  

J. Pathol. 189: 504–513 (1999)

MUTATIONS IN EXONS 5–8 OF THE p53 GENE,

INDEPENDENT OF THEIR TYPE AND LOCATION, ARE
ASSOCIATED WITH INCREASED APOPTOSIS AND
MITOSIS IN INVASIVE BREAST CARCINOMA
-  1,  .   2, - ø3,  . ̈4,
 ́4,  5,  . 6,  2,  . 2 
   1*
1
Department of Surgery, Leiden University Medical Center, Leiden, The Netherlands
2
Department of Pathology and Human Genetics, Leiden University Medical Center, Leiden, The Netherlands
3
Department of Genetics, Institute for Cancer Research, The Norwegian Radium Hospital, Oslo, Norway
4
The Icelandic Cancer Society, Reykjavik, Iceland
5
Tumorgenetik, Max Delbrück Centrum für Moleculare Medizin, Berlin, Germany
6
Department of Pathology, Institute for Cancer Research, The Norwegian Radium Hospital, Oslo, Norway

SUMMARY
In breast cancer, mutations located in the zinc-binding functional domains of the p53 gene have been reported to predict a worse
prognosis and a worse response to treatment with doxorubicin, compared with mutations in other parts within exons 5–8 of the gene.
Similarly, mutations in residues of p53 that directly contact DNA have been associated with a poor prognosis. To investigate whether
these specific p53 mutations are associated with differences in the rate of apoptosis and/or mitosis, or expression of the anti-apoptotic
Bcl-2 protein, these parameters were evaluated in 89 invasive breast cancers with a confirmed p53 mutation in exons 5–8 and in 99
tumours without a p53 mutation in exons 5–8. Neither mutations located in the zinc-binding functional domains nor mutations in residues
that directly contact DNA were associated with alterations in mitotic or apoptotic activity. However, compared with the wild-type p53
tumours, both apoptotic and mitotic indices showed an approximately two-fold increase in the mutant p53 group (p<0·001). The presence
of a p53 mutation was also associated with the presence of tumour necrosis (p<0·001), high tumour grade (p<0·001) and low expression
of Bcl-2 (p<0·001). Our data support the concept that in invasive breast carcinoma, loss of p53 function is involved in enhanced
proliferation rather than decreased apoptosis and that the resulting acceleration of cell turnover may enhance clonal evolution and
tumour progression. Copyright  1999 John Wiley & Sons, Ltd.
KEY WORDS—breast cancer; apoptosis; p53; Bcl-2; proliferation

INTRODUCTION associated with apoptotic pathways,18 including certain

A major function of the p53 tumour suppressor gene
is to prevent replication of cells with damaged DNA or
oncogenic mutations, by mediating a permanent cell-
cycle arrest or by triggering apoptosis.1–4 Abrogation of
p53-dependent apoptosis is believed to play an import-
ant role in tumour development and progression,5–7, as
well as treatment resistance.8–10 p53 binds to DNA in a
sequence-specific fashion and is involved in gene tran-
scription, cell-cycle regulation, centrosome duplica-
tion,11 DNA repair,12,13 senescence and apoptosis,14 and
differentiation and development.15 The exact mechanism
through which p53 induces apoptosis has not yet been
elucidated. In some experimental systems, sequence-
specific transactivation is required.16,17 In fact, an
increasing number of p53-responsive genes are being
*Correspondence to: J. H. van Dierendonck, PhD, Department of
Surgery, Leiden University Medical Center, P1-Q, PO Box 9600, 2300
RC Leiden, The Netherlands. E-mail: dierendonc@surgery.azl.nl
Contract/grant sponsor: E. C. Biomedical and Health Research
Concerted Action; Contract/grant number: BMH1-CT92-0890.
Contract/grant sponsor: Norwegian Cancer Society.
Contract/grant sponsor: Icelandic Cancer Society.
Contract/grant sponsor: Nordic Cancer Union.
CCC 0022–3417/99/130504–10$17.50
Copyright  1999 John Wiley & Sons, Ltd.
members of the Bcl-2 family of apoptosis-regulating
genes.19,20
The p53 gene is the most frequently mutated gene in
human cancer. In breast cancer, p53 mutation has been
found to be associated with aneuploidy, rapid prolifer-
ation, and poor prognosis,21,22 and some evidence has
been provided for an association of p53 mutation with
increased resistance to chemo- and/or hormonal
therapy.23,24
The majority of p53 mutations occur in the central
conserved part of the protein or ‘core domain’ (residues
102–292), which is responsible for sequence-specific
DNA binding.25 In general, these mutations are of the
missense type and lead to loss of DNA binding, which is
believed to be critical for the biological activity of p53.26
However, up to 20 per cent of p53 mutations have been
reported to occur outside of exons 5–8, and these are
predominantly nonsense mutations.
The core domain of p53 consists of three loops
involved in DNA binding.27 Almost 50 per cent of all
missense mutations occur in the L2 domain (residues
163–195) and L3 domain (residues 236–251), which are
within conserved regions III and IV and encode for the
L2 and L3 loops that interact through a tightly bound
Received 22 January 1999
Revised 11 May 1999
Accepted 28 July 1999
 p53 MUTATION AND APOPTOSIS IN BREAST CANCER 505

