Download TP53 mutations occur in 15.7% of ALL and are associated with MYC

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Regular Article
LYMPHOID NEOPLASIA
TP53 mutations occur in 15.7% of ALL and are associated with
MYC-rearrangement, low hypodiploidy, and a poor prognosis
Anna Stengel, Susanne Schnittger, Sandra Weissmann, Sabrina Kuznia, Wolfgang Kern, Alexander Kohlmann,
Torsten Haferlach, and Claudia Haferlach
Munich Leukemia Laboratory, Munich, Germany
TP53 is the most extensively studied gene in cancer. However, data on frequency and
the prognostic impact of TP53 mutations in acute lymphoblastic leukemia (ALL) remain
• TP53 mutations are detected scarce. Thus, we aimed at identifying the mutation frequency of TP53, its association with
in 15.7% of patients with ALL cytogenetic subgroups, and its impact on survival in a large cohort of 625 patients with
ALL. Our data revealed an overall mutation incidence of 15.7%, which increases with age.
and are correlated to a low
Correlation with cytogenetic subgroups showed that mutations were most frequent in
hypodiploid karyotype and to
ALL with low hypodiploidy or MYC-rearrangements. Furthermore, for a large number of
MYC-translocations.
patients, both TP53 alleles were altered, either by 2 TP53 mutations (12%) or by a TP53
• Disruption of both TP53
mutation and a TP53 deletion in the second allele (39%). A high TP53 mutation load was
alleles is associated with
correlated to low hypodiploidy, high hyperdiploidy, and a complex karyotype. Moreover,
adverse prognosis in ALL.
a higher mutation load was found in B-lineage ALL compared with T-lineage ALL. Similar
to other cancers, the median overall survival was significantly shorter in patients with
TP53 mutation compared with patients with wild-type TP53. This effect was especially pronounced when both TP53 alleles were
affected, either by 2 TP53 mutations or by both a mutation and an accompanying TP53 deletion. (Blood. 2014;124(2):251-258)
Key Points
Introduction
Acute lymphoblastic leukemia (ALL) is a biologically and clinically
heterogeneous disease that affects both children and adults. It is
caused by uncontrolled proliferation of immature B- or T-lymphoid
cells and occurs more frequently in the B-lineage (85% to 90% in
children, ;75% in adults) than in the T-lineage (10% to 15% in
children, ;25% in adults).1,2 ALL is the most common childhood
tumor; however, progress in treatment development has led to
a cure rate of .80% in children, in contrast to the situation in
infants and adults, where prognosis remains poor.1 This is, among
other factors, related to the types of genetic alterations of the disease,
as the frequencies of particular genetic subtypes differ in children and
adults and shift from aberrations with a favorable outcome to alterations associated with poor outcome with increasing age.3,4
Genetic alterations described in ALL often affect genes involved
in cell proliferation, cell differentiation, or apoptosis and comprise
activation of proto-oncogenes, inactivation of tumor suppressor
genes, or creation of fusion genes, some of them encoding active
kinases. The mechanisms underlying such alterations include chromosomal rearrangements, chromosome gains or losses, deletions,
mutations, and (de)-methylation of the promoter regions of the
respective genes.1,5,6 Alterations most commonly found in ALL include
t(9;22)(q34;q11) BCR-ABL1 (mainly in adults), hyperdiploidy, t(12;21)
(p13;q22) ETV6-RUNX1 (predominantly in children), and MLL- and
MYC-rearrangements. In addition, multiple other genotypes have been
described.1,4
The most frequently mutated gene in cancer is TP53, which is
well characterized in other hematological malignancies, including
acute myeloid leukemia (AML) and chronic lymphocytic leukemia
(CLL). However, the frequency and prognostic impact of TP53
mutations has only rarely been studied in ALL thus far. Mutations in
or deletion of TP53 are generally associated with advanced stages of
disease,7 insufficient response to therapy,8,9 and a poor prognosis.10,11
TP53 is a transcription factor and functions as key tumor suppressor
and master regulator of various signaling pathways. Its importance
is reflected by the fact that it is altered in ;50% of human cancers.12,13
It is located on the short arm of chromosome 17 (17p13) and encodes
for a nuclear phosphoprotein that is tetrameric in its functional
form.14,15 The TP53 protein plays crucial roles in a variety of cellular
processes, including cell cycle arrest, apoptosis, DNA repair, genomic
stability, cell differentiation, and senescence.16-20 Inactivation of
TP53 in tumors is supposed to be most frequently generated by
missense mutations in the coding region or by deletions of the
TP53 gene (17p13 deletion), although hypermethylation of the
promoter region resulting in a decrease of TP53 transcription has
also been reported.14,21 It was conclusively shown that TP53
deletions are often associated with TP53 mutations of the second
allele.6,13 This supports the “two-hit” hypothesis implying that
both copies of a tumor-suppressor gene have to be altered for
cancer development.6 The cancer-associated TP53 mutations
are very diverse in their locations within the TP53 sequence.
