Vol. 3, 523-530, April 1997 Clinical Cancer Research 523
Incidence and Timing of p53 Mutations during Astrocytoma
Progression in Patients with Multiple Biopsies
Kunihiko Watanabe,’ Kazufumi Sato,
Wojciech Biernat, Osamu Tachibana,
Klaus von Ammon, Nobuyoshi Ogata,
Yasuhiro Yonekawa, Paul Kleihues, and
Hiroko Ohgaki2
Unit of Molecular Pathology, IARC. 150 cours Albert Thomas, 69372
Lyon Cedex 08. France 1K. W.. K. S.. W. B., 0. T.. P. K.. H. 0.1, and
Institute of Neuropathology [P. K.l and Department of Neurosurgery
[K. �‘. A., N. 0.. Y. Y.]. University Hospital. 8091 Zurich.Switzerland
ABSTRACT
Mutations of the p53 tumor suppressor gene are a
genetic hallmark of human astrocytic neoplasms, but
their predictive role in glioma progression is still poorly
understood. We analyzed 144 biopsies from 67 patients
with recurrent astrocytoma by single-strand conforma-
tion polymorphism and direct DNA sequencing. We found
that 46 of 67 patients (69%) had a p53 mutation in at least
one biopsy. In 41 of these (89%), the mutation was al-
ready present in the first biopsy, indicating that p5.3
mutations are early events in the evolution of diffuse
astrocytomas. Double mutations of the p5.3 gene were
observed in three tumors and also present from the first
biopsy. Of 28 low-grade astrocytomas with a p53 muta-
tion, 7 (25 %) showed loss of the normal allele in the first
biopsy. The allele status remained the same in 95 % of the
cases, irrespective of whether the recurrent lesion had the
same or a higher grade of malignancy. Progression of
low-grade astrocytomas to anaplastic astrocytoma or glio-
blastoma occurred at a similar frequency in lesions with
(79%) and without (63%) p53 mutations (P 0.32),
indicating that this genetic alteration is associated with
tumor recurrence but not predictive of progression to a
more malignant phenotype. However, the time interval
until progression was shorter in patients with low-grade
astrocytomas carrying a p53 mutation (P = 0.055).
INTRODUCTION
Low-grade astrocytomas (WHO grade II) are well differ-
entiated and grow slowly but show a consistent tendency to
Received 9/6/96; revised 12/13/96; accepted 12/19/96.
The costs of publication of this article were defrayed in part by the
payment of page charges. This article must therefore be hereby marked
advertise,nent in accordance with 18 U.S.C. Section 1734 solely toindicate this fact.
I Present address: Department of Neurosurgery. Dokkyo UniversitySchool of Medicine, Mibu Tochigi 321-02. Japan.2 To whom requests for reprints should be addressed. Phone: 33-472-73-85-34; Fax: 33-472-73-85-64.
diffusely infiltrate the surrounding brain tissue (1, 2). Therefore,
they almost invariably recur and this is often associated with
progression to higher malignancy, i.e. , anaplastic astrocy-
toma (WHO grade III) or glioblastoma (WHO grade IV).
Astrocytoma progression is associated with sequentially ac-
quired multiple genetic alterations (3, 4). p53 Mutations are
considered an early genetic alteration in the evolution of
diffuse astrocytomas since they occur at a similar overall
incidence of 24-34% in low-grade anaplastic astrocytoma
and glioblastoma (3). However, recent studies indicate that
the frequency ofp53 mutations is much higher (58-83%) in
low-grade astrocytomas which progress to glioblastoma (5,
6). The present study focuses on recurrent low-grade astro-
cytoma with and without evidence of progression. The ob-
jective was to assess the frequency ofp53 mutations in these
two subsets of low-grade astrocytomas and to determine
whether the presence of p53 mutations or p53 protein accu-
mulation is a predictive criterion for clinical outcome, in
particular, the time interval until progression. In addition, we
investigated anaplastic astrocytomas with and without pro-
gression to glioblastoma, since it has been postulated that
low-grade astrocytomas recurring as anaplastic astrocytoma
and those recurring as glioblastoma follow different genetic
pathways (7). Finally, this study addresses the question of the
timing of p53 mutations in the evolution of astrocytic brain
tumors.
