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Vol. 2, 1887-1894, November 1996 Clinical Cancer Research 1887
Apoptosis Loss and Bcl-2 Expression: Key Determinants of Lymph
Node Metastases in T, Breast Cancer’
Angels Sierra,2 Xavier Castellsagu#{233}, Silvia T#{243}rtola,
Agustmn Escobedo, Belen Lloveras,
Miguel A. Peinado, Abelardo Moreno, and
Angels Fabra
Departament Cancer i Metastasis, Institut de Recerca Oncol#{246}gica
[A. S.. S. T., M. A. P., A. F.], Servei d’Epidemiologia i Registre del
Cancer [X. Cl, and Servei d’Oncologia, Institut Catal#{224}d’Oncologia.
Hospital Duran i Reynals IA. El, and Servei d’Anatomia Patol#{244}gica
lB. L., A. M.], Ciutat Sanitaria i Universitaria de Bellvitge, Autoviade Castelldefels, km 2.7, 08907 L’Hospitalet de Llobregat, Barcelona,
Spain
ABSTRACT
The Bcl-2 proto-oncogene extends cell survival but does
not confer any proliferative advantage to cells that express
it. Thus, the loss of apoptosis may have a role in progression
allowing the acquisition of additional mutations. To deter-
mine whether apoptosis loss at diagnosis is associated with
the metastatic advantage of ductal breast carcinomas and to
examine the relationship between Bcl-2 expression, p53, and
tumor cell death status, we examined tumor samples from
116 patients diagnosed with T1 (2 cm or less) breast cancer
with (n = 49) or without (n 67) lymph node metastases.
Apoptosis loss in histological sections was considered when
< 1 % of tumor nuclei were stained with terminal de-
oxynucleotidyl transferase labeled with biotin. We studied
the expression of Bcl-2 and p53 by immunohistochemistry
and in 37 p53 mutations by single-strand conformational
polymorphism analysis and cycle sequencing. Multivariate
logistic regression modeling was used to estimate prevalence
odds ratios (pORs) for apoptosis loss and presence of lymph
node metastases. Patients with marked apoptosis loss in
their tumor cells were about 5 times more likely to present
lymph node metastases than those with no apoptosis loss in
their tumor cells (adjusted pOR, 4.7; 95% confidence inter-
val, 1.4-15.6; trend test, P 0.008). Bcl-2 expression was
strongly associated with both apoptosis loss (pOR, 6.9; trend
test, P < 0.0001) and presence of lymph node metastases
(pOR, 5.7; trend test, P 0.002). These associations were
more evident in histological grade I and II tumors than in
poorly differentiated histological grade III tumors and in
p53-negative tumors than in p53-positive tumors. This study
Received 3/5/96; revised 7/1/96; accepted 8/15/96.
, This study was supported by Grant FIS 94/1524 from the Ministerio de
Sanidad y Consumo Espa#{241}ol and Grant SAF 93/51 1 from Comision
Interministerial de Ciencia y Tecnologia.
2 To whom requests for reprints should be addressed, at DepartamentCancer i Metastasis, Institut de Recerca OncolOgica (I.R.O.), Hospital
Duran i Reynals, Ciutat Sanitaria i Universitaria de Bellvitge, Autoviade Castelldefels, km 2.7, 08907 L’Hospitalet de Llobregat, Barcelona,
Spain. Phone: 34-3-263-00-43: Fax: 34-3-263-22-51.
demonstrates for the first time that the lymphatic progres-
sion of T1 human breast cancer is strongly related to apop-
tosis loss.
INTRODUCTION
The identification of genetic lesions that lead to the devel-
opment and progression of early breast cancer is critical to
understanding the biological heterogeneity of breast carcinomas
and to distinguishing a population of patients who may benefit
from more aggressive therapies when the risk of metastasis is
elevated.
We recently reported that overexpression of Bcl-2 in-
creased the risk of lymph node metastases, particularly in HG3
I and II tumors and progesterone-positive T, (2 cm or less)
breast carcinomas ( 1 ). We showed that the overexpression of
Bcl-2 correlated with a reduction of the number of apoptotic
cells, suggesting that Bcl-2 is important in early progression
because it extends the survival of cells, which may eventually
accumulate or acquire other genetic changes. Similar findings
have been reported recently in colorectal epithelia and neopla-
sia, in which altered activation and expression of Bcl-2 resulted
in attenuated apoptosis (2). Furthermore, progressive inhibition
of apoptosis has been associated with the transformation of
colorectal epithelia to carcinomas (3).
