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Nonhereditary p53 Mutations in T-cell Acute Lymphoblastic Leukemia Are
Associated With the Relapse Phase
By Michael H. Hsiao, Alice L. Yu, Jo Yeargin, David Ku, and Martin Haas
We have previously reported that greater than 60% of human leukemic T-cell lines possess mutations in the p53 tumor suppressor gene. To determine whether T-cell acute
lymphoblastic leukemia (T-ALL) patient samples possess
p53 mutations, we screened peripheral blood- and bone marrow-derived leukemia samples, taken at diagnosis and at
relapse, for p53 mutations. Exons 4 through 9 and selected
intron regions of the p53 gene were analyzed using polymerase chain reaction-single-strandconformation
polymorphism and direct sequencing. p53 mutations were found in
0 of 15 T-ALL diagnosis samples, as compared with 10 of
36 (28Y0)T-ALL relapse samples. To determine whether p53
mutations play a role in the recurrence (relapse) of T-ALL,
t w o special groups of T-ALL patients were studied: (1) a
group of 8 relapse patients whosedisease was refractoryt o
chemotherapeutic treatment, and (2) a group of 6 "paired"
T-ALL cell samples from patients for whom we
possess both
diagnosis and relapse samples. Three of 8 relapsed patients
(37.5Y0) whose disease was refractory t o the reinduction of
remission by chemotherapy possessed missense mutations
of the p53 gene. All 3 caseshad mutationsin exon 5. Among
the paired samples, 3 of 6 patients harbored p53 mutations
at disease recurrence, but possessed only wild-type p53 alleles at diagnosis. One case had mutation onexon 4, l case
in exon 5, and 1 case in exon 8 with loss of heterozygosity.
These data clearly indicate that recurrence of T-ALL is associated with missense mutations in p53. Our results indicate
that (1) mutations of p53 do occur in T-ALL in vivo, and
such mutations are associated with the relapse phase of the
disease; and (2) p53 mutation isinvolved in the progression
of T-ALL. This conclusion is supported by our observation
that the introduction of T-ALL-derived mutant p53 expression constructs into T-ALL cell lines further increases their
growth rate in culture, enhances cell cloning in methylcellulose, and increases tumor formation in nude mice.
0 1994 by The American Societyof Hematology.
P
have also. been found in the precancerous phasesof adenocarcinomas and in adenomatous polypsof patients with familial
polyposis coli,2zsuggesting that p53 mutation may occur as
an early event in carcinogenesis as well.
In our studies on the role of p53 in the pathogenesis of
human T-cell acute lymphoblastic leukemia (T-ALL), we
have shown that greater than 60% of T-ALL cell lines, all
of which had been grownfrom relapse T-ALL cases, possess
mutations on both p53
suggesting that p53 serves
a critical role in the generation of the fully tumorigenic
leukemic T cells. However, our experiments did not show
whether these p53 mutations originated in vivo or whether
the p53 gene had been mutated during cell line establishment
in vitro, as has been shown to occur during the establishment
of some rat embryo fibroblast cell lines.24Gaidano et alZs
and Jonveaux and BergerZ6have reported that samples from
T-ALL patients obtained at diagnosis harbor no mutations
in the p53 gene. In all, these two groups studied 37 T-ALL
diagnosis samples, all of which possessed only wild-type
p53 alleles. Recently, we reported that T-ALL cell lines
established in our laboratory from a patient sample taken at
relapse possess the same p53 mutation found in the in vivo
sample." Furthermore, no additional mutations of p53 OCcurred during establishment of the cell lines. These results
suggest that the establishment of human T-ALL cell lines
need not be associated with the induction of p53 mutations.
To studywhether p53 mutations may be differentially
associated with the relapse versus diagnosis phases of TALL, we used polymerase chain reaction-single-strand conformation polymorphism (PCR-SSCP)*' and direct sequencing of PCR-amplified fragments to study 15 cases of T-ALL
at diagnosis and 36 cases at relapse. The results suggest
that, in the patients studied, mutation of the p53 gene was
associated with the clonal evolution that takes place during
recurrence of the disease.