MATERIALS AND METHODS
Tumor Samples. The surgical specimens of astrocyto-mas were obtained from patients treated in the Department of
Neurosurgery, University Hospital (Zurich, Switzerland) be-
tween 1974 and June 1994. Astrocytomas were graded ac-
cording to the WHO classification into low-grade (usually
fibrillary or gemistocytic) astrocytoma (grade II), anaplastic
astrocytoma (grade III), and glioblastoma (grade IV; Refs. 1
and 2). Patients were divided into five groups: progression
from low-grade astrocytoma to anaplastic astrocytoma
(groups II -p III, 16 patients), from low-grade astrocytoma to
glioblastoma (groups II -� IV, 18 patients), and from ana-
plastic astrocytoma to glioblastoma (groups III -� IV, 10
patients), recurrence from low-grade to low-grade astrocy-
toma (groups II -� II, 13 patients), and from anaplastic to
anaplastic astrocytoma (group III -� III, 10 patients). Thirty
patients were females and 37 were males. The mean age at
first operation was 30.5 ± 13.3 years in group II -p II, 34.6 ±
9.6 years in group II -� III, 33.7 ± 8.4 years in group II -�
IV, 38.8 ± 13.7 years in group III -� III and 39.1 ± 16.7
years in group III -� IV (Table 1 ). The mean observation
period from the first biopsy to recurrence was 84.1 ± 13.2
months in group II -� II, 89.7 ± 21 months in group II -� III,
68.7 ± 8.8 months in group II -� IV, 41.1 ± 4.1 months in
group III .-� III. and 37.1 ± 8.1 months in group III -� IV.
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Table 1 p53 Mutationsa in recurrent astrocytomas
Tumor
Interval between operations (mo)” p53 Mutation
1st 2nd 3rd 4th Nucleotide Amino acidCase Age/sex localization op. op. op. op. TLD Exon Codon substitution substitution
Grade II .-� II1 4/M BG II 1511 43 -2 9/M T II 5511 124 -3 24fF T II 59 11+ 63 6 194 CT)’ -+ CGT Leu -* Arg4 261M FT II 6 II 23 II >43 -5 27fF F 11+ 23 11+ >43 5 175 CGC -� CAC Arg -� His6 3lfM F II 44 II >50 -7 331M T 11+ 14 11+ 205 8 282 COG -+ TGG Arg -* Trp8 341M
9 35/F
F
TO
11+11+II
60
6064
11+11+II
91
91
78
7
8
-
248
273
CGG -+ CAGCGT -� TGT
Arg -* GlnArg -� Cys
10 381M F 11+ 94 11+ > 131 7 248 COG -� CAG Arg -� Gblb 40/F
12 42/F
T
F
11+11+II
59
5953
11+11+II
82
82
97
7
8
-
245
282
GGC-+AGC
CGG -f TGGGly-�Ser
Arg -* Trp
13 54fF T 11+ 25 11+ 43 5 15 1 CCC -� TCC Pro -* 5crGrade II -* III
14 25/M TO II 48 II 21 III >69 -15 251M F II 27 III 3 III 46 -16 26/M F II 63 III 13 III 97 -
17 271M TP 11+ 19 111+ 30 8 273 CGT -� TGT Arg -� Cys18 29/F T 11+ 45 111+ nd. 8 265 CTG - CCG Leu -� Pro19 29/F FT II 35 III- 45 8 287 GAG -� GGG Glu -� Gly20 30/M
21 33/M
P0
F
11+11+II-
36
1 16
(III)(III)II- 14 II- 42 III-
nd.nd.
