Gloria Loredo-Pozos, Erwin Chiquete, Antonio Oceguera-Villanueva, Arturo Panduro, Fernando Siller-Lo´pez, Martha E. Ramos-Márquez Low BRCA1 gene expression is associated with increased invasiveness and influences the response of breast carcinoma (BC) to chemotherapeutics. However, expression of BRCA1 and BRCA2 genes has not been completely characterized in premenopausal BC. We analyzed the clinical and immunohistochemical correlates of BRCA1 and BRCA2 expression in young BC women. We studied 62 women (mean age 38.8 years) who developed BC before the age of 45 years. BRCA1 and BRCA2 mRNA expression was assessed by reverse transcriptase-polymerase chain reaction (RT-PCR) and that of HER-2 and p53 proteins by immunohistochemistry. Body mass index (BMI) C27 (52%) and a declared family history of BC (26%) were the main risk factors. Ductal infiltrative adenocarcinoma was found in 86% of the cases (tumor size [5 cm in 48%). Disease stages I–IV occurred in 2, 40, 55, and 3%, respectively (73% implicating lymph nodes). Women aged B35 years (24%) had more family history of cervical cancer, stage III/IV disease, HER-2 positivity, and lower BRCA1 expression than older women (P-.05). BRCA1 and BRCA2 expression correlated in healthy, but not in tumor tissues (TT). Neither BRCA1 nor BRCA2 expression was associated with tumor histology, differentiation, nodal metastasis or p53 and HER-2 expression. After multivariate analysis, only disease stage explained BRCA1 mRNA levels in the lowest quartile. Premenopausal BC has aggressive clinical and molecular characteristics. Low BRCA1 mRNA expression is associated mainly with younger ages and advanced clinical stage of premenopausal BC. BRCA2 expression is not associated with disease severity in young BC women.

1. Med Oncol
DOI 10.1007/s12032-008-9114-7
ORIGINAL PAPER
Expression profile of BRCA1 and BRCA2 genes in premenopausal
Mexican women with breast cancer: clinical
and immunohistochemical correlates
Gloria Loredo-Pozos Æ Erwin Chiquete Æ
Antonio Oceguera-Villanueva Æ Arturo Panduro Æ
´
´
Fernando Siller-Lopez Æ Martha E. Ramos-Marquez
Received: 2 August 2008 / Accepted: 15 October 2008
Ó Humana Press Inc. 2008
Abstract Low BRCA1 gene expression is associated with
increased invasiveness and influences the response of
breast carcinoma (BC) to chemotherapeutics. However,
expression of BRCA1 and BRCA2 genes has not been
completely characterized in premenopausal BC. We analyzed the clinical and immunohistochemical correlates of
BRCA1 and BRCA2 expression in young BC women. We
studied 62 women (mean age 38.8 years) who developed
BC before the age of 45 years. BRCA1 and BRCA2 mRNA
expression was assessed by reverse transcriptase-polymerase chain reaction (RT-PCR) and that of HER-2 and
p53 proteins by immunohistochemistry. Body mass index
(BMI) C27 (52%) and a declared family history of BC
(26%) were the main risk factors. Ductal infiltrative adenocarcinoma was found in 86% of the cases (tumor size
[5 cm in 48%). Disease stages I–IV occurred in 2, 40, 55,
and 3%, respectively (73% implicating lymph nodes).
Women aged B35 years (24%) had more family history of
cervical cancer, stage III/IV disease, HER-2 positivity, and
´
´
G. Loredo-Pozos Á F. Siller-Lopez Á M. E. Ramos-Marquez (&)
´
Instituto de Enfermedades Cronico-Degenerativas. Centro
Universitario de Ciencias de la Salud, Universidad de
Guadalajara, Sierra Mojada 950. Colonia Independencia,
Guadalajara, Jalisco C.P. 44340, Mexico
e-mail: eloisa@cucs.udg.mx
E. Chiquete
Departamento de Medicina Interna, Hospital Civil de
Guadalajara ‘‘Fray Antonio Alcalde’’, Guadalajara, Mexico
e-mail: erwinchiquete@runbox.com
A. Oceguera-Villanueva
´
Instituto Jaliciense de Cancerologıa, Guadalajara, Mexico
A. Panduro
´
Departamento de Biologıa Molecular, Hospital Civil de
Guadalajara ‘‘Fray Antonio Alcalde’’, Guadalajara, Mexico
lower BRCA1 expression than older women (P 0.05).