53 BELONGS TO THE tumor suppressor class of genes
whose function involves the negative regulation of cell
growth. The loss of function of the p53 gene by deletion,
mutation, or rearrangement may contribute to the genesis or
progression of a wide variety of human cancers.',' Abnormalities in the p53 gene have been demonstrated in human carcinoma of the colorectum,3
l i ~ e r , bladder,8
~.~
and
ovary'; in blast crisis chronic myelogenous and myelocytic
leukemias'",''; in progressed adult T-cell le~kemia".'~;and
in osteogenic sarcoma^.'^^'^ Several lines of evidence suggest
that p53 mutatioddeletion is a late event in the development
of cancer, because (1) in carcinomas of the colorectum, chromosome 17p deletions are associated with the transition from
benign adenoma to malignant
carcinoma'6;( 2 ) the progression
of brain tumors is associated witha clonal expansion of cells
that have acquireda mutation in thep53 gene17;(3) the evolution of chronic-phase chronic myelogenous leukemia (CML)
to myeloid blast crisis18-*"is associated with mutation of the
p53 gene; and (4)the mutation of p53 and the lossof heterozygosity of chromosome 17pand 10 areassociatedwiththe
progression of astrocytomas."However,mutationsin
p53
From the Department of Biology/Cancer Center, and the Department of Pediatrics, University of California, San Diego, La Jolla,
CA.
Submitted September 10, 1993; accepted January 7, 1994.
Supported in part by grants from the US Department of Energy
(DE-FG03-91 ER611 71), the American Cancer Society (CH456),
and the National Cancer Institute, National Institutes of Health
(ROICA56075 and UIOCA28439 [to A.L.Y.]), US Department of
Health, Education, and Welfare.
Address reprint requests to Martin Haas,MD, Department of
Biology, UCSD Cancer Center, 0063, University of California, San
Diego, 9500 Gilman Dr, La Jolla, CA 92093-0063.
The publication costsof this article were defrayedin part by page
charge payment. This article must therefore be hereby marked
"advertisement" in accordance with 18 U.S.C. section I734 solely to
indicate this fact.
0 1994 by The American Society of Hematology.
0006-4971/94/8310-0015$3.00/0
2922
MATERIALSAND METHODS
T-ALL patient samples. Peripheral blood or bone marrow cells
from 51 T-ALL patients at diagnosis or at relapse were donated in
Blood, Vol 83, No 10 (May 15), 1994: pp 29222930
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2923
p53 MUTATIONS IN RELAPSEACUTE T LEUKEMIA
accordance with a protocol approved by the Committee on Investigation Involving Human Subjects at the University of California, San
Diego. Some samples were kindly provided by the tumor cell bank
of the Pediatric Oncology Group at St Jude’s Children’s Research
Hospital. Peripheral blood or bone marrow cells were collected for
the purpose of routine clinical diagnosis, and cells that remained
after the diagnostic procedures were frozen live in liquid nitrogen
for future use. High molecular weight DNA was extracted from one
ampoule (IO7 cells) of each frozen T-ALL patient sample.
SSCP ana!\sis. SSCP analysis was adaptedfromthe
original
SSCP method of Orita et al.” Briefly, 1 0 0 ng of genomic DNA was
usedin a PCRamplification.Eachreactionmixturecontained
in
addition to genomic DNA, 10 pm01 of each primer, 2.5 pmol/L
dNTPs, I pCi of [a-”P]dCTP (Amersham; specific activity, 3,000
Ci/mmol; 1 Ci = 37 GBq), 10 mmol/L Tris-HC1 (pH 8.8). 50 mmol/
L KCI, 1 mmoVL MgCI2,0.01% gelatin, and 0.5 U of Taq polymerase,
in a final volume of 10 pL. Thirty cycles of denaturation (93°C for
2 minutes), annealing (60°C for exons 4, 5, 6, and 8: 68°C for exon
7; 51°C for exon 9: for 2 minutes), and extension (72°C for 1 minute)
were performed. Two microliters of the reaction mixture was diluted
25Xin 0.1% sodium dodecyl sulfate (SDS)/IO mmol/L EDTA and
further mixed I: I with a sequencing stop solution containing 20 mmoll
L NaOH. Samples were heated to 95°C for 5 minutes and chilled on
ice, and 3 pL was immediately loaded onto the gel. Gels were run at
4 W for 16 to 20 hours at room temperature. Autoradiography was
performed with an intensifying screen for 6 to 24 hours. Because not
all of the mutation bands were easily separated from normal germline
bands with good resolution using one gel condition. three gel conditions were used for each sample: (1) 6% acrylamide with 10% (voll
vol) glycerol (better separation with poor resolution): (2) 4% MDE
(AT Biochem, Malvern. PA) with glycerol (intermediate separation
and resolution); and (3) 4% MDE without glycerol (best resolution
withleast separation). Mobility shifts that were detected by SSCP
were confirmed underatleast two different gel conditions, before
sequencing.