Submitted February 27, 2014; accepted April 30, 2014. Prepublished online as
Blood First Edition paper, May 14, 2014; DOI 10.1182/blood-2014-02-558833.
The publication costs of this article were defrayed in part by page charge
payment. Therefore, and solely to indicate this fact, this article is hereby
marked “advertisement” in accordance with 18 USC section 1734.
The online version of this article contains a data supplement.
There is an Inside Blood Commentary on this article in this issue.
BLOOD, 10 JULY 2014 x VOLUME 124, NUMBER 2
© 2014 by The American Society of Hematology
251
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252
BLOOD, 10 JULY 2014 x VOLUME 124, NUMBER 2
STENGEL et al
However, they most frequently result in disruption of the specific
DNA-binding ability of TP53.22
In ALL, TP53 mutations have so far been considered to be rather
infrequent13 (2% to 3%), although other studies suggest a much higher
percentage of TP53 alterations (30% to 40%), if analyses do not merely
focus on TP53 mutations and deletions but also include promoter
hypermethylation.6 Generally, TP53 mutations mainly have been
investigated in children, where they most frequently occur at relapse
and are associated with poor outcome.23,24 The proposed role of
TP53 mutations in disease relapse might be due to the selective
advantage of the respective cells caused by their resistance to
therapy.6 For TP53 mutations in adult ALL, only a few studies are
available that include mostly relapsed cases and small cohorts of
patients.25,26 Thus far, the largest series of adult ALL investigated for
TP53 mutations includes 98 adult patients2 and shows TP53 mutations in 8 patients (8.2%), which is similar to the TP53 mutation
frequency in AML at diagnosis and in CLL at the time of
progression.10,11 Furthermore, the results support previous findings
of an increase of TP53 mutations at disease reappearance.23,25,26
Moreover, TP53 mutations were found to be more frequent in T-lineage
ALL than in B-lineage ALL and were detected in 14% of cases
negative for recurrent fusion genes.2
As data on incidence and prognostic significance of TP53
mutations in ALL remains scarce to date, we aimed to determine the
TP53 mutation frequency, the association with cytogenetic subgroups and age, as well as the impact on survival in a large cohort of
625 ALL patients.
additionally investigated by 24-color fluorescence in situ hybridization
(FISH; Metasystems, Altlussheim, Germany). For classification of abnormalities
and karyotypes, the International System for Human Cytogenetic Nomenclature guidelines (2013) were used.32 In 383 patients, interphase FISH using
probes for TP53 spanning a 167-kb region in 17p13 including the complete
sequence of TP53 was performed to determine the copy number state of TP53
(Metasystems, Altlussheim, Germany). Moreover, interphase FISH was
applied with probes for BCR-ABL1 (Metasystems) and MLL (Abbott,
Wiesbaden, Germany). MYC (Abbott) was analyzed in cases in which
respective rearrangements were suspected by chromosome banding
analysis.
Mutation analysis
For the total cohort of 625 patients, deep-sequencing was applied to identify
and quantify the TP53 mutation load as previously described.33 For this,
DNA was extracted according to standard methods from bone marrow and
peripheral blood cells obtained at the time of diagnosis. Next-generation
amplicon deep-sequencing (Illumina, San Diego, CA; 454 Life Sciences,
Branford, CT) was applied to investigate the mutational hotspot regions of
TP5334 (ENST00000269305, exons 4-11). next generation sequencing data
were analyzed using the GS Amplicon Variant Analyzer Software 2.6 (454
Life Sciences) and Sequence Pilot (version 4.1.1 Build 514 for the 454
platform; version 4.1.1 Build 510 for the Illumina platform, JSI Medicalsystems, Kippenheim, Germany).
Statistical analysis
SPSS (version 19.0.0) software (IBM Corporation, Armonk, NY) was used
for statistical analysis. OS curves were calculated according to Kaplan-Meier
and compared using the 2-sided log-rank test. All reported P values are
2-sided and were considered significant at P # .05. OS was measured from the
date of diagnosis until last follow-up or death.
Methods
Patient cohort
A total of 625 patients newly diagnosed with ALL was studied. Patient samples
were sent between August 2005 and May 2013 for diagnosis to the Munich
Leukemia Laboratory, and patients agreed with the use of laboratory data for
research studies. The study was approved by the institutional review board. The
study was conducted in accordance with the Declaration of Helsinki. The cohort
comprised 353 male and 272 female patients and the median age was 49.5 years
(range 0.1-91.4 years; supplemental Figure 1, available on the Blood Web site).