An inverse correlation has been observed between Bcl-2
and p53 expression in advanced breast cancer (4, 5), as well as
in colorectal adenomas. This suggests a potential down-regula-
tion of Bcl-2 by mutant p513 during tumorigenesis (6), because
p53 and Bcl-2 may participate in a common pathway for the
control of cell survival (7). Furthermore, experimental studies
using breast cancer cell lines have demonstrated that the p53
mutant protein can effectively control the expression of Bcl-2,
probably by suppressing Bcl-2 transcription (8).
Apoptosis is thought to complement mitosis in regulating
cell number. Inhibition of apoptosis by the deregulation of
certain oncogenes and tumor suppressor genes may be associ-
ated with enhanced proliferation as well as decreased cell death
(9). The extended cell life span conferred by Bcl-2 presumably
permits the accumulation of the additional events needed for
tumor progression (10). Indeed, mechanisms of apoptotic cell
death must be closely linked to the function of Bcl-2 and p53 in
the homeostatic regulation of cell populations, and deregulation
of apoptosis may be a relevant event in the natural history of
breast cancer. We were interested in studying in breast carcino-
mas how these gene products correlate with both apoptosis and
tumor progression. Thus, the aim of this study is to assess
3 The abbreviations used are: HG, histological grade; ER, estrogenreceptor: IHC, immunohistochemical: PCNA, proliferating cell nuclear
antigen: pOR, prevalence odds ratio: PR, progesterone receptor; CI,
confidence interval.
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1888 Apoptosis Loss in Ductal Breast Carcinoma
whether loss of apoptosis is associated with the presence of
lymph node metastases at diagnosis, and to investigate the
relationship between Bcl-2 expression, altered p53, and tumor
cell death status in patients with T, (�2 cm) breast tumors.
MATERIALS AND METHODS
Tissue Samples and Patients
We studied a series of 1 16 patients with T, (�2 cm)
invasive ductal breast carcinomas obtained from two pathology
departments (Ciutat Sanitaria i Universitaria de Bellvitge and
Hospital del Mar, Barcelona, Spain) between 1988 and 1993.
All of the tissue blocks routinely embedded in paraffin were cut
in serial sections of 5 �i.m in our laboratory. Sixty-seven patients
did not have lymph node involvement (T,N0), and 49 had lymph
node involvement (T,N,). The two groups did not differ signif-
icantly with regard to clinical factors such as menopausal status
or patient age. The groups had a similar HG and tumor size
distribution. Neither of the two groups received any form of
treatment before surgery.
In Situ Nick-End Labeling of Apoptotic Nuclei
Apoptosis was detected histologically in paraffin embed-
ded sections by in situ labeling of nuclear DNA fragmentation
as described by Gavrieli (1 1). Deparaffinized sections were
incubated with 20 p.g/ml of proteinase K (Sigma Chemical Co.,
St. Louis, MO) for 15 mm before immersion in 0.3 enzyme
units/�.al terminal deoxynucleotidyl transferase and terminal de-
oxynucleotidyl transferase buffer (Boehringer Mannheim,
Mannheim, Germany) with biotinylated CIUTP at 1 :25 for 60
mm at 37#{176}C.Nuclei were visualized with avidin-biotin-peroxi-
dase complex followed by diaminobenzidine staining.
The quantitative measurement of positive cells was evalu-
ated as described elsewhere (1).
The degree of apoptosis was scored in three categories:
>3%, 1-3%, and < 1% stained tumor nuclei. We considered
that tumors had apoptosis loss when less than 1 % of cell nuclei
were stained.
PS3, Bcl-2, PCNA, and Hormone Receptor Expression
Studies
IHC staining of deparaffinized sections was performed in
normal breast, in breast tumor, and in lymph node metastases
from each patient. Each case was analyzed independently by
two investigators, and the number of positive cells was meas-
ured in 20 fields of the tumor.
To determine Bcl-2 protein expression, the monoclonal
anti-Bcl-2 124 (Cambridge Research Biochemicals, Cheshire,
United Kingdom) was used at 1 :200 dilution. Tumors were
considered positive for Bcl-2 expression when more than 5% of
tumor cells showed cytoplasmic staining.
After microwave boiling in 10 mxi sodium citrate (pH 6.0),
the samples were incubated overnight with antihuman p53 an-
tibody diluted in PBS containing 2% BSA. We used two com-
mercially available antibodies: the monoclonal antibody to p53,
clone BP-53-12-l (Biogenex, San Ramon, CA), and monoclonal
antibody Ab-6, clone DO-l (Oncogene Science, Uniondale,
NY), which are known to have a very good performance (I 2-
15). Staining was revealed with biotin-streptavidin-peroxidase
conjugate (Link-Label kit, Biogenex). Specimens showing more
than 25% stained cell nuclei were considered positive for p53
overexpression. Cytoplasmic staining was not considered to
determine positivity.We also studied tumor proliferation and expression of
hormone receptors, detecting expression of PCNA (anti-PCNA,
Santa Cruz Biotechnology, Inc., Santa Cruz, CA), ERs (DAKO,
Copenhagen, Denmark), and PRs (Novocastra Laboratories,
Newcastle, United Kingdom). Tumors with less than 10% of the
cells with nuclear staining were considered negative for the
expression of hormone receptors or of low proliferation.
