Background: TCF3::HLF-positive B-cell acute lymphoblastic leukemia (B-ALL) represents one of the most aggressive and treatment-refractory subtypes of ALL. It is characterized by the t(17;19)(q22;p13) translocation generating the TCF3::HLF fusion, leading to impaired B-cell differentiation, chemoresistance, and early relapse. Despite its classification as a distinct entity in the 2022 WHO and ICC frameworks, comprehensive clinical and molecular characterization remains limited due to its extreme rarity.

Methods: We analyzed 34 consecutive TCF3::HLF-positive B-ALL patients diagnosed between 2012 and 2024 at a single leukemia center, representing the largest cohort to date. Multi-dimensional profiling included clinical data, immunophenotyping, karyotyping, gene mutation screening of 86 leukemia-related genes, and whole transcriptome sequencing (WTS). Comparisons were made with 129 TCF3::PBX1- and 25 TCF3::ZNF384-positive B-ALL cases to contextualize clinical outcomes and molecular features.

Results: TCF3::HLF accounted for 1.6% of all B-ALL cases (n=2,136), with a pediatric predominance (88%, median age 11 years). Three fusion isoforms were identified, but Isoform III was always co-detected with Isoform II and showed significantly lower read support, suggesting an alternatively spliced variant rather than a distinct event. Immunophenotypically, CD33 and/or CD13 expression was present in 88% of cases, implying partial myeloid lineage priming. Karyotyping revealed a high frequency of complex abnormalities; only 59% had visible t(17;19).

Mutational analysis showed a remarkably high frequency of isolated RAS pathway mutations (86%), distinguishing this subtype from other TCF3-fusion leukemias. No concurrent mutations outside the RAS pathway were observed. WTS analysis revealed 2,049 differentially expressed genes compared to other TCF3 fusions, including upregulation of EMT, interferon signaling, and coagulation-related genes (e.g., F3, NOX4), alongside downregulation of B-lineage and neuronal development programs. GSEA and KEGG/GO analyses supported enrichment of migration, adhesion, and immune dysregulation pathways, potentially contributing to leukemic aggressiveness and extramedullary behavior.

Therapeutically, conventional chemotherapy was inadequate: all 12 patients not receiving allo-HSCT relapsed or died within 30 months (median time to relapse: 6.7 months). CAR-T therapy enabled MRD-negative remission in 13 patients, of whom 11 successfully proceeded to allo-HSCT in CR1. Two patients achieving MRD-negative remission after CAR-T without transplant relapsed and failed subsequent salvage, underscoring CAR-T's role as a bridge, not a cure. Allo-HSCT significantly improved overall and event-free survival (both p<0.0001), with 14 of 20 transplanted patients remaining in continuous CR, the longest exceeding 6 years. Post-HSCT relapse occurred in 4 patients, suggesting a need for better maintenance strategies.

Use of blinatumomab showed mixed outcomes—effective in MRD-positive remission but ineffective in frank relapse. Venetoclax failed to induce remission in a multiply relapsed case. Notably, one patient received prophylactic blinatumomab post-HSCT without relapse, suggesting a possible preventive role.

Conclusions: TCF3::HLF-positive B-ALL is an ultra-high-risk leukemia with distinct molecular and immunophenotypic features, a striking predominance of RAS-only mutations, and a unique transcriptional program marked by EMT and immune dysregulation. Allo-HSCT remains the only curative approach, ideally preceded by MRD-negative remission. CAR-T therapy is effective in bridging patients to transplant, but durable remissions remain elusive without allo-HSCT. Given consistent CD33 expression and RAS pathway dependency, this subtype may benefit from CD33-directed or MEK-targeted strategies. Our findings highlight the urgent need for international collaboration to design tailored treatment regimens and prospective trials in this challenging and lethal leukemia subtype.

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2025
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