Sequential monitoring using NGS allows detection of ABL1 KD mutations in patients with chronic Phase CML: Results from the prospective calls trial

Abstract Background BCR :ABL1 kinase domain (KD) mutations are a well-established mechanism of resistance to tyrosine kinase inhibitors (TKIs) in chronic myeloid leukaemia (CML). Early detection of emerging clinically relevant mutations is crucial for timely treatment modification, yet there are currently no standardized recommendations on screening frequency. Traditional Sanger sequencing lacks the sensitivity to detect low-level mutations, potentially delaying clinical intervention. In contrast, next-generation sequencing (NGS) offers superior sensitivity (≥3% variant allele frequency, VAF), enabling earlier detection of resistance-associated mutations that may impact clinical outcomes and guide treatment optimization. Aims To assess the utility of sensitive NGS-based BCR::ABL1 KD mutation screening in a large, prospective cohort of CML patients receiving TKI therapy, and to characterize the mutational landscape, temporal evolution, and clinical impact of NGS-based KD mutations screening results across centres in the UK, Ireland, and France (CALLS trial, NCT03647215). Methods In this prospective, multicentre, non-interventional study, CML patients receiving first-line or subsequent TKI therapy were screened for BCR::ABL1 KD mutations using NGS and results were communicated to the treating hematologist. Patients were enrolled based on European LeukemiaNet (ELN) criteria, and sequential NGS was performed to monitor mutation evolution over time. Mutational burden, frequency, clinical relevance, and treatment outcomes were analysed descriptively. Results Of 415 patients consented, 402 were enrolled and 381 (94.8%) had confirmed chronic phase CML (CP CML) and are the subject of this analysis. At the time of trial enrollement, 160 (42%) patients were treated with imatinib, 101 (26.5%) with dasatinib, 62 (16.3%) with nilotinib, 33 (8.7%) with bosutiinib, 24 (6.3%) with ponatinib and one with asciminib. BCR::ABL1 KD mutations were detected in 37 patients in CP CML (9.7% of enrolled cohort). Among mutation-positive patients, 89.2% (33/37) harboured a single mutation, 5.4% (2/37) had two mutations, and 5.4% (2/37) had three or more mutations. Themost frequently identified mutation was T315I, detected in 12 of 37 patients while other recurrent mutations included F317L, E279K, Y253H, G250E, E255K, and H396R. Several mutations were identified at low VAFs otherwise undetectable by conventional Sanger sequencing, demonstrating the enhanced sensitivity of NGS. Sequential NGS data revealed new KD mutations in 11 patients despite a first negative screening by NGS on the first sample taken as part of the study. The median time between the negative and the first sample tested positive by NGS was 7 months (range: 5-31 months) with 90% acquiring mutations conferring resistance to their current TKI. Of 27 patients with detected mutations in sequential analysis, 12 (44%) ultimately switched TKIs due to lack of response (67%) and intolerance (25%). Among patients with sequential NGS, 70% demonstrated increased VAF over time, suggesting clonal evolution under therapeutic pressure. Notably, 36% of patients who switched TKI achieved improved clinical responses, validating the clinical relevance of mutation-guided therapy. Conclusion NGS-based screening detects clinically relevant BCR::ABL1 KD mutations at low VAFs, enabling earlier intervention than conventional methods. The high prevalence of T315I and frequent detection of resistance-conferring mutations support the routine use of NGS, in line with the 2025 ELN guidelines. Sequential monitoring using NGS allowed detection of KD mutations in patients previously tested negative. Given the absence of evidence-based guidelines on screening frequency in this group, these findings may support the integration of sensitive, routine NGS monitoring at regular interval to optimize clinical management and enable proactive treatment adjustments in order to reduce the risk of delayed intervention in CML patients treated with TKIs.