This document summarizes a study examining the expression of BRCA1 and BRCA2 genes in premenopausal Mexican women with breast cancer. The study analyzed 62 premenopausal women under age 45 with breast cancer to evaluate the clinical and immunohistochemical correlates of BRCA1 and BRCA2 mRNA expression. Low levels of BRCA1 expression were associated with younger patient age and more advanced clinical stage, but BRCA2 expression did not correlate with disease severity. Neither gene expression correlated with tumor characteristics like histology, differentiation, metastasis, or markers like p53 and HER-2.

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 lower BRCA1 expression than older women (P 0.05).
increased invasiveness and influences the response of BRCA1 and BRCA2 expression correlated in healthy, but
breast carcinoma (BC) to chemotherapeutics. However, not in tumor tissues (TT). Neither BRCA1 nor BRCA2
expression of BRCA1 and BRCA2 genes has not been expression was associated with tumor histology, differen-
completely characterized in premenopausal BC. We ana- tiation, nodal metastasis or p53 and HER-2 expression.
lyzed the clinical and immunohistochemical correlates of After multivariate analysis, only disease stage explained
BRCA1 and BRCA2 expression in young BC women. We BRCA1 mRNA levels in the lowest quartile. Premeno-
studied 62 women (mean age 38.8 years) who developed pausal BC has aggressive clinical and molecular
BC before the age of 45 years. BRCA1 and BRCA2 mRNA characteristics. Low BRCA1 mRNA expression is associ-
expression was assessed by reverse transcriptase-poly- ated mainly with younger ages and advanced clinical stage
merase chain reaction (RT-PCR) and that of HER-2 and of premenopausal BC. BRCA2 expression is not associated
p53 proteins by immunohistochemistry. Body mass index with disease severity in young BC women.
(BMI) C27 (52%) and a declared family history of BC
(26%) were the main risk factors. Ductal infiltrative ade- Keywords BRCA1 Á BRCA2 Á Breast carcinoma Á
nocarcinoma was found in 86% of the cases (tumor size Gene expression Á Mexico Á mRNA
[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 Introduction
cervical cancer, stage III/IV disease, HER-2 positivity, and
Breast carcinoma (BC) currently represents a major health
G. Loredo-Pozos Á F. Siller-Lopez Á M. E. Ramos-Marquez (&)
´ ´
problem worldwide. Depending on the population and
´
Instituto de Enfermedades Cronico-Degenerativas. Centro clinical stage, premenopausal women represent 25% of the
Universitario de Ciencias de la Salud, Universidad de people with this type of cancer [1–3]. Many studies have
Guadalajara, Sierra Mojada 950. Colonia Independencia, demonstrated that BC in premenopausal patients has a
Guadalajara, Jalisco C.P. 44340, Mexico
e-mail: eloisa@cucs.udg.mx
more aggressive clinical course than in older patients
[4–6]. In general, breast tumors in young women are bio-
E. Chiquete logically different, with a higher proliferation index and
Departamento de Medicina Interna, Hospital Civil de less histological differentiation, when compared with older
Guadalajara ‘‘Fray Antonio Alcalde’’, Guadalajara, Mexico
e-mail: erwinchiquete@runbox.com
patients [7].
Mutations in BRCA1 and BRCA2 genes are responsible
A. Oceguera-Villanueva for 90% of the inherited BC cases [8]. A reduced expression
´
Instituto Jaliciense de Cancerologıa, Guadalajara, Mexico of BRCA1 has been associated with increased invasiveness
of sporadic or inherited BC [9]; and as is the case for other
A. Panduro
´
Departamento de Biologıa Molecular, Hospital Civil de gene expression markers [10–13], BRCA1 expression influ-
Guadalajara ‘‘Fray Antonio Alcalde’’, Guadalajara, Mexico ences the response of BC to chemotherapeutic agents