Table I—Mutations in exons 5–8 of the p53 gene and immunohistochemical (IHC) staining results

Tumour Codon From base To base Domain p53 IHC

Missense/microdeletions
1* Exon 5 ND
2* Exon 5 4
3* Exon 5 6
4* Exon 8 2
5 132 AAG < GAG 6
6 134 TTT < TGT ND
7 134 TTT < CTT 5
8 134 TTT < TTA 7
9 135 TGC < TTC 5
10 141 TGC < TAC 6
11 163 TAC < TGC L2 4
12 163 TAC < TGC L2 6
13 173 CTG < TTG L2 0
14 173 GTG < TTG L2 4
15 173 GTG < ATG L2 6
16 175 CGC < CAC L2/PSR 4
17 175 CGC < CAC L2/PSR 5
18 179 CAT < AAT L2 3
19 179 CAT < CGT L2 5
20 181 CGC < CAC L2 2
21 193 CAT < CCT L2 4
22 194 CTT < CGT L2 4
23 195 ATC < ATT L2 5
24 196 CGA < CCA 5
25 196 CGA < CCA 6
26 205 TAT < TCT 5
27 213 CGA < CGG 0
28 216 GTG < ATG 6
29 220 TAT < TGT 5
30 235 AAC < AGC 4
31 237 ATG < ATA L3 4
32 238 TGT < TTT L3 5
33 239 AAC < AAA L3 5
34 241 TCC < TTC L3/DNA 4
35 241 TCC < TTC L3/DNA 6
36 242 TGC < TAC L3 3
37 242 TGC < TTC L3 5
38 245 GGC < AGC L3/PSR 2
39 245 GGC < AGC L3/PSR 7
40 248 CGG < CAG L3/DNA 5
41 248 CGG < TGG L3/DNA 5
42 248 CGG < CAG L3/DNA 6
43 248 CGG < CAG L3/DNA 6
44 248 CGG < CAG L3/DNA 6
45 248 CGG < CAG L3/DNA 6
46 248 CGG < CAG L3/DNA 6
47 248 CGG < CAG L3/DNA 6
48 248 CGG < CAG L3/DNA 6
49 248 CGG < TGG L3/DNA 7
50 249 6 bp del L3/PSR 6
51 264 3 bp del 4
52 266 GGA < GAA 4
53 270 TTT < CTT 3
54 272 GTG < ATG 3
55 272 GTG < GTA 7
56 273 CGT < CAT DNA ND
57 273 CGT < CAT DNA 4
58 273 CGT < CAT DNA 6
59 273 CGT < TGT DNA 6
60 273 CGT < TGT DNA 7
61 273 CGT < CAT DNA 7

Copyright  1999 John Wiley & Sons, Ltd. J. Pathol. 189: 504–513 (1999)
506 H.-J. VAN SLOOTEN ET AL.

Table I—Continued

Tumour Codon From base To base Domain p53 IHC

Missense/microdeletions continued
62† 273 CGT < CAT DNA 7
62† 128 CCT < TCT 7
63 273 CGT < CAT DNA 7
64 274 GTT < GCT 2
65 275 TGT < TAT 6
66 278 CCT < TCT 7
67 279–281 6 bp del 7
68 281 GAC < GGC 6
69 281 GAC < CAC 6
70 282 CGG < TGG PSR 5
71 282 CGG < TGG PSR 6
72 285 GAG < AAG 4
73 286 GAA < AAA 6
74 298 GAG < GAT 0
Frameshift
75 133–134 1 bp del 0
76 135 2 bp ins 0
77 151 1 bp del 0
78 174–180 17 bp del 5
79 182 1 bp del 0
80 187 1 bp del 3
81 210 1 bp del 0
82 244–247 8 bp del 0
83 251/252 1 bp del 0
84 262 14 bp del 0
Nonsense
85 144 GAG < TAG/STOP 0
86 192 CAG < TAG/STOP 0
87 196 CGA < TGA/STOP 0
88 271 GAG < TAG/STOP 0
89 280 AGA < TGA/STOP 3

*Mutation has not been verified.

