Adult-onset Still's disease (AOSD) is a rare autoinflammatory disorder featuring high fever, rash, arthralgia, and elevated ferritin. Reliable biomarkers are lacking for AOSD diagnosis in general, and refractory AOSD further exhibits poor treatment response, higher relapse rates, and worse prognosis.This study integrates bioinformatics and clinical validation to identify molecular biomarkers for refractory AOSD, establish a predictive model, and provide a theoretical basis for its future diagnosis.

Four transcriptomic datasets from the GEO database, comprising 77 samples of refractory AOSD, non-refractory AOSD, and healthy controls, were analyzed. Differentially expressed genes (DEGs) were screened using R software, with enrichment analysis focusing on inflammation-related pathways (JAK–STAT, NF-κB, cytokine storm) to identify core inflammatory genes in refractory AOSD. For validation, we performed transcriptomic sequencing on 10 AOSD patients from Tianjin Medical University General Hospital (4 refractory on upadacitinib, 6 non-refractory without upadacitinib). A separate cohort of 157AOSD patients was enrolled for clinical validation. Expression differences of the core inflammatory genes were validated at both molecular and clinical levels. Baseline characteristics, inflammatory markers, and clinical features were compared among groups to assess their association with disease refractoriness.

(1) Differential expression and pathway analysis revealed significant enrichment of inflammation-related pathways (JAK–STAT, cytokine storm) in refractory AOSD compared to non-refractory AOSD and healthy controls. Differentially expressed genes (e.g., DUSP2, HUWE1, IRF9) were predominantly downstream targets of JAK1 and key pro-inflammatory cytokines, suggesting their role as critical regulators in refractory AOSD-associated inflammation.

(2) Clinical validation showed that core inflammatory genes were highly expressed in refractory AOSD patients, consistent with the bioinformatics findings, and their expression levels effectively distinguished refractory from non-refractory cases. No significant differences in age or sex were observed, but inflammatory markers (ferritin, CRP, ESR) were significantly higher in the refractory group than in non-refractory and relapsed non-refractory groups. Peak body temperature, arthralgia, and skin rash were also more severe in the refractory group. Notably, the upregulated genes and aberrant inflammatory markers identified may be relevant targets of upadacitinib, suggesting it may alleviate refractory AOSD by suppressing these inflammatory pathways and cytokine release.

Through integrated transcriptomic features and clinical validation, this study identified a set of inflammatory genes overexpressed in refractory AOSD and confirmed their involvement in the JAK–STAT pathway. These biomarkers, along with elevated inflammatory markers, correlated with disease refractoriness and may serve as diagnostic candidates. The findings also provide molecular insights supporting the potential use of upadacitinib in refractory AOSD.