BRCA1 and BRCA2 expression correlated in healthy, but
not in tumor tissues (TT). Neither BRCA1 nor BRCA2
expression was associated with tumor histology, differentiation, nodal metastasis or p53 and HER-2 expression.
After multivariate analysis, only disease stage explained
BRCA1 mRNA levels in the lowest quartile. Premenopausal BC has aggressive clinical and molecular
characteristics. Low BRCA1 mRNA expression is associated mainly with younger ages and advanced clinical stage
of premenopausal BC. BRCA2 expression is not associated
with disease severity in young BC women.
Keywords BRCA1 Á BRCA2 Á Breast carcinoma Á
Gene expression Á Mexico Á mRNA
Introduction
Breast carcinoma (BC) currently represents a major health
problem worldwide. Depending on the population and
clinical stage, premenopausal women represent 25% of the
people with this type of cancer [1–3]. Many studies have
demonstrated that BC in premenopausal patients has a
more aggressive clinical course than in older patients
[4–6]. In general, breast tumors in young women are biologically different, with a higher proliferation index and
less histological differentiation, when compared with older
patients [7].
Mutations in BRCA1 and BRCA2 genes are responsible
for 90% of the inherited BC cases [8]. A reduced expression
of BRCA1 has been associated with increased invasiveness
of sporadic or inherited BC [9]; and as is the case for other
gene expression markers [10–13], BRCA1 expression influences the response of BC to chemotherapeutic agents

2. Med Oncol
[14, 15], which might in the future have an impact on
treatment choices [16]. Thus, there is increasing interest in
BRCA1 and BRCA2 expression as alternative biologic
markers of cancer behavior and response to treatment [7, 16–
18]. However, to the best of our knowledge, the expression
of these genes has been scarcely characterized in premenopausal BC patients. In this study, we analyzed the clinical
significance of expression of BRCA1 and BRCA2 genes in
premenopausal women with BC. Our hypothesis was that
tumor mRNA levels of these two genes are associated with
clinical and laboratory characteristics of disease severity.
Methods
From January 2003 to June 2005, we studied 62 women
with BC diagnosed before the age of 45 years, who were
treated at the Instituto Jaliciense de Cancerologı´a and at
the Hospital Civil de Guadalajara ‘‘Fray Antonio Alcalde’’
(Guadalajara Jalisco, Mexico). The internal Committee of
Ethics of our centers approved this study. Informed consent
was obtained either from all patients or their legal proxy. A
standardized, structured questionnaire was used to collect
data from the patient regarding demography and relevant
antecedents.
slides coated with silane (Sigma Immunchemicals, St.
Louis, Missouri, USA). The samples were deparaffinised in
xylene and rehydrated via a series of graded alcohols
(absolute alcohol to wash away the xylene, followed by
95% and then 70% alcohol, with 5 min duration each for
rehydration). Sections were taken to water. Epitope
retrieval was carried out in the microwave oven using
epitope retrieval solution at 95°C for 45 min. Then the
sections were rinsed under running water and taken to PBS
buffer for 1 min. Endogenous peroxidase activity was
blocked by incubating the sections in 12 ll of 30%
hydrogen peroxide for 30 min, followed by washing in
PBS buffer. Then anti-HER-2 antibody at 1:100 dilution
was applied for 30 min followed by rinsing in PBS buffer
for 1 min. Visualization reagent was applied for 30 min
and rinsed with distilled water, followed by DAB solution
for 5 min. DAB was removed with distilled water. The
slides were then counterstained with Meyer hematoxylin,
dehydrated in increasing grades of ethanol, cleared in
xylene, and mounted in Depex. A TT known to react with
anti-HER-2 antibody was used as positive control. Slides
exposed to PBS without primary antibody were used as
negative controls in each staining batch. The DAKO Hercep Test Protocol system was used to grade the degree of
membrane staining (0 and 1± = negative; 2± = moderate
positivity; and 3± = intense positivity).
