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

In patient 530447, two potential driver mutations (TET2 and NUP98) were not detectable in the prior banked sample.

“However, two potential driver mutations (TET2 and NUP98) were not detectable in the prior banked sample.”

In patient 967645, del 5 and del 7 were subclonal at diagnosis (present in 54% and 38% of metaphases, respectively).

“Of note, del 5 and del 7 were subclonal at diagnosis (present in 54% and 38% of metaphases, respectively) (Suppl. Table 1).”

TP53 mutations were identified in 9 of 19 evaluable healthy elderly cancer-free donors who had not received prior cytotoxic therapy, with VAFs ranging from 0.01% to 0.37%.

“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).”

The number of identifiable subclones in t-AML is similar to that observed in de novo AML.

“Moreover, the number of identifiable subclones in t-AML was similar to that observed in de novo AML (Fig. 1f and Extended Data Fig. 1B).”

Mutations of ABC transporter genes are enriched in t-AML versus de novo AML.

“Interestingly, mutations of ABC transporter genes, a subset of which have been implicated in chemotherapy resistance, also are enriched in t-AML versus de novo AML.”

In patient 967645, the G155S SNAP25 mutation was identified in the prior banked bone marrow sample with a similar VAF (0.0029%) as that for Y220C TP53.

“Indeed, we identified the G155S SNAP25 mutation in the prior banked bone marrow sample with a similar VAF (0.0029%) as that for Y220C TP53 (Fig 3c).”

TP53 mutations are significantly enriched in t-AML/t-MDS compared with de novo AML/MDS.

“As reported previously, TP53 mutations are significantly enriched in t-AML/t-MDS compared with de novo AML/MDS (Fig. 1g–h and Suppl. Table 5).”

In patient 967645, the same Y220C TP53 mutation was identified in a bone marrow sample obtained before any chemotherapy at a frequency of 0.0027%.

“Using a droplet digital PCR (ddPCR) assay, we identified the same Y220C TP53 mutation in a bone marrow sample obtained before any chemotherapy at a frequency of 0.0027% (average of two independent experiments) (Fig 3b).”

Two other cases of t-AML/t-MDS had clonal TP53 mutations but subclonal del 5 and/or del 7.

“Of note, we found two other cases of t-AML/t-MDS with clonal TP53 mutations but subclonal del 5 and/or del 7 (UPNs 756582 and 837334, Suppl. Table 1).”

In patient 895681, TP53 H179L was identified at a VAF of 0.05% in a bone marrow sample taken prior to the initiation of cytotoxic therapy.

“Using ddPCR, we identified TP53 H179L at a VAF of 0.05% in a bone marrow sample taken prior to the initiation of cytotoxic therapy (Fig 3f).”

The specific TP53 mutation present in the diagnostic t-AML/t-MDS sample was identified in prior-banked specimens in four out of the seven cases tested.

“The specific TP53 mutation present in the diagnostic t-AML/t-MDS sample was identified in prior-banked specimens in four out of the seven cases tested”

Structural variants and somatic copy number alterations were uncommon in these t-AML cases.

“Structural variants and somatic copy number alterations were uncommon in these t-AML cases (Suppl. Table 2 and Extended Data Fig. 1A).”

In patient 530447, both TP53 mutant alleles were present with a VAF of approximately 0.5% in a leukapheresis sample obtained 6 years prior to the development of t-AML.

“Analysis of a leukapheresis sample obtained 6 years prior to the development of t-AML revealed that both TP53 mutant alleles were present with a variant allele fraction (VAF) of approximately 0.5% (Fig. 2b).”

The percentage of transversions, and of all six classes of SNVs, is similar in t-AML, de novo AML, and s-AML.

“the percentage of transversions, and in fact of all six classes of SNVs, was similar in all three cohorts (Fig. 1d–e).”

The mutation burden in the genomic region containing TP53 is similar between t-AML and de novo AML.

“The mutation burden in the genomic region containing TP53 (including silent Tier 1, Tier2, and Tier 3 mutations) is similar between t-AML and de novo AML (Extended Fig. 1C).”

The total number of validated somatic single nucleotide variants (SNVs) and genic (tier 1) somatic SNVs is similar in t-AML, de novo AML, and s-AML.

“the total number of validated somatic single nucleotide variants (SNVs) and genic (tier 1) somatic SNVs identified was similar to that for de novo AML and s-AML (Fig. 1a–b).”