Bound antibody was visualized with biotinylated goat an-
timouse IgG (Vector Laboratories, Burlingame, CA). Vectastain
Elite detection kit (Vector) was used to amplify signal by
avidin-peroxidase, following visualization with diaminobenzi-
dine and counterstaining with methyl green or hematoxylin.
p53 Gene Mutation Detection
Amplification of the p53 Gene. A fragment of the p53
gene including exons 5-9 was amplified in a first step by PCR.
Fifty ng of genomic DNA were added to a tube containing 1 unit
of Taq polymerase (Boehringer Mannheim), 0.4 p.M of each
primer, 125 JiM of each dNTP, and PCR buffer (10 mrvt Tris-HC1
pH 7.8, 50 mM KC1, 1.5 mtvi MgC12, 0.01% gelatin) in a final
volume of 25 �il. The reaction was carried out for 25 cycles in
a thermal cycler (Mi Research) at 94#{176}Cfor 30 s, 63#{176}Cfor 30 s,
and 72#{176}Cfor 1 mm. Second nested PCRs were performed to
amplify two fragments including exons 5-6 and 7-8, which
comprise most of the point mutations detected in human tumors
(16). Second PCRs were performed with 0.1-0.5 p.1 of the first
reaction in the same conditions with the following differences:
the primers were 2 p.M each and the PCR consisted of 35 cycles
in the same conditions as the previous one.
The primers used were as follows. First PCR: 12979U,
GCTGCCGTG1TCCAGTTGCT; l4875D, AGGCATCACT-
GCCCCCTGAT. Exons 5-6: 1 3054U, TACTCCCCTGCCCT-
CAACAAG; 13463D, CTCCTCCCAGAGACCCCAGT. Exons
7-8: 13966U, CTGGCCTCATCTTGGGCCTG; 14587D,
CTCGCTFAGTGCTCCCTGGG.
Single-Strand Conformational Polymorphism Analysis.
For analysis of p53 mutations, we used the single-strand con-
formational polymorphism method (17). The nested PCRs were
carried out as described above in the presence of a radioactive
nucleotide ([a-32P]dCTP, 2 �i.Ci/PCR tube). Five p.1 of the
radioactive PCR product were digested with HpaII. Samples
were diluted 20-fold in formamide-dye loading buffer (18) and
incubated for 3 mm at 95#{176}C.Tubes were cooled on ice and 2 p.1
were loaded on a 6% polyacrylamide/l0% glycerol nondenatur-
ing sequencing gel. Electrophoresis was carried out at room
temperature at 30 W for 12 mm and 6 W for 14 h. The gels were
dried under vacuum at 85#{176}Cand exposed to a X-ray film at
room temperature without intensifier screen.
Cycle Sequencing of the p53 Gene. One-half p.1 of the
nested PCR product was used as a template for sequencing.
Reactions were performed using a commercial cycle sequencing
kit (Perkin-Elmer Cetus) and [a-33P}dATP. One of the primers
used for PCR was added (final concentration, 1 p.M) to the
reaction tube. The other reagents (included in the kit) were used
as suggested by the manufacturer. Sequencing reaction was
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Clinical Cancer Research 1889
performed for 30 cycles under the same conditions as the
corresponding PCR amplification. The final product was diluted
3-fold in formamide-dye loading buffer. Samples were dena-
tured for 3 mm at 95#{176}C,and 2 p.1 of each were analyzed in a
denaturing 6% polyacrylamide/8 M urea sequencing gel for 2 or
5 h at 55 W. The gels were dried under vacuum at 85#{176}Cand
exposed to an X-ray film at room temperature without intensi-
fier screen.
The presence of the mutation was confirmed by sequencing
of the other DNA strand and, when possible, by digestion with
a restriction enzyme (when the mutation created or destroyed a
restriction site).
Statistical Analysis
To compare the distributions across groups of a given
variable, the uncorrected x2 test and, when appropriate, the
Fisher’s exact test were performed. Comparisons between pro-
portions as well as tests for linear trends were done by using the
STATCALC module of the Epi Info program (Centers for
Disease Control; Ref. 19).
