1. Art and Diseño

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p53 Mutations Are Associated With Resistance to Chemotherapy
and Short Survival in Hematologic Malignancies
By Eric Wattel, Claude Preudhomme, Bernard Hecquet, Michael Vanrumbeke, Bruno Quesnel,
Isabelle Dervite, Pierre Morel, and Pierre Fenaux
We analyzed the prognostic value of p53 mutations for response t o chemotherapy and survival in acute myeloid leukemia (AML), myelodysplastic syndrome(MDS), and chronic
lymphocytic leukemia (CLL). Mutations were detected by
single-stranded conformation polymorphism (SSCP) analysis of exons 4 t o 10 of theP53 gene, and confirmed by direct
sequencing. A p53 mutation was found in 16 of 107 (15%)
AML, 20 of 182 (11%) MDS, and 9 of 81 (11%) CLL tested. In
AML, three of nine (33%) mutated cases and 66 of 81 (81%)
nonmutated cases treated with intensivechemotherapy
achieved complete remission(CR) ( P = .005) and none of five
mutated cases and three of six nonmutated cases treated by
low-dose Ara C achieved CR or partial remission (PR) ( P =
.06). Median actuarial survival was 2.5 months in mutated
cases, and 15 months in nonmutated cases ( P < lo-‘). In the
MDS patients who received chemotherapy (intensive chemotherapy or low-dose Ara C), 1 of 13 (8%) mutated cases
and 23 of 38 (60%) nonmutated cases achieved CR or PR ( P
= .004), and medianactuarialsurvivalwas
2.5 and 13.5
months, respectively ( P C lo-’). In all MDS cases (treated
and untreated), the survival difference between mutated
cases and nonmutated cases was also highly significant. In
CLL, 1 of 8 (12.5%) mutated cases treated bychemotherapy
(chlorambucil andlor CHOP andlor fludarabine) responded,
as compared with 29 of 36 (80%)nonmutated cases ( P = .02).
In all CLL cases, survival from p53 analysis was significantly
shorter in mutatedcases (median 7 months) thanin nonmutated cases (median not reached) ( P < IO-’). In 35 of the 45
mutated cases of AML, MDS, and CLL, cytogenetic analysis
or SSCP and sequence findings showedloss of thenonmutated P53 allele. Our findings show that p53 mutations are
a strong prognostic indicator of response t o chemotherapy
and survival in AML, MDS, and CLL. The usual association
of p53 mutations t o loss of the nonmutated P53 allele, in
those disorders, ie, t o absence of normalp53 in tumor cells,
suggests that p53 mutations could induce drug resistance,
at least in part, by interfering with normal apoptotic pathways in tumor cells.
0 1994 by The American Societyof Hematology.
T
dominate in advanced stages of the disease and have been
correlated withshort survival in carcinoma of the breast,
prostate, lung, and s t o m a ~ h . ’ ~In‘ ’most
~ hematologic malignancies, p53 mutations also seem to be a late event in the
disease course: in CML, they are almost exclusively seen
after progression to acute leukemia4;in MDS, they predominate in patients with an excess of marrow blasts7and in CLL
in patients with Binet’s stage C disea~e.”~
In a recent work,
El Rouby et all’found a correlation between p53 mutations
and resistance to chemotherapy in CLL. However, no other
attempt at correlating p53 mutations to response to chemotherapy and survival in hematologic malignancies has been
made, to our knowledge.
In the past 3 years, we looked for p53 mutations in large
series of hematologic malignancies. In this report, we tried
to correlate the presence of those mutations to results of
treatment and survival.
HE p53 GENE IS a tumor suppressor gene often inactivated by deletion andor point mutation in most types
of solid tumors.’.’ In hematologic malignancies, P53 gene
mutations are found in 25% to 30% of Burkitt’s lymphoma”
and chronic myeloid leukemia (CML) in blast crisis: 15%
of chronic lymphocytic leukemia (CLL),3,’ and5% to 10%of
acute myeloid leukemia (AML): myeloysplastic syndrome
(MDS),’ and large cell non-Hodgkin’s lymphoma (NHL),’
but are very rare in acute lymphocytic leukemia (ALL, except in Burkitt’s ALL or in relapse)’ in multiple myeloma’”
and in follicular NHL (except after histologic progression).”
P53 gene mutations are generally missense mutations, involving almost exclusively exons 4 to 8 of the gene.’.’* They
are generally detected by single-stranded conformation polymorphism (SSCP) analysis of DNA or immunocytochemical
analysis of p53 in the nucleus of tumor cells because the
latter method can only detect p53 with a prolonged half-life,
which generally correspond to mutated p53.’
In solid tumors, P53 gene mutations are generally considered to be a late event in carcinogenesis because they preFrom the Service desMaladies duSang, CHU; Laboratoired’Himatologie A, Centre Hospitalier Universitaire; U,,,INSERM, Institut
de Recherche sur le Cancer; and the Department of Biostatistics,
Centre Oscar Lambret, Lille, France.
Submitted April 14, 1994; accepted July IO, 1994.
Supported by the Association de Recherche sur le Cancer, the
Comite‘ du Nord de la Ligue contre le Cancer, the Gefiuc, and the
Centre Hospitalier Universitaire of Lille.
Address reprint requests to P. Fenaux, MD, Service des Maladies
du Sang , CHU-l, Place de Verdun, 59037 Lille. France.
The publication costsof this article were defrayed in part by page
chargepayment. This article must therefore be hereby marked
“advertisement” in accordance with 18 U.S.C. section 1734 solely to
indicate this fact.
