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Int. J. Cancer: 72, 599–603 (1997)
r 1997 Wiley-Liss, Inc.
Publication of the International Union Against Cancer
Publication de l’Union Internationale Contre le Cancer
LOSS OF HETEROZYGOSITY AT THE TP53 GENE: INDEPENDENT OCCURRENCE
FROM GENETIC INSTABILITY EVENTS IN NODE-NEGATIVE BREAST CANCER
Sarab LIZARD-NACOL1*, Jean-Marc RIEDINGER2, Gérard LIZARD3, Anne-Lise GLASSER4, Nolwenn COUDRAY5, Gilles CHAPLAIN6
and Jacques GUERRIN7
1Laboratory of Molecular Genetics, Centre G.F. Leclerc, Dijon, France
2Laboratory of Medical Biology, Centre G.F. Leclerc, Dijon, France
3INSERM CJF 93-10, Hospital of Bocage, Dijon, France
4INSERM U-71, Clermont-Ferrand, France
5Institute of Cellular and Molecular Biology, Strasbourg, France
6Côte d’Or Cancer Registry, BP 728, Dijon, France
7Oncological Service, Centre G.F. Leclerc, Dijon, France
TP53 abnormalities have been reported as an early event in
the process of cellular transformation of human breast cancers, and involved in mammary-tumor evolution, from in situ
to invasive disease. In this study, node-negative (N2) tumors
were examined for TP53 allelic loss in relation to different
genetic instability events, including allelic loss at chromosome 17p13.3 and c-H-ras-1 loci, as well as alteration of the
c-myc and c-erbB-2/neu oncogenes. TP53 allelic loss was
analyzed to determine whether such an abnormality was the
more important, among other genetic events, in the N2
tumors, whether it appeared independently of these genetic
events, and whether accumulation of genetic events arises in
this group of breast tumors. Clinicopathological parameters
were also examined. Loss of heterozygosity (LOH) at the
TP53 gene appears the most frequent alteration detected
(26% vs. 13%, 8%, 9% and 3% for LOH at D17S30 and c-H-ras-1
loci, and amplification of c-myc and c-erbB-2/neu respectively). There was no association between LOH at the TP53
locus and other genetic events. Among clinicopathological
parameters, significant associations were observed only with
estrogen-receptor-negative tumors (p 5 0.05). Our results
demonstrate that LOH at TP53 arises more frequently in the
N2 breast cancer, thus supporting earlier findings suggesting
that TP53 abnormality has a role early in the pathogenesis of
breast lesions. Moreover, the data indicate that accumulation
of many genetic events occurs at a low level in N2 breast
tumors, and that TP53 abnormality occurs independently of
these genetic events. Int. J. Cancer 72:599–603, 1997.
r 1997 Wiley-Liss, Inc.
In breast cancer, a model taking into account accumulation of
relevant mutations and their order of occurrence is still not clearly
defined. However, certain mutations appear to be more important
than others in the process of breast malignant transformation. Loss
of heterozygosity (LOH) at the TP53 gene (Radford et al., 1993), as
well as abnormal TP53 protein expression (Poller et al., 1993),
were found in a pre-invasive form of breast cancer, the ductal
carcinoma in situ, involving TP53 in mammary-tumor evolution
from in situ to invasive disease. The invasive-breast-cancer group
includes 2 sub-groups of patients: those with lymph-node-negative
and those with lymph-node-positive cancers (N2 and N1 respectively). There are fundamental differences in the biological potential between these populations of tumor cells. Pre-invasive breast
cancer is rarely fatal, N2 invasive breast cancer results in about
20% mortality, whereas mortality from cancer in the N1 sub-group
is much higher (Mackey et al., 1990). It is widely held, but not
proven, that the non-invasive phase precedes tumor invasion, that
the N2 invasive phase appears before the N1 form, and that one
leads to the other (Mackey et al., 1990).