Tissue extraction and histological analysis
Immunohistochemical analysis of p53 in tumor tissues
Tumor (TT) and healthy tissue (HT) biopsies or mastectomies were obtained from each patient (from the same
breast and in the same surgical act, one specimen per
patient) for the histological, gene expression, and immunohistochemical analyses. These tissues were obtained
before exposure to any chemotherapeutic agent and in the
premenopausal status. TT and HT were fixed in 10% buffered neutral formalin, dehydrated in alcohols, cleared in
xylene, and embedded into low-melting-point paraffin, as
described elsewhere [19, 20]. All staining procedures for
light microscopy were carried out on 5-lm thick sections
and routine histological examinations were performed for
all tissue samples on sections stained with hematoxylin and
eosin. TT was composed essentially from tumor cells, and
since HT was extracted from the same affected breast, it
was composed from non-macroscopically affected mammary gland and some adipose tissue.
Immunohistochemical analysis of HER-2
(c-erb-B2/c-neu) in tumor tissues
Sections of 4 lm thick were cut from appropriately
selected paraffin blocks containing lesional tissue using a
rotary microtome (Leica RM 2135, Meyer Instruments,
Houston Texas, USA). Blocks were mounted on glass
Immunohistochemical analysis of p53 was performed
according to the manufacturer instructions (Cell Marque,
Rocklin, California, USA). Briefly, sections of 4 lm thick
were cut from appropriately selected paraffin blocks containing lesional tissue using the rotary microtome, mounted
on prewashed glass slides positively charged and heated at
60°C for 1 h. The samples were deparaffinised in xylene
and rehydrated via a series of graded alcohols twice for
4 min each. A 1:10 dilution of epitope retrieval solution
was prepared (20x ImmunoDNA Retriever with Citrate,
Bio SB) and verted in coupling glasses containing samples
slices and were placed inside a pressure cooker set at low
pressure at 95°C for 45 min. Slides were cooled at room
temperature for 20 min and rinsed with PBS (pH 7.0) for
1 min, followed by washing in peroxidase blocker for
20 min. The preparations were incubated with the primary
antibody (DO-7, Cell Marque, 0.5 mM concentration) at
1:400 dilution for 1 h at room temperature and rinsed with
PBS buffer for 1 min. Slides were incubated in the Mouse/
Rabbit ImmunoDetector Biotin LinK visualizing system
and Mouse/Rabbit ImmunoDetector HPR Label for 15 min
each and rinsed with distilled water followed by DAB
solution for 3 min. A TT known to react with anti-p53
antibody was used as positive control. Negative controls

3. Med Oncol
were also included. The preparations were counterstained
with Meyer hematoxylin for 1 min, dehydrated with a
series of distilled water, 96% ethanol, absolute ethanol, and
xylene (15 washes in every step). The mounted glasses
were then covered and observed with the microscope.
RNA extraction and quantification in tumor
and healthy tissues
Isolation of total RNA from TT and HT was carried out
according to the Chomczynski–Sacchi method [21].
Briefly, TT and HT were homogenized using a polytron in
the presence of Trizol. Chloroform was added to separate
aqueous phase and total RNA was precipitated with isopropanol at 4°C overnight. Quantity and intactness of RNA
were routinely tested by determining 260/280 absorbance
analysis and ethidium bromide fluorescence of RNA electrophoresed in 1% formaldehyde agarose gels.
Expression analyses of BRCA1 and BRCA2 genes
in tumor and healthy tissues
BRCA1 and BRCA2 expression in TT and HT was
accomplished essentially as previously described [19]. HT
was analyzed as external efficiency control of the same
affected breast, when compared with gene expression in
TT. Three reverse transcriptase reactions per patient were
carried out in order to obtain the corresponding cDNAs.