The following p53 amplification primers were used? MH41 (5‘
exon 4 primer), 5’-ATCTACAGTCCCCCTTGCCG-3’; MH42 (3‘
exon 4 primer), 5‘-GCAACTGACCGTGCAAGTCA-3‘; MH22 (5‘
MH20 (3’
exon 5 primer), 5’-CTGTTCACTTGTGCCCTGAC-3‘;
exon 5 primer), 5’-CAACCAGCCCTGTCGTCTCT-3’;
MH28 (5’
exon 6 primer), 5‘-GAGACGACAGGGCTGGTT-3’; MH29 (3‘
exon 6 primer), 5’-CCACTGACAACCACCCTT-3‘;
MH30 (5‘ exon
7 primer), 5‘-CCAAGGCGCACTGGCCTC-3’: MH3I (3’ exon 7
primer), 5‘-GAGGCAAGCAGAGGCTGG-3’; MH 19 (5‘ exon 8
primer), 5’-GGGACAGGTAGGACCTGATT-3‘; MH23 (3’ exon 8
primer), 5’-CACCGCTTCTTGTCCTGCTT-3’;
MH34 (5‘ exon 9
primer), 5‘”ITATGCCTCAGATTCACTTTT-3’; MH25 (3’ exon 9
primer), 5’-CATCGAATTCTGGAAACTTTCCACTTGAT-3”.
Direct sequencing of PCR products. Samples that were found
by SSCP analysis to possess p53 mutations were sequenced to identify the nature of the mutation. Solid-phase sequencing of in vitro
amplified genomic DNA was
in
which genomic DNA was
amplified by PCR using biotinylated primers (Operon, Almeda, CA).
One microgram of genomic DNA was used as template in a 100 pL
PCR reaction with 12 pm01of biotinylated primer and 36 pm01
of nonbiotinylated primer. Forty microliters of this reaction was
incubated with magnetic beads conjugated covalently with streptavidin (Dynabeads M280-streptavidin: Dynal, Oslo, Norway) that were
used to selectively immobilize the biotin-labeled PCR product and
allow melting of the DNA duplex, followed by elution of the nonlabeled single strand. The immobilized single-strand DNA was then
used as sequencing template using the Sequenase (US Biochemical
CO, Cleveland, OH) protocol and an internal primer.
RESULTS
p53 mutation is a frequent genetic change in relapse
ALL
T-
We used PCR-SSCP analysis to screen for p53 muta-
Exon 5
Exon 4
P
L
P
L
A
C
A
C
E
N C H A F O
T E A S A N
A M N N N N
E
N S
T M
A N
ri6
P
L
A
C
E
C N F G
E T M C
M A N N
U
yI1
W
h
Yr
Fig 1. PCR-SSCP analysis of p53 mutations in T-ALL patient M m ples. Human placenta DNA was used as normal control. DNA extracted from thecell line CEM was used as a positive control for exon
5. Gel conditions: exon 4,6% acrylamide gel with 10% glycerol; exon
5 (left panel), 4% MDE gel; exon 5 (right panell, 6% acrylarnide gel
with 10% glycerol. Note that 6% acrylamide gel with 10% glycerol
provides the best separation but poor resolution. 4% MDE gel without glycerol provides the best resolution with least single-strand
band separation.
tions in the genomic DNAof 51 patients with childhood
acute lymphoblastic leukemia at different clinical stages (15
diagnosis T-ALL, 36 relapse T-ALL). Significant electrophoretic mobility shifts were detected in 10 of the 36 relapse
T-ALL samples (28%),and in none (0 of 15) of the diagnosis
T-ALL samples (0%). Figure 1 shows examples ofPCRSSCP analyses of exons 4 and 5 . DNA extracted from a
normal human placenta was used as normal control to show
the germline bands. The T-ALL cell line CEM, which harbors a mutation in p53 codon 175,*” was used as a mutant
control of exon 5 to show different mobility shift patterns
under two different gel conditions (see legend to Fig 1 and
Materials and Methods).