Inactivation of the TP53 tumor-suppressor gene is an important
step in the development of the majority of human cancers,
including those of the breast. Functional inactivation occurs most
commonly as a result of mis-sense mutation, but can also result
from nonsense or deletion mutations (Hollstein et al., 1991). A
number of solid tumors contain a wild-type allelic deletion with
mutations in the remaining TP53 allele. In breast cancer, about 30
to 50% of the cases carry a mutant TP53 gene and/or an LOH,
suggesting that loss of the wild-type allele may be a necessary step
in the oncogenic activation of TP53 in this cancer (Deng et al.,
1994). Associations between TP53 abnormalities and genetic
instability, measured as allelic loss on chromosome 17 and
amplification of the c-erbB-2/neu oncogene, have been recently
reported in breast cancer (Eyfjörd et al., 1995). Interestingly, there
was no association between TP53 abnormalities and amplification
of the c-erbB-2/neu oncogene in the in situ lesions, whereas such
an association was found in invasive cancers (Poller et al., 1993).
Although TP53 abnormalities play a role early in human
mammary tumor formation, one may expect that their frequencies
and their associations with other genetic changes are relatively
similar, in the N2 sub-group, to those found in the in situ lesions.
An association between TP53 abnormalities and genetic instability
was often observed in the N1 sub-group of patients. Such an
association has not been reported in the N2 sub-group, so that a
study including the simultaneous analysis of these changes on the
same tumor specimens is of interest.
In the present study, TP53 LOH alterations were analyzed in a
group of 161 patients with N2 breast cancer. Clinicopathological
parameters (hormone-receptor status, pathological grade, tumor
differentiation and cellular density), and biological parameters
(cathepsin-D, cath-D and pS2 protein) were known for a majority
of the patients. TP53 LOH frequencies in this group of tumors were
compared with several genetic instability factors that were more
frequently involved in breast cancer. These abnormalities included
LOH at chromosome 17p13.3 and c-H-ras-1 loci, as well as
amplification of the c-myc and c-erbB-2/neu oncogenes.
PATIENTS AND METHODS
Patients
Tumor specimens from 161 patients with surgically confirmed
node-negative breast cancer were included in this study. The
patients were all women, ranging in age from 30 to 86 years
(median, 61 years). Tumor samples were drawn from a pool of
frozen specimens (liquid nitrogen) originally submitted for steroidreceptor analysis. Clinical information related to each patient was
available. Only 16 patients received adjuvant treatment by chemotherapy and/or tamoxifen therapy. The median clinical follow-up
was 20 months (range, 10 to 36 months). During this period, 9
Contract grant sponsors: Ligue Bourguignone and the Comité Départemental de Saone et Loire de la Ligue Contre le Cancer.
*Correspondence to: Laboratory of Molecular Genetics, Centre GeorgesFrançois Leclerc, 1, rue du Professeur Marion, 21O34 Dijon Cedex, France.
Fax: (33) 3 80 73 77 26.
Received 21 January 1997; revised 18 April 1997
LIZARD-NACOL ET AL.
600
patients developed tumor recurrence, 8 developed metastatic
lesions, and 7 died of breast cancer.
protein) was based on the results reported by Foekens et al. (1990),
as limited concentration of hormone-sensitivity determination.
DNA analysis
Cellular DNA was extracted from tissues and from blood
leukocytes by a standard proteinase-K and phenol/chloroform
method, and Southern blotting of restriction-enzyme-digested
DNA was performed (Southern, 1975). Briefly, 10 µg of DNA were
digested with appropriate restriction endonucleases (Table I), size
fractionated on a 0.8% agarose gel, transferred to a nylon membrane (Hybond N1, Amersham, Aylesbury, UK), and hybridized
overnight at 65°C with randomly primed 32P-labeled probes. The
probes used for the analyses presented in Table I were obtained
from the ATCC (Rockville, MD). Autoradiography was performed
on membrane filters for 2 days at 270°C using Kodak XAR65
films.
Statistical analysis
The association between the different parameters studied was
examined by a standard univariate x2 test. A p value of 0.05 or less
was considered statistically significant.
Determination of allele loss
Paired normal and tumor DNA from each patient was analyzed
using probe-enzyme combinations which identify restrictionfragment-length polymorphism in a large proportion of individuals.
Normal DNA samples that were polymorphic at a given locus were
considered to be ‘‘informative,’’ whereas the homozygotes were
declared ‘‘uninformative’’. The signal intensity of fragments was
determined by visual examination and confirmed by densitometry.
