Combined Bulk and Single‐Cell RNA Sequencing Reveals Alterations in the Immune Microenvironment and Candidate Immune‐Related Genes Associated With Diagnosis and Relapse in Childhood B‐Cell Acute Lymphoblastic Leukemia

Background The association between relapse and remodeling of the bone marrow immune microenvironment in pediatric B‐cell acute lymphoblastic leukemia (B‐ALL) remains incompletely understood. This study aims to characterize the transcriptional and cellular alterations associated with B‐ALL relapse and identify immune‐related genes (IRGs) associated with disease recurrence. Methods Bulk (GSE3910 and GSE28460) and single‐cell RNA sequencing (scRNA‐seq) (GSE130116) data were obtained from the GEO database, whereas IRGs were retrieved from ImmPort database. GSE3910 dataset was analyzed to identify differentially expressed genes (DEGs), followed by functional enrichment and immune infiltration analysis. scRNA‐seq data were used for cell annotation and cell–cell communication analysis to identify key immune shifts. Candidate genes were identified through the intersection of scRNA‐seq DEGs, bulk RNA sequencing (bulk RNA‐seq) DEGs, and IRGs. Gene–gene interaction (GGI) and transcription factor analyses, and external validation using GSE28460 were subsequently performed. Results A total of 544 DEGs were identified from DEG3910. Functional enrichment analysis indicated that relapse was associated with enhanced cell proliferation, immune stress, and inflammatory responses. scRNA‐seq analysis revealed substantial microenvironmental heterogeneity. Cell–cell communication analysis identified alternations in signaling networks during relapse, particularly toward T and natural killer cells. Pathways related to prostaglandin and NEGR signaling were notably enriched in relapse samples. Integrative analysis identified seven IRGs ( ELANE , S100A8 , MNDA , S100A9 , S100A12 , PSAP , and FCN1 ) associated with relapse and were predominantly associated with myeloid cells. Conclusion Relapsed pediatric B‐ALL may be associated with heterogeneous alterations in immune microenvironment and intercellular communication. The identified genes may represent relapse‐associated molecular features and warrant further functional investigation.