It has to be noted that the presented cohort comprises a relatively low proportion
of childhood ALL, which is generally known to constitute a large number of
ALL cases. This study includes 24 cases that were analyzed in a study on ALL
with low hypodiploid/near-triploid karyotype.27 For analysis of the overall
survival (OS), patients with an available detailed immunophenotype who were
aged $18 years were selected from the total cohort (n 5 374). The median
follow-up time was 18.4 months.
Cytomorphology and immunophenotype
Cytomorphology of bone marrow and/or peripheral blood samples was carried
out as previously described.28 Multiparameter flow cytometry analyses were
performed using FC500 or Navios flow cytometers (Beckman Coulter, Miami,
FL). List mode files were analyzed using CXP Software (version 2.0) and
Kaluza (version 1.0) (Beckman Coulter). Diagnoses were assigned according to
the European Group for the Immunological Characterization of Leukemias and
World Health Organization classifications.29,30 Detailed data on immunophenotype were available for 408 patients.
Cytogenetics and FISH
Chromosome preparations and banding analysis were performed for all 625
cases as previously described according to standard methods.31 If required,
cases with an aberrant karyotype in chromosome banding analysis were
Results
Frequency, type of TP53 mutations, and TP53 mutation load
Analysis of the TP53 mutation frequency in the total cohort
revealed 110 mutations in 98/625 (15.7%) of all patients. In 12
patients, 2 concomitant TP53 mutations were found. Investigation
of the type of TP53 mutation showed that the vast majority of
mutations were missense mutations (90/110; 81.8%), followed
by frameshift (8/110; 7.2%), in-frame (5/110; 4.5%), splice-site
(3/110; 2.7%), and nonsense mutations (4/110; 3.6%) (Figure 1).
The mutations were found to be quite diverse with respect to the
location across the coding region of the gene; however, they occurred
preferentially in the DNA-binding domain of TP53 (amino acids
101-300). Moreover, many mutations affected codons described
to code for the “hotspot” residues Arg273, Arg175, Arg282, and
Arg248 (Table 1). Whereas single heterozygous TP53 mutations
were present in 78.6% (77/98) of patients with TP53 mutation,
2 heterozygous TP53 mutations were observed in 12.2% (12/98),
and for 9.2% of patients (9/98), a homozygous TP53 mutation was
detected.
In addition, because next-generation sequencing was used, we
were able to investigate the TP53 mutation load and observed
a range between 2% and 98% (median: 41%) (Figure 2A). For some
patients with 2 TP53 mutations, the respective mutation load
differed considerably between the 2 mutations, whereas in other
patients the respective mutation loads were found to be almost
identical (Figure 2B). Comparison of the type and localization of
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BLOOD, 10 JULY 2014 x VOLUME 124, NUMBER 2
TP53 MUTATIONS IN ALL
253
Figure 1. Location, frequency, and type of TP53 mutations in ALL. The locations and mutation types are depicted. Each circle represents a detected mutation. Numbers
in the gray bar denote the respective exon, and numbers below correlate to the amino acid sequence. Functional TP53 domains are color-coded.
the mutations with the respective mutation load did not reveal any
correlation between these parameters (supplemental Table 1).
Correlation of TP53 mutations with TP53 deletion
The TP53 deletion status was investigated in a subset of 383 patients
by interphase FISH. It was revealed that 64/383 (16.7%) patients
with available TP53 deletion status harbored a TP53 mutation, whereas
the remaining 83.3% were TP53 wild-type (wt). Of the TP53 mutated
patients, 25/64 (39.1%) showed a deletion of the second allele, whereas
no TP53 deletion was found in 39/64 (60.9%) patients with TP53
mutation. Moreover, in only 17/319 (5.3%) TP53wt patients with
available TP53 deletion status, a TP53 deletion was detected
(P , .001). Consequently, no TP53 deletion was observed in 302/319
TP53wt patients (94.7%) (supplemental Figure 2A-B).
Correlation of TP53 mutations with cytogenetic subgroups
The distribution of cytogenetic subgroups in the total cohort of 625
patients resembles the frequencies of specific genotypes previously
reported in patients with ALL,1 although it has to be noted that the
Table 1. Amino acid residues of TP53 affected by mutations
Site of respective mutation
Arg273*
Number of patients with
respective mutation
9
Arg175*, Arg282*
7
Arg248*, Ile195
5
Arg283
4
Tyr220, Tyr234, Met237, Gly245*, Ile332
3
Ser127, Lys132, Arg158, Cys176,
2
Leu194, Val216, Cys238, Ser241,
Cys242, Gly244, Cys275, Glu285, Arg337
Trp53, Arg72, Pro128, Met133, Cys135, Gln136,
1 each
Cys141, Val143, Thr150, Pro151, Lys164,
Gly199, Tyr205, Arg213, Tyr236, Met246,
Arg267, Phe270, Val272, Ala276, Pro278,
Gly279, Asp281, Thr284, Glu286, Arg290,
Lys292, Ile331, Glu349, Met384, Phe385
The number of patients showing 1 of the respective mutations indicated is
depicted.
