Incidence and Timing of p53 Mutations during Astrocytoma ... · Astrocytoma progression is associated with sequentially ac-quired multiple genetic alterations (3, 4). p53 Mutations

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.

Research. on June 5, 2021. © 1997 American Association for Cancerclincancerres.aacrjournals.org Downloaded from

http://clincancerres.aacrjournals.org/

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



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



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


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.




. �;. �

.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




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-




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.

REFERENCES

1. Kleihues, P., Burger, P. C., and Scheithauer, B. W. Histologicaltyping of tumours of the central nervous system. WHO International

Histological Classification ofTumours, 2nd ed. Berlin: Springer-Verlag,1993.

2. Kleihues, P., Burger, P. C., and Scheithauer, B. W. The new WHO

classification of brain tumours. Brain Pathol., 3: 255-268, 1993.

3. Ohgaki, H., Schauble, B., zur Hausen, A., von Ammon, K., andKleihues, P. Genetic alterations associated with the evolution and pro-gression of astrocytic brain tumours. Virchows Arch., 427: 113-118,1995.

4. Collins, V. P. Amplified genes in human gliomas. Semin. CancerBiol., 4: 27-32, 1993.

5. Reifenberger, J., Ring, G. U., Gies, U., Cobbers, L., Oberstrass, J.,

An, H. X., Niederacher, D., Wechsler, W., and Reifenberger, G. Anal-ysis of p53 mutation and epidermal growth factor receptor amplification

in recurrent gliomas with malignant progression. J. Neuropathol. Exp.

Neurol., 55: 822-831, 1996.

6. Watanabe, K., Tachibana, 0., Sato, K., Yonekawa, Y., Kleihues, P.,and Ohgaki, H. Overexpression of the EGF receptor and p53 mutationsare mutually exclusive in the evolution of primary and secondaryglioblastomas. Brain Pathol., 6: 217-224, 1996.

7. van Meyel, D. J., Ramsay, D. A., Casson, A. G., Keeney, M.,

Chambers, A. F., and Cairncross, J. G. p53 mutation, expression, and

DNA ploidy in evolving gliomas: evidence for two pathways of pro-gression. J. Natl. Cancer Inst., 86: 101 1-1017, 1994.

8. Brustle, 0., Ohgaki, H., Schmitt, H. P., Walter, G. F., Ostertag, H.,and Kleihues, P. Primitive neurocctodermal tumors after prophylacticcentral nervous system irradiation in children. Association with an

activated K-ras gene. Cancer (Phila.), 69: 2385-2392, 1992.

9. Zariwala, M., Schmid, S., Pfaltz, M., Ohgaki, H., Kleihues, P., andSchafer, R. p53 gene mutations in oropharyngeal carcinomas: a com-parison of solitary and multiple primary tumours and lymph-node me-tastases. Int. J. Cancer, 56: 807-81 1. 1994.

10. Weston, A., Ling Cawley, H. M., Caporaso, N. E., Bowman, E. D.,Hoover, R. N., Trump, B. F., and Harris, C. C. Determination of theallelic frequencies of an L-myc and a p53 polymorphism in human lung

cancer. Carcinogenesis (Lond.), 15: 583-587, 1994.

11. Beckman, G., Birgander, R., Sjalander. A., Saha. N., Holmberg,

P. A., Kivela, A., and Beckman, L. Is p53 polymorphism maintained bynatural selection? Hum. Hered., 44: 266-270, 1994.

12. Kraus, J. A., BolIn, C., Wolf, H. K., Neumann, J., Kindermann, D.,Fimmers, R., Forster, F., Baumann, A., and Schlegel, U. TP53 alter-ations and clinical outcome in low grade astrocytomas. Genes Chromo-

somes & Cancer, 10: 143-149, 1994.

13. Schlegel, U. p53: an important or most overvalued tumor gene?

Laryngorhinootologie, 73: 65 1-653, 1994.

14. al Sarraj, S., and Bridges, L. R. p53 immunoreactivity in astrocy-tomas and its relationship to survival. Br. J. Neurosurg., 9: 143-149,1995.

15. Chozick, B. S., Pezzullo, J. C., Epstein, M. H., and Finch, P. W.Prognostic implications of p53 overexpression in supratentorial astro-cytic tumors. Neurosurgery, 35: 831-837, 1994.

16. Goh, H-S., Yao, J., and Smith, D. R. p53 Point mutation andsurvival in colorectal cancer patients. Cancer Res., 55: 5217-5221,

1995.

17. Kawajiri, K., Nakachi, K., Imai, K., Watanabe, J., and Hayashi, S.Germ line polymorphisms ofp53 and CYP1AJ genes involved in humanlung cancer. Carcinogenesis (Lond.), 14: 1085-1089, 1993.