†Tumour has two mutations.
p53 IHC: 0=no staining; 7=intense staining; ND=not done; L2/L3=zinc-binding domains; DNA=residues that
directly contact DNA; PSR=protein stabilizing residues.

zinc atom to provide stable DNA contacts.27 The core
domain also interacts with various cellular proteins,28
including the p53-binding protein 53BP2. The latter
protein interacts with the L2 and L3 loops, thereby
inhibiting sequence-specific binding of p53,29 but also
seems to interact in a competitive fashion with the
anti-apoptotic Bcl-2 protein.30 Apart from a classifica-
tion of mutations according to their location in various
functional domains, mutations are divided into class I
mutations, affecting residues that directly contact DNA
(e.g. hotspots Arg-248 and Arg-273) and class II muta-
tions, affecting residues crucial for stabilizing the
structural integrity of the domain (e.g. hotspot Arg-175).
Recently, Børresen and co-workers reported that in
breast cancer, mutations in the L2/L3 domains were
associated with significantly decreased overall survival
rates (p=0·012) as well as with increased chemoresist-
ance, when compared with patients with mutations
located outside these domains.31,32 Moreover, in most
cases, patients with mutations in L2–L3 who responded
to primary chemotherapy had a relapse of their disease
Copyright  1999 John Wiley & Sons, Ltd.
within months. In a similar study, Berns et al. showed
that mutations in residues of p53 that directly contact
DNA predicted a poor outcome, but not mutations in
L2/L3.33
The above data suggest that the biochemically defined
functional domains of p53 have biological relevance in
terms of disease outcome, and that specific p53 muta-
tions contribute to the development of more aggressive
and possibly treatment-resistant tumours. We therefore
hypothesized that tumours carrying mutations within
the L2/L3 domains, or in residues directly contacting
DNA, would show an altered profile of prolifera-
tive activity and/or apoptotic activity (or expression of
Bcl-2 protein) compared with tumours carrying other
mutations.
We selected 89 tumours with as well as 99 tumours
without a p53 mutation, as confirmed by DNA mutation
analysis, from a total of almost 600 tumours screened
for mutations within exons 5–8 of p53 (as a part of a
European collaborative study on genetic changes in
breast cancer). In these tumour series we analysed the
J. Pathol. 189: 504–513 (1999)
 p53 MUTATION AND APOPTOSIS IN BREAST CANCER
levels of immunohistochemically detectable p53 and
Bcl-2 protein expression, mitotic counts (as performed
according to standard pathological procedures), the
frequency of apoptotic cells [using terminal transferase
(TdT)-mediated dUTP nick end labelling], and the
extent of tumour necrosis.
MATERIALS AND METHODS
Patients
Material was collected from a cohort of 600 breast
tumours analysed for the presence of p53 mutations, as
a part of a collaborative European study of genetic
changes in breast cancer.31 Material from 188 tumours
was available for analysis. Of these tumours, 89 con-
tained a confirmed p53 mutation. As reported pre-
viously,31 clinico-pathological parameters, including
patient age, tumour size, nodal status, and oestrogen
and progesterone receptor status, were not significantly
associated with specific p53 mutations. None of the
patients received preoperative chemo- and/or radio-
therapy, excluding the possibility that the results have
been influenced by cytotoxic treatments.
Mutations in exons 5–8 of the p53 gene
In Table I, the specific p53 gene alterations in these
breast carcinomas are listed, together with the results of
immunohistochemical detection of p53 protein expres-
sion. DNA isolation, mutation analysis and sequencing
were performed as previously described.31,34,35 Exons 5
through 8 (codons 126–306), of the p53 gene were
screened for mutations. Because almost all codons of
exons 5–8 are located within the sequence-specific DNA-
binding domain (codons 102–292), only one mutation
occurred outside this domain in the present cohort.
Missense mutations occurred in 74 out of 89 tumours.
The codons most frequently affected by missense
mutation were codons 248 and 273. All non-missense
mutations occurred within the sequence-specific DNA-
binding domain and one-third of the non-missense
mutations resulted in a stop codon. Three tumours with
missense mutations in exon 5 were excluded from analy-
sis pertaining to the biological relevance of the zinc-
binding domains, because their localization within or
outside the L2 domain could not be verified by sequence
analysis. In the remaining tumours with missense muta-
tions, 33 out of 71 occurred in the L2 and L3 loops. One
tumour containing two different missense mutations
outside the zinc-binding domains was counted as one
case. Four tumours with non-verified missense muta-
tions in exon 5 or 8 were excluded from analysis
pertaining to residues that directly contact DNA. In the
remaining tumours, 20 out of 70 mutations occurred in
residues directly contacting DNA (codons 120, 241, 248,
273, 276, 280 and 283).
Detection of apoptotic cells
Apoptotic cells were visualized in paraffin-embedded,
formalin-fixed tissue sections, using terminal transferase
(TdT)-mediated dUTP nick end labelling of DNA strand
Copyright  1999 John Wiley & Sons, Ltd.
507
breaks (TUNEL) according to a protocol previously de-
scribed,36 using the enzyme TdT (Boehringer Mannheim,
Mannheim, Germany) and biotinylated dUTP
(Boehringer Mannheim). Although TUNEL is sensitive to
tissue fixation, we and others have observed that ad-
equate pretreatment leads to reliable results.36–38 Using a
Zeiss ‘Axioscop’ microscope (Carl Zeiss, Oberkochen,
Germany) with a checkerboard grid in the ocular, tissue
sections were analysed at a magnification of 400.
Because the number of apoptotic events per high-
power field was very low, we decided to count apoptotic
events in 20 randomly distributed fields per section. Only
diaminobenzidine-stained cells that also displayed an
apoptotic morphology (cell shrinkage and chromatin
condensation and/or nuclear fragmentation) were
included, whereas necrotic areas were excluded from the
analysis. To correct the resulting apoptosis count (AC)
for variations in tumour cell density, the number of
tumour cells was counted in four randomly distributed
high-power fields on haematoxylin and eosin (H&E)-
stained sections. In our series, the AC per 20 high-power
fields ranged from 0 to 304. The mean tumour cell count
per high-power field ranged from 67 to 762.
The amount of necrosis in each section was scored
semiquantitatively on H&E-stained sections at a magni-
fication of 100 by an experienced breast cancer path-
ologist (MJvdV), using four categories: none, low (<25
per cent per field), intermediate (25–50 per cent per
field), and high (>50 per cent per field).
Immunohistochemical analysis
Expression of Bcl-2 and p53 was determined immu-
nohistochemically on paraffin-embedded, formalin-fixed
sections using anti-Bcl-2 monoclonal antibody (MAb)
clone 124 (Boehringer Mannhein) and anti-p53 MAb
DO-7 (DAKO) and a microwave antigen retrieval
method.23,39 Sections were scored semiquantitatively as
described before (for p53 see ref. 23; for Bcl-2 see ref.
39), taking into account both the staining intensity and
the fraction of tumour cells showing positive staining.
Briefly, p53 staining intensity was scored negative (0),
weakly positive (1), moderately positive (2), or strongly
positive (3). The fraction of tumour cells showing the
most intense staining was estimated to be 1–25 per cent
(1), 25–50 per cent (2), 50–75 per cent (3), and 75–100
per cent (4). In tumours completely lacking p53 staining
(p53 staining intensity=0), the fraction of stained
tumour cells was scored as 0 per cent (0). Addition of the
two scores resulted in a total of seven p53 scores: 0 and
2–7. For Bcl-2 a similar scoring system was used, but
tumours were grouped into separate categories, not by
addition or multiplication of scores, but by classifying
them according to the fraction of cells showing the most
intense staining. This scoring system resulted in Bcl-2
scores ranging from 0 to 6. The mitotic count was
determined by an experienced breast cancer pathologist
(MJvdV) according to a standard procedure: paraffin-
embedded, formalin-fixed tissue sections were H&E-
stained and the number of mitoses per 10 high-power
fields (400) was counted using a Zeiss ‘Axioscop’
microscope (Carl Zeiss).
J. Pathol. 189: 504–513 (1999)
508 H.-J. VAN SLOOTEN ET AL.