Several well-defined driver gene mutations (i.e., DNMT3A and NPM1) were significantly less common in t-AML.

“On the other hand, several well-defined driver gene mutations (i.e., DNMT3A and NPM1) were significantly less common in t-AML.”

In all cases, the TP53 mutation was clonal in the t-AML/t-MDS diagnostic sample.

“Of note, in all the cases, the TP53 mutation was clonal in the t-AML/t-MDS diagnostic sample.”

The number of small insertions or deletions (indels) in genic regions is similar in t-AML, de novo AML, and s-AML.

“Likewise, the number of small insertions or deletions (indels) in genic regions was similar in t-AML, de novo AML, and s-AML (Fig. 1c).”

In two of the four cases, the prior banked sample was obtained prior to the initiation of chemotherapy.

“In two of the four cases, the prior banked sample was obtained prior to the initiation of chemotherapy.”

The majority of the TP53 mutations identified in healthy elderly are known pathogenic mutations previously implicated in cancer.

“Interestingly, the majority of the TP53 mutations identified are known pathogenic mutations previously implicated in cancer.”

All patients in Extended Data Table 3 had one or more clonal TP53 mutation in their diagnostic t-AML/t-MDS samples.

“All patients had one or more clonal TP53 mutation in their diagnostic t-AML/t-MDS samples (530447 had biallelic mutations).”

TP53 mutations are associated with decreased overall survival in t-AML/t-MDS.

“TP53 mutations are associated with decreased overall survival in t-AML/t-MDS”

Upon treatment with N-ethyl-N-nitrosourea, Tp53+/− HSPCs display a competitive advantage.

“Regardless, upon treatment with N-ethyl-N-nitrosourea, Tp53+/− HSPCs display a competitive advantage.”

Somatic TP53 mutations were present in 19 cancer-free individuals.

“Somatic TP53 mutations in 19 cancer-free individuals.”

In mice treated with vehicle control, there was a non-significant trend towards increased Tp53+/− donor contribution to hematopoiesis.

“In mice treated with vehicle control, we observed a non-significant trend towards increased Tp53+/− donor contribution to hematopoiesis (Fig 4b–e).”

A HSPC carrying a functional TP53 mutation was present prior to cytotoxic therapy exposure, later giving rise to the malignant t-AML/t-MDS clone.

“Thus, as with patient 967645, a HSPC carrying a functional TP53 mutation was present prior to cytotoxic therapy exposure, later giving rise to the malignant t-AML/t-MDS clone (Fig 3g).”

• AssumptionRests on an assumption: TP53 mutations detected in peripheral blood leukocytes or bone marrow samples reflect the presence of the same mutations in hematopoietic stem/progenitor cells (HSPCs).

Although the total mutation burden is similar, a distinct subset of mutated genes is present in t-AML/t-MDS.

“Thus, although the total mutation burden is similar, a distinct subset of mutated genes is present in t-AML/t-MDS.”

Loss of TP53 provides a selective advantage for neoplastic growth.

“loss of TP53 provides a selective advantage for neoplastic growth18.”

• cites [18] Hollstein, M., Sidransky, D., Vogelstein, B. & Harris, C. C. p53 mutations in human cancers. Science 253, 49–53 (1991) sourceNot checked yet

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The vast majority of TP53 mutations in t-AML/t-MDS have previously been identified as pathogenic.

“the vast majority of these mutations have previously been identified as pathogenic10.”

• cites [10] Petitjean, A. et al. Impact of mutant p53 functional properties on TP53 mutation patterns and tumor phenotype: lessons from recent developments in the IARC TP53 database. Hum. Mutat. 28, 622–629 (2007) sourceNot checked yet

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Cytotoxic therapy does not directly induce TP53 mutations.

“These data suggest that cytotoxic therapy does not directly induce TP53 mutations.”

• Also stated in: § The Role of TP53 Mutations in the Origin and Evolution of Therapy-Related AML · hedged

  “Thus, it is not likely that chemotherapy directly induces TP53 mutations.”

These observations suggest a central role for TP53 mutations in the pathogenesis of many cases of t-AML/t-MDS.

“These observations suggest a central role for TP53 mutations in the pathogenesis of many cases of t-AML/t-MDS.”

TP53 mutations precede the development of the characteristic cytogenetic abnormalities of t-AML/t-MDS.

