Genetic evolution in CML patients from diagnosis to TKI failure: An analysis of 121 paired diagnosis and treatment failure samples

Abstract

Abstract Background and purpose:Somatic mutations in blood cancer genes have recently been investigated inchronic myeloid leukemia (CML) and are associated with resistance to tyrosine kinase inhibitors (TKIs).These findings suggest that some mutations are acquired following treatment, while others are pre-existed at diagnosis. However, the mechanism of TKI treatment failure remains to be incompletely understood. This study aimed to analyze gene mutations in paired diagnostic and treatment failure samples for the same individuals, and to compare genetic alterations andtheir variant allele frequencies (VAF) between diagnosis and treatment failure to characterize the genetic evolution during TKI therapy. Methods: All the newly diagnosed CML patients were treated with frontline TKIs and underwent scheduled peripheral blood BCR::ABL1 transcript level monitoring for at least 12 months in the central laboratory at Chang Gung Memorial Hospital, Taiwan. Treatment responses were evaluated based on the ELN 2020 recommendations. We also included failure after second- or third-line TKI switch. Between April 2002 and June 2024, 311 CML patients met any criteria for TKI failure were enrolled. Among them, 121 CML patients with paired diagnosis and failure samples available were analyzed: 83 after frontline TKIs (72 for imatinib, 11 for second-generation TKIs), 20 after second-line TKIs, and 18 third-line TKIs. Somatic mutations were identified using Illumina TruSight Myeloid or Sophia Myeloid Solution next-generation sequencing (NGS) panels (161 samples). For patients without available gDNA samples, we used the RNA-based gene panel (72 genes) for NGS assay (81 samples) according to the method of Branford et al. (J Mol Diagn 2022). Twenty matched DNA and RNA samples were simultaneously compared for concordance validation, and showed a 95.2% (of 168 variants) concordance rate. Results: The median time from diagnosis to failure of the 121 patients was 16.2 months. Sixty-seven patients (55.4 %) had at least one mutation, with totally 93 mutations detected at diagnosis. Forty-seven mutations were acquired, including 16 emerging following second- or third-line TKIs, whereas 28 mutations were lost at treatment failure. Sixteen patients at diagnosis and 34 patients at TKI failure had multiple mutations. ASXL1 mutations occurred most frequent at both diagnosis (24.8%) and treatment failure (19.8%). Co-occurrence of ASXL1 and ABL1 mutations was observed in 5 patients. RUNX1 mutations were not detected at diagnosis but emerged in 5 patients at TKI failure, 2 of them had co-occurring RUNX1 andABL1 mutations. Another two patients had BCORL1 co-existed with ABL1 mutations. A significant decrease in VAF of mutated genes involving epigenetic regulators (42 ASXL1, 5 KDM6A, 3 KMT2D, 11 DNMT3A, and 11 TET2) from diagnosis (median 29.5%) to TKI failure (median 13.6%) (P = 0.0467). In contrast, VAF increased in either transcription factor (TF) (5 RUNX1, 5CUX1,4GATA2, 2STEBP1, and one each for CEBPA, IKZF1, PFH6, and ETV6) or tumor suppressor (TS) (7 BCORL1, 5 WT1, and one each for TP53, CDKN2A, andFBXW7) genes, with a median VAF rising from 0% at diagnosis to 25.7% at time of failure for TF (P = 0.0162) and 5.4% to 27.0% (P = 0.0654) for TS. Mutations in cohesin complex and signal pathway genes remained unchanged between diagnosis and TKI failure (Median VF 49.8% vs 50.1%, and 48.1% vs 49.6%, respectively). Of the 121 patients, 23 progressed to blastic phase and 25 died. Patients with TS mutations at TKI failure had inferior 10-year overall survival (OS) of 57.8% (P = 0.015) and 10-year progress-free survival (PFS) of 43.3% (P = 0.002) compared to patients without mutations (10-year OS: 78.8% and 10-year PFS: 80.2%). Similarly, patients with TF mutations had shorter 10-year OS of 58.3% (P = 0.162) and 10-year PFS of 56.5% (P = 0.012) compared to patients without mutations (10-year OS: 77.7% and 10-year PFS: 79.0%). Conclusions:Our study demonstrates that TKI failure reflects a complex pattern of clonal evolution, characterized by stable mutant clones of cohesin complex and signaling genes, loss of epigenetic mutant clones and emergence of TF and TS mutations. Notably, the latter are strongly associated with adverse outcomes and should be considered in therapeutic decision-making.