Because the tumor sample used contained a certain number of
non-neoplastic cells, Southern blotting techniques may have limited resolution for LOH in comparison with PCR-based techniques.
Allelic loss was therefore considered if the intensity in the tumor
was less than 50% of that in its corresponding normal tissue.
Determination of gene amplification
Restriction-enzyme-digested tumor DNA was compared with
matching lymphocyte DNA in the same agarose gels. Blots of these
gels were first hybridized with the oncogene probes. Rehybridation of the same blots with the human a-actin probe
provided a control for the amount of DNA transferred to the nylon
membranes. The oncogene and control-gene autoradiographs were
measured by visualization examination and/or densitometry of
Southern blots.
RESULTS
LOH analysis
Because the amounts of normal and tumoral tissues available
were not sufficient, not all of the samples were tested for LOH at all
the different chromosomal loci. Thus, after digestion with appropriate restriction endonucleases, informative cases identified in a total
number of patients were: 30/117 for TP53 at 17p13.1, 72/86 for
D17S30 at 17p13.3, and 72/126 for c-H-ras-1 at 11p15 (Fig. 1).
Among these informative cases, LOH was observed in 8 cases
(26%) for TP53, 10 cases (13%) for D17S30, and 6 cases (8%) for
c-H-ras-1 (Fig. 2).
Oncogene alterations
Amplification of the 12-kb c-myc fragment was detected in 4
tumors, and a rearrangement of this gene was found in another
tumor by the presence of an additional 4.5-kb-EcoRI restriction
fragment. This band was absent in DNA from the patient’s
lymphocytes, indicating that this alteration was specific for the
tumor tissue (Fig. 3). Alterations of the c-myc gene were observed
in a total of 3% of cases (5/161), and amplification of the
c-erbB-2/neu gene in 9% of cases (15/161) (Fig. 2). None of the
tumors examined showed c-erbB-2/neu gene rearrangement. Analysis of transcription levels of the c-erbB-2/neu gene was performed
in 3 of the 15 amplified tumors. High levels of a 4.8-kb transcript
RNA analysis
Total RNA was isolated from the frozen tumor tissue as
described by Chomczynski and Sacchi (1987), run on 1.2% agarose
gels with formaldehyde, blotted and hybridized as described for
DNA analysis.
Hormone-receptor assay
The estrogen(ER)- and progesterone(PgR)-receptor contents
were determined in cytosolic tumors using ER-EIA and PgR-EIA
methods respectively (Abbott, North Chicago, IL). The cutoff level
used for ER and PgR was 20 fmol/mg of cytosolic proteins.
Assay of cath-D and pS2 protein
Cath-D and pS2 levels were determined in breast-tumor cytosols
using immunoradiometric assay (Elsa-Cath-D and Elsa pS2 respectively), according to the instructions of the manufacturer (CIS Bio,
Gif sur Yvette, France). The Cath-D assay measured the total
amount of Cath-D (52-, 48- and 34-kDa species). The cutoff level
used was 40 pmol/mg of cytosolic protein (Kute et al., 1992). The
choice of a cutoff value for the pS2 protein (1 ng/mg of total
TABLE I – DNA PROBES USED
Locus
Probe
Chromosomal
location
Restriction
enzyme
TP53
D17S30
c-H-ras-1
c-myc
c-erbB-2/neu
php53B
pYNZ22
J77
pTyc 7.4
MAC117
17p13.1
17p13.3
11pter-p15.5
8q24
17q11.2-q12
ScaI
BamHI
BamHI
EcoRI
EcoRI
FIGURE 1 – Southern-blot analysis of paired tumor (T) and normal
lymphocyte (L) DNA, demonstrating loss of heterozygosity (LOH) in
N2 breast tumor. Loci detected were: c-H-ras-1, D17S3O (multiple
alleles detected polymorphism with bands between 0.5 kb and 1.3 kb),
and TP53. For more details of probes and restriction enzymes used, see
Table II.
