Clonal hierarchies and dynamics. (A) Co-occurrence of CH associated with an increased risk of MDS/AML development. A Venn diagram shows the number of patients with Chr1q⁺, U2AF1^S34, and TP53 mutations (left). Chr1q+ and U2AF1^S34 or TP53 mutations were mutually exclusive in patients who developed malignancies (MDS, AML, or squamous cell carcinoma [SCC]). A patient with Coats plus syndrome had both TP53 and U2AF1^S34 mutations without developing cancer (NIH-47). A graph shows the co-occurrence of Chr1q⁺ and U2AF1^S34 with somatic mutations in other MDS-related genes (right). Chr1q⁺ and U2AF1^S34 were always dominant clones, as a measure of VAF (right). Somatic mutations in other MDS-related genes, mainly in RUNX1, ETV6, and splicing factors, were likely subclonal. In contrast to U2AF1^S34, all patients with Chr1q⁺ and CH in MDS-related genes developed MDS. TP53 mutations rarely co-occurred with somatic mutations in other MDS-related genes. (B) Clonal hierarchies defined by scDNA-seq analysis (left) and longitudinal analysis of mutant clones in granulocytes or total PB defined by bulk ECS (right) in 2 patients with multiple PPM1D or POT1 mutations (NIH-33 and BR-01). NIH-33 (MAA and liver disease at the age of 12) and BR-01 (MAA at age 18 years) had developed no malignancy at the last follow-up and are alive. ∗indicates PPM1D VAF in total PB from NIH-33; ∗∗indicates PPM1D VAF in PB mononuclear cells from NIH-33. An orange bar indicates the time NIH-33 was under low-dose danazol treatment. (C-D) Clonal hierarchies defined by scDNA-seq analysis (left) and longitudinal analysis of mutant clones in total PB defined by bulk ECS (right) in 3 patients with U2AF1^S34 mutations (NIH-02, NIH-21, and NIH-39) (C) and in 1 patient with Chr1q+ and U2AF1^Q157R mutation (NIH-11) (D). Blue and orange bars indicate the times when patients were under regular-dose or low-dose danazol treatment, respectively. NIH-21 (MAA and liver disease at the age 60 years) and NIH-39 (mild cytopenias at the age 26 years) had the U2AF1^S34 mutation at VAF >20% in PB but no MDS/AML at the last follow-up. In contrast, NIH-02 (MAA and pulmonary fibrosis at the age of 39) developed MDS 2 years after the initial screening, and NIH-11 (MDS at the age 54 years), with a detectable Chr1q⁺ and U2AF1^Q157R mutation at assessment, evolved to MDS with an excess of blasts (MDS-EB) and AML at 5.5 years of follow-up and died. Single-cell proteogenomic analysis evidenced that AML evolution coincided with the emergence of a KRAS mutation and monosomy 7 in CD34⁺ HSC clones harboring the Chr1q⁺ and U2AF1^Q157R mutations (supplemental Figure 16). D, dominant clone; MAA, moderate aplastic anemia; S, subclonal clone; scDNA-seq, single-cell proteogenomic sequencing.