0 1994 by The American Society of Hematology.
0006-4971/94/8409-002$3.00/0
3148
MATERIALS AND METHODS
Patients
Between January 1991 and December 1993, welooked for p53
mutations by single-stranded conformation polymorphism (SSCP)
analysis of exons 4 to 10 of the P53 gene, a sensitive method for
the detection of P53 gene mutations,I8 in 503 cases of hematologic
malignancies diagnosed at our institution, including 107 cases of
AML, 93 cases of ALL, 182 cases of MDS, 81 cases of CLL, and
40 cases of myeloma. In those patients, results of SSCP and sequence analysis of the P53 gene have already been published in
part,5-7.9.~n.~9.20 Because p53 mutations were very rare inALLand
myeloma (3 of 93 and 1 of 40 cases, respectively) comparisons
between mutatedand nonmutated cases for initial characteristics,
response to chemotherapy and survival were only made in AML,
MDS, and CLL in the present study.
AML and MDS were classified according to French-AmericanBritish (FAB) criteria2’,2zand CLL according to Binet’s classificat i ~ n . ’No
~ documented MDS phase was seen in any of the cases of
AML. No familial history of Li Fraumeni syndrome or of clustering
Blood, Vol 84, No 9 (November l), 1994: pp 3148-3157
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31 49
p53 MUTATIONS AND RESISTANCETOCHEMOTHERAPY
of malignancy (especially hematologic) was found in the AML,
MDS, and CLL studied. Cytogenetic analysis was successfully performed at the time of DNA analysis in 103 of the 107 AML, 144
of the 182 MDS, and 40 of the 81 CLL using conventional banding
techniques. Complex cytogenetic abnormalities were defined by the
presence of at least three chromosome rearrangements. Cytogenetic
results, in patients with p53 mutations, have been published with
SSCP and sequencing data?”9.10.19.20
DNA was extracted from circulating leukocytes in acute leukemias
with greater than 60% blasts and in CLL, and from bone marrow
(BM) cells in other cases of acute leukemia and in MDS. DNA
samples were obtained at diagnosis in all cases of AML and MDS.
In CLL, DNA samples were obtained at diagnosis in 43 cases, and
during the disease course in 38 cases; 18 of the CLL had received
(or were receiving) chemotherapy when DNA samples were taken.
Treatment
AML. Patients were generally treated by conventional intensive
anthracyclin-cytosine arabinoside (Ara C) chemotherapy, according
to successive multicenter trials (LAM 86 and LAM 90 trials). Patients entering complete remission (CR) received consolidation chemotherapy with an anthracycline (or derivative) and Ara C, but a
few patients with an identical HLA sibling were allografted in first
CR. Elderly patients often received less intensive chemotherapy with
low-dose Ara C (10 mg/m2/12 h subcutaneously during 3 weeks,
followed by identical 14-day courses in case of response), but a few
of them in poor general condition received no treatment apart from
supportive care.
MDS. Treatment was not randomized, but based on age, general
condition, and disease course. Some relatively young patients with
refractory anemia with excess blasts (RAEB), RAEB in transformation, or chronic myelomonocytic leukemia (CMML) received intensive anthracyclin-Ara C chemotherapy before or after progression
toAML. Some older patients with RAEB, RAEB-T, or CMML
received low-dose Ara C (same dosage as for AML), whereas the
remaining patients received supportive care only or treatments other
than chemotherapy (androgens, retinoids, or growth factors). In
AML and MDS, complete remission (CR), partial remission (PR),
resistance to chemotherapy, and death in aplasia were defined according to cancer and leukemia group B (CALGP) criteria?“
CLL. Stage A patients with stable disease were untreated. Stage
B and C patients and stage A patients with progressive disease
were treated by one ofthe following approaches: continuous or
intermittent chlorambucil (with or without prednisone); cyclophosphamide, doxorubicin, vincristine, prednisone (CHOP) regimen with
low -dose adriamycin (25 mg/m2on day 1 of each course)23;fludarabine (25 mg/m2/d, in 5-day courses). Stage B and C patients generally received “intensive” chemotherapy (CHOP or fludarabine),
whereas stage A patients with progressive disease or elderly patients
generally received chlorambucil (often followed by CHOP or fludarabine in the absence of response). In CLL, CR was defined by
the absence of organomegaly, a lymphocyte count < 4 X lo9& a
granulocyte count > 1.5 X lo9& and normal BM examination on
core biopsies. PR was defined by a decrease of at least 50% in the
diameter of enlarged lymph nodes and a decrease of the lymphocyte
count by 75%.23
Analysis of p53 Gene Mutations
The methods used for the detection of p53 mutations have been
analyzed in detail elsewhere, and will only be summarized
here.5.7.19.20 For exons 5 to 9 of the P53 gene, three genomic regions
were amplified: region 1, encompassing exons 5 and 6 and intron
5 , and measuring 408 bp; region 2, encompassing exons 7 and 8
and intron 7, and measuring 610 bp. Because SSCP analysis seems
to require fragments of less than 400 bp,I8 region 2 was digested after
amplification and before SSCP analysis by Dra 1 enzyme because a
Dra 1restriction site is present in intron 7. This led to two fragments
encompassing exon 7 and exon 8, respectively, and measuring 392
and 218 bp;finally region 3, encompassing exons 8 and 9, and
intron 8, and measuring 398 bp was amplified. Exons 4 and 10 were
amplified separately.
