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

Wong TN, Ramsingh G, Young AL, Miller CA, Touma W, Welch JS, Lamprecht TL, Shen D, Hundal J, Fulton RS, Heath S, Baty JD, Klco JM, Ding L, Mardis ER, Westervelt P, DiPersio JF, Walter MJ, Graubert TA, Ley TJ, Druley TE, Link DC, Wilson RK - Nature (2014)

Bottom Line: 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.

View Article: PubMed Central - PubMed

Affiliation: Department of Medicine, Division of Oncology, Washington University, St Louis, Missouri 63110, USA.

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.

Show MeSH

Related in: MedlinePlus

Droplet digital PCR verification of selected somatic TP53 mutations identified in peripheral blood of cancer-free individualsDroplet 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.
© Copyright Policy - permissions-link
Related In: Results  -  Collection

License
getmorefigures.php?uid=PMC4403236&req=5

Figure 10: Droplet digital PCR verification of selected somatic TP53 mutations identified in peripheral blood of cancer-free individualsDroplet 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.

Mentions: To determine whether HSPCs harboring TP53 mutations are present in healthy individuals, we analyzed peripheral blood leukocytes from 20 elderly (68—89 years old) cancer-free donors who had not received prior cytotoxic therapy. We limited our sequencing to exons 4–8 of TP53 since the majority of pathogenic mutations in TP53 are located in these exons. Using our unique adaptor sequencing assay, we identified TP53 mutations in 9 of 19 evaluable cases, with VAFs ranging from 0.01% to 0.37% (Extended Table 4). Of note, since we did not sequence the entire coding region of TP53, it is likely that our study underestimates the true frequency of healthy elderly individuals harboring HSPCs with TP53 mutations. Droplet digital PCR confirmed the presence of the TP53 mutation in all three cases that were tested (Extended Fig. 6). Interestingly, the majority of the TP53 mutations identified are known pathogenic mutations previously implicated in cancer. These data suggest that functional TP53 mutations may confer (even in the absence of cytotoxic therapy) a subtle competitive advantage that results in modest HSPC expansion over time.


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

Wong TN, Ramsingh G, Young AL, Miller CA, Touma W, Welch JS, Lamprecht TL, Shen D, Hundal J, Fulton RS, Heath S, Baty JD, Klco JM, Ding L, Mardis ER, Westervelt P, DiPersio JF, Walter MJ, Graubert TA, Ley TJ, Druley TE, Link DC, Wilson RK - Nature (2014)

Droplet digital PCR verification of selected somatic TP53 mutations identified in peripheral blood of cancer-free individualsDroplet 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.
© Copyright Policy - permissions-link
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4403236&req=5

Figure 10: Droplet digital PCR verification of selected somatic TP53 mutations identified in peripheral blood of cancer-free individualsDroplet 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.
Mentions: To determine whether HSPCs harboring TP53 mutations are present in healthy individuals, we analyzed peripheral blood leukocytes from 20 elderly (68—89 years old) cancer-free donors who had not received prior cytotoxic therapy. We limited our sequencing to exons 4–8 of TP53 since the majority of pathogenic mutations in TP53 are located in these exons. Using our unique adaptor sequencing assay, we identified TP53 mutations in 9 of 19 evaluable cases, with VAFs ranging from 0.01% to 0.37% (Extended Table 4). Of note, since we did not sequence the entire coding region of TP53, it is likely that our study underestimates the true frequency of healthy elderly individuals harboring HSPCs with TP53 mutations. Droplet digital PCR confirmed the presence of the TP53 mutation in all three cases that were tested (Extended Fig. 6). Interestingly, the majority of the TP53 mutations identified are known pathogenic mutations previously implicated in cancer. These data suggest that functional TP53 mutations may confer (even in the absence of cytotoxic therapy) a subtle competitive advantage that results in modest HSPC expansion over time.

Bottom Line: 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.

View Article: PubMed Central - PubMed

Affiliation: Department of Medicine, Division of Oncology, Washington University, St Louis, Missouri 63110, USA.

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.

Show MeSH
Related in: MedlinePlus