Table II—p53 mutation and clinico-pathological characteristics of the tumours

Wt p53 Mutant p53 Total p value

Age (years)
c50 28 (42·4) 38 (57·6) 66 (36·1)
>50 65 (55·6) 52 (44·4) 117 (63·9) 0·09
Tumour size
T1 24 (50·0) 24 (50·0) 48 (27·0)
T2 39 (47·0) 44 (53·0) 83 (46·6)
T3 14 (51·9) 13 (48·1) 27 (15·2)
T4 12 (60·0) 8 (40·0) 20 (11·2) 0·77
Node status
Negative 34 (47·2) 38 (52·8) 72 (43·6)
Positive 54 (58·1) 39 (41·9) 93 (56·4) 0·17
Distant metastasis*
Negative 73 (49·0) 76 (51·0) 149 (93·1)
Positive 5 (45·5) 6 (54·5) 11 (6·9) 0·82
Tumour grade
I 20 (100) 0 (0·00) 20 (10·75)
II 34 (69·39) 15 (30·61) 49 (26·34)
III 41 (35·04) 76 (64·96) 117 (62·90) <0·001
Mitosis count
0–9 45 (75·00) 15 (25·00) 60 (32·26)
10cx<20 16 (55·17) 13 (44·83) 29 (15·59)
d20 34 (35·05) 63 (64·95) 97 (52·15) <0·001
Bcl-2 expression
None–low 43 (39·45) 66 (60·55) 109 (57·98)
Medium–high 54 (68·35) 25 (31·65) 79 (42·02) <0·001

*Distant metastasis present at the time of diagnosis. In 5/188 cases, information on patient age, tumour size, node
status, and distant metastasis could not be retrieved.