LOH AT TP53 LOCUS IN NODE-NEGATIVE BREAST CANCER
were detected in the tumor cells in comparison with those obtained
from normal tissue (Fig. 4). No co-amplification of the c-myc and
c-erbB-2 genes was found in the same tumor.
Thus, among all these genetic alterations tested, LOH at the
TP53 locus was the most frequently alteration found in the N2
breast tumors analyzed (Fig. 2).
LOH at the TP53 gene and genetic alterations
There was no association between LOH at the TP53 and other
loci for which LOH was detected, even between LOH at the TP53
601
and D17S30 loci, both localized on the short arm of chromosome
17 (17p13.1 and 17p13.3 respectively). On the contrary, an inverse
relationship was found between these 2 loci ( p 5 0.0195). An
inverse relationship was also found between LOH at TP53 and
amplification of c-erbB-2/neu ( p 5 0.0132). No association was
observed between LOH at the TP53 locus and oncogene amplifications (Table II).
LOH at the TP53 gene and clinicopathological characteristics
of the tumors tested
LOH at the TP53 gene was associated only with estrogenreceptor-negative tumors ( p 5 0.05). Recent biological parameters, such as cath-D and pS2 protein, known to be involved in the
N2 breast cancer, have also been analyzed in this study. Cath-D
values ranged between 13 and 114 pmol/mg of protein. The levels
of pS2 varied from 0 to 80 ng/mg of protein. There were no
statistically significant associations between LOH at the TP53
locus and the levels of cath-D or of pS2 (Table III).
Relationship between other genetic events and clinicopathological
characteristics of the patients tested
LOH at the D17S30 locus was significantly associated with
poorly differentiated tumors ( p 5 0.05), and LOH at the c-H-ras -1
gene was strongly associated with elevated pathological grade
( p 5 0.01) (Table III). c-myc-oncogene alterations were significantly associated with poorly differentiated tumors ( p 5 0.025), as
well as with elevated tumor-cell density ( p 5 0.025), and with
progesterone-receptor-negative tumors ( p 5 0.05). A significant
correlation was also found for c-erbB-2/neu-oncogene amplification with poorly differentiated tumors ( p 5 0.01) and with elevated
pathological grade ( p 5 0.05). No significant correlations of any of
FIGURE 2 – Frequencies of genetic alterations detected in the 161 N2
breast tumors analyzed: TP53 (26%), D17S30 (13%), c-H-ras-1 (8%),
c-myc (3%) and c-erbB-2/neu (9%).
FIGURE 4 – DNA: representative Southern-blot analysis of paired
tumor (T) and normal lymphocytes (L) corresponding to sample 2
showing amplification of the c-erbB-2/neu gene in N2 breast tumor
(lane 2). RNA: representative Northern-blot analysis of c-erbB-2/neu
and of b-actin corresponding to the same sample, showing a high level
of c-erbB-2/neu transcript in comparison with normal tissue (N).
TABLE II – RELATIONSHIP BETWEEN ABSENCE OF LOH AT TP53 AND GENETIC
ALTERATIONS IN THE N2 BREAST TUMORS ANALYZED
FIGURE 3 – Southern-blot analysis of c-myc gene in N2 breast tumor.
DNA from tumors (T) or peripheral-blood cells (L) were digested with
EcoRI. Additional restriction fragments are found in tumor tissue from
patient 2. Hybridization with b-actin probe provides a control for the
amount of DNA transferred to the nylon membranes. The size of each
band is indicated on the left.
TP53
N
D17S30
c-H-ras-1
c-myc
c-erbB-2/neu
With LOH
Without LOH
p
8
22
0 (0)
10 (45)
0.0195
0 (0)
6 (27)
0.0986
0 (0)
5 (23)
0.1396
1 (12)
14 (64)
0.0132
N, total number of cases; number in parentheses, percent of cases
with genetic alteration.
LIZARD-NACOL ET AL.