> 174
878
277238273
TGT -* 1TFTGT -� CGTCGT -* TGT
Cys -� PheCys -� ArgArg -� Cys
22 33fF TO (II) 173 II 27 111+ 298 8 280 AGA -+ AAA Arg -+ Lys23 35/F F II 52 111+ 128 8 273 CGT -� TGT Arg -#{247} Cys24 351M T II 34 III 53 -25 37fF F 11+ 14 111+ 22 8 273 CGT-�TGT Arg-�Cys26 39fM F 11+ 24 111+ 26 8 273 CGT -+ TGT Arg -+ Cys27 401M T II 47 III 75 -
28 481M F 11+ 21 111+ nd. 5 175 CGC -� CAC Arg -� His29 62/M T 11+ 49 111+ 103 5 175 CGC -� CAC Arg -� His
Grade 11 -� lv
30 23/F F II- 62 IV- 71 8 301 2-bp del Frameshift31 241M F II 112 IV 130 -
32 25/ F T II 16 II 65 IV nd. -33 25fM F II- 7 IV- 3 IV- 70 5 175 CGC -� CAC Arg -� His34 271M FT 11+ 37 11+ 33 IV+ 85 5 141 TGC -� TAC Cys -‘ Tyr35 29fF F II- 25 II- 39 IV- 67 5 163 TAC -* TGC Tyr -� Cys36 29/F F 11+ 133 IV+ 27 (IV) >160 8 273 CGT -�TGT Arg -�Cys37 29fF T II- 50 IV- 53 8 278 CCT-�CTT Pro -Leu38 301M F II- 53 IV- 67 8 300 89-bp del Frameshift39 32/F BG II 11 IV+ 19 5 163 TAC -*TGC Tyr -�Cys40 351M FT 11+ 4 (II) 56 IV- 71 5 175 CGC -+CAC Aig -* His41 39/M F II- 36 IV- 39 8 275 15-bp del Frameshift42 39/F T 11+ 56 IV+ 60 8 275 TGT -*TFF Cys -�Phe43 40/M F II 87 IV 93 -
44 40/F T 11+ 16 IV+ 28 7 256 l-bp del Frameshift45 41/F P 11+ 49 111+ 4 IV+ >53 6 205 TAT -* TGT Tyr -p Cys46 49fF F 11+ 33 IV- 36 8 278 CCT - ACT Pro -* Thr47 5 l/M T 11+ 23 IV+ 28 (IV) 58 8 273 CGT -� TGT Arg -* Cys
Grade III .-� III48 201M F 111+ 34 111+ 34 6 209 2-bp ins Frameshift49 26/F P 111+ 14 111+ 49 8 273 CGT -+ TGT Arg -� Cys50 28/M F III 40 III 44 -
5 1 31/F T 111+ 29 111+ 30 6 212 10-bp del Frameshift52 39fF TO 111+ 12 111+ 23 7 248 CGG - GGG Arg -� Gly53 40/M T III- 27 III- 32 8 273 CGT -#{247} TGT Arg -� Cys54 411M T 111+ 18 111+ 54 7 248 CGG -*TGG Arg -*Trp55 42/NI 0 III- 1 1 III- >57 8 280 AGA -p ACA Arg -� Thr56 57/M P0 III 24 III 29 -57 64fF P 111+ 48 111+ >59 8 273 CGT -� TGT Arg -� Cys
524 p53 Mutations during Astrocytoma Progression
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Clinical Cancer Research 525
3 The abbreviations used are: LI, labeling index; SSCP, single-strand
conformation polymorphism.
Table 1-continued
Tumor
Interv al betwe en o perations (mo) p53 Mutation
1st 2nd 3rd 4th Nucleotide Amino acidCase Age/sex localization 0�#{149}b op. op. op. TLD Exon Codon substitution substitution
Grade ill -p IV58 12fF BG III- 51 IV- 104 8 280 AGA -� AAA Arg -� Lys59 25/F TP III 24 IV 32 -60 30fF FP III- 24 LV- 32 8 273 CGT -* TGT Arg -� Cys61 321M fP III 22 IV 25 -
62 341M H’ 111+ 21 (III) 2 IV+ 51 5 175 CGC -*CAC Arg -sHis63 44/M TP (III) 6 (III) 12 III- 1 LV- 24 8 280 AGA -* AAA Arg -� Lys64 45/F T III 22 IV 25 -65 45/F F III 6 IV 12 -66 501M FT 111+ 20 IV+ 30 6 21 1 ACT -� GCT Thr - Ala67 74/M T III 29 IV 36 -
a � mutations in glioblastoma patients 30-47 and 58-67 were previously reported in an article on primary and secondary glioblastomas by
Watanabe et al. (6).b mo, months; op., operation; BG, basal ganglia; F, frontal; 0, occipital; P, parietal; T, temporal; M, male; F, female; TLD, total length of disease
(mo) from first biopsy to death of the patient; del, deletion; ins, insertion mutation; nd., not determined. II, low-grade astrocytoma without p53mutations; III, anaplastic astrocytoma without p.53 mutations; IV, glioblastoma without p53 mutations; II, low-grade astrocytoma with p53 mutations;HI, anaplastic astrocytoma with p.53 mutations; IV, glioblastoma with p53 mutations; Roman numerals in parentheses signify that the histologicalgrade was determined but that the material available was insufficient for genetic analysis. + , presence of the p53 wild-type base; - , absence of thep53 wild-type base.
p53 Immunohistochemistry. An ascites preparation of
the IgGl anti-human p53 monoclonal antibody PAb 1801
(Cambridge Research Biochemicals, Gadbrook, United King-
dom) was diluted 1 :300 in PBS and applied to formalin-fixed
paraffin-embedded sections. The incubations were carried
out for 1 h at room temperature after blocking of nonspecific
binding with normal rabbit serum (DAKO A/S, Glostrup,
Denmark, diluted 1: 10 in PBS) for 15 mm. The reaction was
visualized using a Vectastain avidin-biotin complex kit and
diaminobenzidine (Vector Laboratories, Burlingame, CA).