Direct sequence analysis was performed onthe 10 samples
found to be positive for p53 mutations based on significant
SSCP shifts. Mostof the mutations foundweremissense
mutations with single-base changes thatencoded single
amino acid substitutions in the p53 protein (Table 1). The
majority of the mutations found are located in exon 5. One
patient, PJN, had an intron 4 mutation. Although its biologic
significance has not been studied further, its detection shows
that cells harboring this mutation were selected for; hence,
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2924
HSlAO ET AL
Table 1. Frequency and Nature of p53 Mutations in Primary l-ALL Samples
Identification of Mutation
T-ALL Patient Sample
SSCP Codon
Seq Change
Genotype
aa Change
%
Blasts
Diagnosis
0/15 (0%)
Relapse 10/36
(28%)
SMN
FAN
ONN
ASN
GCN
SRN
FMN
HAN
BTN
PJN
Ex 4 110
Ex 5 135
Ex 5 135
Ex 5 135
Ex 5 141
Ex 5 161
Ex 5 167
170
Ex 5 175
Ex 8 282
ln4$
CGT
TGC
TGC
TGC
TGC
GCC
CAG
ACG
CGC
CGG
C-T
- ClT
-- AGC
-- AGC
TAC
TC
-- lACC
CGG
- ATG
- GGC
- GGG
Arg
Cys
Cys
Cys
Cys
Ala
Gln
Thr
Arg
Arg
--
-
---
Heterozygous
Heterozygous
Heterozygous
Homozygous"
Heterozygous
Heterozygous
Leu
Ser
Ser
Tyr
Phe
Thr
Arg
Met
Gly
Gly
Homozygous"
Heterozygous
Homozygoust
95
92
90
91
90
93
Wild-type allele at this locus undetectable on sequencing gel, but no LOH by codon 72 polymorphism (see Fig 7).
t Homozygous mutation verified by LOH determination (see Fig 7).
* C - T mutation occurs in intron 4 at position 13049 (Ref: Genebank sequence accession no. X54156).
this intronic mutation may possess biologic significance. Figures 2 and 3 showrepresentativeexamples of thedirect
sequencing data. Figure2 demonstrates two relapse T-ALL
samples (GCN and HAN, respectively) with heterozygous
missense mutations in exon 5. Interestingly, three separate
cases possess mutations at codon 135. Two of these cases
(FAN and ONN) possess a heterozygous mutation with nucleotide changefrom TGC to A/TGC ( 13YY6"?, whereas
a homozygous mutation with nucleotide change from TGC
TAC ( 13YYs
"3 was found in the peripheral blood-derived
leukemic cells of patient ASN (Fig 3).
The apparent heterozygous nature of the mutations summarized in Table 1 may be caused by contamination of the
leukemia samples with normal cells. Table 1 shows that at
the time of DNA extraction the percent blast cells present
in the patients' cell samples was at least
90%.The minority
(wild-type) alleles detected could not have been caused by
contamination with normal cells at the less than 10% level,
-
because in a mixture of mutant and wild-type DNA molecules the minority component is detectable on SSCP or sequencing gels only when present at 20%
the level or higher.'"
Hence, we conclude that the heterozygous natureof the mutations presented in Table 1 and in the corresponding figures
representmutations in oneallele of theleukemiacells,
whereas the other allele remains wild-type. To verify this
conclusion, we have tested whether unrearranged (ie, germline) T-cell receptor (TCR) @-chainDNA could be demonto
stratedinthesamplestested.Southernblotscontaining
BumHI-digestedgenomicT-ALLDNAwerehybridized
with a probe specific for the human T@ locus. All
6 samples
showed rearranged TCR@ bands, whereas
in only one (ONN)
patient sample could a band of apparent germline size be
shown (data not shown). Hence, the p53 mutations shown
in Table 1 represent bona fide heterozygous mutations that
are not caused by contamination
of the leukemia cell samples
with normal cells.
EXON 5
PLACENTA
GCN
C T ACGT A G
PLACENTA
C T A G
HAN
C T A G
c\
G
T f
I
I
*
CODON 141
CODON 175
Fig 2. DNA sequence identification of exon 5 mutations detected by SSCP. Shown are codon 141 and 175 heterozygous
mutations. Human placenta
DNA sewves as normal control.
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2925
p53 MUTATIONS IN RELAPSEACUTE T LEUKEMIA
EXON 5
PLACENTA
ASN
Fig 3. Verificationofmutations
in exon 5 that
were found by SSCP analysis. Shown are three codon 135 mutations in T-ALL patient samples ASN,
ONN, and FAN, as determined by directsequencing
of PCR-amplified DNA. Patient ASN was homozygous for the mutation; patient samples ONN and
FAN possess the same heterozygous mutations.