602
TABLE III – RELATIONSHIP BETWEEN GENETIC ALTERATIONS AND CLINICOPATHOLOGICAL PARAMETERS
IN THE N2 BREAST TUMORS ANALYZED
Parameters
Tumor differentiation
Well 1 moderately
Poorly 1 undifferentiated
p
Tumor grade
I 1 II
III
p
Tumor-cell density
1
2
3
p
Hormonal receptors
ER2
ER1
p
PR2
PR1
p
Cath-D
#40 pmol/mg protein
.40 pmol/mg protein
p
pS2
#1 ng/mg protein
.1 ng/mg protein
p
TP53
n 5 30
D17S30
n 5 72
c-H-ras-1
n 5 72
c-myc
84
66
5/20 (25)
3/10 (30)
0.7703
3/40 (7)
7/32 (21)
0.05
4/46 (8)
2/26 (7)
0.8824
0 (0)
5 (7)
0.01
4 (4)
11 (16)
0.01
123
22
6/23 (26)
2/7 (28)
0.4254
7/48 (14)
3/24 (12)
0.8096
0/50 (0)
6/22 (27)
0.01
3 (2)
2 (9)
0.1153
10 (8)
5 (22)
0.03
24
103
34
1/4 (25)
5/19 (25)
2/7 (28)
0.9901
1/7 (14)
6/43 (13)
3/22 (13)
0.9989
1/11 (9)
3/40 (7)
2/21 (9)
0.9591
0 (0)
1 (1)
4 (11)
0.025
2 (8)
9 (8)
4 (11)
0.8566
41
120
4/7 (57)
4/23 (17)
0.05
3/13 (23)
5/17 (29)
0.6477
3/22 (13)
7/50 (14)
0.9672
5/33 (15)
5/39 (12)
0.7757
2/27 (7)
4/45 (8)
0.8257
3/32 (9)
3/40 (7)
0.7748
2 (4)
3 (2)
0.4486
4 (5)
1 (1)
0.05
4 (12)
10 (8)
0.7801
6 (8)
7 (7)
0.9511
91
57
2 (2)
3 (5)
0.3152
9 (9)
1 (1)
0.0550
4 (4)
1 (1)
0.5868
3 (3)
1 (1)
0.5734
5 (5)
3 (5)
0.9517
48
94
0 (0)
5 (5)
0.1038
5 (10)
3 (3)
0.0773
2 (4)
3 (3)
0.7655
1 (2)
3 (3)
0.7058
4 (8)
2 (2)
0.0821
N
73
88
c-erbB-2/neu
N, total number of cases; n, number of informative cases for the locus analyzed; number in parentheses,
percent of cases with genetic alteration.
these genetic alterations were observed with the levels of cath-D or
pS2.
DISCUSSION
In breast cancer, the number of genetic events needed for tumor
initiation, progression and metastasis, depends on several parameters and remains unclear. It is unknown which, if any, of the
genetic alterations underly this change, or whether their combinations are essential to the malignant process. TP53 abnormalities
have been reported as an early event in the process of cellular
transformation in certain malignancies (Hasegawa et al., 1995)
including human breast cancers (Radford et al., 1993). In a
pre-invasive form of breast cancer, the ductal carcinoma in situ,
these abnormalities appear to occur independently of other genetic
instability events (Poller et al., 1993).
In this study, we evaluated TP53 status in the N2 breast tumors,
examining frequencies of TP53 LOH alterations and their relationships to other genetic events. These events include LOH at
chromosome 17p13.3 and c-H-ras-1 loci as well as alteration of the
c-myc and c-erbB-2/neu oncogenes.
The c-H-ras-1 gene, located at 11p15, is frequently (30%)
involved in deletion or LOH in breast cancer (Champène et al.,
1995); however, this alteration has never been determined in N2
patients. The c-myc gene, which encodes a nuclear-DNA-binding
protein, has been reported to be amplified in 15 to 31% of N1 breast
carcinomas (Escot et al., 1986; Berns et al., 1992), and rearrangement of c-myc was observed in 5% of cases (Escot et al., 1986).
However, few data are available for c-myc alterations in N2
patients. Because the c-ras and c-myc oncogenes were both
involved in cellular transformation and/or apoptosis pathway in
cooperation with TP53 (Finlay et al., 1989), we analyzed these 2
genes in the N2 breast tumors. The c-erbB-2/neu gene, which
encodes a transmembrane protein with extensive homology with
the epidermal-growth-factor receptor (Dobashi et al., 1991), has
been found to be amplified and over-expressed in 10- to 50% of N1
breast cancers. In N2 breast cancer, the prevalence is somewhat
lower (10 to 20%), but the prognostic value of c-erbB-2/neu is now
well established in this group of patients (Press et al., 1993).