*These are the known hotspot mutation residues located in the DNA-binding
domain of TP53.40
cohort used in our study includes both children and adults encompassing relatively few childhood cases (0.1-91.4 years) (Figure 3 and
supplemental Figure 3). As indicated above, TP53 mutations were
found in 15.7% of all patients analyzed in the total cohort. However,
when TP53 mutations were analyzed in the different cytogenetic
subgroups, a clear correlation to ALL with low hypodiploidy (22/24;
91.7%) and MYC-translocated ALL (25/40; 62.5%) was observed
(Table 2). Additionally, TP53 mutations were found to appear aboveaverage in ALL with complex karyotype (16/69; 23.2%) and in
MLL-translocated ALL (6/37; 16.2%). In contrast, TP53 mutations were clearly underrepresented in ALL with t(9;22)(q34;q11)
(7/162; 4.3%), rare in ALL with high hyperdiploidy (3/38; 6.1%),
ALL with normal karyotype (13/101; 12.9%) and ALL with other
cytogenetic abnormalities (6/139; 4.3%), and were even absent
in ALL with the ETV6-RUNX1 translocation t(12;21) (p13;q22)
(0/15; 0%). Taken together, statistically significant correlations of
TP53 mutations with a low hypodiploid karyotype and also with
MYC-translocations were observed.
Frequency of TP53 mutations in Burkitt leukemia, B-lineage
ALL, and T-lineage ALL
For 408 patients, detailed data on immunophenotype was available
(T-lineage: n 5 105, 25.7%; B-lineage: n 5 267, 65.4%; Burkitt:
n 5 36, 8.8%); hence, analysis of the frequency of TP53 mutations
in the respective subgroups was performed. Mutations of TP53
occurred in 58.3% of all Burkitt leukemias analyzed (21/36) and
were also detected in a substantial fraction of B-lineage ALL (41/267;
15.4%). Compared with these findings, TP53 mutations were less
frequent in T-lineage ALL (8/105; 7.6%).
Age dependence of TP53 mutations and TP53 deletions
The cohort was split into subgroups of patients ,60 years (n 5 417)
and patients $60 years (n 5 208). A clear interrelation of TP53
mutation frequency with increasing age was observed (TP53mut ,60
years 5 10.8% (45/417) vs $60 years 5 25.5% (53/208); P , .0001).
To analyze the TP53 mutation frequency in childhood ALL compared
with adult ALL in more detail, the cohort was further split into the
following subgroups: infant ALL (,1 years), childhood ALL (1-14
years), TYA ALL (teenage and young adults; 15-24 years), adult ALL
(25-59 years), and older ALL (.60 years) (supplemental Figure 4A-B).
The highest frequency of TP53 mutations was observed for patients
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254
STENGEL et al
Figure 2. Mutation load in patients with TP53 mutations. (A) Numbers of
patients with the respective TP53 mutation load percentages are depicted. It
was investigated by next-generation deep-sequencing and varied between 2%
and 98% (median: 41%). (B) TP53 mutation load percentages in the 12 patients
that were identified to carry 2 TP53 mutations. The mutation type is depicted as well.
.60 years (25.2%), although a relatively high incidence was also
detected for adult ALL (12.7%) and childhood ALL (9.6%).
Moreover, the frequency of TP53 deletions was analyzed in patients
with available TP53 deletion status ,60 years (n 5 249) and in
patients $ 60 years (n 5 134). A similar correlation was observed,
although it has to be noted that TP53 deletions were generally found
to occur less frequently than TP53 mutations (TP53del ,60 years
vs $60 years 5 8.4% [21/249] vs 15.7% [21/134; P , .0001]).
Taken together, our data suggested an age dependence of TP53
mutation/deletion frequency.
Impact of TP53 mutations and their mutation load and TP53
deletions on survival
OS was significantly shorter in patients with a TP53 mutation
(TP53mut) compared with patients without (TP53wt) (median 11.5
months vs not reached, P , .0001) (Figure 4A). Similarly, OS was
clearly shorter in patients with a TP53 deletion (TP53del) compared
with patients without (TP53notdel) (median 18.5. months vs 63.1
months, P 5 .005) (Figure 4B). Next, it was investigated whether both
TP53 alleles have to be altered to cause the negative effect on prognosis.