Statistical methods
The presence of differences in the distribution of
clinico-pathological characteristics between patient
groups with or without a confirmed p53 mutation was
tested for by
2 analysis. The relationship between
various tumour parameters and mean levels of apoptosis
and mitosis was analysed using one-way analysis of
variance. For necrosis, the cut-off was none and low,
versus intermediate and high. The cut-off values used for
the other prognostic factors (p53, Bcl-2, and mitosis
count) were identical to the values used in previous
analyses.23,39 Statistical analysis was done using SPSS
software (SPSS Inc., Chicago, IL, U.S.A.).
RESULTS
We wanted to investigate whether specific mutations
in p53 were associated with differences in proliferative
and apoptotic activity or expression of Bcl-2 in human
breast tumours. So the subset of 89 tumours with
mutated p53 was stratified according to the mutation
type. The specific gene alterations and the scoring results
of immunohistochemical detection of p53 are listed in
Table I. Missense mutations in the p53 gene often
lead to increased protein stability, resulting in an
immunohistochemically (IHC) detectable nuclear
accumulation of the mutant protein. As shown in
Table I, in 12 out of 15 cases frameshift and nonsense
mutations within the DNA-binding domain resulted in
Copyright  1999 John Wiley & Sons, Ltd.
total absence of p53 staining, despite the fact that the
DO-7 antibody recognizes an epitope between amino
acids 1 and 45 in the N-terminal domain of human
wild-type and mutant p53.40
Various clinico-pathological characteristics of
patients with or without a confirmed p53 mutation in
their tumours are listed in Table II. As shown, high
tumour grade, high mitotic counts, and absent or low
Bcl-2 expression were strongly associated with the
presence of a p53 mutation, whereas age, tumour size,
nodal status, and the presence of distant metastasis
were not significantly associated with p53 status.
To analyse the effect on proliferative and apoptotic
activity of mutation location (e.g. in the zinc-binding
domains, in residues directly contacting DNA, or in
residues critical for protein stabilization), mean levels of
mitosis and apoptosis were determined for the subset of
tumours carrying a missense mutation, stratified accord-
ing to mutation location. To investigate whether muta-
tion type (missense vs. frameshift/nonsense mutations)
was associated with changes in proliferation and cell
death parameters, a similar calculation was made for all
tumours with a verified p53 mutation, stratified accord-
ing to mutation type. As shown in Table III, neither the
location of a p53 mutation nor the type of mutation was
associated with changes in the mean level of apoptosis or
mitosis.
Table IV shows data on apoptosis and mitosis, com-
paring tumours with no p53 mutation detected within
exons 5–8 and tumours with a verified mutation: both
J. Pathol. 189: 504–513 (1999)
 p53 MUTATION AND APOPTOSIS IN BREAST CANCER 509

Table III—Type or location of missense mutations in exons 5–8 of p53 is not associated with alterations in the
levels of apoptotic or mitotic activity

p53 mutation N Mean 95% CI p value

Apoptotic index Inside L2–L3 30 0·56 0·38–0·74

Outside L2–L3 35 0·69 0·42–0·96 0·43
Mitotic count Inside L2–L3 31 16·9 13·1–20·7
Outside L2–L3 37 20·9 17·4–24·4 0·12
Apoptotic index Nonsense 14 0·48 0·23–0·72
Missense 67 0·62 0·45–0·78 0·45
Mitotic count Nonsense 15 17·6 11·2–24·0
Missense 71 19·4 16·8–22·0 0·58
Apoptotic index Non-direct DNA 46 0·64 0·41–0·86
Direct DNA contact 20 0·56 0·40–0·72 0·68
Mitotic count Non-direct DNA 51 18·4 15·4–21·4
Direct DNA contact 19 21·7 15·7–27·7 0·27
Apoptotic index Non-protein stabilizing 58 0·63 0·46–0·80
Protein stabilizing residues 6 0·60
0·36–1·56 0·91
Mitotic count Non-protein stabilizing 60 19·1 16·2–21·9
Protein stabilizing residues 7 18·9 10·8–26·9 0·96

Tumours with verified p53 mutations were stratified according to mutation type, e.g. missense mutations located in- or
outside the L2–L3 zinc-binding domains, missense or nonsense mutations, missense mutations located in residues that do
or do not directly contact DNA, as well as missense mutations located in residues critical for protein stabilization. In 19/188
and 8/188 cases, TUNEL-staining results and mitotic counts, respectively, were not evaluable. As a result, the number of
cases analysed in each group varies from 64 to 86.
Apoptotic index=[number of apoptotic cells per 20 high-power fields (400)/tumour cell density]100%; mitotic
count=number of mitosis per 10 high-power fields (400); N=number of cases; 95% CI=95 per cent confidence interval.