Reverse transcriptase-polymerase chain reaction (RT-PCR)
was performed using 2 lg of total RNA transcribed in
0.05 M Tris–HCl pH 8.3, 40 mM KCl, 7 mM MgCl2
buffer containing 0.05 mg/ml of random hexamers, 1 mM
dNTPs mix 0.05 U/ml RNase inhibitor and 200 U/ml
Moloney murine leukemia virus (M-MLV) reverse transcriptase. Samples were incubated for 10 min at 70°C and
then for 60 min at 37.5°C. Reverse transcriptase was further inactivated by heating the sample tubes at 95°C for
10 min. cDNAs were used immediately for reaction or
were stored at -20°C until use. Gene amplifications were
performed in a PCR buffer of 50 mM Tris–HCl pH 9.0, and
50 mM NaCl containing a mix of 100 mM dNTPs and 1 U
Taq DNA polymerase. Final oligonucleotide primer concentration were 10 lM. Primer sequences were BRCA1
sense 50 -ACAGCTGTCTGGTGCTTCTGTG-30 , antisense
50 -CATTGTCCTCTGTCCAGGCATC-30 ; BRCA2 sense
50 -CTTGCCCCTTTCGTCTATTTG-30 , antisense 50 TACG
GCCCTGAAGTACAGTCTT-30 , GAPDH sense 50 -CGGA
TGCAACGGATTTGGTCGTAT-30 antisense 50 -AGCCTT
CTCCATGGTGGTGAAGAC-30 [14]. The pair of primers
for BRCA1, BRCA2, and GAPDH amplified 106, 350, and
567 bp fragments, respectively. PCR was run in an automated thermal cycler with initial denaturation at 95°C for
10 min followed by 40 cycles each at 95°C for 15 sec,
61°C for 1 min, and 72°C for 1 min and a final extension
for 5 min at 72°C. Levels of expression of all transcripts
were quantified with a photodocumentation system. We
used water as ‘‘blank reaction’’ since the RT-PCR assay
and a sample of mammary adipose tissue as negative
control since RNA extraction.
Statistical analysis
Pearson chi-square and Fisher exact tests were used to
assess proportions in nominal variables for bivariate and
homogeneity (when more than two variables) analyses. To
compare quantitative variables distributed between two
groups, Student t test was performed in distributions of
parametric variables. Pearson correlation was used in
continuous variables. BRCA1 and BRCA2 mRNA levels are
reported as relative units with respect to the mRNA
expression of the housekeeping gene GAPDH. No patient
was excluded in any comparison analysis. To find independent predictors of the expression of BRCA1 in the
lowest quartile, multivariate analyses were constructed by
forward stepwise logistic regression, after selection of
candidate variables by means of bivariate analyses (selected if P 0.1). Adjusted odds ratios (OR) with the
respective 95% confidence intervals (CI) are provided. The
fitness of the model was evaluated by the Hosmer-Lemeshow goodness-of-fit test, which was considered as reliable
if P [ 0.2. All P-values are two-sided and considered
significant when P 0.05 in final analyses. SPSS version
13.0 for WindowsTM (SPSS Inc., Chicago, IL) was used in
all calculations.
Results
We studied 62 premenopausal women with BC. Mean age
was 38.8 years (range 22–45 years). A high body mass
index (BMI) and a declared family history BC were the
main risk factors (Table 1). There were 15 (24%) women
aged B35 years, who had significantly more history of
cervical cancer, stage III/IV disease (80% vs. 51%,
P = 0.048) and HER-2 protein positivity than older women
(Table 1). The right mammary gland was affected in 61% of
cases. In most patients the histological type of BC was
infiltrating ductal carcinoma, and in near half of the cohort
the tumor size in its greatest measurement was [5 cm.
Advanced disease was present in the majority of cases; 73%
with affection to lymph nodes. Well-differentiated tumors
were not observed (Table 1). HER-2 protein expression was
present in 16 (26%) tumors: 3 had moderate positivity (2±)
and 13 had intense positivity (3±). p53 was expressed in 32
(52%) tumors: 9 were weakly positive, 12 moderately
positive, and 11 were highly positive.

