The prognostic significance of the TCF3::PBX1 fusion in pediatric B-cell acute lymphoblastic leukemia (B-ALL) remains controversial, with studies reporting variable treatment responses and no consensus on its independent impact on outcomes. This uncertainty hinders the development of precise risk stratification strategies. While this fusion accounts for approximately 4%–6% of pediatric ALL cases in the literature, its clinical and molecular heterogeneity demands deeper investigation.

To characterize the epidemiological and molecular profile of pediatric B-ALL patients with TCF3::PBX1 fusion treated at a single Brazilian reference center, and to compare these findings with international literature, focusing on challenges in risk assessment.

A retrospective observational study was conducted including patients diagnosed with B-ALL at Hospital da Criança de Brasília between 2012 and 2022. Eligibility required confirmed TCF3::PBX1 fusion by cytogenetics or molecular methods. Clinical and laboratory data were collected from electronic medical records. RNA sequencing was performed on available samples to assess gene expression and detect co-occurring fusions and variants.

TCF3::PBX1 fusion was detected in 5.71% of the cohort (n=21/368), consistent with international incidence. Median age was 5-years, with slight female predominance. Elevated WBC count (>50,000 mm3) occurred in 28.57%. At diagnosis, 61.9% were high risk, 23.8% intermediate, 4.8% low; others unclassified. After induction, 38% were high risk, 33.3% intermediate, 4.8% low; remainder unclassified. Relapse occurred in 33.3%, mostly early (85.7%), with mean time to relapse of 0.88 years. Mortality was 14.3%. RNA-seq identified 35 genes differentially expressed between diagnosis and relapse, including MYC, CCND2, FGFR2, JAK2, STAT5B, GATA1, IKZF1, CEBPE, BCOR, AFF3, and BCL9, indicating deregulation of proliferation, signaling, hematopoietic differentiation, and epigenetic regulation. WT1 overexpression at diagnosis was associated with relapse.

Risk category shifts from diagnosis to post-induction illustrate stratification challenges. Despite a high proportion initially classified as high risk, many migrated to lower categories, yet relapse remained frequent and often early. This mirrors literature describing heterogeneous outcomes for TCF3::PBX1 despite its historical classification as non-adverse. The persistence of high relapse rates, including in non-high-risk groups post-induction, suggests current stratification relying on clinical and MRD data may underestimate relapse potential. The molecular alterations identified ‒ particularly WT1 overexpression and pathway deregulation— highlight the value of integrating genomic and transcriptomic profiling to improve prediction accuracy. B-ALL with TCF3::PBX1 fusion poses significant risk-stratification challenges. Our findings support the incorporation of comprehensive molecular characterization into clinical protocols to refine prognosis and enable more personalized treatment strategies.