For this purpose, the OS was analyzed in more detail by dividing the
cohort in (1) patients with mutations and/or deletions in both alleles
(including: TP53mut 1 TP53del, homozygous TP53mut, 2 TP53mut;
TP53 2hits, n 5 29), (2) patients with TP53del only without TP53mut
(TP53del sole; n 5 16), (3) patients with one TP53mut (n 5 22), and
(4) patients with TP53wt (n 5 188). The results revealed that for
patients with TP53 2hits, a drastically reduced OS was observed compared with patients with TP53del, one TP53mut, and TP53wt patients
(TP53 2hits [7.1 months] vs TP53del sole [not reached], P 5 .005;
TP53 2hits vs TP53mut sole [63.5 months], P 5 .026; TP53 2hits vs
TP53wt [75.5 months]; P 5 .001) (Figure 4C). To exclude a bias
of the differently treated patients with Burkitt/MYC1 ALL and
t(9;22) ALL (Ph1), we performed additional OS analyses excluding
BLOOD, 10 JULY 2014 x VOLUME 124, NUMBER 2
these patients. The impact of TP53 alterations on OS in this subset
was comparable with the total cohort. The respective data for median
OS were: 12.2 vs 75.5 months for TP53mut patients vs TP53wt
patients (P , .0001); 18.5 vs 60.2 months in patients with TP53del
compared with patients without TP53del (P 5 .02); 11.5 months
for patients with 2 TP53 alterations (2hits) vs not reached for
patients with TP53del sole (P 5 .007); vs 63.1 months for cases
with TP53mut sole (P 5 .030); vs 60.2 months for TP53wt patients
(P 5 .002) (supplemental Figure 5). The results clearly show that the
negative effect on prognosis is dependent on alterations of both
TP53 alleles.
When the OS was investigated with respect to the age of the
patients, it was observed that in patients ,60 years, the median OS
was not reached in TP53wt patients (n 5 231) compared with 45.3
months in patients harboring the TP53mut (n 5 31; P 5 .04). For
patients $60 years, the median OS was found to be 37.0 months vs
6.4 months (n 5 91 and n 5 36; P , .0001) (Figure 5A-B).
In univariate Cox regression analysis, the following factors had
an adverse impact on OS: TP53 2hits (hazard ratio [HR] 5 3.7;
P , .0001), age (,60 years vs $60 years, HR 5 2.9; P 5 .0001),
MLL-translocation (HR 5 2.3; P 5 .005), MYC-translocation
(HR 5 3.3; P , .0001), and low hypodiploid ALL (HR 5 2.5;
P 5 .004) (Table 3). Multivariate Cox regression analysis, including
the parameters identified to be associated with shorter OS in univariate analysis, revealed an independent adverse impact on OS for
TP53 2hits (HR 5 2.4; P , .0001), age (,60 years vs $60 years,
HR 5 2.8; P , .0001), MLL-rearrangement (HR 5 2.1; P 5 .017),
and MYC-rearrangement (HR 5 2.1; P 5 .025) (Table 3). Taken
together, it can be concluded that a complete loss of wt TP53 is
associated with short OS in patients with ALL, independent of age
or the cytogenetic subgroups analyzed.
Correlation of TP53 mutation load with cytogenetic subgroups
and immunophenotype
Analysis of the TP53 mutation load in regard to the respective
karyotypes revealed a clear correlation with a high mutation load
of .50% for patients with a complex karyotype, ALL with low
hypodiploidy, and ALL with high hyperdiploidy (median 51.6%,
66.9%, and 67.0%, respectively). In contrast, the mutation load of
patients with MLL-translocated ALL was found to be ,50% in all
cases (median: 26.7%) (Figure 6A). When the mutation load was
Figure 3. Frequency of specific karyotypes of patients with ALL. Analysis of
karyotypes in the total cohort of 625 patients. Percentages are given for each
cytogenetic subgroup. BCR-ABL1 t(9;22)(q24;q11) translocation (n 5 162),
normal karyotype (n 5 101), complex karyotype ($4 aberrations) (n 5 69),
MYC-rearrangements (n 5 40), high hyperdiploidy (51-68 chromosomes) (n 5 38),
MLL-rearrangements (n 5 37), low hypodiploidy (,40 chromosomes) (n 5 24),
t(12;21)(p13;q22) (n 5 15), and other cytogenetic abnormalities (n 5 139).
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BLOOD, 10 JULY 2014 x VOLUME 124, NUMBER 2
TP53 MUTATIONS IN ALL
ALL and TP53 mutation was too low (n 5 8) to draw definite
conclusions.