To estimate the magnitude of the association of each din-
ical and immunohistochemical variable with apoptosis loss or
lymph node metastases, pORs were calculated using uncondi-
tional logistic regression (20). This measure of association es-
timates the excess odds for loss of apoptosis or metastases for
each category with regard to an arbitrary reference category of
a given variable. Values above 1 indicate an excess odds for
apoptosis loss or metastases, values close to 1 indicate no
association, and values below I indicate a reduction in the odds
for that category with regard to the reference category. pORs
estimated through logistic regression modeling have the advan-
tage of allowing for multivariate analyses in which the effects of
several variables can be taken into account simultaneously. Uni-
and multivariate analyses are therefore presented. All logistic
regression models were fitted by means of the maximum like-
lihood estimation of parameters (20) using the statistical pack-
age GLIM (21). For all analyses, an a value of 0.05 was
established to determine statistical significance and, accord-
ingly, 95% CIs around pORs are reported.
RESULTS
Apoptosis Loss, Bcl-2 Expression, and p53 Expression
in T1 Breast Tumors. Table 1 presents the distributions of
apoptosis loss, Bcl-2 expression, and p53 expression (p53-IHC)
according to selected clinical and pathological variables in our
series of I 16 T, breast cancer patients.
Apoptosis loss, as defined by the detection of less than I %
of stained cell nuclei, was observed more frequently in low-
grade tumors (HG I and II) than in poorly differentiated tumors
(HG III; trend test, P < 0.0001) and in estrogen-positive tumors
than in estrogen-negative tumors (x2test, P = 0.02).
Apoptosis loss and Bcl-2 expression were highly corre-
lated. The proportion of tumors with apoptosis loss increased
with increasing values of Bcl-2 expression in tumor cells (trend
test, P < 0.0001). In contrast, the proportion of tumors with
apoptosis loss was significantly larger in p53-negative tumors
than in p53-positive tumors, showing an inverse association
(40.3 versus 16.7%, respectively; P = 0.02).
As with apoptosis loss, Bcl-2 expression was associated
with the expression ofERs (P < 0.0001) and, inversely, with the
HG of the tumor (trend test, P = 0.02). Furthermore, Bcl-2 was
expressed more frequently in tumors expressing PRs (P = 0.02).
No significant associations were observed between Bcl-2 ex-
pression and age, hormonal status, or PCNA (Table 1).
p53 expression was strongly and linearly related to HG but
in the opposite direction of Bcl-2 and apoptosis loss. Thus, p53
was found to be expressed more frequently in HG III tumors
than in HG I and II tumors (trend test, P < 0.000]) and in
estrogen-negative tumors than in estrogen-positive tumors (P <
0.0001).
Immunohistochemical Correlates of Apoptosis Loss.
Table 2 summarizes the results of the uni- and multivariate
analyses for the relationship between apoptosis loss and expres-
sion of pS3, Bcl-2, hormone receptors, and HG. The crude
analysis showed that ERs, Bcl-2, and, inversely, HG and p53-
IHC were the main variables significantly associated with ap-
optosis loss. However, after correcting for the effects of HG and
Bcl-2, the magnitude of the associations found with p53 and
ERs was markedly reduced and no longer significant. In the
multivariate analysis, HG and Bcl-2 expression remained the
two variables most strongly and significantly associated with
apoptosis loss. Furthermore, these associations showed a strong
dose-response relationship but in opposite directions. Whereas
the likelihood of apoptosis loss decreased with increasing HG
(trend test, P = 0.0009), the likelihood of apoptosis loss in-
creased with increasing levels ofBcl-2 expression (P < 0.0001).
Of note are (a) the statistically significant 7-fold increased
likelihood of apoptosis loss for tumors with high Bcl-2 expres-
sion (>50% ofcells) as compared to Bcl-2-negative tumors; and
(b) the fact that even moderate levels of Bcl-2 expression
(between S and 50% of cells) were associated with a significant
4-fold increased likelihood of apoptosis loss.
Fig. 1A shows that the percentage of tumors with loss of
apoptosis was significantly larger in Bcl-2-positive tumors than
in Bcl-2-negative tumors (50.0 versus 14.9%, respectively: P <
0.0001), and in p53-negative tumors than in p53-positive tumors
(40.3 versus 16.7%, respectively; P 0.02). Fig. lB shows that
the association between Bcl-2 and apoptosis loss is only signif-
icant in p53-negative tumors (P = 0.004); in p53-positive tu-
mors, the association is markedly reduced and not statistically
significant (P 0.1).
