Longitudinal single-cell analysis identifies a chemotherapy-suppressed proliferative program associated with adverse prognosis in neuroblastoma.

Background: High-risk neuroblastoma frequently relapses after intensive therapy, but the treatment-associated malignant-neuroblast programs that inform baseline tumor risk remain incompletely defined. We used longitudinal single-cell/single-nucleus RNA-seq data to identify post-treatment transcriptional changes in malignant neuroblasts and evaluated their prognostic relevance in diagnostic bulk RNA-seq cohorts. Methods: We analyzed a longitudinal single-cell/single-nucleus RNA-seq atlas of diagnostic (DX) and post-treatment (PTX) neuroblastoma samples. Neuroblast cells were aggregated into donor- and treatment-state-level pseudo-bulk profiles, and paired differential expression analysis was performed to identify PTX-regulated malignant-neuroblast programs. PTX-down and PTX-up gene sets were functionally characterized by Hallmark over-representation analysis and Hallmark pathway activities were further estimated by GSVA in bulk cohorts. We then projected the PTX-down program onto diagnostic bulk RNA-seq cohorts by calculating a PTX-down proliferation score as the mean cohort-wise z-score of PTX-down genes. Prognostic value was evaluated in a primary diagnostic bulk RNA-seq validation cohort and three independent external diagnostic bulk RNA-seq cohorts. Clinical model increment, cross-cohort meta-analysis, clinical feature association, and Hallmark correlation analyses were performed. Results: Paired pseudo-bulk analysis showed that PTX malignant-neuroblast profiles had reduced activity of a proliferative-replication program enriched for E2F targets, G2/M checkpoint, MYC targets, DNA repair, and mitotic spindle pathways, together with relative enrichment of adaptive-state features related to hypoxia, inflammatory signaling, apoptosis, and epithelial-mesenchymal transition. In the primary validation cohort, high PTX-down proliferation score was associated with inferior OS and EFS and remained prognostic after adjustment for age, COG-defined high-risk classification, MYCN status, and INSS stage. Adding the PTX-down score improved C-index and 1-, 3-, and 5-year time-dependent AUC beyond both COG and clinical models. Across four bulk RNA-seq cohorts, higher PTX-down score was consistently associated with adverse OS (random-effects pooled HR = 1.83, 95% CI 1.20-2.80; P = 0.005). The score was also higher in tumors with MYCN amplification, stage 4 disease, and age ≥ 18 months. Core-gene and Hallmark correlation analyses defined the PTX-down score as an E2F/G2M/MYC-driven proliferative axis. Conclusion: Longitudinal single-cell analysis identifies a chemotherapy-suppressed proliferative program in malignant neuroblasts. Projection of this program onto diagnostic bulk tumors captures high-risk biology, improves prognostic discrimination, and predicts adverse outcome in neuroblastoma.