Timeline of mutation acquisition. Multiple patients had additional analyses after diagnosis by next-generation or Sanger sequencing. Shown are the BCR-ABL1 transcript levels over time (black diamond symbol; left y-axis). White diamonds indicate BC. Variants are plotted according to their time point of detection and percentage variant allele frequency (VAF; right y-axis). Lines that extend from 0 VAF indicate the time point before the first detection of the variant. BCORL1 and PHF6 are located on the X chromosome and represent a single-gene copy (all male patients). The VAFs of the ABL1 KD mutations were derived from Sanger sequencing by isolating the BCR-ABL1 allele, but the VAFs are normalized for diploid number. Fusions and intra- and intergenic deletions were not quantified and are plotted with symbols above their time point of detection. (A-H) Eight patients with BC before 12 months. In 6 of these patients, variants were present at diagnosis. ABL1 KD mutations were acquired in 7 of the 8 patients. (I-N) Six patients with BC after 12 months. Variants were present at diagnosis in 4 and ABL1 KD mutations were acquired in 3 of the 6 patients. The graphs demonstrate that the VAF decreased or increased over time for some variants, which corresponded with BCR-ABL1 levels. In some cases, the VAF changes suggest clonal competition. (N) Patient 3 had DNMT3A and TET2 mutations detected in a remission sample when BCR-ABL1 transcripts were undetectable, and the pattern of clonal selection is consistent with their presence in a BCR-ABL1⁻ clone. IS, International Scale.