Apoptosis Loss, Bcl-2, p53, and Likelihood of Lymph
Node Metastases. Table 3 summarizes estimated crude and
adjusted pORs for lymph node involvement in relation to degree
of apoptosis loss and expression of Bcl-2 and p53. The main
finding of these analyses was that both apoptosis loss and Bcl-2
expression were significantly and strongly associated with the
presence of lymph node metastases. Patients with tumors with
very little apoptotic activity (< I % of apoptotic cells) were
about S times as likely to present lymph node metastases as
patients with tumors with a clear apoptotic activity (>3% of
apoptotic cells). Similarly, patients with evident Bcl-2 expres-
sion (>50% of cells) were almost 6 times as likely to have their
lymph nodes affected as patients with tumors with virtually no
Bcl-2 expression. Furthermore, the magnitude of these associ-
ations showed a highly significant dose-response relationship.
Thus, the likelihood of presenting lymph node metastases in-
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Table I Distribution of apoptosis loss, Bcl-2 expression, and p53 expression according to main clinicalpatients with 1, breast tumors
and pathological characteristics in
Apoptosis loss Bcl-2 p53-IHC
No. No. No.
Variable tested % positive tested % positive tested % positive
Numberofpatients n = Ill n = 116 ii = 110
P = 0.95
58.342.6I 2.5
P < 0.0001
P = 0.33
75.059.6
45.0
P - 0.02
P = 0.65
P = 0.02
8.320.850.0
P < 0.0001
Age
<50 30 36.7 30 43.3 29 31.0
51-60 27 25.9 29 69.0 27 25.9>60 39 41.0 42 64.3 40 22.5Unknown 15 15 14Trend test P = 0.65 P = 0.10 P = 0.43
Hormonal status
Premenopausal 40 35.0 40 52.5 39 28.2Postmenopausal 61 34.4 66 62.1 62 24.2Unknown 10 10 9
x2 testHistological Grade
I 24 24 24II 47 52 48III 40 40 38Trend test
ER
Negative (<10%) 19 10.5 19 10.5 19 68.4Positive (�lO%) 73 38.4 78 69.2 74 24.3Unknown 19 19 17,<2 test
PRNegative (<10%) 62 65 61Positive (�l0%) 42 44 43
Unknown 7 7 6
x2 testPCNA
Negative (< 10%) 58 36.2 60 63.3 56 28.6
Positive (�l0%) 48 35.4 48 54.2 47 27.7
Unknown 5 8 7
x2 test P = 0.93 P = 0.34 P = 0.92
Bcl-2
Negative (�5%) 47 14.9 48 35.4
6-50% 39 43.6 37 24.3>50% 25 60.0 25 20.0Trend test P < 0.0001 P = 0.17
p53-JHC
Negative (<25%) 77 40.3 79 60.8Positive (�25%) 30 16.7 31 45.2Unknown 4 6
x2 test P = 0.02 P = 0.14
33.9
35.7
P = 0.85
P < 0.000147.7
70.5
P = 0.02
P < 0.0001
29.530.2
P = 0.94
1890 Apoptosis Loss in Ductal Breast Carcinoma
creased with increasing number of tumors cells showing apop-
tosis loss or Bcl-2 expression. Further stratified analyses by HG
showed that these associations were only observed in HG I and
II tumors (data not shown).
As shown in Table 3, patients with tumors showing both
apoptosis loss and Bcl-2 expression had an almost 8-fold increased
likelihood of presenting lymph node metastases. The magnitude of
this association was even larger among patients with HG I and II
tumors (adjusted pOR, 12.7; 95% CI, 2.6-62.4).
Table 4 further shows that the association between Bcl-2/
apoptosis loss and presence of lymph node metastases was
restricted and even larger in patients with p53-negative tumors.
The effect of Bcl-2 and apoptosis loss among patients with
p53-positive tumors was weak and not statistically significant.
HG and tumor size were not associated with the presence
of lymph node metastases (data not shown).
p53 Expression and Mutations in Breast Tumors. Nu-
clear p53 staining of primary tumors assessed by IHC staining
was present in 28.2% (31 of I 10) of the specimens. We could
study p53 mutations in 37 specimens (33.6%). The correlation
between p53 mutations and p53 expression by IHC staining was
indeed extremely high (x2 test, P = 0.00001).
All tumors having p53 mutations (n = 12) were correctly
classified by IHC staining. From 25 tumors with no p53 gene
mutation, 19 (76%) were correctly classified by IHC staining.
Most of the p53 mutations characterized in this study have
been previously described either in breast or other tumors. It is
noteworthy that in two cases we found a single base substitution
resulting in a stop signal at codon 192. This change should result
in a truncated protein. This prediction was confirmed by the fact
that in both cases, antibody PAb24O (which recognizes a region
comprising amino acid residues 156-214) failed to detect over-
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Table 2 Crude and adjusted pORs for apoptosis loss (< 1% of tumor
cells) according to hormonal and IHC variables in patients with T,A
, 32/64
psO.02
31177
“ Adjusted by HG and Bcl-2 expression.
p53 +TOTAL �53
Bcl-2 +p53+ Bcl-2 -
Bcl-2 +PS3-
Bcl-2 -
0 10 20 30 40 50 60
% of tumors with apoptosis loss
Fig. I Percentage of tumors with apoptosis loss (< I % of dead cells)according to Bcl-2 expression (>6% of tumor cells stained) and p53
expression (>25% of tumor nuclei). A. percentages according to overallBcl-2 status and overall p53 status; B, percentages according to differentcombinations of p53 and Bcl-2 status.