Table IV—Mutation of p53 in exons 5–8 is associated with increased levels of apoptosis and mitosis,
compared with wild-type p53

No. of
p53 status cases Mean 95% CI p value

Apoptotic index Wild type 88 0·28 0·21–0·34

Mutant 81 0·59 0·45–0·73 <0·001
Mitotic count Wild type 94 10·0 8·1–11·9
Mutant 86 19·1 16·7–21·4 <0·001

Apoptotic index and mitotic count were determined as described in Table III. In 19/188 and 8/188 cases, TUNEL-staining
results and mitotic counts, respectively, were not evaluable. As a result, the number of cases analysed varies from 169 to 180.

the apoptotic index and the mitotic counts show a
two-fold increase in the mutant p53 group compared
with the group containing mainly wild-type p53
(p<0·001). Figure 1 shows examples of TUNEL staining
in a tumour with wild-type p53 and low levels of
apoptosis, and in a tumour with mutant p53 and very
high levels of apoptosis.
Relationships between the apoptotic index and necro-
sis, Bcl-2 expression, mitotic counts and tumour grade
are shown in Table V. Tumours with high mitotic counts
(d20) or high tumour grade (grade III) also had signifi-
cantly increased levels of apoptosis, whereas tumours
expressing medium to high levels of Bcl-2 had a signifi-
cantly lower level of apoptosis than tumours with low or
no detectable Bcl-2. The presence of necrosis was not
significantly correlated with the level of apoptosis.
In line with the data shown in Table IV, the immuno-
histochemically detected accumulation of p53 protein
was also positively correlated with increased levels of
Copyright  1999 John Wiley & Sons, Ltd.
apoptotic cell death (p<0·001) and mitosis (p<0·001). In
addition, in conjunction with DNA mutation analysis of
p53 mutations, p53 protein accumulation strongly cor-
related with decreased expression of Bcl-2 (p<0·001) and
high tumour grade (p<0·001). It has been reported that
a dose–effect relationship exists between p53 expression
and induction of either cell cycle arrest or apopto-
sis.41,42. Combining data on DNA mutation analysis
and IHC allowed us to analyse the relationship between
expression of either ‘wild-type’ or mutant p53 protein
and levels of apoptosis and mitosis. In the group of
tumours with mutant p53, p53 protein expression was
not significantly associated with levels of apoptosis or
mitosis (Table VI), or with Bcl-2 expression (not shown).
However, in the group of tumours with ‘wild-type’ p53,
11 out of 99 expressed high levels of p53 protein; these
tumours showed a relative increase in levels of mitosis
(Table VI), as well as a relative decrease in Bcl-2
expression (not shown). This finding suggests that these
J. Pathol. 189: 504–513 (1999)
510 H.-J. VAN SLOOTEN ET AL.

DISCUSSION

The data presented in this study indicate that in

Fig. 1—(A) TUNEL staining of an invasive ductal carcinoma with
wild-type p53 (i.e. no mutation within exons 5–8) and a low level of
apoptosis (AI<0.01 per cent) (400, reduced to 70 per cent in
printing). (B) TUNEL staining of an invasive ductal carcinoma with
mutant p53 and a very high level of apoptosis (AI=4·75 per cent)
(400, reduced to 70 per cent in printing)
cases may represent a group of tumours with a p53
mutation located outside exons 5–8, or with an accumu-
lation of inactivated wild-type p53 protein.
invasive breast cancer, localization and type of mutation
of p53 do not seem to be associated with significant
changes in the frequencies of apoptosis or mitosis or the
expression of the anti-apoptotic gene bcl-2. Moreover, in
contrast to the paradigm that p53 inactivation promotes
tumour development primarily by reducing tumour
apoptosis, p53 mutation was accompanied by an
increased incidence of both apoptosis and mitosis.
Animal work has provided evidence that abrogation
of p53-dependent apoptosis promotes tumour devel-
opment and progression,5–7 as well as treatment
resistance.8–10 In general, in breast tumours, p53 muta-
tions have been found to be associated with aneuploidy,
high proliferative activity, and poor prognosis,21,22,43,44
as well as with increased chemo- and hormonal
resistance.23,24
A number of recent studies suggested that mutations
in specific regions of the p53 gene may be of particular
biological significance. Børresen and co-workers
reported that missense mutations in the zinc-binding
L2/L3 domains of the p53 gene were especially associ-
ated with a significant decrease in overall survival rates
(n=76),31 and possibly with increased chemoresist-
ance.32 In a similar study (n=53), it was shown that
missense mutations in residues that directly contact
DNA, but not mutations in L2/L3, were associated with
poor disease-free survival.33 However, in a series of
node-positive and steroid receptor-positive breast cancer
patients, p53 mutation predicted a worse prognosis, but
specific mutations in the L2/L3 domains were not an
independent indicator for prognosis (n=42).45 Bergh
et al. performed complete sequencing of the p53 gene in
a series of 316 breast cancer patients,24 but their data on
the relationship between specific mutations and patient
survival are not compatible with those reported by
Børresen et al. It is difficult to explain these different
reported observations, but an evident problem with
these types of studies is that in breast cancer the
proportion of tumours with a p53 mutation is relatively

