TP53 mutations are highly enriched in therapy-related acute myeloid leukemia (t-AML) compared to de novo AML. The study sequenced genomes from 22 t-AML cases and found a similar mutation burden but a higher frequency of TP53 mutations compared to de novo AML. Further sequencing of 149 genes in 89 additional t-AML/t-MDS cases confirmed TP53 as the most commonly mutated gene. Some t-AML patients were found to have TP53 mutations in samples collected before cytotoxic therapy, suggesting these pre-existing clones are selected for by therapy. Experiments in mouse models also showed HSPCs with TP53 mutations have a competitive advantage after chemotherapy.

1. Role of TP53 mutations in the origin and
evolution of therapy-related
acute myeloid leukaemia(t-AML)
T N. Wong, Richard K. Wilson et al
Department of Medicine, Division of Oncology,
Washington University, St Louis
Siteman Cancer Center, Washington University, St Louis
Nature, December 2015
1
Mohsin
Graduate student AIIMS

2. Introduction
Therapy-related
acute myeloid leukaemia(t-AML)
• Therapy-related acute myeloid leukaemia (t-AML) and therapy-
related myelodysplastic syndrome(t-MDS) are complications of
cytotoxic chemotherapy and/or radiotherapy (1-5 years)
• WHO Classification of Tumours of Haematopoietic and Lymphoid-
Tissues - therapy-related myeloid neoplasms (t-MN)
• Several features that distinguish t-AML from de novo AML
• abnormalities of chromosomes 5 or 7
• complex cytogenetics and a reduced response to chemotherapy
• Higher incidence of TP53 mutations
2

3. • T-MN 10 to 20 percent of all cases of AML, MDS, and MDS/MPN
• Median age 61, incidence varies (cytotoxic agents(alkylating
agents/Topoisomerase II Inhibitors), specific agents, timing of
exposure, and dose )
• Some patients may have a heritable predisposition to the
development of t-MN
• clinical presentation of t-MN is variable but most patients have
symptoms similar to patients with de novo AML or MDS
• Pancytopenia and symptoms related to this
3

4. Methods
• Patient characteristics: 22 selected cases of t-AML to have
minimal numbers of cytogenetic abnormalities
• additional 89 cases of t-AML/t-MDS were randomly selected
fromthose samples with sufficient tumour and skin DNA
4

5. Whole-genome sequencing and variant detection
• Illumina DNAsequencing was used to generate sequence
• Validation and extension sequencing with variant
detection,custom sequence capture arrays from Roche
Nimblegen that targeted variants detected by whole-genome
sequencing
• Detection of somatic TP53 mutations in cancer-free subjects.
Amplicons were prepared from healthy control genomic DNA
samples using primers designed to amplify exons 4–8 of TP53
• Generation and analysis ofTp53 +/- bone marrow chimaeras.
Tp53 +/- ,Mice type mice were inbred on a C57BL/6 strain.
5

6. • Flowcytometry. Flow cytometry data were collected on a
Gallios 10-colour, 3-laser flow cytometer (Beckman Coulter)
• Cells were stained by standard protocols with the following
antibodies (eBiosciences)
• Ly5.1 (A20, CD45.1), Ly5.2 (104, CD45.2), Ly6C/G (RB6-
• 8C5, Gr-1), CD3e (145-2C11), CD45R(RA3-6B2, B220), CD11c
(N418), TER-119, CD117 (ACK2, c-Kit) and Ly-6A/E (D7, Sca).
6

7. Aim
• To understand better how prior cytotoxic
therapy contributes to the high incidence of
TP53 mutations and karyotypic abnormalities
in t-AML/t-MDS
7

8. RESULTS
(To know the mutational spectrum of t-AML)
• Genomes of 22 cases of t-AML were sequenced
• compared to whole-genome sequence data previously
reported for de novo AML And secondary AML(s-AML) arising
from MDSfor which patients did not receive chemotherapy
except hydroxyurea
• The Cancer Genome Atlas Research Network. Genomic and epigenomic landscapes of adult
de novo acute myeloid leukemia. N. Engl. J. Med. 368,2059–2074 (2013)
• Walter, M. J. et al. Clonal architecture of secondary acute myeloid leukemia. N. Engl. J.
Med. 366, 1090–1098 (2012)
• Walter, M. J. et al. Clonal diversity of recurrently mutated genes in myelodysplastic
syndromes. Leukemia 27, 1275–1282 (2013)
• 23% had rearrangements of MLL (also known as KMT2A),
23% had complex cytogenetics, and 36% had normal
cytogenetics 8

9. • 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 (a & b)
• Number of small insertions or deletions (indels) in genic regions
was similar in t-AML, de novo AMLand s-AML (c)(c)
9SNVs, InDels in t-AML and de novo AML
The mutational burden in t-AML is similar to de novo AML

10. • The percentage of transversions (where a purine is substituted for a
pyrimidine, A->C vice versa), and in fact of all six classes of SNVs,
was similar in all three cohorts
• these data show that the mutation burden of t-AML genomes
is similar to that of de novo AML genomes.
10Transversions

