AUTHORS
Ruidong Zhang, PhD, Mingying Zhang2, Peijing Qi, Doctor of Medicine3, Ying Wu, Master of Medicine4, Jiaole Yu, Doctor of Philosophy5, Yuanyuan Zhang, N/A6, Wei Lin, Doctor of Medicine7, Jia Fan, Master of Medicine8, Pengli Huang, Master of Medicine9, Bei Hou, PhD10, Jie Yang11, Huiqing Liu, PhD12, Mengjia Liu, PhD13, Zhan Chen, N/A14, Kexin Zhu, N/A15, Chengxian Dou, N/A16
AFFILIATIONS
1 Leukemia Department, Beijing Children’s Hospital of Capital Medical University, Beijing, China
2 Leukemia Department, Beijing Children’s Hospital of Capital Medical University, Beijing, China
3 Leukemia Department, Beijing Children’s Hospital of Capital Medical University, Beijing, China, Beijing, China
4 Leukemia Department, Beijing Children’s Hospital of Capital Medical University, Beijing, China, Beijing, China
5 Hematology Center, Beijing Children’s Hospital of Capital Medical University, Beijing, China, Beijing, China
6 Leukemia Department, Beijing Children’s Hospital of Capital Medical University, Beijing, China, Beijing, China
7 Leukemia Department, Beijing Children’s Hospital of Capital Medical University, Beijing, China
8 Leukemia Department, Beijing Children’s Hospital of Capital Medical University, Beijing, China
9 Leukemia Department, Beijing Children’s Hospital of Capital Medical University, Beijing, China
10 Leukemia Department, Beijing Children’s Hospital of Capital Medical University, Beijing, China
11 Leukemia Department, Beijing Children’s Hospital of Capital Medical University, Beijing, China
12 Leukemia Department, Beijing Children’s Hospital of Capital Medical University, Beijing, China
13 Leukemia Department, Beijing Children’s Hospital of Capital Medical University, Beijing, China
14 Shanghai Rightongene Biotechnology, Shanghai, China
15 Shanghai Rightongene Biotechnology, Shanghai, China
16 Shanghai Rightongene Biotechnology, Shanghai, China
ABSTRACT
Background: In pediatric B-cell acute lymphoblastic leukemia (B-ALL), relapses are the leading causes of treatment failures, and measurable residual disease (MRD) identifies patients at high relapse risk. While blinatumomab has transformed first-line therapy, primary resistance and the cellular dynamics governing treatment response remain poorly understood. We assessed blinatumomab’s efficacy and safety in newly diagnosed MRD-defined high-relapse-risk B-ALL and used single-cell multiomics to elucidate immune microenvironment dynamics influencing response.
Methods: In this prospective, single-center study (ChiCTR2200062866), 49 children with newly diagnosed MRD-defined high-relapse-risk B-ALL received blinatumomab. MRD was monitored via MFC, polymerase chain reaction (PCR), and next-generation sequencing (NGS). High-relapse risk was defined as MFC MRD ≥1.0% on day 15, or positive MRD (MFC/PCR MRD ≥0.01% or NGS MRD ≥0.0001%) at postinduction (D33), preconsolidation, or premaintenance.
To investigate the mechanisms underlying treatment resistance and response, we integrated bulk RNA sequencing and single-cell multiomics (scRNA-seq, scBCR-seq, and scTCR-seq). Thirty-one longitudinal bone marrow samples from eight patients were sequenced at baseline, days 14 and 28 of blinatumomab treatment. Patients were categorized into a deep molecular response (DMR) group ( n = 5; D28 NGS MRD < 0.0001%) and a non-deep molecular response (NDMR) group ( n = 3; D28 NGS MRD ≥ 0.0001%). Single-cell data processing was performed using Seurat, Monocle2, and STARTRAC, complemented by malignant clone identification via InferCNV, cell-cell communication analysis via CellChat, transcription factor network evaluation via SCENIC, and in vitro T-cell functional co-culture assays with pharmacological inhibitors.
Results: Clinical evaluation over a median follow-up of 39.4 months demonstrated a 3-year overall survival (OS) of 100%, an event-free survival (EFS) of 97.96% (95% CI, 94.0%–100.0%), and a low cumulative incidence of relapse (CIR) of 2.04% (95% CI, 0.0%–6.0%), with no treatment-related deaths in 49 high-risk of relapsed patients. Safety profiles were favorable, showing low rates of Grade 3 cytokine release syndrome (CRS; 4.1%) and neurotoxicity (2.0%), with no Grade 4 events.
Multiomics integration mapped the cellular and molecular trajectories of response versus resistance. Malignant B cells in DMR patients followed a canonical differentiation pathway and activated immune-related signaling, whereas NDMR patients exhibited a differentiation block (with abnormal hematopoietic stem cell and pre-B cell accumulation) and metabolic rewiring favoring oxidative phosphorylation. T-cell subset profiling revealed that DMR patients experienced rapid, sustained expansion of functionally active, highly cytotoxic subsets (CXCR6+CD8+ and MKI67+CD8+) expressing robust GZMA/B/K and coordinated metabolic-immune profiles. Conversely, patients with NDMR exhibited a disconnection in metabolic-signaling, and their inability to generate proliferative effector clones.
Trajectory and clonal analyses suggested that a structural T-cell differentiation cascade (Teff → CXCR6+CD8+ → MKI67+CD8+) is essential for achieving deep molecular remission. Mechanistically, SCENIC and in vitro validation established RUNX3 and EZH2 as subset-specific master transcription regulators facilitating this functional rejuvenation while balancing activation with exhaustion mitigation. Furthermore, cell-cell communication analysis identified the LGALS9-mediated signaling pathway (via CD44 and CD45 axes) as a vital link that not only enhances T-cell effector functions but also restricts leukemia cell survival, an axis prominently induced on day 14 in the DMR group but inactive in the NDMR group. Longitudinal tracking indicated that the residual disease observed on day-14 reflects the biological time lag associated with immune remodeling rather than treatment failure, supporting the importance of a full 28-day treatment course to overcome immune latency and achieve total tumor clearance.
Conclusion: Blinatumomab exhibits high efficacy, favorable safety profile, and long-term survival benefits in pediatric high-relapse-risk B-ALL. Single-cell multiomics reveals that a RUNX3/EZH2-driven T-cell maturation trajectory and LGALS9-mediated tumor-immune crosstalk are potential mediators of deep molecular response, providing mechanistic insights to overcome clinical immunotherapy resistance.
DISCLOSURES
Mingying Zhang: No relevant conflicts of interest to declare.