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, 518 (7540), 552-555

Role of TP53 Mutations in the Origin and Evolution of Therapy-Related Acute Myeloid Leukaemia

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Role of TP53 Mutations in the Origin and Evolution of Therapy-Related Acute Myeloid Leukaemia

Terrence N Wong et al. Nature.

Abstract

Therapy-related acute myeloid leukaemia (t-AML) and therapy-related myelodysplastic syndrome (t-MDS) are well-recognized complications of cytotoxic chemotherapy and/or radiotherapy. There are several features that distinguish t-AML from de novo AML, including a higher incidence of TP53 mutations, abnormalities of chromosomes 5 or 7, complex cytogenetics and a reduced response to chemotherapy. However, it is not clear how prior exposure to cytotoxic therapy influences leukaemogenesis. In particular, the mechanism by which TP53 mutations are selectively enriched in t-AML/t-MDS is unknown. Here, by sequencing the genomes of 22 patients with t-AML, we show that the total number of somatic single-nucleotide variants and the percentage of chemotherapy-related transversions are similar in t-AML and de novo AML, indicating that previous chemotherapy does not induce genome-wide DNA damage. We identified four cases of t-AML/t-MDS in which the exact TP53 mutation found at diagnosis was also present at low frequencies (0.003-0.7%) in mobilized blood leukocytes or bone marrow 3-6 years before the development of t-AML/t-MDS, including two cases in which the relevant TP53 mutation was detected before any chemotherapy. Moreover, functional TP53 mutations were identified in small populations of peripheral blood cells of healthy chemotherapy-naive elderly individuals. Finally, in mouse bone marrow chimaeras containing both wild-type and Tp53(+/-) haematopoietic stem/progenitor cells (HSPCs), the Tp53(+/-) HSPCs preferentially expanded after exposure to chemotherapy. These data suggest that cytotoxic therapy does not directly induce TP53 mutations. Rather, they support a model in which rare HSPCs carrying age-related TP53 mutations are resistant to chemotherapy and expand preferentially after treatment. The early acquisition of TP53 mutations in the founding HSPC clone probably contributes to the frequent cytogenetic abnormalities and poor responses to chemotherapy that are typical of patients with t-AML/t-MDS.