18. Jin, X., Wu, X., Roth, J. A., Amos, C. I., King, T. M.. Branch, C.,Honn, S. E., and Spitz, M. R. Higher lung cancer risk for youngerAfrican-Americans with the Pro/Pro p53 genotype. Carcinogenesis

(Lond.), 16: 2205-2208, 1995.

19. Wu, W. J., Kakehi, Y., Habuchi, T., Kinoshita, H., Ogawa, 0.,

Terachi, T., Huang. C. H., Chiang, C. P., and Yoshida, 0. Allelicfrequency ofp53 gene codon 72 polymorphism in urologic cancers. Jpn.J. Cancer Res., 86: 730-736, 1995.

20. von Deimling, A., Eibl, R. H., Ohgaki, H., Louis, D. N., vonAmmon, K., Petersen, I., Kleihues, P., Chung, R. Y., Wiestler, 0. D.,and Seizinger, B. R. p53 mutations are associated with lip allelic lossin grade II and grade III astrocytoma. Cancer Res., 52: 2987-2990,1992.

21. Louis, D. N. The p53 gene and protein in human brain tumors.J. Neuropathol. Exp. Neurol., 53: 11-21, 1994.

22. Newcomb, E. W., Madonia, W. J., Pisharody, S., Lang, F. F.,

Kosbow, M., and Miller, D. C. A correlative study of p53 proteinalteration and p53 gene mutation in glioblastoma multiforme. BrainPathol., 3: 229-235, 1993.

23. Lang, F. F.. Miller, D. C., Pisharody, S., Kosbow, M., and

Newcomb, E. W. High frequency of p53 protein accumulation withoutp53 gene mutation in human juvenile pilocytic, low grade and anaplastic

astrocytomas. Oncogene, 9: 949-954, 1994.

24. Louis, D. N., von Deimling, A., Chung, R. Y., Rubio, M. P.,

Whaley, J. M., Eibl, R. H., Ohgaki, H., Wiestler, 0. D., Thor, A. D., andSeizinger, B. R. Comparative study of p53 gene and protein alterationsin human astrocytic tumors. J. Neuropathol. Exp. Neurol., 52: 3 1-38,

1993.

25. Sidransky, D., Mikkelsen, T., Schwechheimer, K., Rosenblum,M. L., Cavanee, W., and Vogelstein, B. Clonal expansion of p53 mutantcells is associated with brain tumour progression. Nature (Lond.), 355:846-847, 1992.




26. Hainaut, P. The tumor suppressor protein p53: a receptor to geno-

toxic stress that controls cell growth and survival. Curr. Opin. Oncol., 7:76-82, 1995.

27. Seth, A., Palli, D., Mariano, J. M., Metcalf, R., Venanzoni, M. C.,

Bianchi, S., Kottaridis, S. D., and Papas. T. S. P53 gene mutations inwomen with breast cancer and a previous history of benign breastdisease. Eur. J. Cancer, 30A: 808-812, 1994.

28. Oda, H., Nakatsuru, Y., and Ishikawa, T. Mutations of the p53 geneand p53 protein overexpression are associated with sarcomatoid trans-formation in renal cell carcinomas. Cancer Res., 55: 658-662, 1995.

29. Spruck, C. H., III, Rideout, W. M., III, Olumi, A. F., Ohneseit, P. F.,

Yang, A. S., Tsai, Y. C., Nichols, P. W., Horn, T., Hermann, G. G.,Steven, K., Ross, R. K., Yu, M. C., and Jones, P. A. Distinct pattern ofp53 mutations in bladder cancer: relationship to tobacco usage. CancerRes., 53: 1162-1166, 1993.

30. Takeshima, Y., Inai, K., Bennett, W. P., Metcalf, R. A., Welsh,J. A., Yonehara, S., Hayashi, Y., Fujihara, M., Yamakido, M., Akiyama.M., Tokuoka, S., Land, C. E., and Harris, C. C. p53 mutations in lungcancers from Japanese mustard gas workers. Carcinogenesis (Lond.),15: 2075-2079, 1994.

31. Shibata, A., Ohneseit, P. F., Tsai, Y. C., Spruck, C. H., Nichols,

P. W., Chiang. H. S., Lai, M. K., and Jones, P. A. Mutational spectrumin the p53 gene in bladder tumors from the endemic area of black footdisease in Taiwan. Carcinogenesis (Lond.), 15: 1085-1087, 1994.

32. Lantos, P. L., VandenBerg, S. R., and Kleihues, P. Tumours of the

nervous system. In: D. I. Graham and P. L. Lantos (eds.), Greenfield’sNeuropathology, 6th ed., Vol. 2, Chap. 9, pp. 583-879. London: Arnold,1996.

