Subtype-specific immunophenotypic and cytogenetic patterns complement flow-cytometric detection of measurable residual disease in B-lymphoblastic leukemia.

Measurable residual disease (MRD) is a key prognostic marker in B-lymphoblastic leukemia (B-ALL). Because immunophenotypic and cytogenetic features vary by genetic subtype and treatment phase, the clinical value of an integrated MRD strategy using multiparametric flow cytometry (MFC), fluorescence in situ hybridization (FISH), and immunoglobulin clonality next-generation sequencing (NGS) warrants evaluation. This retrospective single-center study included 136 patients with B-ALL and 491 bone marrow MRD assessments. MRD was evaluated by an 8-color MFC panel adapted from the modified EuroFlow protocol, targeted FISH probes with an exploratory integrated interpretation for low-level signals, and IGH/IGK NGS. Immunophenotypic marker expression and associations with genetic subtypes were analyzed in MFC MRD-positive samples. MRD levels were significantly correlated across modalities: MFC-FISH (ρ = 0.762), MFC-NGS (ρ = 0.674), and FISH-NGS (ρ = 0.341). Using NGS as the molecular comparator, MFC showed higher sensitivity than FISH (41.0% vs. 31.8%) with specificity of 100.0% vs. 96.9%. Discordant MFC/FISH cases showed subtype-associated patterns, with MFC-/FISH+ findings more frequently observed in cases with numerical chromosomal abnormalities (e.g., hyperdiploidy) and immunophenotypic features resembling hematogones. Immunophenotypic marker expression varied by subtype, with near absence of CD10 in KMT2A-rearranged B-ALL and increased CD81 expression in TCF3::PBX1 cases. MRD positivity by any method was associated with significantly worse relapse-free survival. MFC, FISH, and NGS demonstrate complementary but variable performance in MRD detection across B-ALL subtypes. Immunophenotypic and cytogenetic heterogeneity influence the sensitivity of individual modalities, supporting the use of an integrated, multimodal approach for MRD surveillance.