CODON 135
p53 mutation i s somatically acquired and is associated
with the recurrence of T-ALL. Ten of 36 relapse T-ALL
cases studied (28%) possess p53 mutations in the p53 gene.
Because these are random T-ALL patient samples collected
during a 5-year period. there is no means of determining
whether the mutations found are germline or somatically
acquired, nor what role, if any, p53 mutations play in the
recurrence of T-ALL. To determine the pathologic significance of p53 mutations in the recurrence (relapse) of TALL, a group of 6 "paired" T-ALL cell samples from
patients for whom we possess both diagnosis and relapse
samples were studied. PCR-SSCP and direct sequencing
showed that none of the samples taken at diagnosis possessed p53 mutations, whereas 3 of 6 (50%) of the "paired"
T-ALL samples studied harbored missense mutations at the
relapse phase of the disease (Table 2). Figure 4 shows that
a mutation was present in the leukemic cells of patient
SMN at relapse. This mutation was absent from SMN's
leukemic cells at diagnosis. PCR-direct sequencing data
identify the nature of this mutation as a heterozygous missense mutation with a nucleotide change from CGT to CTT
( 1 IOArE+ Leu). A heterozygous missense mutation with a
nucleotide change from GCC to ACC
(161""
was
found in the leukemic cells of patient SRN at relapse but not
at diagnosis (Fig 5). Furthermore, a homozygous missense
mutation was detected in the leukemic cells of patient BTN
at relapse but not at diagnosis. Figure 6 shows this mutation
as found by SSCP (arrow). Direct sequencing data establish
the nucleotide change at relapse, a codon change from CGG
to GGG (282Arg "ly).
We also studied whether p53 mutations were present in
the cells of a group of 8 T-ALL relapse patients whose
-
-
Table 2. p53 Mutations in Paired Diagnosis/Relapse T-ALL Patient Samples
Identification of Mutation
SSCP (exon1
Patient
AHNlD
AHNIR
BTNID
BTNIR
LMNID
LMNIR
SZNID
SZN/R
SRNID
SR NIR
SMNID
SMNm
4
5
6
7
Sequencing
8
I
+
+
9
Codon
Sequence
282
CGG
- GGG
161
GCC
- ACC
110
CGT
- ClT
Abbreviations: /D, patient sample obtained at diagnosis; /R, patient sample obtained at relapse.
Homozygouslheterozygous mutations, genotype verified by codon 72 polymorphism (see Fig 7).
aa Change
Arg
- Gly
- Thr
Arg - Leu
Ala
Genotype'
Homozygous
Heterozygous
Heterozygous
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2926
HSIAO ET AL
Exon 4
P
L
A
C
E
N
T
A/
B
T
N
D/
B
T
N
R/
L
M
N
D/
L
M
N
R/
S
M
N
D/
S
M
N
R/
A
H
N
D/
A
H
N
R
Fig 4. Identificationand localization of mutations in exon
SMN/D
G A T C
P
v
l
c r -
Y
L
-
SMN/R
G A T C
=x,
-
"
"
--=S"
~
r
T,
=
-=
r
-/cT
UT\
CODON 110
diseasewasrefractorytochemotherapeutictreatment.30.3'
p53 exons 4 through 9 were screened for mutations using
PCR-SSCPanddirectsequencing.Threeof
8 patients
(37.5%) in this group possess p53 mutations as detectedby
PCR-SSCP. The p53 mutations in the groupof refractory TALL cases all mapped to exon 5. Direct sequencing showed
a homozygous missense mutation in codon 135 witha nucleotide change from TGC to TAC (135cys'Ty?
in patient ASN
(Table 3). Leukemic cells of patients
SRN andHANharbored heterozygous missense mutations in codon 161 (GCC
to ACC,161 A'r '
and in codon175(CGCtoGGC,
175Arg'C'y), respectively (Table
3). Taken together, the presence of p53 mutations in 33%to 50% of relapse T-ALL
cells in the different groups of patient samples studied suggests that p53 gene mutation is associated with the tumorigenic progression of relapse T-ALL cells.
-
4. in "paired" diagnosislrelapse
T-ALL samples, and verification
of the mutation in codon 110 in
patient SMN at
relapse by direct
sequencing of PCR-amplifiedgenomic DNA.
DNA
extracted from
e human
normal
placenta was
used as control. Gel conditions
for SSCP analysis: 6% acrylamide gel with 10% glycerol.