In the N2 tumors analyzed in this study, LOH at the c-H-ras-1
gene was observed in only 8% of informative cases. This small
proportion of LOH at the c-H-ras-1 gene suggests that LOH at this
locus may not be a causal factor in breast carcinogenesis but rather
a marker for tumor aggressiveness (Champène et al., 1995). We
found an alteration of the c-myc gene in 3% of cases, and an
amplification of the c-erbB-2/neu gene in 9% of cases. The
prevalence of these 2 oncogene alterations in our study is in
agreement with earlier data concerning the N2 patient group (Berns
et al., 1992).
In our group of patients, it is still too soon after surgery to
investigate the prognostic significance of the genetic alterations
tested. Nevertheless, these alterations are known to be associated
with other cellular parameters reflecting poor prognosis. We have
therefore made comparisons with a variety of factors: clinical
(steroid-receptor status), morphological (histoprognostic grade and
tumor differentiation) and biological (Cath-D and pS2). Cath-D is a
protease which facilates cancer-cell migration and invasion, and
pS2 is an estrogen-regulated protein found indicative for hormonal
sensitivity. Both of these 2 proteins have been reported in N2 breast
tumors (Kute et al., 1992; Foekens et al., 1990). Our results (LOH
at the D17S30 and c-H-ras-1 loci and amplification of c-myc and
c-erbB-2/neu) confirm earlier observations (Berns et al., 1992;
Press et al., 1993; Coles et al., 1992), and show significant
associations between the genetic events tested and some clinicopathological factors (poorly differentiated tumors, elevated pathological grade, elevated tumor-cell density and progesterone-receptornegative tumors).
LOH AT TP53 LOCUS IN NODE-NEGATIVE BREAST CANCER
Among the different genetic alterations analyzed, LOH at the
TP53 gene is the most frequent alteration detected (26%). LOH at
chromosome 17p13.3, near to the TP53 gene localization (17p13.1),
was the second most prevalent alteration observed in these tumors
(13%). No association was found between LOH at these 2 loci,
suggesting that the use of the pYNZ22 probe provides additional
information about regions of loss on the short arm of chromosome
17, and agrees with Coles et al. (1990), who reported the existence
of (a) tumor-suppressor gene(s) located distally to the TP53 gene.
There was no association between LOH at the TP53 locus and other
genetic events, or with cath-D and pS2 levels. These findings
demonstrate that this alteration arises not only more frequently in
the N2 breast cancer, but also independently of other genetic
events.
We have shown that no LOH at the TP53 gene was found in
benign breast samples among the heterozygous patients tested
(Lizard-Nacol et al., 1995). However, mutations in the TP53 gene,
determined by the PCR-SSCP method, have been identified in 8%
of the benign breast tissues (data not shown). These results are in
agreement with the findings of Millikan et al. (1995) and Younes et
al. (1995), and suggest that point mutations in the TP53 gene occur
603
before loss of the wild-type allele in breast carcinomas (Coles et
al., 1992). In view of the multi-step nature of carcinogenesis, an
initial genetic change in breast-tumor cells may be an alteration at
TP53, beginning with point mutation in the pre-cancerous lesions,
followed by LOH in the more developed lesions. Thus, additional
genetic changes, such as oncogene amplifications and/or LOH at
another chromosomal locus, might contribute to breast-tumor
progression.
Our study supports earlier findings suggesting that TP53 abnormality has a role early in the pathogenesis of breast lesions.
Moreover, the data indicate that accumulation of many other
genetic events occurs at a low level in N2 breast tumors, and that
TP53 abnormalities occur independently of these genetic events.
ACKNOWLEDGEMENTS
This work was supported by the Ligue Bourguignonne and the
Comité Départemental de Saône et Loire de la Ligue contre le
Cancer. We thank Mrs. M. Arnal and Mrs. L. Hahnel for technical
assistance.
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