Table 2. Patients with TP53 mutations within cytogenetic
subgroups
Cytogenetic subtype (patients
mutated/studied)
Low hypodiploid
TP53 mutation
(% of respective
subgroup)
P value (TP53
mutated patients
vs total cohort)
91.7
,.0001
62.5
,.0001
23.2
,.0001
4.3
,.0001
12.9
n.s.
16.2
n.s.
6.1
n.s.
4.3
n.s.
0.0
n.s.
(n 5 22/24)
MYC-rearrangement
(n 5 25/40)
Complex karyotype
(n 5 16/69)
BCR-ABL1 fusion gene
(n 5 7/162)
Normal karyotype
(n 5 13/101)
MLL-rearrangement
(n 5 6/37)
High hyperdiploid
(n 5 3/38)
Other cytogenetic
abnormalities
(n 5 6/139)
ETV6-RUNX1 fusion
gene (n 5 0/15)
n.s., not significant.
analyzed in relation to the immunophenotype, a high mutation load
was detected in patients with B-lineage ALL (median: 54.8%)
(Figure 6B). In contrast to this, patients with Burkitt ALL showed a
comparably low TP53 mutation load (median: 33.6%). For T-lineage
ALL, a median mutation load of 43.6% was observed. However, it
has to be noted that the mutation load in this subgroup showed a wide
variation (2% to 82%), and the number of patients with T-lineage
Figure 4. OS of patients with TP53 mutation and/or
deletion. OS was analyzed in patients with (TP53 mut;
n 5 67) and without (TP53 wt; n 5 322) TP53 mutation
(A); in patients with (TP53 del; n 5 34) and without
(n 5 217) TP53 deletion (B); and in patients with
TP53 mutation without TP53 deletion (TP53 mut
sole; n 5 22), patients with TP53 deletion without
TP53 mutation (TP53 del sole; n 5 16), in patients
that showed alterations in both alleles (TP53 2hits,
n 5 29) and in patients without aberrations in TP53
(TP53 wt; n 5 188) (C).
255
Discussion
To our knowledge, this study comprises the largest cohort of patients
with ALL analyzed for TP53 alterations so far. The data clearly
showed that (1) TP53 mutations occur more frequently thanpreviously
published (15.7% vs 2% to 3%13; 8% to 9%2,6), (2) TP53 mutations are
predominantly associated with ALL with low hypodiploidy and
MYC-translocated ALL, (3) the TP53 mutation/deletion frequency
increases with age, (4) TP53 mutations are associated with short
survival independent of age and specific cytogenetic alterations, and
(5) the adverse impact on survival occurs only when the wt TP53
is lost. The reason why TP53 mutations were found at a higher
frequency in ALL patients than in earlier studies might be due to the
cohort analyzed, as previous studies mainly focused on children and
our cohort includes a large variety of ALL subgroups, including, eg,
Burkitt/MYC1 ALL. Alternatively, the higher mutation frequency
might be caused by differences in sensitivity of the sequencing
methods used (in our study, the limit-of-detection for TP53 mutations
was approximately 3% using the 454 Life Sciences technology and
approximately 5% when using the Illumina assay). Moreover,
previous analyses often focused on sequencing exons 5 to 8 of TP53
(the DNA-binding domain), whereas 10% to 30% of the cancerassociated mutations in TP53 are known to be located outside this
hotspot region.6,35 7.3% [8/110] of TP53 mutations detected were
not located in exons 5 to 8, indicating a very high preference for TP53
mutations to occur in the hotspot region for ALL. The overall mutation
frequency observed in our study clearly remains lower compared with
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256
BLOOD, 10 JULY 2014 x VOLUME 124, NUMBER 2
STENGEL et al
Figure 5. OS of patients with TP53 mutation according
to age. OS was analyzed in patients ,60 years (A) and
in patients $60 years (B).
the landscape in many solid tumors, eg, ;50% in gastric cancer,36 and
almost 100% in high-grade serous carcinoma of the ovary.12 However,
the incidence of TP53 mutations found in our analyses is in the range of
other hematologic neoplasms, including AML10,37 or CLL.11,38 TP53
was found to be predominantly inactivated by missense mutations,
whereas deletions and insertions occurred rarely. This corroborates
previous results regarding TP53 mutations in ALL and other human
cancers.21,39,40 It has to be noted that a high number of mutations
(31/110; 28.2%) detected in our study affect the 6 known hotspot residues (R175, G245, R248, R249, R273, and R282). These
results nicely resemble previous data from different types of
cancer, proposing that ;30% of TP53 mutations cluster in these
residues.12,41
Further, analysis of TP53 deletions revealed a frequent correlation
with TP53 mutations in the second allele, thus underlying the “2-hit”
idea for tumor-suppressor genes and their function in cancer
development.6 Accordingly, previous data for patients with AML
further support this hypothesis, as .40% of patients with a TP53
mutation were found to carry an additional TP53 deletion of the
second allele.37,42 On the other hand, for patients with AML, a high
coincidence of TP53 mutations with a complex aberrant karyotype
was observed, in contrast to the present study, in which TP53
mutations were most frequently found in ALL with low hypodiploidy
(91.7%) and MYC-translocations (62.5%). However, in ALL with
complex karyotype, TP53 mutation frequency was still above average
(23.2% vs 15.7%).