/D and /R refer t o DNA samples
extracted the
from T-ALL cells of
same patients at diagnosis and
at relapse, respectively.
c
Loss of heterozygosity at the p53 locus is an infrequent
event in relapse T-ALL.. In human solid tumors, mutations
of the p53 gene are frequently accompanied by the loss of
heterozygosity of chromosome 17~13.1,the locus to which
thep53genemaps.Wehavepreviouslyreportedthat,
in
leukemia cell lines, both alleles of the p53 gene were often
independently mutated without concomitant loss of heterozygosity at the 17~13.1locus. To investigate whether loss
of heterozygosity of p53 is a frequent event during recurrence of T-ALL, we used the known polymorphism at codon
72 the
human
of
p53
This
polymorphism
enis
coded by sequences in exon 4 of the p53 gene.The frequency
of the 72"9- and the 72P"-encoding alleles (CGC and CCC,
respectively) in the human population is 0.68 and 0.32, respectively.36 Thus,44% of the human population are heterozygous at this locus, whereas 46% and
10%encode homozy-
Exon 5
P
Fig 5. Identificationand localization of mutations in exon
5, in "paired" diagnosislrelapse
T-ALL samples, and verification
of the mutationin codon 161 in
patient SRN at relapse by direct
sequencing of PCR-amplified genomic DNA. DNA extracted from
a normalhuman placenta and
DNA extracted from CEM cells
were used as normal and mutant
controls in SSCP analysis. Gel
condition for SSCP 4% MDE gel
with 10% glycerol.
L
A
C
E
S
N C Z
T E N
A M D
S
Z
N
~
A
H
N
D
A B B S S
H T T R R
N N N N N
~ D / R D I R
SRN/D
SRNm
G A TGCA T C
C
C
GA
CODON 161
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p53 MUTATIONS
ACUTE
IN RELAPSE
2927
T LEUKEMIA
EXON 8
H
T
B
2
G
Fig 6. SSCP analysis and direct sequencing of mutations in
exon 8 of ”paired” diagnosislrelapse T-ALL
samples.
Human
placenta DNA was used asa normal control (not shown) and
DNA extracted from the breast
carcinoma line MDA-MB-231
(HTB-26),“ which carries a mutation in p53 exon 8, was used as
a positive control for SSCP. Gel
condition for SSCP:4% MDE gel.
B
T
N
~
B
T
N
R
A
H
N
~
A
H
N
R
L L S
M M Z
N N N
I D R I
’,,LamL4U
S
Z
N
D
S
R
N
R
S
R
N
I
S
M
N
D
S
M
N
R I DGATC
R
m
.
m””.
”WvomqUBB,
“
”
-
”
.
-
m
GATC
I
d
-
..
L+*
CODON 282
gous (arg/arg)- or homozygous(pro/pro)-containingp53
relapse T-ALL patients, frequently harbor mutations in the
proteins, respectively.
p53 gene.23In contrast, T-ALL patient samples have been
We applied PCR-SSCP analysis of exon 4 to determine
r e p ~ r t e d ~to
’ . lack
~ ~ such mutations. This suggested that p53
in “paired” diagnosis
the polymorphic status at codon 72
mutations that are foundin cell linesmay have been induced
and relapse leukemia samples taken from the same patients. during in vitro establishmentof the lines. However, in vitro
Five of the 6 “paired” leukemia samples were heterozygous mutation of p53 during the establishment of acute leukemia
at codon 72 (4 of these are shown in Fig 7), whereas 1 of
cell lines seemed unlikely because we have found in other
the 6 “paired” patient samples,LMN, showed the homozyexperiments that, during establishment of multiple T-ALL
gous arg/arg genotype at this position (Fig 7). Only1 of the
cell lines, the p53 gene remained stably unaltered
when com3 leukemia cases that harbored a p53 mutation at relapse
pared with its state in the patients’ leukemia cells.27 This
(Table 2), patientBTN,also underwent loss of heterozygosin thecurrentexperimentsby
discrepancywasresolved
ity upon leukemia relapse, by losing the arg-encoding p53
screening 15 untreated (“diagnosis”) and 36 recurrent (“reallele. As can be seen, the band of the amplified 72P”’-encod-lapse”) T-ALL cases, 12 of which were donated, pairwise,
ingallelesofpatient
BTN’s relapsesampleis of double
bythesamepatientsatthediagnosisandattherelapse
intensity compared with the cases that were heterozygous at phases of their disease.