After amplification of the DNA segment considered, I pL of the
reaction mixture for region 1, region 3,and exons 4 and IO was
mixed with 19 p L of 0.1% sodium dodecyl sulfate (SDS) 20 mmol/
L EDTA solution. For region 2, 1 pL of the reaction mixture was
first digested by 3 U of Dra 1 in 10 pL final-volume reaction, and
diluted in 10 pL of SDS EDTA solution. Then 3 pL of the diluted
reaction was mixed with 3 pL of a solution of 95% formamide 20
mmoVL EDTA, 0.05% bromophenol blue, and 0.05% xylene cyano],
heated at 8 0 T , and applied (2 pL/lane) to a 5% polyacrylamide gel
containing 90 mmoVL TRIS-borate pH 8.3, 4 mmoVl EDTA, and
10% glycerol. Electrophoresis was performed at 35 W for 5 to 6
hours at room temperature, with a fan for cooling.
For direct sequencing, asymmetric polymerase chain reaction using a 50 to 100 reduction of one of the primers was performed. The
resulting single-stranded DNA was purified and sequenced by the
dideoxychain termination method, using the sequenase kit (US Biochemical, Cleveland, OH) and analyzed on a polyacrylamide gel
containing 7 mol/L urea.
Statistical Analysis
The prognostic value of p53 mutations and other initial parameters
on response to chemotherapy and survival was analyzed for each
disorder (AML, MDS, CLL). Response rates to chemotherapy were
compared by Student’s and chi-square tests. Survival curves were
drawn using the Kaplan Meier method, and compared with the logrank test. Correlations between prognostic factors established by
univariate analysis were studied by analysis of variance or chi-square
test, depending on the nature of parameters. A multivariate analysis
of factors that had shown prognostic value for survival in univariate
analysis was also performed, using a Cox model.25
RESULTS
Ident$cation of p53 Mutations
AML. A P53 gene mutationwasdetectedby
SSCP in
16 of the 107 (15%) patients analyzed by SSCP (Table 1).
In those 16 cases, sequence analysis found a missense mutation in 11 cases, involving exon 4 (1 case), exon 5 (3 cases),
exon 6(1 case), exon 7 ( 2 cases), and exon8 (4cases). In the
remaining 5 cases, the mutation was astop codon mutation in
exon 5 in 1 case, a l-bp deletion in 2 cases (exon 4, l case;
exon 8, 1 case) a l-bp insertion in 1 case (in exon 7) and a
mutation at the splicing acceptor site preceding exon 8 in
the remaining case.
In 10 of the 16 mutated cases, cytogenetic analysis found
17pmonosomy, resulting from monosomy 17, del17p,
i(17q), or from unbalanced t(5;17)or t(7;17) translocations.26
Except in one case, 17p monosomywas part of complex
cytogenetic abnormalities. In the remaining 6 mutatedcases,
cytogenetic analysis found normal 17p in 3 cases and was
not successfully performed in 3 cases. In 3 of those 6 cases
(including two patients with cytogenetically normal 17p and
one patient where no successful karyotype was obtained),
SSCP and sequencing results showed complete disappearance of the residual germline bands, strongly suggesting loss
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3150
UPN
WATTEL ET AL
SexJAge
FAB Type/
Binet's
Stage
p53 Mutations
Loss of
Normal p53
Allele
Exon
Codon
Nucleotide
Deleted C (CTA +TA)
CGT TGT
GTG + ATG
AML
1209
541
20
F152
F167
F131
M2
M2
M2
8
8
8
264
273
272
C792
53
C961
F168
MI55
MI75
M4
M2
MO
4
5
5
110
175
166
111
410
FP3
MI54
M4
M2
5
7
1168
C319
498
1128
MI40
MI30
MI54
F180
MI85
MI61
M2
M2
M2
M1
M2
M2
CGT GGT
CGC CAC
TCA + TAA
(stop codon)
7
237
ATG ATA
Mutation at exon 8 splicing acceptor
site: AG AT
8
273
CGT CTT
5
162
ATC + AGC
5
135
TGC AGC
238
TGT TAT
7
193
CAT CGT
6
122
Deleted G (GTG GT)
4
178
452
F160
MI79
M6
M5
8
7
MDS
1380
662
285
MI57
MI53
MI53
RAEB
RAEB-T
RAEB
7
8
MI40
F130
MI76
MI74
F128
MI57
F165
MI56
FP5
MI14
F165
MI57
FI75
MI63
FD9
F150
MI78
RAEB-T
RAEB
RAEB
RAEB
RAEB-T
RAEB-T
RAEB
RAEB
RAEB
RAEB
RAEB-T
RAEB
RAEB-T
RAEB
RAEB
CMML
RAEB
5
8
7
7
5
8
8
7
5
7
7
7
8
5
5
8
6
MI67
F155
MI63
MI63
MI74
M167
FP3
FP0
MI62
C
B
B
C
A
C
A
C
C
8
4
5
7
6
5
8
8
8
434
14
54
210
1005
11
12
31
6
1118
55
4
C641
c439
C1270
692
C1290
CLL
29
35
C1080
16
C l 005
C1013
28
52
C1066
-
+
-+
-
+
+
-
-+
+
282
255-256
-
CGG TGG
Inserted A between
codon 255 and 256
+
-
238
TGT TAT
273
CGT CAT
p 53 rearrangement by
Southern analysis
CTG GTG
130
GAG + AAG
285
CGG CAG
285
TGC -t TAC
242
