Role of TP53 mutations in the origin and evolution of therapy-related acute myeloid leukaemia

Abstract

Therapy-related acute myeloid leukaemia (t-AML) and therapy-related myelodysplastic syndrome (t-MDS) are well-recognized complications of cytotoxic chemotherapy and/or radiotherapy1. There are several features that distinguish t-AML from de novo AML, including a higher incidence of TP53 mutations2,3, abnormalities of chromosomes 5 or 7, complex cytogenetics and a reduced response to chemotherapy4. 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.

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Figure 1: The mutational burden in t-AML is similar to de novo AML.
Figure 2: Biallelic TP53 mutations are early mutational events in the AML cells of UPN 530447.
Figure 3: HSPC clones harbouring somatic TP53 mutations are detected in patients before cytotoxic therapy exposure.
Figure 4: Heterozygous loss of TP53 confers a clonal advantage to HSCs after exposure to ENU.

Accession codes

Data deposits

Sequence information on the 22 t-AML whole-genome sequencing patients and one exome sequencing patient (UPN 967645) has been deposited in dbGaP under accession number phs000159.

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Acknowledgements

We thank A. Schmidt, B. McKethan and R. Miller for technical assistance, and K. Odell and J. Tucker-Davis for animal care. We thank P. Goodfellow and J. Ivanovich for providing peripheral blood leukocyte genomic DNA from cancer-free individuals. This work was supported by National Institutes of Health grants PO1 CA101937 (D.C.L.) and U54 HG003079 (R.K.W.) and by a grant from the Leukemia & Lymphoma Society (D.C.L.).

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Authors

Contributions

T.N.W. and G.R. designed and performed the research, analysed the data, and wrote the manuscript. A.L.Y. and T.E.D. developed and optimized the amplicon-based random primer sequencing assay. C.A.M., D.S., J.H., R.S.F., L.D., E.R.M. and R.K.W. contributed to the generation and analysis of the whole-genome or targeted sequencing. W.T., T.L.L., S.H., J.M.K. and P.W. collected and processed clinical data and tissue samples. J.D.B. performed statistical analyses of the clinical data. J.S.W., J.F.D., M.J.W., T.A.G. and T.J.L. contributed to data analysis. D.C.L. supervised all of the research and edited the manuscript, which was approved by all co-authors.

Corresponding author

Correspondence to Daniel C. Link.

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The authors declare no competing financial interests.

Extended data figures and tables

Extended Data Figure 1 Whole-genome sequencing analysis of t-AML.

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 (bottom) show variant allele frequency and read depth in the tumour sample. Variant alleles in the founding clone are depicted in green, while variants in subclones are depicted in orange or purple. Top, 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 TP53 mutations are associated with decreased overall survival in t-AML/t-MDS.

a, Overall survival in TP53 mutated (n = 13) and TP53 wild-type (n = 39) t-AML patients. b, Overall survival in TP53 mutated (n = 24) and TP53 wild-type (n = 35) t-MDS patients.

Extended Data Figure 3 Model of how cytotoxic therapy shapes clonal evolution in t-AML/t-MDS.

Age-related mutations in 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 harbouring a TP53 mutation have a competitive 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 probably accounts for the high incidence of cytogenetic abnormalities in t-AML/t-MDS and poor response to chemotherapy.

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 grey) are ligated to each amplicon. Step 3: the indexed amplicons are amplified to generate multiple reads possessing the same barcode (that is, 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 tumour sample (UPN 895681) with a known TP53 somatic mutation (chromosome 17: 7519119 T to A) at a VAF of 37% was mixed with normal genomic DNA sample at the indicated ratio, and conventional (left) or unique adaptor next-generation sequencing (middle and right) was performed, as described in Methods. DNA degradation with time may result in errors that 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 analysed in this study were C to A transversions, we also analysed the data after removing C to A calls (right). 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 Clonal evolution in case 314666.

a, Clinical course of case 341666. Chemo, chemotherapy; DLBCL, diffuse large B-cell lymphoma; XRT, radiotherapy. b, Unique adaptor sequencing was performed on genomic DNA derived from leukapharesis samples obtained 3 years before the diagnosis of t-MDS for the two clonal mutations present in the diagnostic t-MDS 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 ddPCR verification of selected somatic TP53 mutations identified in peripheral blood of cancer-free individuals.

ac, ddPCR was performed on genomic DNA isolated from the peripheral blood of cancer-free individuals (middle) 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 VAFs not above background levels for the mutation of interest (right); the negative control for TP53 Y220C is shown in Fig. 3b. a, The diagnostic t-AML sample from patient 967645 was used as a positive control for TP53 Y220C. b, c, For TP53 V173M (b) and TP53 I195T (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 grey. The number of droplets in each gate is indicated.

Extended Data Table 1 Clinical summary of the 22 t-AML whole-genome sequencing cases
Extended Data Table 2 Clinical summary of the combined 111 t-AML/t-MDS cases
Extended Data Table 3 Previously banked tissue samples in patients with t-AML/t-MDS with clonal TP53 mutations
Extended Data Table 4 Somatic TP53 mutations in 19 cancer-free individuals

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Wong, T., Ramsingh, G., Young, A. et al. Role of TP53 mutations in the origin and evolution of therapy-related acute myeloid leukaemia. Nature 518, 552–555 (2015). https://doi.org/10.1038/nature13968

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