4. Med Oncol
Table 1 Main characteristics of the study cohort
Variables
All patients
(n = 62)
Persons aged B35 years
(n = 15)
Persons aged [35 years
(n = 47)
P-Value*
0.66
Risk factors
BMI C 27, n (%)
32 (52)
7 (47)
25 (53)
Declared family history of BC, n (%)
16 (26)
3 (20)
13 (28)
Tumor size
0.74
0.33
Greatest diameter 2 cm, n (%)
3 (5)
1 (7)
2 (4)
Greatest diameter 2–5 cm, n (%)
31 (50)
5 (33)
26 (55)
28 (45)
9 (60)
19 (40)
54 (87)
13 (87)
41 (87)
Lobular, n (%)
3 (5)
1 (7)
2 (4)
Medullary, n (%)
2 (3)
0 (0)
2 (4)
Mixed, n (%)
3 (5)
1 (7)
2 (4)
Poorly differentiated, n (%)
12 (19)
4 (27)
8 (17)
Moderately differentiated, n (%)
50 (81)
11 (73)
39 (83)
0 (0)
0 (0)
0 (0)
Greatest diameter [5 cm, n (%)
Tumor histology
Infiltrating ductal, n (%)
0.82
Histological grade**
Well differentiated, n (%)
0.46
Clinical staging
0.03
Stage I, n (%)
1 (2)
1 (7)
0 (0)
Stage II, n (%)
25 (40)
2 (13)
23 (49)
Stage III, n (%)
34 (55)
11 (73)
23 (49)
Stage IV, n (%)
2 (3)
1 (7)
1 (2)
32 (52)
8 (53)
24 (51)
0.89
16 (26)
7 (47)
9 (19)
0.03
Immunohistochemistry
Positivity to p53, n (%)
Positivity to HER-2, n (%)
* P-value for differences between patients aged B35 years and older women; Pearson chi-square or Fisher exact test, as corresponded
** Grade of cellular differentiation
BC Breast carcinoma; CC Cervical carcinoma; BMI Body mass index
Mean BRCA1 (0.583 vs. 0.344 relative units, respectively; P 0.001) and BRCA2 (0.286 vs. 0.149 relative
units, respectively; P = 0.006) mRNA levels were higher
in TT than in HT. No correlation was observed between TT
and HT with respect to BRCA1 or BRCA2 expression.
However, mRNA levels of BRCA1 correlated with those of
BRCA2 in HT, but not in TT (Fig. 1); which denotes a
differential deregulation in mRNA expression of these
genes in tumor cells. BRCA1 mRNA was not detected in 4
(6.5%) tumors, and 15 (24%) fell in the lowest quartile of
the sample. On the other hand, BRCA2 mRNA was negative in 19 (31%) specimens, which coincided exactly with
the lowest quartile of the sample. TT of women aged
B35 years had significantly lower levels of BRCA1 gene
expression, as compared with older women (Fig. 2). Also,
women aged B35 years had more cases of BRCA1
expression in the lowest quartile of the sample, when
compared with their older counterparts (53% vs. 19%,
respectively; P = 0.002). However, there were no other
significant differences in mean tumor mRNA levels of
BRCA1 and BRCA2 genes according to other clinical and
histological characteristics (Figs. 2 and 3). After multivariate analysis adjusted for differentiation grade,
histological type, lymph nodes implication, age, relevant
antecedents, p53 and HER-2 proteins expression; the only
factor independently associated with BRCA1 mRNA levels
in the lowest quartile was clinical staging, when considered
as a continuous variable (OR: 3.22, 95% CI: 1.01–10.27).
Discussion
We found that in premenopausal women with BC a low
BRCA1 gene expression is associated with a younger age
and clinical staging. This molecular marker was not associated with other characteristics of disease severity as p53
or HER-2 expression, grade of differentiation, lymph nodes
implication and other factors previously linked to this

5. Med Oncol
Fig. 1 Correlation between BRCA1 and BRCA2 expression in
healthy (a) and tumor tissue (b). Relative BRCA1 or BRCA2/GAPDH
mRNA levels are depicted in y axes. Centered line represents
correlation fit; outliers represent the mean 95% confidence interval
marker [9, 16–18, 22]. Nevertheless, it is not unusual that
several markers are associated with some clinical and
molecular variables in several studies, but not, or only to
some extend in other reports, especially when considering
young women [23–27]. These discrepancies could be due
to different sample sizes and age cut-offs used when
selecting patients for analyses. As was shown in the present
report, an age B35 years seems to distinguish among the
young premenopausal women, those with the worse clinical and molecular profile. It is well known that women
younger than 35 years had a high frequency on inherited
gene variants that render them with a high probability of
developing BC [1, 18]. Race differences and disease
severity may also account for the heterogeneity of the
results of studies designed to explore the clinical significance of gene expression profile in defining prognosis in
young BC women.