33. Lane, D. P. Cancer. p53, guardian of the genome. Nature (Lond.),

358: 15-16, 1992.

34. Hartwell, L. Defects in a cell cycle checkpoint may be responsible

for the genomic instability of cancer cells. Cell, 71: 543-546, 1992.

35. Schmidt, E. E., Ichimura, K., Reifenberger, G., and Collins, V. P.CDKN2 (p16/MTSJ) gene deletion or CDK4 amplification occurs in themajority of glioblastomas. Cancer Res., 54: 6321-6324, 1994.

36. Jen, J., Harper, J. W., Bigner, S. H., Bigner, D. D., Papadopoulos,N., Markowitz, S., Willson, J. K., Kinzler, K. W., and Vogelstein, B.

Deletion of p16 and p15 genes in brain tumors. Cancer Res., 54:6353-6358, 1994.

37. Ueki, K., Rubio, M.-P., Ramesh, V., Correa, K. M., Rutter, J. L.,

von Deimling, A., Buckler, A. J., Gusella, J. F., and Louis, D. N.MTSI/CDKN2 gene mutations are rare in primary human astrocytomaswith allelic loss of chromosome 9p. Hum. Mol. Genet., 3: 1841-1845,

1994.

38. von Deimling, A., Louis, D. N., von Ammon, K., Petersen, I.,

Wiestler, 0. D., and Seizinger, B. R. Evidence for a tumor suppressor

gene on chromosome 19q associated with human astrocytomas, oligo-dendrogliomas, and mixed gliomas. Cancer Res., 52: 4277-4279, 1992.

39. Fulls, D., Brockmeyer, D., Tullous, M. W., Pedone, C. A., and

Cawthon, R. M. p.53 mutation and loss of heterozygosity on chromo-somes 17 and 10 during human astrocytoma progression. Cancer Res.,52: 674-679, 1992.

40. Venter, D. J., and Thomas, D. G. Multiple sequential molecularabnormalities in the evolution of human gliomas. Br. J. Cancer, 63:753-757, 1991.

41. von Deimling, A., Louis, D. N., von Ammon, K., Petersen, I., Hoell,

T., Chung, R. Y., Martuza, R. L., Schoenfeld, D. A., Yasargil, M. G.,Wiestler, 0. D., and Seizinger, B. R. Association of epidermal growthfactor receptor gene amplification with loss of chromosome 10 in humanglioblastoma multiforme. J. Neurosurg., 77: 295-301, 1992.

42. Henson, J. W., Schnitker, B. L., Correa, K. M., von Deimling, A.,

Fassbender, F., Xu, H-J., Benedict, W. F., Yandell, D. W., and Louis,

D. N. The retinoblastoma gene is involved in malignant progression ofastrocytomas. Ann. Neurol., 36: 714-721, 1994.

43. Ueki, K., Ono, Y., Henson, J. W., Efird, J. T., von Deimling, A., andLouis, D. N. CDKN2/pl6 or RB alterations occur in the majority ofglioblastomas and are inversely correlated. Cancer Res., 56: 150-153,

1996.

44. Reifenberger, G., Reifenberger, J., Ichimura, K., Meltzer, P. 5., and

Collins, V. P. Amplification of multiple genes from chromosomalregion l2q13-14 in human malignant gliomas: preliminary mapping ofthe amplicons shows preferential involvement of CDK4, SAS, andMDM2. Cancer Res., 54: 4299-4303, 1994.



1997;3:523-530. Clin Cancer Res K Watanabe, K Sato, W Biernat, et al. progression in patients with multiple biopsies.Incidence and timing of p53 mutations during astrocytoma

Updated version

http://clincancerres.aacrjournals.org/content/3/4/523

Access the most recent version of this article at:

E-mail alerts related to this article or journal.Sign up to receive free email-alerts

Subscriptions

Reprints and

[email protected] at

To order reprints of this article or to subscribe to the journal, contact the AACR Publications

Permissions

Rightslink site. Click on "Request Permissions" which will take you to the Copyright Clearance Center's (CCC)

.http://clincancerres.aacrjournals.org/content/3/4/523To request permission to re-use all or part of this article, use this link


http://clincancerres.aacrjournals.org/content/3/4/523http://clincancerres.aacrjournals.org/cgi/alertsmailto:[email protected]://clincancerres.aacrjournals.org/content/3/4/523http://clincancerres.aacrjournals.org/

Documents

Incidence and Timing of p53 Mutations during Astrocytoma ... · Astrocytoma progression is associated with sequentially ac-quired multiple genetic alterations (3, 4). p53 Mutations