Table V—Correlation between apoptotic index (AI), Bcl-2 expression, mitotic count, and tumour grade

No. of Mean AI
cases (%) 95% CI p value

Necrosis (none–low) 147 0·41 0·32–0·49

Necrosis (medium–high) 19 0·62 0·42–0·82 0·09
Bcl-2 (none–low) 92 0·52 0·39–0·65
Bcl-2 (medium–high) 71 0·33 0·25–0·41 0·02
Mitotic count 0–9 49 0·26 0·17–0·35
Mitotic count 10cx<20 28 0·33 0·20–0·47
Mitotic count d20 90 0·55 0·42–0·68 0·004*
Grade I 17 0·17 0·05–0·30
Grade II 41 0·29 0·19–0·39
Grade III 109 0·52 0·41–0·63 0·005*

The mean apoptotic index was calculated as described in Table III.

*Kruskal–Wallis one-way analysis of variance.
Copyright  1999 John Wiley & Sons, Ltd. J. Pathol. 189: 504–513 (1999)
 p53 MUTATION AND APOPTOSIS IN BREAST CANCER
Table VI—‘Wild-type’ p53 protein accumulation is correlated with increased levels of mitosis
p53 status p53 IHC
Apoptotic index Wild type Negative
Wild type Positive
Mitotic count Wild type Negative
Wild type Positive
Apoptotic index Mutant Negative
Mutant Positive
Mitotic count Mutant Negative
Mutant Positive
Tumours were divided into four groups according to their p53 status, as determined by DNA mutation analysis, and
the presence of p53 protein accumulation, determined by immunohistochemical staining. The mean apoptotic index and
mitotic count in these four groups were analysed as described in Table III.
small. Therefore, the number of tumours with a specific
mutation is usually not sufficient for proper analysis,
especially when most patients have not been treated in a
standardized way. An additional problem is that up to
20 per cent of p53 mutations have been reported to
occur outside exons 5–8. As a result, when only exons
5–8 are screened,31–33,45 a small number of patients with
a p53 mutation located outside exons 5–8 will be missed.
For this reason, the reported correlations between
specific p53 mutations and survival of breast cancer
patients have to be interpreted with caution. Our failure
to detect any differences in proliferative and/or cell death
activity in tumours with and without specific p53 muta-
tions seems at odds with the clinical evidence for a
relationship between specific mutations in p53 and
biologically more aggressive breast carcinomas.
Unexpectedly, we found p53 mutation to be associ-
ated with an increase of both apoptotic and mitotic
activity. This association was highly significant and it
seems, therefore, unlikely that the present data have
been seriously skewed by the fact that only exons 5–8
were screened. Moreover, the same association was
found when p53 protein accumulation was used as a
(more crude) measure of p53 mutation. Although p53
inactivation is generally believed to promote tumour
development and progression by reducing tumour apop-
tosis, the effects of p53 mutation on tumour tissue are by
no means clear. Transgenic mouse models used to
investigate the relationship between p53 inactivation,
apoptosis, and tumourigenesis can be grouped into four
categories: (i) models in which abrogation of p53-
dependent apoptosis was found to promote tumour
development and progression;5–7 (ii) models in which
tumour development and progression were accompanied
by a decrease in tumour cell apoptosis that was, how-
ever, independent of p53 status;46,47 interestingly, only
the reduction in basal levels of tumour apoptosis was
reported to occur independently of p53, whereas sensi-
tivity to induction of apoptosis by irradiation remained
dependent on the presence of an intact p53 gene;47 (iii)
models in which loss of p53 function resulted in an
increase in the tumour growth rate without altering the
rate of tumour apoptosis;48–50 and (iv) models in which
p53 inactivation was accompanied by genomic instabil-
Copyright  1999 John Wiley & Sons, Ltd.
511
No. of
cases Mean 95% CI p value
81 0·27 0·20–0·34
7 0·36 0·04–0·67 0·49
86 9·2 7·4–10·9
8 19·6 7·9–31·3 0·002
22 0·40 0·30–0·51
58 0·67 0·48–0·86 0·09
25 15·7 11·3–20·1
59 19·9 17·1–22·6 0·10
ity and increased apoptosis and mitosis.11,51,52 Of inter-
est, genomic instability and apoptosis are frequent
events in all tissues of p53-deficient young mice. Despite
extensive genomic alterations, p53
/
mice mature
without discernible abnormalities until tumours start to
appear, which suggests that many of the genetically
aberrant cells are eliminated through p53-independent
apoptosis.11 In line with this finding, premalignant
mammary glands of mutant p53/neu double transgenic
mice showed significantly higher rates of apoptosis than
similar tissue of mice transgenic for neu only.51
Apart from analysing the relationships between p53
mutation and either proliferation or cell death, we
investigated whether proliferative activity was associated
with cell death activity. Our data show that in invasive
breast cancer, independently of p53 status, high mitotic
activity strongly correlates with increased apoptotic
activity and high tumour grade. Similar relationships
between proliferation and apoptosis and tumour grade
were reported for ductal carcinoma in situ (DCIS),53
as well as for other tumour types.54–59 Consistent
with these clinico-pathological findings, Hundley et al.
reported that although established mammary tumours
originating in mice transgenic for myc or ras (MMTV/
myc and MMTV/ras tumours, respectively) had equal
growth rates, MMTV/myc tumours showed high levels
of apoptosis and proliferation, whereas MMTV/ras
tumours had low levels of apoptosis and proliferation.60
This observation illustrates that in tumours with equal
growth rates, profound differences can exist in the rate
of cell turnover and supports the concept that a tight
link exists between cell proliferation and apoptosis.
Expression of Bcl-2-like survival genes can block
p53-dependent apoptosis,61,62 whereas the presence of
either mutant or wild-type p53 leads to reduced Bcl-2
expression.19,63,64 In line with previous data,36 lack
of expression of the apoptosis inhibitor Bcl-2 was
associated with increased apoptotic activity and muta-
tion of p53, but altered expression of Bcl-2 could not be
linked to specific p53 mutations. It has been reported
that a dose–effect relationship exists between p53 expres-
sion and induction of either cell-cycle arrest or apoptosis
or differentiation.41,42 Combining data on DNA muta-
tion analysis and immunohistochemical staining allowed
J. Pathol. 189: 504–513 (1999)
512 H.-J. VAN SLOOTEN ET AL.