A higher than expected frequency of p53 positivity
status in TT was found in our cohort (52%; 53% in those
aged B35 years and 51% in women aged C 35 years), and
a similar picture in HER-2 only in women aged B35 years.
Maru, et al. [24] have reported 50% cases of p53 expression in women aged B30 years; and Gammon, et al. 44%
in a cohort of women aged B45 years [28]. In Mexican
women it has been reported that a very high frequency of
p53 positivity status (as high as 78.5% in young women
with advanced disease), a high frequency of lymph nodes
involvement and advanced clinical stages, either in premenopausal or older women [29, 30]. Thus, it appears that
these characteristics are unusual with respect to other
populations, but common in a Mexican cohort. Inherited
influence for the development of BC might be a significant
pathogenic factor in the patients included in our report.
With the objectives and methodology applied in the present
study, we could not determine the exact role of BRCA1 and
BRCA2 gene mutations on disease behavior. Nevertheless,
we could expect a high frequency of predisposing gene
variants in our patients, as suggested by the family history
of BC (26%) and the age cut-off that was considered.
mRNA levels of BRCA1 and BRCA2 genes were higher
in TT than in HT. This finding may be attributable to the
fact that HT was composed from cells with a relatively low
proliferation index. BRCA1 expression correlated with that
of BRCA2 in HT, but not in tumors. This finding may
denote a pathological deregulation in mRNA expression of
these genes in tumor, but not in healthy cells. Women aged
B35 years had significantly lower levels of BRCA1 mRNA
and they had more cases of BRCA1 expression in the
lowest quartile of the sample, as compared with older
women. This is in accordance with previous observations
regarding a more ‘‘deleterious’’ gene expression profile in
young versus older women [9, 16–18, 22]. However, in a
multivariate analysis the only factor independently associated with BRCA1 mRNA levels in the lowest quartile was
clinical staging, even when adjusting for age, either as a
continuous or a discrete (patients aged B35 years versus
older women) variable. These observations satisfied our
hypothesis in that low BRCA1 mRNA levels are independently associated with disease severity. On the other hand,
we could not find any relationship between BRCA2 gene
expression profile and clinical, histological, or molecular
variables. Indeed, an important limitation of our study is
the small sample size, which may be underpowered to
detect some small, but pathophysiologically important
differences in disease behavior.

6. Med Oncol
Fig. 2 BRCA1 expression according to several clinical and immunohistochemical variables (n = 62, in every comparison). Relative
BRCA1/GAPDH mRNA levels in tumor tissues are depicted in y axes.
Filled bars represent arithmetic means; error bars represent standard
error of means (±2)
Fig. 3 BRCA2 expression according to several clinical and immunohistochemical variables (n = 62, in every comparison). Relative
BRCA2/GAPDH mRNA levels in tumor tissues are depicted in y axes.
Filled bars represent arithmetic means; error bars represent standard
error of means (±2)
In conclusion, young premenopausal women with BC
have aggressive clinical, histological, and molecular characteristics. BRCA1 expression is a useful marker of disease
severity in very young women with BC. BRCA2 expression
seems not to be a good molecular marker of severity in
premenopausal BC.

7. Med Oncol
Acknowledgments All the authors have contributed to the conception and design of the work and the analysis of the data in a manner
substantial enough to take public responsibility for it; each one
believes the manuscript represents valid work; and each has reviewed
the final version of the manuscript and has approved it for publication.
The authors had full access to all the data in the study and take
responsibility for the integrity of the data and the accuracy of the data
analysis. The authors of this article declare that the article is original
and has not been published or submitted for publication elsewhere, and
that there is no any affiliation with any organization with a direct or
indirect financial interest in the subject matter discussed in the manuscript that may affect the reporting of the work submitted.
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