this codon. The status of codon 72 in the alleles carried by
Among the 15 diagnosis T-ALL cases studied here, none
the leukemia cell lines MOLT-4 (arg/arg) and CEM (arg/
possess p53 mutations in exons 4 through 9. Adding these
pro), which we have previously shown by immunoprecipita- 15
diagnosis
cases
to
those
reported
by
a total
of
tion to harbor alleles encoding homozygous (arg/arg) and
52 diagnosis T-ALL cases have now been reported to lack
heterozygous(arg/pro)p53proteins,respectively,”
are
p53 mutations, supporting the notion that mutation of the
shown as internal controls. Subsequent DNA sequencing has p53 gene is not a causal event in the induction of childhood
shown that the arginine allele was indeed lost during the
T-ALL. In contrast to diagnosis T-ALL cases, among 36
recurrence (relapse)of disease in patient BTN (Fig 7, right). relapse cases studied, 10 (28%) harbored mutations in the
p53 gene. Most(9 of 10)of these mutant p53 alleles specified
DISCUSSION
missense mutations that gave rise to nonconservative amino
Wehave previously shown that acute lymphoblastic Tacid exchanges, whereas1 occurred in intron 4. The majority
cellleukemiacelllines,all
ofwhichwerederivedfrom
of these mutations were mapped to the highly conserved, TTable 3. p53 Mutation inT-ALL Cases Refractoryto Chemotherapy
Identification of Mutation
SSCP (exon)
Patient
ASN
CJN
GENt
HAN
HJN
HMN
JRN
SRN
4
5
+
6
Change
7
Sequencing
8a8
Change
9 Sequence
Codon
TGC135
CysTAC
-
Homozygous
Tyr
CGC175
-ArgGGC
Heterozygous
Gly
161
Ala
GCC
-
- ACCHeterozygous - Thr
HomozygouJheterozygous mutations, verified by status of codon72 polymorphism.
t This patient‘s leukemia was refractow to chemotherapy, but relapse was not formally established.
Genotype*
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HSlAO ET AL
2928
Exon 4
M
B B
L C T T
T E N N
4 M I D I R I
0
A
H
N
D /
A
H
N
R I
L
M
N
D /
L
M
N
R I
S
R
N
D /
S
R
N
R /
S
Z
N
D /
S
Z
N
R
BTNlD
CTAG
m
CTAG
CODON 72
antigen binding region of the p53 protein,3*suggesting that
the mutations were biologically significant by conveying a
selective growth advantage to the cells so endowed. In recent
work (M. Hsiao. E. Dorn, J. Yeargin, and M. Haas, manuscript submitted), we have observed that mutated p53 genes
thathadbeen
cloned from relapse T-ALL cells and constructed into retroviral expression vectors indeed possess
dominant oncogenic activity whenintroduced into T-ALL
cells, as assayed by in vitro and in vivo criteria. Thus, p53
mutations in relapse T-ALL cells most probably play a biologically significant role in the disease.
The lack of p53 mutations in any of the "diagnosis"
samples suggests, but does not prove beyond a reasonable
doubt, that mutation of the p53 gene in relapse samples is
specifically associated with the progression of the leukemic
phenotype. To prove that p53 mutations in relapse leukemia
represent clonal evolution events, we compared the status of
the p53 gene in diagnosis and in relapse samples in a pairwise fashion. Six pairs of leukemia cell samples, each derived from the same patients at different points in the clinical
course, were studied for the status of the p53 gene. All six
diagnosis samples lacked detectable p53 mutations in exons
4 through 9, whereas in 3 of the 6 relapse samples (50%)a
mutation had occurred in the p53 gene (Table 2 ) . Clearly,
p53 mutation is associated with the clonal evolution of relapse leukemia in a significant fraction of cases. This result
supports our previous finding that a significant fraction of
acute leukemia cell lines, all derived from relapse cases,
possess mutations in the p53 gene.
In some human solid tumors mutation of one p53 allele
is often accompanied by the loss of the other p53
However, in acute leukemia cell lines loss of heterozygosity
is an infrequent finding:3 and in the acute leukemia patient
samples reported here, only 1 patient (BTN) among the 47
evaluable polymorphic cases displayed loss of heterozygosity of the p53 locus, as determined by the status of the
polymorphic marker at codon 72. Although in greater than
50% of acute leukemia cell lines each p53 allele carried an
independent mutation, in the current set of patient samples
only 1 relapse case possessed two independent mutations
Fig 7. Detection of allelic loss
at the p53 locus
in
"paired"
diagnosislrelapse patient samples by SSCP analysis
of the polymorphic
marker
at codon
72.