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

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

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

5. Med Oncol
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 signifi-
cance 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 expres-
sion 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 pre-
menopausal 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
Fig. 1 Correlation between BRCA1 and BRCA2 expression in lowest quartile of the sample, as compared with older
healthy (a) and tumor tissue (b). Relative BRCA1 or BRCA2/GAPDH women. This is in accordance with previous observations
mRNA levels are depicted in y axes. Centered line represents regarding a more ‘‘deleterious’’ gene expression profile in
correlation fit; outliers represent the mean 95% confidence interval
young versus older women [9, 16–18, 22]. However, in a
multivariate analysis the only factor independently asso-
marker [9, 16–18, 22]. Nevertheless, it is not unusual that ciated with BRCA1 mRNA levels in the lowest quartile was
several markers are associated with some clinical and clinical staging, even when adjusting for age, either as a
molecular variables in several studies, but not, or only to continuous or a discrete (patients aged B35 years versus
some extend in other reports, especially when considering older women) variable. These observations satisfied our
young women [23–27]. These discrepancies could be due hypothesis in that low BRCA1 mRNA levels are indepen-
to different sample sizes and age cut-offs used when dently associated with disease severity. On the other hand,
selecting patients for analyses. As was shown in the present we could not find any relationship between BRCA2 gene
report, an age B35 years seems to distinguish among the expression profile and clinical, histological, or molecular
young premenopausal women, those with the worse clini- variables. Indeed, an important limitation of our study is
cal and molecular profile. It is well known that women the small sample size, which may be underpowered to
younger than 35 years had a high frequency on inherited detect some small, but pathophysiologically important
gene variants that render them with a high probability of differences in disease behavior.