Sections were counterstained with hematoxylin. Formalin-
fixed paraffin-embedded glioblastoma sections, which had
previously been found to contain p53 point mutations by
DNA sequence analysis, were used as positive controls. The
fraction of p53-immunoreactive tumor cells was determined
at high-power magnification (X400). Data from 5 to 10
tumor areas were pooled, with at least 1000 counted cells per
specimen. The percentage of immunoreactive tumor cells was
recorded as the p53-LI.2
SSCP Analysis and Direct DNA Sequencing for p5.3
Mutations. DNA was extracted from paraffin sections as de-
scribed previously (8). For samples with positive immuno-
staining with PAb 1801, the same areas were chosen for DNA
extraction. For samples with negative immunostaining, DNA
was extracted from the lesion which was a histologically typicaltumor area avoiding the peripheral infiltration zone. For 13
tumors with p53 mutations which contained both positive and
negative regions immunoreactive to PAb 1 801 (four low-grade
astrocytomas from cases 20, 41 , 42, and 45, six anaplastic
astrocytomas from cases 21, 26, 18, 19, 45, 60, and three
glioblastomas from cases 36, 39, and 60), DNA was extracted
from both regions and analyzed separately.
Prescreening for mutations by PCR-SSCP analysis in cx-
ons 4-8 of the p53 gene was performed as described previously
(6, 9). Briefly, PCR was carried out with 2 p.1 of DNA solution,
2.5 pmol of each primer, 50 p.M deoxynucleotide triphosphates,
1 p.Ci of [a-33P}dCTP (Amersham, Buckinghamshire, United
Kingdom; specific activity, 3000 Ci/mmol), 10 nmi Tris (pH
8.8), 50 mM KC1, 1 mtvi MgCI2, and 0.2 units of Taq polymerase
(Perkin Elmer-Cetus, Paris, France) in a final volume of 10 p.1.
Thirty-five cycles of denaturation (94#{176}C)for 50 s, annealing
(60#{176}Cfor exons 6-8, and 55#{176}Cfor exons 4 and 5) for 60 s, and
extension (72#{176}C)for 70 s were carried out in an automated DNA
Thermal Cycler (Perkin Elmer-Cetus). Two p.1 of 0.2 M NaOH
and 9 p.1 of sequencing stop solution (United States Biochemical
Corp., Cleveland, OH) were added to the 1.5-pA PCR reaction
mixture. Samples were heated at 95#{176}Cfor 10 mm and immedi-
ately loaded onto a 6% polyacrylamide nondenaturing gel con-
taming 6% glycerol. Gels were run at 40 W for 3 h with cooling
by fan at room temperature, dried at 80#{176}C,and autoradiographed
for 12 to 48 h. For the samples which did not contain mutations
in exons 4-8, additional sequence analyses were carried out for
exons 9-1 1 . The primer sequences for SSCP and sequencing
have been described previously (6, 9).
Samples that showed mobility shifts in the SSCP analysis
were further analyzed by direct DNA sequencing. After PCR
amplification as described above, 10 p.1 of PCR products were
digested with 2 units of shrimp alkaline phosphatase and 10
units of exonuclease I at 37#{176}Cfor 15 mm. After inactivation of
these enzymes at 80#{176}Cfor 15 mm, sequencing primer (15 pmol)
and 2 p.1 of 5X Sequenase buffer [200 mrsi Tris-HC1 (pH 7.5),
100 mr�i MgCl2, and 250 mr�i NaC1) were added. Template-
primer mixture was heated at 100#{176}Cfor 3 mm and then placedin ice-cold water. 0. 1 M DTT, 3 units of Sequenase version 2.0
(United States Biochemical Corp.), and 0.5 p.Ci of [a-33P]dATP
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30 37 41 42 46 60
C ‘III IV’’ II IV ‘ II IV II IVII IV II IV
� :� � � --�- � � ,� - �,� $m�#{248} s..__� �‘ � ‘---a , ..