Other studies suggested a much higher percentage of TP53
alterations in ALL (30% to 40%), if analyses do not merely focus on
TP53 mutations and deletions but include promoter hypermethylation.14
In this case, methylation of the TP53 promoter was even found to
constitute the most frequent molecular event in ALL (32% methylated
Table 3. Univariate and multivariate Cox regression analysis for OS
Univariate
Multivariate
HR
P value
HR
P value
TP53 2hits
3.7
,.0001
2.4
,.0001
Age ,60 vs $60 y
2.9
,.0001
2.8
,.0001
MYC translocated
3.3
,.0001
2.1
.025
MLL translocated
2.3
.005
2.1
.017
Low hypodiploid
2.5
.004
—
n.s.
Complex karyotype
—
n.s.
—
—
BCR-ABL1 rearrangement
0.5
.009
0.6
.03
n.s., not significant.
TP53 2hits: TP53 mutation 1 TP53 deletion, homozygous TP53 mutation,
2 TP53 mutations; low hypodiploid: 32-39 chromosomes; complex: .3 cytogenetic
aberrations.
samples vs 8.8% missense mutations), hypothesizing that promoter
methylation in different genes is common in ALL. However, an impact
of this on OS and prognosis could not be demonstrated to date. Because
we focused on TP53 mutations/deletions and not on methylation, the
role of this potentially common event in ALL remains to be investigated.
It was proposed that hypermethylation of the promoter regions of tumorsuppressor genes such as TP53 induces transcriptional silencing and is
related to tumor progression. Thus, further analyses of the clinical
and biological role of methylated genes in ALL might support the
development of new markers and therapies.6
Moreover, our data suggest a correlation of TP53 mutations
with Burkitt ALL and B-lineage ALL compared with T-lineage
ALL, in which TP53 mutations were found to be rather infrequent.
These results are, however, in contrast to a previous study in which
TP53 mutations were found to be more frequent in T-lineage than in
B-lineage ALL (4/36, 11.1% vs 4/62, 6.4%), although the different
size and composition of the cohort has to be considered.2
Interestingly, our results demonstrate a clear association of
TP53 mutations with ALL with low hypodiploidy (22/24; 91.7%)
and MYC-translocated ALL (25/40; 62.5%). This corroborates
recent studies demonstrating a very high frequency (91.2% and
93.1%, respectively) of TP53 alterations in low-hypodiploid ALL
(32-39 chromosomes).27,43 In contrast, patients with near-haploid
ALL (24-31 chromosomes) were found to show a different genomic profile, namely alterations in genes involved in the RAS
signaling pathway and the lymphoid transcription factor gene
IKZF3.43 Regarding MLL-translocations (16.2% TP53 mutation
in our study), previous studies proposed a correlation to TP53
mutations in a cohort of children with relapsed ALL.23 Moreover,
we showed that TP53 mutations are rare in patients with BCRABL1-positive ALL (7/162; 4.3%), which are known to constitute
the largest cytogenetic subgroup in adult ALL1 (;25%) and are
associated with poor prognosis, although higher remission rates
can be achieved by treatment with tyrosine-kinase inhibitors.44
As TP53 mutations seem to be almost mutually exclusive of BCRABL1 rearrangements in ALL, these 2 aberrations might function
as independent prognostic markers, both defining genetic subgroups
with a high prognostic significance.
In addition, analysis of the TP53 mutation load in regard to
cytogenetic subgroups revealed a rather low load for patients with
MLL- and BCR-ABL1-translocations, indicating that in these
cases, not the TP53 mutation but rather the MLL- and BCR-ABL1translocations promote the impact on prognosis. By contrast, patients with a complex karyotype, ALL with low hypodiploidy and
ALL with high hyperdiploidy show a drastically higher TP53
mutation load. For patients with a low hypodiploid karyotype, this
From www.bloodjournal.org by guest on June 12, 2017. For personal use only.