Fig. 2—Model illustrating the relationships between p53 mutation, levels of apoptosis
and mitosis, and Bcl-2 expression. Loss of functional p53 results in loss of cell-cycle
control (G1/S and G2/M checkpoints and centrosome duplication) and p53-dependent
apoptosis. This leads to (i) genomic instability and (ii) rapid proliferation and a
concomitant increase in p53-independent apoptosis, resulting in an increase in cell
turnover. These two mechanisms may synergistically enhance the rate at which mutations
accumulate in p53-mutated tumours, accelerating clonal evolution and promoting rapid
tumour progression and development of resistance against cytotoxic treatments. Also,
abrogation of p53-dependent apoptosis may lead to an inherent increase in resistance to
various cytotoxic treatments

us to evaluate whether the level of (mutant) p53 protein
expression affects the level of apoptosis and mitosis and
Bcl-2 expression. We found that in p53-mutated tumours,
p53 protein levels were not associated with alterations in
the levels of apoptosis, mitosis or Bcl-2, indicating that
the expression level of mutant p53 protein affects neither
proliferative nor cell death activity. In contrast, tumours
that expressed high levels of p53 protein in the absence of
a confirmed p53 mutation showed increased mean mitosis
levels and decreased expression of Bcl-2. This finding is
most likely explained by the assumption that the latter
tumours contain mutations in p53 outside the exons 5–8
screened in this study, or alternatively by accumulation
of an inactivated form of ‘wild-type’ p53 (e.g. p53 protein
bound to Mdm-2).
In conclusion, the data from this study support a
model (Fig. 2) in which inactivation of p53 results in loss
of cell-cycle control, leading to genomic instability and
increased proliferative activity. Concomitantly, frequent
p53-independent apoptosis occurs, resulting in increased
tumour cell turnover rates. Regarded in the perspective
of Nowell’s paradigm on clonal evolution of tumour cell
populations,65 a tumour phenotype with accelerated cell
turnover coupled to genomic instability theoretically
enhances the rate at which mutations accumulate,
allowing rapid clonal evolution and the development
of more aggressive and/or treatment-resistant clones. It
is important to note that although tumours with a
p53 mutation generally have elevated levels of tumour
apoptosis, they may in fact be relatively resistant to
cytotoxic treatments, due to the abrogation of p53-
dependent apoptosis. Our data do not support the
hypothesis that differences in proliferative activity
and/or spontaneous apoptotic cell death are responsible
for the previously reported worse prognosis of patients
with p53 mutations located in the L2/L3 domain, or in
residues that directly contact DNA. However, we cannot
exclude the possibility that tumours with mutations in
L2/L3 are more resistant to anti-cancer therapy, thereby
contributing to poorer patient survival.
ACKNOWLEDGEMENTS
We thank R. Keijzer, Department of Surgery,
LUMC, The Netherlands and N. J. Kuipers-
Dijkshoorn, Department of Pathology, LUMC, The
Netherlands, for their helpful assistance with the
retrieval of tissue blocks and immunohistochemical
staining of the tumour material. We thank Jon
Gunnlaugur Jonasson, Department of Pathology,
University Hospital, Iceland, for supplying tissue
blocks. This work was supported by the E.C. Biomedical
and Health Research Concerted Action (Contract
BMH1-CT92-0890), the Norwegian Cancer Society, the
Icelandic Cancer Society, and the Nordic Cancer Union.
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