11. RESULTS
whether the pattern of genes frequently mutated in
t-AML/t-MDS is distinct from that observed in de novo AML/MDS
• To define better pattern and frequency of specific mutations in
t-AML/t-MDS, we sequenced a panel of 149 AML/MDS-related
genes in an additional 89 patients with t-AML or t-MDS
• TP53 mutations are significantly enriched in t-AML/t-MDS
compared with de novo AML/MDS
• A distinct subset of mutated genes is present in t-AML/t-
MDS
11

12. • TP53 is the most commonly mutated gene in t-AML/t-MDS,
with 33.3% of patients affected in our series
• Multivariate analysis revealed that TP53 mutations were
associated with poor risk cytogenetics and a worse prognosis-
hallmarks of t-AML/t-MDS
12
• suggest a central role for TP53 mutations in the pathogenesis of many cases of
t-AML/t-MDS
• mechanism by which TP53 mutations are selectively enriched in t-AML/t-MDS
is unclear

13. • Individual HSPCs accumulate somatic mutations as a function
of age, such that by age 50, there are on average five coding
gene mutations per HSPC
• these observations suggest a model in which rare HSPCs
carrying age-related TP53 mutations are resistant to
chemotherapy and expand preferentially after treatment
13

14. • t-AML containing clonal TP53 mutations, HSPCs harbouring
the specific TP53 mutation will be present long before the
development of overt t-AML??
• seven cases of t-AML/t-MDS with specific TP53 mutations for
which we had leukapheresis or bone marrowspecimens
banked
14

15. • Patient 530447 developed t-AML after an autologous stem
cell transplant for refractory Hodgkin’s lymphoma (Fig). The t-
AML sample carried mutations of
• TP53, missense mutations of TET2 and NUP98, a silent
mutation of CSMD1, and a subclonal KRAS mutation.
• Analysis of a leukapheresis sample obtained 6 years before
the development of t-AML revealed that both TP53 mutant
alleles were present and CSMD1 mutation was also present
(passenger mutation)However, two
• potential driver mutations (TET2 and NUP98) were not
detectable,
• So, TP53 mutations preceded the development of t-AML
15

16. • t-AML 5 years after the diagnosis of marginal zone lymphoma
The diagnostic t-AML sample contained a homozygous TP53
• these data provide evidence that an HSPC harbouring a
• TP53 Y220Cmutation preferentially expanded after
chemotherapy with
• the subsequent acquisition of del(5q) and then del(7q) (Fig.
3d).
• these data suggest that TP53 mutations
• precede the development of these characteristic cytogenetic
abnormalities
• of t-AML/t-MDS.
• patient 895681 developed t-MDS 5.5 years after the 16
HSPC clones harbouring somatic TP53 mutations are detected in patients
before cytotoxic therapy exposure.

17. whether HSPCs harbouring TP53 mutations are present
in healthy individuals
• peripheral blood leukocytes from 20 elderly (68–89 years old)
cancer-free donors who had not received prior cytotoxic
therapy
• sequencing to exons 4–8 of TP53 since the majority of
pathogenic mutations in TP53 are located in these exons
• TP53 mutations dectected in 9 of 19 evaluable cases,
• 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
17

18. To test directly the hypothesis that functional TP53 mutations confer
a competitive advantage after chemotherapy
• generated mixed bone marrow chimaeras containing both
wild-type and Tp531/2 cells
• mice treated with vehicle control (placebo) - a non-significant
trend towards an increased Tp53+/-
• upon treatment with N-ethyl-N-nitrosourea (ENU), Tp53
HSPCs show a competitive advantage. Importantly, a previous
study similarly showed that Tp53 ½ HSCs also have a
competitive advantage after irra1/2 18

19. TN Wong et al. Nature 000, 1-4 (2014) doi:10.1038/nature13968
Biallelic TP53 mutations are early
mutational events in the AML cells of UPN 530447.

20. References & More Information
• WHO Classification of Tumors of Hematopoietic and Lymphoid Tissues ,
4th
edition, S. Swerdlow, E. Campo, N. Lee Harris, E. Jaffe, S. Pileri, H. Stein,
J. Thiele, J. Vardiman, IAR C, Lyon, France, 2008
• 2010 Hematopoietic and Lymphoid Neoplasm Case Reportability and
Coding Manual, C. Hahn Johnson, M. Adamo, S. Peace, NCI SEER, 2009
• Advances in Understanding and Management of Myeloproliferative
Neoplasms, Alessandro M. Vannucchi, Paola Guglielmelli and Ayalew
Tefferi, CA Cancer J Clin 2009;59;171-191; Apr 15, 2009
• Proposed Classification of Lymphoid Neoplasms for Epidemiollogic
Research from the International Lymphoma Epidemiology Consortium
(Inter-Lymph), L. Morton, J. Turner, J. Cernan, Blood, DOI 10.1182/2006-
11-0515672; Mar 27, 2007
20

21. • Thank you
21

22. Conclusion
• The new hematopoietic and lymphoid
neoplasm rules go into effect for cases
diagnosed January 1, 2010, and after
• Email address for questions
askseerctr@imsweb.com
22