Cell linesMOLT4 and CEMwere
usedascontrols.
MOLT-4 cells
are homozygous I~odon72'~'"~).
whereas CEM cells are heterozygous172"g/72p") for thispolymorphic marker?' The leukemic
cells of patient BTN were observed to undergo loss of heterozygosity at the p53locus (left
panel), which was verified by direct sequencing(right panel).Gel
conditionsfor SSCP: 4% MD€ gel.
(FMN,Table 1). These two mutations occurred on the same
allele, as determined by molecular cloning (not shown). This
discrepancy between leukemia cell lines and patient relapse
samples suggests either that relapse leukemia cells possessing independent mutations in both alleles are favored
during establishment in vitro, or that, in the lines, the second
allele became mutated upon establishment.
Because the failure of chemotherapeutic treatment of acute
childhood leukemia is often caused by the development of
a drug-resistant clone, acute leukemias of patients who are
refractory to the induction of remission might possess mutated p53 genes, as suggested by work of Cabanillas et 81'"
and Chin et aL3' To test the possible correlation between
refractoriness toremission induction and p53 mutation in
acute T-cell leukemia, we tested a set of 8 refractory cases
for the status of p53. Three of the 8 cases (37%) possess a
mutation in the p53 gene (Table 3). Interestingly, in these 3
refractory cases, the mutations were all located in exon 5, a
hotspot of p53 mutations in human ~ancer.~"
The possibility
that these exon 5 mutations specifically affect the drug-resistant status of the leukemia cells raises interesting questions.
In each of the groups of relapse leukemia cases examined,
the incidence of cases found to harbor p53 mutations probably represents a lower limit. Both the SSCP and the direct
sequencing techniques fail to detect mutations that are present in less than 20% of the cell populations:' In addition,
mutations maybepresentin
exons other than exons 4
through 9 examined, or in introns. Alternatively, the frequency of mutations that we have found in relapse leukemias
may represent only a fraction of the cases in which the wildtype suppressor function of p53 is inactivated. Relapse cases
that lack detectable p53 mutations may have lost some of
the p53 functions by inactivation of the p53 protein through
complex formation with other cellular proteins, eg, mdm2.42.43 Studies of the inactivation of p53 function in relapse
leukemias by means other than mutations are in progress.
It is important to consider why acute leukemia patient
samples as well as cell lines infrequently display LOHat
the p53 locus, whereas human solid tumors often show LOH
at the p53 locus on chromosome 17p. The difference may
From www.bloodjournal.org by guest on June 18, 2017. For personal use only.
p53 MUTATIONS IN RELAPSE ACUTE T LEUKEMIA
lie in the nature of acute leukemia cells as compared with
cells of malignant solid tumors. The generation of malignant
human solid tumors requires a series of 6 to 8 or more
genetic alterations in the same ce11“; the malignant cells
are “transformed” and transplantable, and possess many in
vitro characteristics of “tumor cells.” In contrast, in acute
leukemias, malignancy is engendered by cells that have undergone fewer genetic alterations4 and that have few, if
any, characteristics of “tumor cells.”47 Specifically, acute
leukemia cells are “malignant,” ie, life-threatening, even
though they merely display the characteristics of differentiation-inhibited, proliferating precursor cells (ibid), and may
lack many or most of the in vitro criteria of “tumor cells,”
We propose that, in acute leukemia cells, mutation of each
p53 allele provides the leukemic cell with a growth advantage through the e l i n a t i o n of normal suppressor functions
and, especially, through the acquisition of dominant oncogenic functions (M. Hsiao, E. Dom, J. Yeargin, and M. Haas,
manuscript submitted, and Dittmer et ala). Thus, whereas
the acquisition of each dominantly acting mutated p53 allele
is significant for the potentiation of the leukemic phenotype
of acute leukemia cells, in solid tumors a fully tumorigenic
and metastatic phenotype necessitates the loss of each functional p53 allele, hence the frequent loss of the functional
allele through LOH.
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From www.bloodjournal.org by guest on June 18, 2017. For personal use only.
1994 83: 2922-2930
Nonhereditary p53 mutations in T-cell acute lymphoblastic leukemia
are associated with the relapse phase
MH Hsiao, AL Yu, J Yeargin, D Ku and M Haas
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