“Together, these data suggest that TP53 mutations precede the development of these characteristic cytogenetic abnormalities of t-AML/t-MDS.”

Prior chemotherapy does not induce genome-wide DNA damage.

“indicating that prior chemotherapy does not induce genome-wide DNA damage.”

There are approximately 10,000 HSCs in humans.

“current estimates that there are approximately 10,000 HSCs in humans12”

• cites [12] Catlin, S. N., Busque, L., Gale, R. E., Guttorp, P. & Abkowitz, J. L. The replication rate of human hematopoietic stem cells in vivo. Blood 117, 4460–4466 (2011) sourceNot checked yet

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The mutation burden of t-AML genomes is similar to that of de novo AML genomes.

“Collectively, these data show that the mutation burden of t-AML genomes is similar to that of de novo AML genomes.”

Since the study did not sequence the entire coding region of TP53, it likely underestimates the true frequency of healthy elderly individuals harboring HSPCs with TP53 mutations.

“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.”

44% of healthy individuals at 50 years of age may have at least one HSPC that carries a randomly generated, functional TP53 mutation.

“we predict that 44% of healthy individuals at 50 years of age may have at least one HSPC that carries a randomly generated, functional TP53 mutation (see extended Methods).”

• AssumptionRests on an assumption: The calculation that 44% of healthy individuals at age 50 have at least one HSPC with a functional TP53 mutation assumes that age-related TP53 mutations are randomly distributed among HSPCs and that the average mutation rate and HSC number are accurate.

The unique adaptor sequencing assay can detect a variant allele with a frequency of 0.009%.

“Using tumor DNA with a known TP53 mutation, we show that this assay can detect a variant allele with a frequency of 0.009% (Extended Fig. 4b–c).”

Individual HSPCs accumulate somatic mutations as a function of age, such that by age 50, there are on average 5 coding gene mutations per HSPC.

“We recently reported that individual HSPCs accumulate somatic mutations as a function of age, such that by age 50, there are on average 5 coding gene mutations per HSPC11.”

• cites [11] Welch, J. S. et al. The origin and evolution of mutations in acute myeloid leukemia. Cell 150, 264–278 (2012) sourceNot checked yet

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Current next-generation sequencing (NGS) technology is limited in the detection of rare variant alleles due to an intrinsic sequencing error rate of ~0.1%.

“Current next-generation sequencing (NGS) technology is limited in the detection of rare variant alleles due to an intrinsic sequencing error rate of ~ 0.1%19.”

• cites [19] Kandoth, C. et al. Mutational landscape and significance across 12 major cancer types. Nature 502, 333–339 (2013) sourceNot checked yet

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An average 50 year old has 1.6 × 10−7 mutations/position.

“Thus, we would predict an average 50 year old to have 1.6 × 10−7 mutations/position.”

• AssumptionRests on an assumption: The somatic mutational burden is aging-related.
• AssumptionRests on an assumption: The estimated rate of mutagenesis is 3.2 × 10−9 mutations/nucleotide/year (95% CI 2.4–4.0 × 10−9) for the average nucleotide in the exome.

Additional mutations, including mutation of the second TP53 allele, are needed for transformation to AML or MDS.

“Clearly, additional mutations, including mutation of the second TP53 allele, are needed for transformation to AML or MDS.”

There is increasing evidence that cancers undergo clonal evolution under the selective pressure of chemotherapy.

“There is increasing evidence that cancers undergo clonal evolution under the selective pressure of chemotherapy 22.”

• cites [22] Greaves, M. & Maley, C. C. Clonal evolution in cancer. Nature 481, 306–313 (2012) sourceNot checked yet

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Functional TP53 mutations may confer (even in the absence of cytotoxic therapy) a subtle competitive advantage that results in modest HSPC expansion over time.

“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.”

• AssumptionRests on an assumption: TP53 mutations detected in peripheral blood leukocytes or bone marrow samples reflect the presence of the same mutations in hematopoietic stem/progenitor cells (HSPCs).

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.

“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.”

DNA degradation with time (guanosine oxidation, cytosine deamination) interferes with rare allele detection.