Clinical Cancer Research 1891
breast tumors
Variables
Apoptosis loss, pOR (95% CI)
Crude Adjusted”
Histological Grade
I 1.00 (reference) 1.00 (reference)II 0.53 (0.20-1.43) 0.63 (0.22-1.82)
III 0.10 (0.03-0.35) 0.13 (0.04-0.47)Trend test P = 0.0001 P = 0.0009
ERNegative (< 10%) 1.00 (reference) 1.00 (reference)Positive (� 10%) 5.29 ( I .15-24.42) 1. 18 (0.20-7.1)
PRNegative (< 10%) 1 .00 (reference) 1 .00 (reference)
Positive (�l0%) 1.08 (0.48-2.47) 0.67 (0.25-1.76)Bcl-2
Negative (�5%) 1.00 (reference) I .00 (reference)6-50% 4.42 (1.59-12.27) 3.85(1.31-11.35)>50% 8.57 (2.76-26.63) 6.94 (2.12-22.68)
Trend test P < 0.0001 P < 0.0001p53-IHC
Negative (<25%) 1.00 (reference) 1.00 (reference)Positive (�25%) 0.30(0.10-0.86) 0.57 (0.17-1.91)
expression, whereas positivity was clearly demonstrated with
the other antibody (Ab-6), which binds to residues 37-45.
Another case presenting the same discordance between the two
antibodies had a mutation in codon 257. This change
(CTG-*CAG, Leu-Gln) might affect the protein conforma-
tion and render unavailable the PAb24O antibody epitope.
On the other hand, 3 of 37 cases showed microsatellite
instability when analyzed by PCR amplification of a CA repeat-
containing sequence (data not shown).
Proliferation of Breast Tumors and Apoptosis. PCNA
staining was not associated with apoptosis loss, Bcl-2, or p53
(Table 1). However, further analyses stratified by p53 tumor
status (Fig. 2) showed that in p53-positive tumors, PCNA stain-
ing was more frequently observed in Bcl-2-negative tumors than
in Bcl-2-positive tumors (62.5 versus 23.15, respectively; P
0.03). This inverse association, however, was not observed in
p53-negative tumors (P = 0.68). PCNA staining was markedly
expressed in poorly differentiated HG III tumors (61 .5%) as
compared to low-grade tumors (31.8% in HG I tumors and
36.2% in HG II tumors). This association was significant and
showed a dose-response relationship indicating that the percent-
age of tumors expressing PCNA increased with increasing de-
grees of undifferentiation (i.e., HG; trend test, P - 0.01). No
association was found between p53 and PCNA or between p53
and nodal involvement in either low-grade or high-grade tu-
mors.
DISCUSSION
This is, to our knowledge, the first study demonstrating in
a large, homogenous series of patients with T, breast tumors that
apoptosis loss is directly and strongly associated with the pres-
ence of lymph node metastases. Our study indicates that breast
cancer patients with tumors with both apoptosis loss and Bcl-2
TOTAL �
0 10 20 30 40 50 60
% oftumors with apopto�is loss
B
overexpression show large excess odds for lymph node metas-
tases (pOR, 7.6). This association is larger in HG I and II tumors
(pOR, 12.7) and in p53-negative tumors (pOR, 14.6). We also
show that the main variables associated with apoptosis loss are
Bcl-2 expression and, inversely, the HG of the tumor.
The associations reported here are large, statistically sig-
nificant, and independent of other variables and showed a clear
dose-response relationship. The statistical robustness of our
findings, coupled with the well-defined inclusion criteria that
restricted the analysis to patients with tumors no larger than 2
cm, rules out both an important role of selection bias and the
possibility that the observed associations might be due to chance
or to the masked effect of other parameters included in the study
(i.e. , age, menopausal status, HG, tumor size, and expression of
hormone receptors).
