A Three-Component Immune Framework Stratifies Treatment Response and Escalation in Pediatric Hemophagocytic...

Pediatric hemophagocytic lymphohistiocytosis (HLH) is a hyperinflammatory syndrome that can be classified into three principal etiologies (primary HLH [pHLH], Epstein-Barr-virus-driven HLH [EBV-HLH], and secondary HLH [sHLH]) and is associated with disparate outcomes. Treatment escalation decisions remain largely empirical, guided by clinical presentation and conventional laboratory results rather than by an integrated biomarker framework.

We prospectively enrolled 180 consecutive pediatric patients with HLH at Children's Hospital 1, Ho Chi Minh City, Vietnam (October 2022-October 2025): pHLH, n = 40; EBV-HLH, n = 46 (CD27 intact, n = 19; CD27 dim, n = 27); and sHLH, n = 94. A 15-analyte multiplex cytokine panel and six-marker lymphocyte subset flow cytometry were performed at diagnosis (T0) and Day 7 (T1). Logistic regression models were developed and internally validated using bootstrap optimism correction (1000 iterations), 5-fold stratified cross-validation, and a temporal split.

Overall mortality was 31.1% (EBV-HLH 67.4%, pHLH 45.0%, and sHLH 7.4%). Three discrete cytokine and subset phenotypes emerged: EBV-HLH showed massive elevations in IFN-γ and sCD25, with profound CD4+ and double-negative T-cell depletion; pHLH showed elevated tumor necrosis factor-α with preserved subsets; sHLH had low-amplitude profiles with the highest CD4+ counts. Day-7 IFN-γ T1/T0 ratio (threshold 0.376) stratified mortality 6- to 27-fold within each etiology. Cytokine drivers of escalation were etiology specific (sCD25 in pHLH, sCD25+IL-18 in EBV-HLH, and IL-10/IL-6 in sHLH). The integrated three-component model achieved an apparent AUC of 0.963, a bias-corrected AUC of 0.947 (95% CI: 0.92-0.97), a 5-fold CV AUC of 0.942 ± 0.05, and a temporal validation AUC of 0.946-all three methods were convergent.

Integrating etiology, cytokine phenotype, and lymphocyte subset profile provides biology-grounded prediction of mortality and treatment escalation in pediatric HLH. Multicenter external validation is the next essential step.