American Society of Hematology

Figure 1.

$Figure 1. In AML, somatically acquired TP53 mutations characterize preleukemic stem cells, are initiating genetic events, and mediate resistant disease. (A) FACS analysis of bone marrow of an NSGS mouse engrafted with 1 × 106 unpurified, human TP53-mutated AML cells showing both blast cells as well as maturation into granulocytes. (B) Engrafted human cells with a blast-cell and B-lymphocyte phenotype. (C) Graft composition of mouse bone marrow. Human blasts were characterized by a sideward scatter (SSC) low/CD34+/CD45dim/CD19− phenotype, granulocytes by a SSChigh/CD33+/CD34−/CD19−, and B lymphocytes by an SSClow/CD34−/CD33−/CD19+ phenotype. The horizontal bar depicts mean TP53 variant allele frequencies (VAFs). (D) Highly purified peripheral blood CD45+/CD3+ cells were obtained at AML diagnosis, and the TP53 VAFs were assessed using ultradeep sequencing. Samples 7071 and 5273, respectively, exhibited 2 different somatic TP53 mutations. Note that in each case analyzed and scored positive, the TP53 VAF exceeded the minute impure fraction of sorted T lymphocytes, thereby excluding results biased because of contamination of AML cells. (E) Data obtained from colony-forming unit–granulocyte, monocyte (CFU-GM) colonies derived from specimen 5652 revealed the TP53 mutation as the initiating event (positive in 38 of 38 colonies), followed by an ASXL1 mutation (37 of 38), IDH2 mutation (20 of 38), and RUNX1 mutation (19 of 38). All cooperating mutations developed sequentially in the TP53-mutated clone. The exact mutation type is shown in Table 1. (F) Loss of heterozygosity at the TP53 locus of samples from UPN 7317. In CFU-GM colonies, a heterozygous TP53 c.681_681dupT mutation is shown, whereas in bulk leukemia cells, the wild-type allele was lost, resulting in a hemizygous state. (G) Quantitative assessment of the TP53 mutational load by the ultradeep sequencing, indicating comparable levels between diagnostic specimens and those obtained at relapsed or refractory (R/R) phase (P = .578 by the exact permutation test for related samples). B, blast cells; CR, complete remission; Dg, diagnosis; G, granulocytes; h, human; L, lymphocytes; m, mouse; SD, standard deviation.$

In AML, somatically acquired TP53 mutations characterize preleukemic stem cells, are initiating genetic events, and mediate resistant disease. (A) FACS analysis of bone marrow of an NSGS mouse engrafted with 1 × 10⁶ unpurified, human TP53-mutated AML cells showing both blast cells as well as maturation into granulocytes. (B) Engrafted human cells with a blast-cell and B-lymphocyte phenotype. (C) Graft composition of mouse bone marrow. Human blasts were characterized by a sideward scatter (SSC) low/CD34⁺/CD45^dim/CD19⁻ phenotype, granulocytes by a SSChigh/CD33⁺/CD34⁻/CD19⁻, and B lymphocytes by an SSClow/CD34⁻/CD33⁻/CD19⁺ phenotype. The horizontal bar depicts mean TP53 variant allele frequencies (VAFs). (D) Highly purified peripheral blood CD45⁺/CD3⁺ cells were obtained at AML diagnosis, and the TP53 VAFs were assessed using ultradeep sequencing. Samples 7071 and 5273, respectively, exhibited 2 different somatic TP53 mutations. Note that in each case analyzed and scored positive, the TP53 VAF exceeded the minute impure fraction of sorted T lymphocytes, thereby excluding results biased because of contamination of AML cells. (E) Data obtained from colony-forming unit–granulocyte, monocyte (CFU-GM) colonies derived from specimen 5652 revealed the TP53 mutation as the initiating event (positive in 38 of 38 colonies), followed by an ASXL1 mutation (37 of 38), IDH2 mutation (20 of 38), and RUNX1 mutation (19 of 38). All cooperating mutations developed sequentially in the TP53-mutated clone. The exact mutation type is shown in Table 1. (F) Loss of heterozygosity at the TP53 locus of samples from UPN 7317. In CFU-GM colonies, a heterozygous TP53 c.681_681dupT mutation is shown, whereas in bulk leukemia cells, the wild-type allele was lost, resulting in a hemizygous state. (G) Quantitative assessment of the TP53 mutational load by the ultradeep sequencing, indicating comparable levels between diagnostic specimens and those obtained at relapsed or refractory (R/R) phase (P = .578 by the exact permutation test for related samples). B, blast cells; CR, complete remission; Dg, diagnosis; G, granulocytes; h, human; L, lymphocytes; m, mouse; SD, standard deviation.

This Feature Is Available To Subscribers Only