Our results are in agreement with previous reports showing
that Bcl-2 expression was particularly common in carcinomas
with HG I or II and strongly correlated with ER and PR
expression (4-5, 14, 22-24). Furthermore, our findings are not
in conflict with those reported by others showing an inverse
relationship between abnormal p53 protein expression and Bcl-2
protein expression, which in turn is associated with increased
cell proliferation and poor prognosis in breast cancer patients (4,
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1892 Apoptosis Loss in Ductal Breast Carcinoma
Table 3 Crude an d adjusted pORs for lymph node invo lvement according t o apoptosis loss, Bc 1-2, and p5 3 expression
Variable ,,
T,N,
% n
T,N(,
%
Crude
pOR
Adjusted”
pOR 95% CI
Apoptosis loss
>3% of apoptosis I I 22.4 22 35.5 1 .00 1 .00 Reference
1-3% of apoptosis 15 30.6 24 38.7 1.25 1.48 0.52-4.24
<1% of apoptosis 23 46.9 16 25.8 2.87 4.70 1.41-15.65
Unknown 0 5Trend test P = 0.03 P = 0.008
Bcl-2
Negative (�5%) 14 28.6 35 52.2 1.00 1.00 Reference6-50% 17 34.7 23 34.3 1.85 2.25 0.82-6.13
>50% 18 36.7 9 13.4 5.00 5.71 1.80-18.14
Trend test P = 0.001 P = 0.002p53-IHC
Negative (<25%) 35 71.4 44 65.7 1.00 1.00 ReferencePositive (�25%) I I 22.4 20 29.9 0.69 0.50 0.17-1.43Unknown 3 3
x2 test P = 0.40 P = 0.190Bcl-2 (apoptosis loss)
�5% (�l%) 13 26.5 27 43.5 1.00 1.00 Reference�5%(<l%)>5% (�l%)
1
13
2.0
26.5
6
19
9.7]
30.6 1 1.16 1.20 0.42-3.44>5% (<1%) 22 44.9 10 16.1 4.57 7.59 2.06-27.89
Unknown 0 5Trend test P = 0.003 P = 0.002
“ Adjusted by HG, ER expression, and p53-IHC. Effect of p53 further adjusted by Bcl-2.
Table 4 Adjusted p ORs for lymph nod e involvement according to Bcl-2 overexpre ssion and a popt osis loss stratified by p53 expression
Variable
p53-negative tumors p53-positive tumors
n
T,N,
% n
T,N0 Adjusted” T,N,
n % n
T,N0 Adjusted”
% pOR 95% CI% pOR 95% CI
Bcl-2
Negative �s5%) 9 25.7 22 50.0 1.00 Reference S 45.5 12 60.0 1.00 Reference6-50% 13 37.1 15 34.1 4.76 1.27-17.88 3 27.3 6 30.0 0.75 0.07-7.87
>50% 13 37.1 7 15.9 8.10 1.97-33.24 3 27.3 2 10.0 1.83 0.13-26.72
Trend test P = 0.0019 P = 0.7 13
Bcl-2 (apoptosis loss):s5% (�l%) 8 22.9 16 38.1 1.00 Reference S 45.5 1 1 57.9 1.00 Reference:s;5% (<1%)
>5% (�l%)
1
9
2.9
25.7
5
13
11.9 0.28 0.02-4.96
31.9 2.41 0.59-9.83
0 0.0
3 27.3
1
6
5.3�
31.61 0.32 0.02-5.05
>5% (<1%) 17 48.6 8 19.0 14.62 2.96-72.23 3 27.3 1 5.3 5.73 0.19-177.49
Unknown 0 2 0 1Trend test P = 0.0005 P = 0.58
“ Adjusted by HG and ER expression.
5, 14, 22). Indeed, these observations strengthen our hypothesis
that different pathways of progression are involved in low- or
high-grade breast tumors. We suggest that in low-grade tumors,
the overexpression of Bcl-2 protein is triggering the progression
in the absence of p53 mutations. This effect may be mediated by
the ability of Bcl-2 to extend cell survival with no direct influ-
ence on cellular proliferation. The lengthening of the life span of
the cells may favor the accumulation of genetic alterations or
simply the occurrence of other secondary changes, such as an
activation of other oncogenes, such as c-myc, or even the ac-
quisition of major genetic alterations, such as loss or mutation of
the p53 gene.
Our findings are different from the results from other
reports, in which no relationship was demonstrated betweenBcl-2 immunoreactivity and nodal status, presence of metasta-
ses, or disease-free survival (5, 25). This may be due to the strict
selection criteria used in our study. The other series included
heterogeneous groups of patients with small and large tumors
(T,-T4), often with high proliferation rates, which may reveal
different mechanisms of progression as compared to those op-
erating in Bcl-2-positive tumors with low proliferating activity
(15).