TGC + TGG
135
CGT CAT
273
CGT + CAT
273
ATG ATA
237
GTG CTG
173
CGG -t CAG
248
GGC AGC
245
246
ATG GTG
CCT + CAT
278
ACC TCC
139
ATC AGC
162
CC insertion
301
TAT + TGT
220
+
-
+
-+
+
+
+
-
+
281
130
239
219
135
273
273
273
GAC TAC
26-bp deletion
CTC + GTC
AAC AGC
CCC CTC
TGC TAC
CGT .+CAT
CGT
CTT
CGT + CTT
+
+
+
+
+
-
+
+
+
-
+
+
+
+
+
-
+
34
8
1
LD Arac
Anthr-AraC
LD Arac
LD Arac
Anthr-AraC
Resistance
Resistance
3
7
No treatment
Anthr-AraC
Anthr-AraC
LD Arac
LD Arac
Anthr-AraC
Anthr-AraC
Anthr-AraC
LD Arac
Resistance
CR
Resistance
+
+
Anthr-AraC
No treatment
LD Arac
No treatment
Anthr-AraC
Anthr-AraC
No treatment
Anthr-AraC
No treatment
Anthr-AraC
LD Arac
No treatment
LD Arac
No treatment
No treatment
LD Arac
LD Arac
Resistance
CLB
CHOP, Fludarabine
CLB
Fludarabine, CHOP
No treatment
CHOP, fludarabine
CLB, CHOP
Fludarabine
CHOP,
fludarabine
Resistance
Resistance
Response
Resistance
+
+
+
-
+
-
+
+
+
+
-
+
+
+
-
+
+
+
+
c
2
2
2
3
CR
CR
Resistance
Resistance
Death i n
aplasia
CR
+
+
+
+
(mod
Resistance
Resistance
Death in
aplasia
Resistance
Resistance
Resistance
Anthr-AraC
Anthr-AraC
Anthr-AraC
Anthr-AraC
No treatment
-
Survival
(mosl
Response
+
+
+
Treatment
Response
Duration
4
8
4
6
2
3
1
5
6.5
2.5
3
1
8
2
2
8
2
8
3
5
6
3
2
3
3
5
3
3
Resistance
Resistance
Resistance
Resistance
Resistance
Resistance
Resistance
Resistance
Resistance
Resistance
Resistance
Resistance
Resistance
Abbreviations: UPN, unique patient number; anthr-AraC, anthracycline-cytosine arabinoside; LD Arac, low-dose Ara C;CLB,
1
9
5
3+
9
9
4+
4
4+
1
31
8
1
chlorambucil.
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3151
p53 MUTATIONS AND RESISTANCE TO CHEMOTHERAPY
of the remaining nonmutated P53 allele. Thus, in 13 of the
16 mutated cases, it is highly probable that no normal p53
was synthesized in tumor cells. By contrast, 17p monosomy
was seen in only 7 of the 90 nonmutated cases that were
karyotyped.
MDS. Twenty of the 182 MDS (1 1%) studied had a p53
mutation detected by SSCP (Table 1). Mutations consisted
of a missense point mutation in 18 cases, involving exon 5
(5 cases), exon 6 ( 1 case), exon 7 (7 cases), and exon 8 (5
cases). One patient had a 2-bp (CC) insertion in exon 8.
The remaining patient had a rearrangement of the P53 gene
detectable by Southern analysis. Southern blot findings in
this patient have previously been p~blished.~’
As for AML, 12 of the 20 mutated MDS cases had cytogenetically detectable 17p monosomy, through monosomy 17,
del 17p, t(5;17), or t(7;17). In three additional patients, SSCP
and sequencing results showed disappearance of the residual
germline bands, also strongly suggesting loss of the remaining P53 allele in spite of cytogenetically normal 17p (1
case) or in the absence of cytogenetic data (2 cases). Thus,
there was probably no normal p53 in the malignant cells
in 15 mutated cases. All patients with 17p monosomy had
complex cytogenetic findings. 17p monosomy was seen in
only 4 of the 126 nonmutated cases that were karyotyped.
CLL. Nine of the 81 patients (1 1%) had a mutation:
missense mutation in 8 cases, involving exon 5 (2 cases),
exon 6 ( l case), exon 7 (1 case), and exon 8 (4cases), and
a 26-bp deletion in exon 4 (between codons 81 and 90) in
the remaining patient (Table 1).
Four of the 9 mutated cases had monosomy 17, and 3
additional mutated cases, where no cytogenetic analysis
was performed, had no residual wild-type bands by SSCP
and sequence analysis. This strongly suggested loss of the
remaining P53 allele and the absence of normal p53 in
tumor cells in those 7 cases. All patients with 17p monosomy had complex cytogenetic findings. None of the 35
nonmutated patients that were karyotyped had 17p monosomy.
Patient Characteristics and Outcome
AML. Comparisons between initial characteristics of the
16 mutated cases and the 91 nonmutated cases with followup data are shown in Table 2. Mutated cases, when compared
with nonmutated cases, were characterized by significantly
older age (mean, 60 v 49 years, P =.04), a higher incidence
of complex cytogenetic abnormalities (84% of the cases v
19%in nonmutated cases, P = l X
and of “unfavorable” cytogenetic abnormalities [ie, all cytogenetic abnormalities except t(8;21), inv(l6), and t(15;17)], 92% V 50%
in nonmutated cases, P =.01.