“DNA degradation with time (i.e. guanosine oxidation, cytosine deamination) is known to interfere with rare allele detection34”

• cites [34] Schmitt, M. W. et al. Detection of ultra-rare mutations by next-generation sequencing. Proc. Natl Acad. Sci. USA 109, 14508–14513 (2012) sourceNot checked yet

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VAFs calculated by droplet digital PCR were highly concordant with VAFs obtained through unique-adaptor NGS.

“VAFs calculated in this method were highly concordant with VAFs obtained through unique-adaptor NGS.”

44% of 50 year-old individuals harbor one or more HSCs with a functional TP53 mutation.

“Based on this simulation, we predict that 44% of 50 year-old individuals harbor one or more HSCs with a functional TP53 mutation.”

• AssumptionRests on an assumption: The somatic mutational burden is aging-related.
• AssumptionRests on an assumption: The estimated rate of mutagenesis is 3.2 × 10−9 mutations/nucleotide/year (95% CI 2.4–4.0 × 10−9) for the average nucleotide in the exome.
• AssumptionRests on an assumption: Somatic mutations are not randomly distributed but biased towards C to T/G to A transitions.
• AssumptionRests on an assumption: An individual possesses approximately 10,000 distinct HSCs.

Only a minority of patients with Li Fraumeni syndrome, most of whom harbor germline heterozygous TP53 mutations, develop AML or MDS.

“Consistent with this observation, only a minority of patients with Li Fraumeni syndrome, most of whom harbor germline heterozygous TP53 mutations, develop AML or MDS 23.”

• cites [23] Birch, J. M. et al. Relative frequency and morphology of cancers in carriers of germline TP53 mutations. Oncogene 20, 4621–4628 (2001) sourceNot checked yet

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This model provides a potential mechanism for the high incidence of TP53 mutations in t-AML/t-MDS.

“This model provides a potential mechanism for the high incidence of TP53 mutations in t-AML/t-MDS24.”

• cites [24] Casás-Selves, M. & Degregori, J. How cancer shapes evolution, and how evolution shapes cancer. Evolution 4, 624–634 (2011)Not checked yet

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HSPCs that acquire heterozygous TP53 mutations as a function of normal aging are subject to Darwinian selection upon exposure to cytotoxic therapy, ultimately resulting in the expansion of HSPCs with these mutations.

“Here, we show that HSPCs that acquire heterozygous TP53 mutations as a function of normal aging also are subject to Darwinian selection upon exposure to cytotoxic therapy, ultimately resulting in the expansion of HSPCs with these mutations.”

• AssumptionRests on an assumption: TP53 mutations detected in peripheral blood leukocytes or bone marrow samples reflect the presence of the same mutations in hematopoietic stem/progenitor cells (HSPCs).
• AssumptionRests on an assumption: The murine Tp53+/− bone marrow chimera model is representative of human t-AML/t-MDS and human HSPC response to chemotherapy.

Age-related mutations in hematopoietic stem/progenitor cells (HSPCs) result in a genetically heterogeneous population of HSPCs, including rare HSPCs with heterozygous TP53 mutations in some individuals.

“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.”

Subsequent acquisition of additional driver mutations results in transformation to t-AML/t-MDS.

“Subsequent acquisition of additional driver mutations results in transformation to t-AML/t-MDS.”

The presence of TP53 mutations likely accounts for the high incidence of cytogenetic abnormalities in t-AML/t-MDS and poor response to chemotherapy.

“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.”

Droplet digital PCR confirmed the presence of the TP53 mutation in all three cases that were tested.

“Droplet digital PCR confirmed the presence of the TP53 mutation in all three cases that were tested (Extended Fig. 6).”

The high frequency (nearly 50%) of elderly individuals with detectable heterozygous TP53 mutations in their circulating leukocytes far exceeds the prevalence of AML or MDS in this age group.

“The high frequency (nearly 50%) of elderly individuals with detectable heterozygous TP53 mutations in their circulating leukocytes far exceeds the prevalence of AML or MDS in this age group.”

Identifying only variant alleles present in droplets also lacking the reference allele greatly increased the specificity of variant calls.

“This greatly increased the specificity of our calls by removing droplets in which one of the two DNA strands may have been chemically altered.”

The TP53 mutation in the founding clone likely contributes to the frequent cytogenetic abnormalities and poor response to chemotherapy that are typical of t-AML/t-MDS.

“The TP53 mutation in the founding clone likely contributes to the frequent cytogenetic abnormalities and poor response to chemotherapy that are typical of t-AML/t-MDS.”