Acquired thiopurine resistance and mismatch repair aberrations in paediatric B-cell acute lymphoblastic leukaemia relapse: A real-world data integrating mutational signatures, clones and clinical thiopurine dosing patterns.

Acquired thiopurine resistance and mismatch repair (MMR) deficiency are increasingly recognised drivers of relapse in paediatric B-cell acute lymphoblastic leukaemia (B-ALL); however, their real-world genomic prevalence and link to maintenance dosing patterns remain poorly characterised. Paediatric B-ALL relapse cases (2018-2024) were subjected to whole exome sequencing (WES; 200-250× depth; n = 70) and deep targeted sequencing (700-900×; n = 5). Per-sample frequencies of resistance gene mutations, single-base mutational signatures, tumour mutational burden-high (≥10 mutations/Mb), microsatellite instability-high (MSI-H) and thiopurine S-methyltransferase (TPMT)/nucleoside diphosphate-linked moiety X-type motif 15 (NUDT15) status were determined. Clinical variables included the median weekly 6-mercaptopurine (6-MP) dose (mg/m2, time-weighted) and duration of 6-MP dose interruptions. A total of 18/75 (24%) relapse cases showed SBS87 (thiopurine-related), SBS6/15 (MMR-related) or Thio-deficient mismatch repair (Thio-dMMR) mutational signature. Thiopurine resistance clones in cytosolic 5'-nucleotidase II (NT5C2) (13), phosphoribosyl pyrophosphate synthetase 1 (PRPS1) (2) or tumor protein p53 (TP53) (7) were noted in 19/75 (25%) cases; germline or somatic clones in MMR genes were noted in 19/75 (25%). Correlation analysis revealed that both low median weekly 6-MP dose (<45 mg/m2) and ≥4 weeks of dose interruptions were significantly associated with higher hazard for SBS87 or Thio-dMMR signature (hazard ratio [HR] 7.09 [95% confidence interval (CI): 1.77-28.34], p = 0.006, and HR 6.21 [95% CI: 1.66-23.32], p = 0.007 respectively). This real-world study demonstrates that suboptimal median 6-MP dosing and prolonged dose interruptions during maintenance are significantly associated with thiopurine resistance and MMR deficiency-related mutational signatures at relapse. These findings underscore the importance of optimised dose intensity and minimal interruptions during maintenance therapy in paediatric B-ALL.