Of the 107 cases, 6 elderly patients in poor general condition received no chemotherapy and 101 were treated with
chemotherapy, including 14 mutated and 87 nonmutated
cases. Nine mutated cases received intensive chemotherapy,
but only 3 (33%) achieved CR, of short duration (4, 4, and
5 months, respectively). By comparison, in nonmutated
cases, 66 of the 81 (81%) patients treated by intensive chemotherapy achieved CR ( P =.005), and median CR duration
was 13 months. Five mutated cases were treated with low-
Table 2. Initial Characteristics and Outcome of AML Patients with
a p53 Mutation, as Compared With Nonmutated Cases
No. of Cases (%)
Nonmutated
Mutated
Cases
No. of patients
Mean age (range)
Male/female
FAB type
MO
M1
M?
M3
M4
M5
M.3
M7
Karyotype
No. patients analyzed
Normal
t(8;21),inv(16),t(15;17)
+8, -7
(single)
Others (single)
Complex abnormalities
Results of treatment
No. of treated patients
Intensive chemotherapy
CR (%)
Resistance
Death in aplasia
Median CR duration
(mod
Low-dose Ara C
CR
PR
Resistance
Total responses (%)
Median actuarial survival
(mos)
All treated patients
Intensive chemotherapy
Low-dose Ara C
16
60 2 17
(30-85)
9i7
1
1
10
0
2
1
1
0
(62.5%)
P
Value
Cases
91
49 5 19
(3-81)
51l40
3
18
27
6
17
16
3
1
.04
NS
NS
(30%)
13
1 (8%)
0
0
1
1 1 (84%)
90
31 (34%)
14 (16%)
10
18
17 (19%)
14
9
3 (33%)
4
2
4
87
81
66
10
5
13
5
0
0
5
3 (21%)
6
2
1
3
69
2.5
5
3
15
15
8.5
(81%)
10-4
,005
.06
(79%)
10-3
40-5
,001
.03
Abbreviation: NS, not significant.
dose Ara C, but none achieved CR or PR, as compared with
3 of the 6 nonmutated cases treated by this approach (P
= .06).
In the 101 patients who received chemotherapy, actuarial
survival was significantly shorter in mutated cases (median,
2.5 months) than in nonmutated cases (median, 15 months)
(P <
(Fig 1). The difference was still significant when
the analysis was restricted to patients treated by intensive
chemotherapy and to patients treated by low-dose Ara C,
respectively (Table 2).
Apart from p53 mutations, age and karyotype were the
only prognostic factors of response to chemotherapy and of
survival in treated patients; older age and complex cytogenetic abnormalities (versus other karyotypes) were associated with a lower response rate to chemotherapy (P <
and P <
respectively) andwith shorter survival (P <
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WATTEL ET AL
3152
Table 4. Initial Characteristics and Outcome of MDS Patients With
a p53 Mutation, as Compared With Nonmutated Cases
lil
loo
00
!S
All patients
No. of patients
Median age (range)
20
- 7
””-”,Mutation
I
I
0
0
1
I
1
10
20
30
I
,
40
,
,
,
,
50
60
70
80
,
I
90 100
Time (months)
Fig 1. Actuarial survival of the l01 AML patients treated by chemotherapy; mutated cases (n = 14) versus nonmutated cases (n =
87) ( P < 10-9.
and P <
On the other hand, cytogenetics hadno
significant prognostic value for response to chemotherapy
and survival if patients with “unfavorable” karyotypes (as
defined above) were compared with patients withnormal
karyotype or t(8;21), inv(16), t(15;17). FAB classification
also had no prognostic value. In treated patients, a significant
correlation was found between p53 mutations and a complex
karyotype ( P <
and
to a lesser extent between p53
mutations and age ( P =.03). In Cox’s multivariate analysis,
presence versus absence of a complex karyotype emerged
as the most powerful prognostic factor of survival, followed
by presence of a p53 mutation and age (Table 3).
MDS. As seen in Table 4, mutated cases of MDS were
characterized by significantly lower age (mean, 57 v 65
years) ( P =.04); a higher incidence of high-risk MDS, ie,
RAEB and RAEB-T ( P = 5 X
a higher incidence of
unfavorable karyotypes, ie, all abnormal karyotypes except
isolated del 5q (89% v 34%) ( P
and a higher incidence of complex cytogenetic abnormalities (83% v 16%)
(P <
Table 3. Multivariate Analysis of Prognostic Factors
for Survival (Cox Model)
Characteristic and Order of Entrance
in Regression
PValue
Chi-square
Model
AML (treated patients)
( 1 ) karyotype
(2) p53 mutations
(3)age
MDS (treated patients)
,008
( 1 ) karyotype
(2)p53 mutations .02
22.9
20.1
5q+8,
(single)
-7
Others (single)
Complex abnormalities
Progression to AML (%)
Median actuarial survival
(mod
Patients treated with
chemotherapy
No. of patients
Mean age
Male/female
FAB type
RA
RARS
CMML
RAEB
RAEB-T
Karyotype
No. cases analyzed
Normal
Nonmutated
Cases
20
57 t 18
( 14-79)
1218
162
65 2 14
( 19-89)
98/64
0
0
1
13
6
18
2
0
0
1
15 (83%)
1 1 (55%)
3
13
54 2 18
914
5q
+8,
-7
(single)
Others (single)
Complex abnormalities
Results of treatment
Intensive chemotherapy
CR
Resistance
Death in aplasia
Median CR duration
Low dose Ara C
CR
PR
Resistance
Total responses (%)
Median actuarial survival
(mod
7
1
6
0
6
0
0
6
1 (8%)
2.5
P Value
.04
NS
29
7
57
50
19
5x10‘
126
78
5
10
13
20
29
27
10
(16%) <l0
(18%) .02
<l0
38
57 -t 15
2411 4
NS
NS
0
0
9
18
11
NS
31
15
0
4
1
11
.01
21
13
7
1
10
17
3
7
7
23 (60%)
13.5
.03
.01
,004
Abbreviation: NS, not significant.