Tumor size has been considered one of the clinical factors
associated with axillary lymph nodal status (26). However, the
interest in additional prognostic indicators in breast cancer is
increasing, especially the interest in predicting whether a patient
Research. on September 10, 2020. © 1996 American Association for Cancerclincancerres.aacrjournals.org Downloaded from
+ - #{247}
p53+ p53-
Clinical Cancer Research 1893
%oftumors 70
PCNA +
BcI-2
60
50
40
30
20
10
0
Fig. 2 Percentage of tumors that express PCNA according to p53 andBcl-2 status. Tumors expressing PCNA in more than 10% ofcells were
considered positive (see “Materials and Methods” for details).
with T1 breast cancer will develop lymph node metastases. Our
findings contribute to this aim by providing an alternative mech-
anism of lymph node involvement for small and low-grade
tumors. However, long-term follow-up studies designed to de-
termine the effect of apoptosis loss and Bcl-2 overexpression on
prognosis in T1 breast cancer patients are needed to confirm this
fact.
The relationship between p53, Bcl-2, and apoptosis is still
under active investigation. In this regard, Bcl-2 can abrogate the
p53-dependent cell death triggered by wild-type p53 (27, 28).
Once p53 mutations occur, the deregulation of cell number by
an apoptotic mechanism may not be required because the mu-
tated p53 protein assures cell proliferation. Bcl-2 and p53 gene
products have been linked to a common apoptotic pathway (29).
Moreover, there is an overlap between Bcl-2 and either wild-
type or mutant p53 functions, which could participate in the
control of cell death. We and others (4, 5) have found an inverserelationship between Bcl-2 expression and the presence of p53
mutations, suggesting a possible negative regulatory effect of
Bcl-2 transcription by mutated p53 protein as it has been shown
in human breast cancer cell lines (8).
In our series, higher proliferation rates were found in
p53-mutated tumors in which the Bcl-2 protein was absent, as
has been reported by others (30, 3 1). We measured cell prolif-
eration as the overexpression of the PCNA protein, which has
been demonstrated to be up-regulated by mutated p53 in human
cancer cell lines (32); our data of increased PCNA expression in
a subset of p53-mutated tumors are concordant with this. The
loss of p53-dependent checkpoint may serve to rapidly fix
stochastic mutations that are involved with angiogenesis, inva-
siveness, or metastatic potential (33). Indeed, our results further
suggest that p53 overexpression is far more common in HG III
tumors. Tumors with p53 mutations have an aggressive pheno-
type; this finding is in agreement with previous reports, which
have associated mutated p53 with a high proliferative activity
(34), a high HG (35), and the absence of ERs and PRs (36). The
mutated p53 protein may provide an enhanced proliferation and
the acquisition of properties for a more aggressive phenotype.
On the other hand, because p.53 wild-type gene inactivation
occurs in over one-half of human cancers, it is also possible that
loss of p53-mediated down-regulation of Bcl-2 gene expression
might account for the overproduction of Bcl-2 seen in tumors.
Bcl-2 is a member of a family of genes that can control the
apoptotic threshold of a cell. The dysfunction of other Bcl-2
family members like Bax, Bdl-xL, Bdl-x5, or Bad (37-39) may
be also involved in this mechanism, and they are now under
investigation. The protein product of the bay gene can form
heterodimers with Bcl-2 and abrogate its ability to suppress
apoptosis (40), which could be attributed to reductions of the
levels of these proteins. Experiments in vitro demonstrate that a
temperature-sensitive p53 cDNA transfected in a murine leuke-
mia cell line regulates the expression of Bcl-2 and Bax (41).
Thus, the ratio of Bcl-2 and Bax proteins contributes to apop-
totic cell death control in several tissues (42, 43), and it might be
relevant in breast tumors (44).
In conclusion, the overexpression of Bcl-2 with apoptosis
loss in low-grade T1 tumors identifies a population of breast
cancer patients who can benefit from more aggressive therapies
because these patients may have up to I 3-fold excess odds of
presenting lymph node metastases.
The knowledge of the genes involved in cell death control
could provide important targets for therapeutic intervention and
might allow effective vectors for gene therapy targeted to in-
ducing selective apoptosis in cells.
ACKNOWLEDGMENTS
We gratefully acknowledge G. Aiza, 1. CoIl, S. Ma#{241}as,and L.
Moreno for their skillful technical assistance. We thank the Comit#{233}de
Cancer de Mama from Ciutat Sanitaria i Universitaria de Bellvitge for
their valuable advice in the clinical selection of patients. We are also
grateful to Dr. R. Colomer (Servicio de Oncologia, Hospital La Paz,
Madrid, Spain) for helpful suggestions and critical reading of the manu-
script.
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1996;2:1887-1894. Clin Cancer Res A Sierra, X Castellsagué, S Tórtola, et al. lymph node metastases in T1 breast cancer.Apoptosis loss and bcl-2 expression: key determinants of
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