecular Phenotypes Across Pediatric, Adolescent, and Adult Osteosarcoma

<b>Background/Objectives</b>: Osteosarcoma exhibits bimodal age distribution with distinct clinical behaviors between pediatric and adult patients. Despite genomic evidence supporting age-related molecular heterogeneity, systematic transcriptomic characterization remains lacking. This study aimed to delineate age-associated transcriptional differences and develop a pediatric-specific prognostic signature. <b>Methods</b>: Bulk RNA sequencing was performed on tumor specimens from 70 osteosarcoma patients stratified into pediatric (≤14 years, <i>n</i> = 37), adolescent (15-18 years, <i>n</i> = 22), and adult (≥19 years, <i>n</i> = 11) groups. Differential expression, functional enrichment, and immune infiltration analyses were conducted. A pediatric-specific signature was validated in the TARGET-OS cohort (<i>n</i> = 87). <b>Results</b>: Pediatric osteosarcoma exhibited a hyperproliferative phenotype, enriched in E2F targets, G2M checkpoint, and DNA replication pathways. Adolescent tumors showed heightened immune-inflammatory signatures, while adult tumors activated osteogenic differentiation programs. Regarding the immune microenvironment, only adolescent tumors demonstrated active immune infiltration; pediatric and adult groups exhibited immunologically "cold" features. We identified a 10-gene pediatric-specific transcriptomic signature that declined with increasing age. High signature scores were significantly associated with inferior overall survival (hazard ratio [HR] = 5.6, 95% confidence interval [CI]: 1.2-26.2, <i>p</i> = 0.01) and progression-free survival (HR = 2.1, 95% CI: 1.1-4.2, <i>p</i> = 0.03). These findings showed concordant trends in the independent TARGET-OS cohort. <b>Conclusions</b>: Pediatric, adolescent, and adult osteosarcoma harbor distinct transcriptional profiles representing biologically different disease entities. The pediatric-specific 10-gene signature may serve as a clinically actionable biomarker for risk stratification and guide age-adapted therapeutic strategies.