13.1
6.9
5.2
CLL (all patients)
10(1)p53 mutations
,005
(2)karyotype
1.2 x
1 ob
3x
Malelfemale
FAB type
RA
RARS
CMML
RAEB
RAEB-T
Karyotype
No. cases analyzed
Normal
Mutated
Cases
24
7.8
Seven of the mutated cases received noChemotherapy.
Seven received intensive anthracyclin-Ara C chemotherapy:
one (14%) achieved CR, of 3-month duration, but six had
resistant disease. By comparison, 21 of the nonmutated cases
From www.bloodjournal.org by guest on February 6, 2015. For personal use only.
3153
p53 MUTATIONS AND RESISTANCE TO CHEMOTHERAPY
received intensive chemotherapy, of whom 13 (62%)
achieved CR (P =.03), and median CR duration was 10
months. Six of the mutated cases were treated with low-dose
Ara C, but they all had resistant disease, whereas 10 of the
17 nonmutated cases (59%) treated with low-dose Ara C
achieved CR or PR (P =.01). Overall, one mutated case
(8%) responded to chemotherapy, as compared with 23 of
38 (60%) of the nonmutated cases (P =.W).
Eleven of the mutated cases (55%) progressed to AML,
as compared with 32 (18%) of the nonmutated cases (P
= .02). Actuarial survival was significantly shorter in mutated
cases, as compared with nonmutated cases, for all patients
Table 5. Initial Characteristics and Outcome of CLL With a p53
Mutation, as Compared With Nonmutated Cases
No. of Patients (%)
Nonmutated
Mutated
Cases
All patients
No. of patients
Mean age (range)
M/F
Binet’s stage
A
B
C
Karyotype (40 cases)
Normal
Single abn
Complex abn
Median actuarial survival
(mod
Patients treated with
chemotherapy
No. of patients
Mean age
M/F
Binet‘s stage
A
B
C
Karyotype (20 cases)
Normal
Single abnormalities
Complex abnormalities
Results of treatment
Chlorambucil
PR
No response
CHOP and/or fludarabine
CR
PR
No response
Total responses (%)
Median actuarial survival
(mod
9
66 ? 6
(55-73)
6/3
2
Cases
72
64 ? 10
(37-85)
4713 1
NS
NS
52
7
13
.01
27
5
2 X 10-4
1
4
3
2
5
0
8
64 rt 5
513
1
-30-5
36
63 ? 9
21/15
NS
NS
.03
2
5
23
5
8
0
14
10-3
0
4
2
3
18
14
1
2
6*
0
0
6
1 (125)
6.5
I
0
2
2
4
3
n
4
8
~
?
1
~
Time (months)
100
NR
7
A
1
20
No mutation
!, Mutation
0
B
0
10
20
30
40
50
60
70
80
90
Time (months)
Fig 2. (A) Actuarial survival of the 182 MDS cases (treated or
untreated); mutated cases (n = 20) versus nonmutated (n = 162)
cases ( P lo-‘). (B) Actuarial survival of the 51 MDS patients who
received chemotherapy; mutated cases (n = 13) versus nonmutated
cases (n = 38) ( P <
0
4
21t
3
12
6
29 (80)
34
.02
<10-4
Abbreviations: NR, not reached; NS, not significant.
One patient receivedCHOP after resistance to chlorambucil.
t Three patients received CHOP or fludarabine after resistance to
chlorambucil.
(median, 3 v 27 months; P < lo-’)and for patients who
received chemotherapy (median, 2.5 v 13.5 months; P <
lo-’) (Fig 2, A and B).
In the patients who received chemotherapy, presence of a
complex karyotype was the only other prognostic factors of
response to treatment (P =.04) and of survival (P <
However, cytogenetic analysis had no prognostic value for
response to treatment and survival if patients with “unfavorable” karyotypes (as defined above) were compared to patients with normal karyotype or del 5q. Age and FAB classi-
From www.bloodjournal.org by guest on February 6, 2015. For personal use only.
WATTEL ET AL
3154
fication also hadno
prognostic value for response to
treatment and survival in the patients who received chemotherapy. As for AML, p53 mutations were highly correlated
In Cox’s multivariate
to a complex karyotype ( P <
analysis, complex cytogenetic findings also emerged as the
most powerful prognostic factor of survival in MDS patients
who received chemotherapy (Table 3).
In all the 182 MDS patients, FAB classification ( P <
IO-‘) and complex karyotype ( P < lo-‘) had prognostic
value for survival in addition to p53 mutations. p53 mutations were strongly correlated to karyotype and FAB classification, as seen above.
CLL. Three of the nine mutations were found at diagnosis, and six in DNA samples obtained 9 to 48 months after
diagnosis (median, 21 months). Before DNA analysis, two
of the six cases had received chlorambucil, and in two cases,
progression from stage A to C had occurred. In nonmutated
cases, DNA samples were taken at diagnosis in 40 cases,
and after 6 to 196 months (median, 30) in the other 32
patients. Sixteen of them had received, or were receiving
treatment (chlorambucil in 12 cases and CHOP in 4 cases)
when DNA samples were collected.