.. - . $.4h.e .- . . .� � -�
a-.
II In lv
II IvACGT ACGT
-CGA
-C
526 p53 Mutations during Astrocytoma Progression
Fig. 1 SSCP autoradiographs of exon 8 of the p53 gene in pairs ofastrocytic tumors from the same patient. Case numbers are as in Table
I . Roman numerals indicate WHO histological tumor grade (II. low-grade astrocytoma; III, anaplastic astrocytoma: IV, glioblastoma). In allcases, the mobility shift indicative ofap53 mutation was already present
in the first biopsy. C. control DNA.
or [ct-33PIdCTP were added to samples, which were then di-
vided into four wells containing each termination mixture. Sam-
ples were incubated at 37#{176}Cfor 10 mm and mixed with 4 p.1 ofstop solution (United States Biochemical Corp.). After being
heated at 80#{176}Cfor 2 mm, samples were loaded onto a 6%polyacrylamide/7 M urea gel. Gels were dried at 80#{176}Cand
autoradiographed for 12 to 48 h.
Statistical Analyses. Student’s paired t test was used to
evaluate differences in the p53-LI on first biopsy and recurrence
in each group. The x2 test was used to analyze differences in theincidence of p53 mutations in each group. Student’s unpaired
test was used to evaluate differences in the interval of recurrence
and total length of disease of each group with or without p.53
mutation or p53 protein accumulation. Fisher’s exact test was
carried out to compare the incidence of loss of the p.53 wild-type
allele in patients with and without progression at the recurrence.
The log rank test was carried out for analysis of the Kaplan-
Meier curve for time until progression in patients with and
without p53 mutation or p53 accumulation.
RESULTS
p53 Mutations. SSCP-PCR and direct DNA sequence
analyses showed that 46 of 67 (69%) patients had a p53 muta-
tion in at least one biopsy (Table 1). The frequency of mutations
expressed as fraction of all biopsies of the same histological
grade ranged from 58% in low-grade astrocytomas (WHO grade
II), to 67% in anaplastic astrocytomas (WHO grade III), and
72% in glioblastomas (WHO grade IV). In 41 of 46 (89%)
astrocytomas containing p53 mutations, the mutation was al-
ready detectable in the first biopsy (Table 1 and Figs. I and 2).
In only four patients, acquisition of the p.53 mutations occurred
during recurrence (grade II -� II, case 3) or progression (grade
II -+ III, cases 19, 22, and 23; grade II -� IV, case 39).
Three tumors contained two p53 mutations (Table 1 , cases
8, 1 1 , and 20). Forty-three of a total of 49 mutations identified
(88%) were missense mutations, others were deletions, and one
was an insertion. Screening was carried out for exons 4-1 1 but
all mutations were located in exons 5-8 (Table 1). G:C -� A:T
transitions were most frequent (69%) and 81% of these were
located at CpG sites.
Polymorphism at codon 72 (Arg-Pro or Pro-Pro instead of
Arg-Arg) in exon 4 was detected in 17 of 67 (25%) patients.
Among these, 12 patients (18%) showed an Arg-Pro polymor-
ACGT ACGTExon 6 Li’- �
C II Ill IV �“L. . �
� -� .-
_mM. �� �r �
414545 �. ..�
�.
� _Fig. 2 SSCP and DNA-sequencing autoradiographs of exon 6 of thep53 gene in a patient (Table I , case 45) who had subsequent surgical
biopsies for low-grade astrocytoma (WHO grade II), anaplastic astro-
cytoma (grade III). and glioblastoma (grade IV) at the age of4l, 45. and45 years. The same mobility shift was observed in all three biopsies
(left). DNA-sequencing autoradiographs (right) showed a TAT -* TGTmutation in codon 205 in all three tumors.
Fig. 3 DNA-sequencing autoradiographs of exon 5 of the p53 gene ina patient (Table 1. case 40) who had surgical biopsies for low-gradeastrocytoma (WHO grade II) at the age of 35 and glioblastoma (grade
IV) at the age of 40 years. The same CGC -* CAC mutation in codon175 was found in both tumors. In low-grade astrocytoma. the wild-type
base G is present along with the mutation (A). but it is lost during
progression to glioblastoma.
phism and 5 (7%) patients a Pro-Pro polymorphism. This fre-
quency is similar to the prevalence of p53 codon 72 polymor-
phism in normal Caucasians ( 10, 1 1 ). There was no significant
difference in the frequency of this polymorphism among the
astrocytoma subgroups.