Figures

Extended Data Figure 1
Extended Data Figure 1. Whole genome sequencing analysis of tAML
a, Somatic copy number alterations in the 22 cases of t-AML. Blue indicates copy number loss. Red indicates copy number gain. b, Representative clonality plots for 8 cases of t-AML are shown. Scatter plots (lower panel) show variant allele frequency and read depth in the tumor sample. Variant alleles in the founding clone are depicted as green, while variants in subclones are depicted as orange or purple. The upper panels contain kernel density plots of the VAF data (green line) along with the posterior predictive densities (grey line) from the mathematical model used to segregate clusters. c, Frequency of Tier 1 silent, Tier 2, and Tier 3 mutations in 1 Mb increments across chromosome 17 in de novo AML and t-AML. The TP53 genomic locus is identified.
Extended Data Figure 2
Extended Data Figure 2. TP53 mutations are associated with decreased overall survival in t-AML/t-MDS
a, Overall survival in TP53 mutated (n=13) and TP53 wildtype (n=39) t-AML patients. b, Overall survival in TP53 mutated (n=24) and TP53 wildtype (n=35) t-MDS patients.
Extended Data Figure 3
Extended Data Figure 3. Model of how cytotoxic therapy shapes clonal evolution in t-AML/t-MDS
Age-related mutations in hematopoietic stem/progenitor cells (HSPCs) result in the production of a genetically heterogeneous population of HSPCs, including rare HSPCs with heterozygous TP53 mutations in some individuals. During chemotherapy and/or radiotherapy for the primary cancer, HSPC clones harboring a TP53 mutation have a selective growth advantage, resulting in expansion of that clone. Subsequent acquisition of additional driver mutations results in transformation to t-AML/t-MDS. Of note, the presence of TP53 mutations likely accounts for this high incidence of cytogenetic abnormalities in t-AML/t-MDS and poor response to chemotherapy.
Extended Data Figure 4
Extended Data Figure 4. Validation of the unique adaptor sequencing method
a, Unique adaptor sequencing approach. Step 1: Genomic DNA is amplified with TP53-specific primers (green) with subpopulation-specific variant alleles highlighted in red. Step 2: Randomly indexed adapters (tan and gray) are ligated to each amplicon. Step 3: The indexed amplicons are amplified to generate multiple reads possessing the same bar code (i.e., read families). Step 4: After sequencing, reads are aligned and grouped by read families to generate an error-corrected consensus sequence. Sequencing errors (yellow) are randomly distributed amongst read families, while true variant alleles (red) are present in all members of a given read family. b, A tumor sample (UPN 895681) with a known TP53 somatic mutation (chromosome 17: 7519119 T to A) at a variant allele frequency (VAF) of ~37% was mixed with normal genomic DNA sample at the indicated ratio, and conventional (left panel) or unique adaptor next generation sequencing (middle and right panels) was performed, as described in Methods. DNA degradation with time may result in errors which are then amplified during PCR, providing a source of false positive calls. This is particularly true for C to A transversions. Since none of the TP53 mutations analyzed in this study were C to A transversions, we also analyzed the data after removing C to A calls (right panel). The TP53 variant allele is circled in blue. c, The threshold of detection for the variant allele with each sequencing method is shown.
Extended Data Figure 5
Extended Data Figure 5. Clonal evolution in case 314666
a, Clinical course of case 341666. b, Unique adaptor sequencing was performed on genomic DNA derived from a leukapharesis samples obtained 3 years prior to the diagnosis of t-MDS for the two clonal mutations present in the diagnostic t-AML sample. Genomic DNA from a patient lacking these variants was used as a control. The blue circle indicates the position of the variant SNV. c, Proposed model of clonal evolution to t-MDS in this case.
Extended Data Figure 6
Extended Data Figure 6. Droplet digital PCR verification of selected somatic TP53 mutations identified in peripheral blood of cancer-free individuals
Droplet digital PCR was performed on genomic DNA isolated from the peripheral blood of cancer-free individuals (middle panel) for whom unique-read adaptor sequencing suggested the presence of the indicated TP53 mutation. Controls represent peripheral blood DNA from cancer-free elderly individuals with variant allele frequencies not above background levels for the mutation of interest (right panel); the negative control for Y220C TP53 is shown in Fig. 3b. The diagnostic t-AML sample from patient 967645 was used as a positive control for Y220C TP53 (a). For V173M TP53 (b) and I195T TP53 (c) double-stranded genomic blocks (gBlocks) were synthesized containing the mutation of interest and mixed with gBlocks of wild-type sequence. Droplets containing only the variant TP53 allele are highlighted in orange, droplets containing the wild type TP53 allele (with or without the variant TP53 allele) are highlighted in blue; empty droplets are gray. The number of droplets in each gate is indicated.
Figure 1
Figure 1. The mutational burden in t-AML is similar to de novo AML
a, Total number of validated tier 1–3 somatic SNVs in t-AML (n=22), de novo AML (n=49), and s-AML (n=8). The mean ages of the t-AML, de novo AML, and s-AML cohorts were 55.7, 51, and 54.6 years respectively. b, Number of validated tier 1 somatic SNVs. c, Number of validated tier 1 small insertions/deletions (indels). d, Percentage of tier 1–3 somatic SNVs that are transversions. e, Mutational spectrum for all validated tier 1–3 somatic SNVs. f, Number of distinct clones per sample inferred from the identification of discrete clusters of mutations with distinct variant allele frequencies. g, Percentage of cases of t-AML (n=52) or de novo AML (n=199) harboring non-synonymous mutations of the indicated gene. h, Percentage of cases of t-MDS (n=59) or de novo MDS (n=150) harboring non-synonymous mutations of the indicated gene. ABC Fm: ABC family genes; NA: not available; s-AML: AML following MDS. +P<0.05 by one-way Anova. *P<0.05 by Fisher's Exact test. Data represent the mean ± SD.
Figure 2
Figure 2. Bi-allelic TP53 mutations are an early mutational event in the AML cells of UPN 530447
a, Clinical course of case 530447. b, Unique adaptor sequencing of a leukapheresis sample obtained 6 years prior to the diagnosis of t-AML for each of the five clonal somatic SNVs identified in the diagnostic t-AML sample. Genomic DNA from a patient lacking these variants served as a control. The blue circle indicates the position of the variant SNV. c, Proposed model of clonal evolution to t-AML in this case.
Figure 3
Figure 3. HSPC clones harboring somatic TP53 mutations are detected in patients prior to cytotoxic therapy exposure
a, Clinical course of case 967645. b, Dot plots of droplet digital PCR of a diagnostic t-AML sample from case 967645, a bone marrow sample from this patient obtained 5 years prior to the development of t-AML (prior to any cytotoxic therapy), or a control sample from a patient lacking a mutation in TP53. Droplets containing only the Y220C TP53 allele are highlighted in orange, droplets containing wild type TP53 (with or without Y220C TP53) are highlighted in blue; empty droplets are gray. The number of droplets in each gate is indicated. Data are representative of two independent experiments. c, Dot plots of droplet digital PCR data for G155S SNAP25 using the same genomic DNA as in b. d, Proposed model of clonal evolution to t-AML in case 967645. e, Clinical course of case 895681. f, Dot plots of droplet digital PCR data of the diagnostic t-AML sample from case 895681, a bone marrow FFPE sample from this patient obtained 3.5 years prior to the development of t-MDS (prior to any cytotoxic therapy), or a control FFPE sample obtained from a patient lacking a mutation in TP53. The labeling scheme is the same as in b. g, Proposed model of clonal evolution to t-AML in case 895681; the diagnostic t-MDS sample contained a subclonal ETV6 mutation.
Figure 4
Figure 4. Heterozygous loss of TP53 confers a clonal advantage to HSCs after exposure to ENU
a, Experimental schema. Bone marrow chimeras were generated by transplanting a seven to one ratio of wild type to Tp53+/− bone marrow into irradiated syngenic recipients. Following hematopoietic reconstitution (5 weeks) mice were treated with ENU or vehicle control as indicated. b–d, Shown is the percentage of total leukocytes (b), Gr-1+ neutrophils (c), or B220+ B cells (d) that were derived from Tp53+/− cells. e, Percentage of Kit+ lineage Sca+ (KSL) cells in the bone marrow 12 weeks after ENU exposure that were derived from Tp53+/− cells. Data represent the mean ± SEM of 11 and 14 mice in the ENU and vehicle cohorts respectively. Peripheral chimerism was analyzed using 2-way Anova and KLS chimerism with ANCOVA.

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