As seen in Table 5 , mutated CLL were characterized at
the time of DNA analysis by a higher incidence of B and C
Binet’s stages (78% v 28% in nonmutated cases, P =.01)
and by a higher incidence of abnormal karyotypes (100% v
23%, P = 2 X IO-‘).
One of the nine mutated cases remained stable and required no treatment. The other patients received chlorambucil and/or CHOP, andor fludarabine. Only one of the eight
mutated cases who received chemotherapy responded (to
chlorambucil). By comparison, 36 nonmutated cases did not
require therapy. Thirty-six cases required therapy, and29
cases (80%)responded. The difference in response rate with
mutated cases was significant ( P =.02).
In the 8 1 CLL cases, median actuarial survival from DNA
analysis was 7 months in mutated cases and not reached in
nonmutated cases ( P <
(Fig 3). The difference was
l00
00
“p’...
I
l
- L-
. .
l
I
I
“l
-
80
-
5
40
-
I
-
I
I
Mutation
I
I”””“”
I
I
E
No mutation
l
I
A
.->
m
v)
20
I
~
0 ,
0
I
I
5
10
15
I
I
I
20 25
30
35
,’
I
1
I
I
40 45 50
55
I
1
60
Time (months)
Fig 3. Actuarial survival of the 81 CLL patients (treated oruntreated); mutated cases (n = 9) versus nonmutated cases In = 72) (P
< 10-5).
also significant when the analysis was restricted to treated
cases (Table 5).
No prognostic factor of response to treatment other than
the presence of a p53 mutation was seen. However, abnormal
cytogenetic findings were correlated with shorter survival ( P
= 2 X 10”) in treated patients.
In the 81 CLL cases, Binet’s stage C ( P =.04) and abnormal karyotype ( P = 1 X
were significantly associated
with short survival. Correlations between p53 mutations and
advanced Binet’s stage ( P =.01) and p53 mutations and
abnormal karyotype ( P = l X
were seen. In Cox’s
multivariate analysis, p53 mutations emerged asthemost
significant prognostic factor of survival inall CLL cases,
followed by karyotype (Table 3).
DISCUSSION
In the present study, with additional patients tested, the
incidence of p53 mutations remained similar to thatobserved
in our initial papers, and reported by other groups: 10% to
15% in AML and CLL,3.s.’9.27.2R
14o to 3% in ALL (if Burkitt’s ALL and relapsing patients are not included), and myeloma.y~’0~2Y
In MDS, the incidence of 11% of mutations was
higher than the 3% incidence we had initially reported.’ This
was probably because of the fact that, after our first report,
we analyzed almost exclusively high-risk MDS cases (RAEB
and RAEB-T). As in our earlier reports and in the literature,
p53 mutations were generally missense mutations involving
exons 4 to 8 of the gene. Because p53 mutations were rare in
multiple myeloma and ALL, analysis of correlations between
p53 mutations and response to chemotherapy and survival
was restricted to AML, MDS, and CLL.
Twenty-six of the 45 mutated cases of AML, MDS and
CLL had cytogenetically detectable 17p monosomy andthus,
probable loss of the normal residual P53 allele. In 9 additional mutated cases where no cytogenetic analysis was performed or no 17p monosomy was detected by cytogenetic
analysis, SSCP, and sequence analysis showed loss of bands
corresponding to the nonmutated P53 allele, also strongly
suggesting loss of this allele. These findings confirmed the
correlation between p53 mutationand deletion of the remaining P53 allele our group and other groups had observed
in hematologic malignan~ies.~”.~~.~”.’~
As a consequence, in
those disorders, no normal p53 is synthesized by the malignant clone in most of the mutated cases.
In AML, MDS, and CLL, p53 mutations were significantly
associated toknown high-risk factors. These included, in
AML, the absence of “favorable” karyotypes [ie, t(8;21),
t( 15;17), inv( 16)],and a much higher incidence of “unfavorable” karyotypes, particularly of complex cytogenetic abnormalities. In MDS, mutated cases were characterized by
a higher incidence of RAEB and RAEB-T, the absence of
RA and RARS, and also a much higher incidence of abnormal karyotypes, particularly of complex cytogenetic findings. In CLL, mutated cases predominantly had Binet’s stage
B and C disease, and also complex cytogenetic findings. In
other reports that correlated p53 mutations with clinical and
hematologic features, mutations also predominated inpatients with high-risk features, particularly in CLL, large cell
lymphoma and MDS.3.X,i2
Furthermore, in CML, p53 muta-
From www.bloodjournal.org by guest on February 6, 2015. For personal use only.
p53 MUTATIONS AND RESISTANCETOCHEMOTHERAPY
tions were almost exclusively seen after progression to
AML4; in follicular lymphoma, they were rarely seen before
progression to large cell lymphoma''; in ALL (with the exception of Burkitt's ALL), they predominated in relapsing
patient^.'^.^'
The fact that p53 mutations are mainly seen in advanced
or relapsing hematologic malignancies suggests that they are
associated with poor response to chemotherapy. However,
to our knowledge, very few studies have addressed this issue.
In several types of solid tumors, p53 mutations (or p53 overexpression detected by immunocytochemistry) have been associated with poor prognostic factors, including a large tumor mass and/or a highly proliferative tumor, and short
s ~ r v i v a l . ' ~ "However,
~ . ~ ~ if two reports correlated p53 overexpression to resistance to hormone therapy in prostatic carcinomaI4and radiation therapy in breast car~inoma:~respectively, no analysis of response to chemotherapy in relation
to p53 mutation or overexpression was made in those papers.