Loss of the wild-type allele of chromosome l7p was de-
termined from DNA-sequencing autoradiographs (Fig. 3).
Among the cases with p53 mutations in at least two biopsies, 38
of 40 (95%) showed the same l’7p allele status at first biopsy
and recurrence (Table 1). Loss of the wild-type allele during
progression was found in only 2 of 40 (5%) tumors (Table I,
cases 40 and 46; Fig. 3). Of 28 low-grade astrocytomas with a
p53 mutation, 7 (25%) showed loss of the normal allele in the
first biopsy. The allele status remained the same in 95% of the
cases, irrespective of whether the recurrent lesion had the same
or a higher grade of malignancy.
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Clinical Cancer Research 527
. �;. �
.1 ...L:...
�1*,�
��,. .. J�4.,
�
�j. ;5-
C,. �. � ,:, CC,�
.. ‘,:: �
,� a,..
�‘C .
� �...v, �
a�.,I���4%!.11S: :‘Fig. 4 Change of p53 protein accumulation during progression fromlow-grade astrocytoma (A: LI. 4%) to glioblastoma (B: LI, 27%). Bothbiopsies contained the same lS-bp deletion in exon 8 of the p13 gene(Table 1, case 41).
p53 Protein Accumulation. Immunoreactivity to the
monoclonal antibody PAb 1801 was restricted to nuclei of
neoplastic cells (Fig. 4). Thirty-two of 47 (68%) recurring
low-grade astrocytomas (groups II -� II, II -+ III, and II -� IV)
and 13 of 20 (65%) recurring anaplastic astrocytomas (groups
III -� III and III -p IV) were p53 positive from the first biopsies
(Table 2). An additional 8 of 67 (12%) cases of low-grade or
anaplastic astrocytomas which were initially p53 negative be-
came p53 positive after recurrence, but none of the initially
p53-positive cases became negative. The mean p53-LI was
2.5% in low-grade astrocytomas, 8.2% in anaplastic astrocyto-
mas, and 10.8% in glioblastomas. The p53-LI increased signif-
icantly during progression: in groups II -� III from 3.5 to 10.4%
(P < 0.05) and in groups II -* IV from 3.7 to 1 1 .3% (P
0.0001, Table 2; Fig. 4). No significant increase was observed in
recurrent astrocytomas without progression: groups II -� II and
III -� III (Table 2). The p53-LI of anaplastic astrocytoma at first
biopsy was significantly higher in groups III -� IV (7.6%) than
in groups III --s III (4.6%, P < 0.05, Table 2).Correlation between p53 Mutations and p53 Protein
Accumulation. Concordance between p53 mutations and p53
protein accumulation was found in 107 of 144 (74%) biopsies
(both positive, 58%; both negative, 17%). Nine tumors showed
the presence ofp53 mutation without p53 protein accumulation.
Of these, three had frameshift mutations. Twenty-six tumors
(18%) showed p53 protein accumulation in the absence of a p53
mutation. In 14 astrocytomas with a p53 mutation, areas with
and without p53 immunoreactivity were separately analyzed by
SSCP. The p53 mobility shift was present irrespective of p53
immunoreactivity.
Predictive Value of p13 Alterations. Progression of
low-grade astrocytoma to anaplastic astrocytoma or glioblas-
toma occurred at a similar frequency in lesions with (79%) and
without (63%) p53 mutations (P = 0.32). We found a reduced
time until progression in patients with low-grade astrocytomas
carrying a p53 mutation (Fig. 5) but the difference was at the
margin of statistical significance (P = 0.055). Time until pro-
gression in patients with low-grade astrocytomas with p53 pro-
tein accumulation was similar to those without p53 protein
accumulation (P = 0.35).
The mean time until recurrence in patients with astrocy-
toma carrying a codon 175 mutation was 31 ± 10 months, i.e.,
somewhat shorter than those with other mutations (mean, 42 ±
4 months) but the difference did not reach statistical significance
(P = 0.45).
Loss of the wild-type allele at any stage was more frequent
in tumors which subsequently progressed: 2 of 15 (13%) cases
from groups II -#{247} II and III -� III and I 3 of 30 (43%) cases from
groups II --s III, II -+ IV, and III -� IV (P = 0.053).