BLOOD, 10 JULY 2014 x VOLUME 124, NUMBER 2
TP53 MUTATIONS IN ALL
257
Figure 6. Correlation of TP53 mutation load with
cytogenetic subgroups and with immunophenotype. (A) For each patient, the mutation load was
determined and correlated to the respective karyotype.
The number of patients with the respective mutation
load is shown in regard to cytogenetic aberrations.
(B) The TP53 mutation load was determined in each
patient and the obtained value was correlated to the
respective immunophenotype. The number of patients
with the respective mutation load is shown in regard to
Burkitt-ALL, T-lineage ALL, or B-lineage ALL.
could be explained by the frequent loss of chromosome 17 in these
cells (20/22 in the present series). Moreover, analysis of the mutation
load in regard to the respective immunophenotype revealed a high
load in patients with B-lineage ALL; however, the reason for this
remains to be investigated.
The frequency of TP53 mutations/deletions was clearly found to
increase with age. It has been noted before that patients harboring
a hypodiploid ALL have a high risk of treatment failure,4,45 and it can
be speculated that this is at least partly related to the high association
with TP53 mutations. Additionally, it is known that TP53 mutations
are generally associated with poor prognosis in all types of cancer.
The higher TP53 mutation frequency in older patients might be one
reason for the general poor outcome of adult ALL. However, MYCtranslocations and low hypodiploid ALLs were not found to occur
predominantly in older patients in contrast to MLL-translocations
that have only been observed in age decades 4 to 8 of TP53-mutated
patients (supplemental Figure 6). Regarding the OS, a negative effect
of TP53 alterations was observed, which was still clearly detectable if
patients with Burkitt/MYC1 ALL or Ph1 ALL were excluded from
the analyses who receive different treatment protocols compared
with other ALL patients. Moreover, patients with alterations in both
TP53 alleles showed the shortest OS of all patients analyzed and
hence define in the best way the subgroup with a very poor prognosis.
Thus, the presence of TP53 mutations/deletions is an important
prognostic parameter for patients with ALL, which has to be
considered to be analyzed in all patients receiving therapy according
to standard protocols for ALL.
Acknowledgments
We thank all coworkers at the Munich Leukemia Laboratory
for approaching together many aspects in the field of leukemia
diagnostics and research by their dedicated work. The authors would
like to thank all physicians for providing and caring for patients as
well as collecting data. Centers and investigators (contributing
more than 10 cases) are listed in order of number of cases provided: Städtisches Klinikum München Schwabing (C. Wendtner),
Klinikum Nürnberg (K. Schäfer-Eckart), Universitätsklinikum Köln
(K.-A. Kreuzer), Klinikum Augsburg (C. Schmid), Universitätsklinikum Frankfurt (H. Serve), Universitätsklinikum Freiburg (J. Duyster),
Asklepios Klinik Altona Hamburg (C. Meyer zum Büschenfelde),
St. Antonius Krankenhaus Eschweiler (P. Staib), Universitätsklinikum des Saarlandes Homburg (M. Pfreundschuh), Universitätsklinikum Marburg (A. Neubauer), Rechts der Isar der Technischen
Universität München (C. Peschel), and St. Marien-Krankenhaus
Siegen (W. Gassmann).
Authorship
Contribution: C.H. designed the study; C.H. and A.S. interpreted the
data; A.S. wrote the manuscript; S.W., S.Z., and A.K. did molecular
analyses; C.H. was responsible for chromosome banding and FISH
From www.bloodjournal.org by guest on June 12, 2017. For personal use only.
258
BLOOD, 10 JULY 2014 x VOLUME 124, NUMBER 2
STENGEL et al
analyses; S.S. was responsible for molecular genetic analyses; W.K. was
responsible for immunophenotyping; and T.H. was responsible for
cytomorphologic analyses. All authors read and contributed to the final
version of the manuscript.
Conflict-of-interest disclosure: C.H., S.S., W.K., and T.H.
declare part ownership of MLL Munich Leukemia Laboratory.
A.S., S.W., S.Z., and A.K. are employed by the Munich Leukemia
Laboratory. The remaining author declares no competing financial
interests.
Correspondence: Claudia Haferlach, Munich Leukemia Laboratory, Max-Lebsche-Platz 31, 81377 München, Germany; e-mail:
[email protected].
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From www.bloodjournal.org by guest on June 12, 2017. For personal use only.
2014 124: 251-258
doi:10.1182/blood-2014-02-558833 originally published
online May 14, 2014
TP53 mutations occur in 15.7% of ALL and are associated with MYC
-rearrangement, low hypodiploidy, and a poor prognosis
Anna Stengel, Susanne Schnittger, Sandra Weissmann, Sabrina Kuznia, Wolfgang Kern, Alexander
Kohlmann, Torsten Haferlach and Claudia Haferlach
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