Recently, El Rouby et all7 found that in CLL, the response
rate to chemotherapy (chlorambucil, fludarabine, or combination chemotherapy) was very low in patients with a p53
mutation (one of seven responses) as compared with patients
without p53 mutation (27 of 29 responses). The prognostic
value of p53 mutations for response to chemotherapy persisted after adjustment for risk factors (including age, sex,
and Rai's stage). In this report, we clearly confirmed the
prognostic value of p53 mutations for response to chemotherapy in CLL. p53 mutations were correlated with disease
stage (according to Binet's classification) and with abnormal
karyotypes (especially complex cytogenetic findings), but
p53 mutations emerged as the strongest prognostic factor for
survival in Cox's multivariate analysis.
In AML, p53 mutations were associated with significantly
poorer response to intensive chemotherapy and to low-dose
Ara C, and the only three mutated cases that responded to
chemotherapy had short remission. The survival difference
between mutated and nonmutated cases was highly significant. In the mutated group, which included more elderly
patients, a higher proportion of cases received low-dose Ara
C, which is less effective than intensive chemotherapy in
AML.34However, the survival difference between mutated
and nonmutated cases persisted when patients treated by
intensive chemotherapy and low-dose Ara C were analyzed
separately. A high correlation between p53 mutations and
complex cytogenetic findings was found, and in Cox's multivariate analysis, complex karyotype emerged as the most
powerful prognostic factor for survival before p53 mutations.
Previous studies had established the prognostic value of cytogenetics in AML, and especially the poor prognosis of
complex cytogenetic finding^.^^.^^
As for AML, MDS with a P53 gene mutation had significantly lower response rates to chemotherapy and shorter
survival than nonmutated cases. p53 mutations were also
strongly correlated to the presence of a complex karyotype,
and complex cytogenetic findings emerged as the strongest
prognostic factor of survival by multivariate analysis. Our
previous experience had shown that karyotype was a major
independent prognostic factor of response to intensive chem ~ t h e r a p yand
~ ~ of survival3sin MDS.
3155
Our present findings showed that the presence of p53
mutations in the hematologic malignancies we studied was
a strong prognostic factor of response to chemotherapy,
whichwas highly correlated to the presence of complex
cytogenetic findings. Thus, it is unclear whether p53 mutations were one of the causes of resistance to chemotherapy,
because they occurred in a context of numerous other genetic
abnormalities that could have played, rather than p53 mutations, a major role in this resistance. On the other hand,
p53 protein seems to be involved in DNA repair, and its
inactivation increases the probability of mutations of other
genes.39Therefore, p53 mutations could have been the cause
rather than the consequence of the multiple chromosomal
abnormalities to which they were associated.
If p53 mutations were one of the factors of resistance to
chemotherapy in the diseases we studied, the mechanisms
whereby p53 mutations induced resistance to chemotherapy
can only be hypothesized. First, it has been shown that normal p53 suppressed the multidrug resistance (mdrl) gene
promoter, whereas mutated p53 could stimulate it.40Expression of the mdr, gene has been correlated to resistance to
chemotherapy inmany tumor types, including AML4' and
MDS.4' Therefore mutated p53, by activating mdr, expression, could interfere with response to chemotherapy. However two recent reports in AML43 and CLL" and our report
in MDSMfailed to show a correlation between p53 mutations
and mdr, gene expression. A second hypothesis can be made
by considering the role of p53 in programmed cell death
(apoptosis). It has recently been suggested that some chemotherapeutic agents, including anthracycline derivative^^^ and
Ara CM could induce leukemic cell death, at least in part,
by triggering apoptosis. p53 appears to be required for the
induction of apoptosis induced by irradiation or heat shock
in cell lines.47Total absence of p53 through mutation of one
P53 allele and loss of the other P53 allele, as found in most
of our mutated cases, could have explained their resistance
to chemotherapy. Because loss of only one allele of the
wild-type p53 already can induce increased resistance to
apoptosis>' this explanation could also hold for patients who
had retained one normal P53 allele.
Finally, the role of p53 mutations in drug resistance could
be regarded as minor in hematologic malignancies because
p53 mutations are, overall, relatively rare in those disorders.
On the other hand, p53 mutations are frequently seenin
relapsing hematologic malignancies or in cell lines
etablished from leukemic patients in relapse.48Using a very
sensitive method of p53 mutation detection, Wada et a148
found that the p53 mutations present in several leukemic
cell lines were already present at diagnosis invery small
proportion of cells andin a larger percentage of cells in
relapse. The same type of clonal expansion of p53 mutant
cells during disease course had also been observed in brain
tum0rs.4~Therefore, the possibility exists that a relatively
large number of leukemias (and possibly of other hematologic malignancies) carry a very small cell population with
mutated p53 at diagnosis, undetectable by conventional
methods (especially SSCP). This population could be resistant to chemotherapy, which would facilitate relapse with a
predominantly p53 mutated cell population. If those findings
From www.bloodjournal.org by guest on February 6, 2015. For personal use only.
31 56
WATTEL ET AL
were confirmed, therole of p53 mutations in drug resistance
in hematologic malignancies wouldbe greater than expected.
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From www.bloodjournal.org by guest on February 6, 2015. For personal use only.
1994 84: 3148-3157
p53 mutations are associated with resistance to chemotherapy and
short survival in hematologic malignancies
E Wattel, C Preudhomme, B Hecquet, M Vanrumbeke, B Quesnel, I Dervite, P Morel and P
Fenaux
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