DISCUSSION
In this study, we analyzed a series of 144 recurrent astro-
cytic brain tumors in patients with two or more biopsies. Mu-
tations of the p.5.3 gene were detected in 58% of low-grade
astrocytomas, 67% of anaplastic astrocytomas, and 72% of
glioblastomas, i.e., at a significantly higher frequency than
reported in previous studies (24-34%) using tumors from mdi-
vidual patients without evidence of recurrence or progression
(3). Our results are consistent with recent reports which showed
a mutation frequency of 58% (6) and 83% (5) in low-grade
astrocytomas which had progressed to anaplastic astrocytoma or
glioblastoma. In the present study, we found a high frequency of
p53 mutations irrespective of histological evidence of progres-
sion (Table 1), suggesting that p53 mutations are associated with
recurrence of astrocytomas rather than progression. The mean
observation period was similar in groups II -� II and groups II
.-� III, or II -� IV, but the possibility cannot be ruled out that
additional cases in group II -� II would eventually have pro-
gressed if patient survival or clinical follow-up had been longer.
Our study provides further evidence that p53 mutations
constitute an early event in the evolution of astrocytomas, since
the incidence of mutations did not differ significantly among
low-grade anaplastic astrocytomas and glioblastomas, and in
89% of the cases with a mutation, this genetic alteration was
already detected in the first biopsy.
In three previous reports (12-14), the presence of p53
mutations or p53 accumulation had no effect on the clinical
outcome of the patients whereas in the study by Chozick et al.
(15), immunoreactivity for p53 protein in low-grade astrocyto-
mas was associated with shorter patient survival. We found a
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528 p53 Mutations during Astrocytoma Progression
Table 2 p53 Mutations and p53 protein accumulation in recurrent astrocytic brain tumors
WHO grade No. of cases p53 Mutation p53 Protein accumulation p53-LI (%)
II -* II 13 6 (46%) -� 7 (54%) 7 (54%) -� 7 (54%) 1 .8 ± 2.0 -� 1.2 ± I .3II -* III 16 8 (50%) - 1 1 (69%) 1 1 (69%) -s 14 (88%) 3.5 ± 3.8 -� 10.4 ± l3.2’�II -b IV 18 14 (78%) -.* 15 (83%) 14 (78%) -#{247} 16 (89%) 3.7 ± 5.1 -s I 1.3 ± 8.6”
III -e III 10 8 (80%) -� 8 (80%) 6 (60%) -“ 6 (60%) 4.6 ± 4.8 -“ 4.7 ± 4.4HI --#{247}IV 10 5 (50%) -+ 5 (50%) 7 (70%) -“ 10 (100%) 7.6 ± 10.8 -“ 8.8 ± 5.3
ap < �
bp 0.0001.
100
Time till progression (months)
Fig. 5 Kaplan-Meier analysis showing a shorter time interval untilprogression (months) for low-grade astrocytomas with a p53 muta-tion (- - -,n 2 1) compared with astrocytomas without a mutation(-, n = 11: P = 0.055).
reduced time until progression in patients with diffuse low-
grade astrocytomas carrying a p.53 mutation (Fig. 5), but the
difference was at the margin of statistical significance (P
0.055).
Goh et al. (16) reported that the location of mutations
within the p53 gene affected survival of colorectal cancer pa-
tients and that carcinomas with a codon 175 mutation were
particularly aggressive. We observed that mutations in this
codon (Table 1, cases 5, 28, 29, 40, and 62) were associated with
a shorter time interval until recurrence; however, the differences
were not statistically significant (P 0.45), possibly due to the
low number of cases. It has also been suggested that the pres-
ence of a Pro-Pro polymorphism in codon 72 of exon 4 is
associated with susceptibility to the development of lung (17,
18) and urological cancer (19). In the present study, the preva-
lence of the Pro-Pro polymorphism in patients with recurrent
astrocytic brain tumors was similar to that in the normal Cau-
casian population ( 10, 1 1), and there was no difference between
astrocytoma subgroups.
It has been discussed whether low-grade astrocytomas al-
ways progress to glioblastoma via anaplastic astrocytoma or,
alternatively, directly without the anaplastic astrocytoma as an
intermediate malignant phenotype. In a report by van Meyel et
a!. (7), low-grade astrocytomas recurring as anaplastic gliomas
were characterized by p.5.3 mutations whereas those recurring as
glioblastoma typically had intact p53 genes, suggesting the
presence of two different pathways of progression. Our results,
based on a larger number of patients, as well as those by
Reifenberger et a!. (5), show that low-grade astrocytoma that
recurred as either anaplastic astrocytoma or glioblastoma con-
tamed p53 mutations at similar frequencies.
An association between loss of heterozygosity on chromo-
some l7p and p53 mutations has been observed in a variety of
human tumors, including brain neoplasms (20, 2 1). However,
the timing of the loss of the wild-type allele during tumor
progression has received little attention. In the present study, 38
of 40 cases (95%) with a p53 mutation in at least two biopsies
showed the same p53 allele status irrespective of tumor progres-
sion. Only 2 of 40 (5%) tumors showed loss of the wild-type
allele in the second or third biopsy. However, loss of the
wild-type allele at any stage was more frequent in tumors which
subsequently progressed (13% versus 43%, P 0.053).
As in previous studies (22-24), p53 protein accumulation
was more frequently observed than were p.5.3 mutations (Table
2). The fraction of cells immunoreactive to PAb 1801 (p53-LI)
increased significantly during progression from low-grade to
anaplastic astrocytoma and glioblastoma (Table 2). A similar
observation was made in a recent study by Reifenberger et a!.
(5) in recurrent astrocytomas with progression. This may reflect
a clonal expansion of mutated cells during the multistep process
of malignant transformation, as suggested by Sidransky et a!.
(25). However, p53 immunoreactivity does not necessarily in-
dicate the presence of a mutation but may also reflect genotoxic
stress or additional genetic alterations (26). In 14 tumors with
p53 mutations, we were able to dissect from histological slides
areas with and without immunoreactivity to PAb 1801 and
found that the p53 mutation was present in both areas. No
significant change in the p53-LI was found in astrocytomas
without evidence of progression at recurrence (groups II -“ II
and III .-* III). The p53-LI at first biopsy was not predictive inlow-grade astrocytomas but found to be higher in anaplastic
astrocytomas which progressed to glioblastoma (7.6%) than in
those without progression (4.7%; P < 0.05).
Three of a total of 67 patients had tumors containing
double p53 mutations, and, in all cases, these were already
present in the first biopsy (Table 1, cases 8, 1 1, and 20),
suggesting that they occurred at an early stage of malignant
transformation. Double mutations are infrequent in astrocyto-
mas (5, 20, 24) and nonneural tumors. Although some have been
identified in apparently sporadic human neoplasms (5, 20, 24,
27, 28), others were found at sites suggestive of exposure to
environmental mutagens, e.g. , bladder cancer in heavy smokers
(29), lung cancer in patients with a history of exposure to
mustard gas (30), and urothelial cancer from the endemic area of
black root disease in Taiwan (31). With the exception of ther-
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Clinical Cancer Research 529
apeutic X-irradiation, no carcinogenic environmental factors
have been unequivocally identified as being involved in the
etiology of human brain tumors (32). It appears, therefore, more
likely that in our cases, the first mutation was biologically less
significant and only capable of inducing clonal expansion of
astrocytes and that the second mutation was necessary to cause
malignant transformation (27, 28).
One of the major functions of the p5.3 gene is to inhibit
progression through G1 into the S-phase in response to DNA
damage (33). Thus, loss of p53 function eliminates a cell cycle
checkpoint, leads to genomic instability, and facilitates addi-
tionai genetic alterations (34). Typical alterations associatedwith the progression of low-grade astrocytomas to anaplastic
astrocytomas and glioblastomas include deletion of the p16 gene
(35-37), loss of heterozygosity on chromosomes 10 and l9q
(38-41), inactivation of the RB gene (42, 43), and CDK4
amplification (35, 44). Amplification and overexpression of the
epidermal growth factor receptor gene in the absence of p53
mutations are characteristic of primary glioblastoma which rap-
idly develops de novo, without clinical or morphological cvi-
dence of a less malignant precursor lesion (6).
It should be noted that in the present study, 12 of 47
low-grade astrocytomas (26%) did not contain p5.3 mutations
(Table 1) and in nonrecurrent lesions, this fraction appears to be
even higher (3). Since most p53 mutations in human neoplasms
have been found in exons 5- 8, the screening from exons 4 to 11
in the present study should have covered most, if not all,
p53 mutations present. Thus, additional genetic alterations in-
volving as yet unidentified genes are likely to play a role in the
evolution of low-grade astrocytomas and their progression to
glioblastoma.
ACKNOWLEDGMENTS
We are grateful to Beatrice Pfister, Ursula Recher, MarianneKonig, Angelika Ruf, Mireille Laval, and Nicole Lyandrat for their
excellent technical assistance and Dr. Pierre Hainaut, IARC (Lyon,
France) for critically reading the manuscript.
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