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

7. Med Oncol
Acknowledgments All the authors have contributed to the concep- 14. Egawa C, Motomura K, Miyoshi Y, Takamura Y, Taguchi T,
tion and design of the work and the analysis of the data in a manner Tamaki Y, et al. Increased expression of BRCA1 mRNA predicts
substantial enough to take public responsibility for it; each one favorable response to anthracycline-containing chemotherapy in
believes the manuscript represents valid work; and each has reviewed breast cancers. Breast Cancer Res Treat. 2003;78:45–50. doi:
the final version of the manuscript and has approved it for publication. 10.1023/A:1022101310500.
The authors had full access to all the data in the study and take 15. Zhou C, Smith JL, Liu J. Role of BRCA1 in cellular resistance to
responsibility for the integrity of the data and the accuracy of the data paclitaxel and ionizing radiation in an ovarian cancer cell line
analysis. The authors of this article declare that the article is original carrying a defective BRCA1. Oncogene. 2003;22:2396–404. doi:
and has not been published or submitted for publication elsewhere, and 10.1038/sj.onc.1206319.
that there is no any affiliation with any organization with a direct or 16. Al-Mulla F, Abdulrahman M, Varadharaj G, Akhter N, Anim JT.
indirect financial interest in the subject matter discussed in the man- BRCA1 gene expression in breast cancer. A correlative study
uscript that may affect the reporting of the work submitted. between real-time PCR and immunohistochemistry. J Histochem
Cytochem. 2005;53:621–9. doi:10.1369/jhc.4A6544.2005.
17. Rakha EA, El-Sheikh SE, Kandil MA, El-Sayed ME, Green AR,
Ellis IO. Expression of BRCA1 protein in breast cancer and its
References prognostic significance. Hum Pathol. 2008;39:857–65. doi:
10.1016/j.humpath.2007.10.011.
1. Ganz PA, Greendale GA, Petersen L, Kahn B, Bower JE. Breast 18. Klauber-DeMore N. Tumor biology of breast cancer in young
cancer in younger women: reproductive and late health effects of women. Breast Dis. 2005–2006;23:9–15.
treatment. J Clin Oncol. 2003;21:4184–93. doi:10.1200/JCO. 19. Ramos-Marquez ME, Grijalva G, Armendariz-Borunda J. Duc-
2003.04.196. tular hyperplasia is characterized by an over expression of c-Myc
2. Choi DH, Lee MH, Bale AE, Carter D, Haffty BG. Incidence of in bile duct ligation ? furan injured rats: possible role of inter-
BRCA1 and BRCA2 mutations in young Korean breast cancer leukin-6. Hepatol Res. 2002;22:127–38. doi:10.1016/S1386-
patients. J Clin Oncol. 2004;22:1638–45. doi:10.1200/JCO.2004. 6346(01)00121-8.
04.179. 20. Salgado S, Garcia J, Vera J, Siller F, Bueno M, Miranda A, et al.
3. Yankaskas BC. Epidemiology of breast cancer in young women. Liver cirrhosis is reverted by urokinase-type plasminogen acti-
Breast Dis. 2005–2006;23:3–8. vator gene therapy. Mol Ther. 2000;2:545–51. doi:10.1006/mthe.
4. Walker RA, Lees E, Webb MB, Dearing SJ. Breast carcinomas 2000.0210.
occurring in young women (35 years) are different. Br J Cancer. 21. Chomczynski P, Sacchi N. Single-step method of RNA isolation
1996;74:1796–800. by acid guanidinium thyocyanate-phenol-chloroform extraction.
5. Adami HO, Malker B, Holmberg L, Persson I, Stone B. The Anal Biochem. 1987;162:156–9. doi:10.1016/0003-2697(87)
relation between survival and age at diagnosis in breast cancer. N 90021-2.
Engl J Med. 1986;315:559–63. 22. Baldassarre G, Battista S, Belletti B, Thakur S, Pentimalli F,
6. Chung M, Chang HR, Bland KI, Wanebo HJ. Younger women Trapasso F, et al. Negative regulation of BRCA1 gene expression
with breast carcinoma have a poorer prognosis than older women. by HMGA1 proteins accounts for the reduced BRCA1 protein
Cancer. 1996;77:97–103. doi:10.1002/(SICI)1097-0142(1996 levels in sporadic breast carcinoma. Mol Cell Biol. 2003;23:
0101)77:197::AID-CNCR16[3.0.CO;2-3. 2225–38. doi:10.1128/MCB.23.7.2225-2238.2003.
7. Armes JE, Trute L, White D, Southey MC, Hammet F, Tesoriero 23. Choi DH, Kim S, Rimm DL, Carter D, Haffty BG. Immunohis-
A, et al. Distinct molecular pathogenesis of early-onset breast tochemical biomarkers in patients with early-onset breast
cancers in BRCA1 and BRCA2 mutation carriers. A population- carcinoma by tissue microarray. Cancer J. 2005;11:404–11. doi:
based study. Cancer Res. 1999;59:2011–7. 10.1097/00130404-200509000-00008.
8. Pherson K, Steel C, Dixon J. Breast cancer-epidemiology, risk 24. Maru D, Middleton LP, Wang S, Valero V, Sahin A. HER-2/neu
factors and genetics. BMJ. 2000;321:624–8. doi:10.1136/bmj. and p53 overexpression as biomarkers of breast carcinoma in
321.7261.624. women age 30 years and younger. Cancer. 2005;103:900–5. doi:
9. Thompson ME, Jensen RA, Obermiller PS, Page DL, Holt JT. 10.1002/cncr.20850.
Decreased expression of BRCA1 accelerates growth and is often 25. Jmor S, Al-Sayer H, Heys SD, Payne S, Miller I, Ah-See A, et al.
present during sporadic breast cancer progression. Nat Genet. Breast cancer in women aged 35 and under: prognosis and sur-
1995;9:444–50. doi:10.1038/ng0495-444. vival. J R Coll Surg Edinb. 2002;47:693–9.
˜
10. Munoz-Gonzalez D, Zeichner-Gancz I, Candelaria M, Ramirez- ´
26. Guerra I, Algorta J, Dıaz de Otazu R, Pelayo A, Farina J. ˜
Ugalde MT, Perez-Sanchez M, Cervantes-Vazquez G, et al. Her- Immunohistochemical prognostic index for breast cancer in
2/neu expression as a predictive factor for response to anthra- young women. Mol Pathol. 2003;56:323–7. doi:10.1136/mp.
cycline-based chemotherapy in a Mexican population of locally 56.6.323.
advanced breast cancer patients. Med Oncol. 2005;22:23–8. doi: 27. Ibrahim EM, Ezzat AA, Rahal MM, Raja MM, Ajarim DS.
10.1385/MO:22:1:023. Adjuvant chemotherapy in 780 patients with early breast cancer:
´ ´
11. Fernandez-Sanchez M, Gamboa-Dominguez A, Uribe N, Garcıa- ´ 10-year data from Saudi Arabia. Med Oncol. 2005;22:343–52.
Ulloa AC, Flores-Estrada D, Candelaria M, et al. Clinical and doi:10.1385/MO:22:4:343.
pathological predictors of the response to neoadjuvant anthracy- 28. Gammon MD, Hibshoosh H, Terry MB, Bose S, Schoenberg JB,
cline chemotherapy in locally advanced breast cancer. Med Brinton LA, et al. Cigarette smoking and other risk factors in
Oncol. 2006;23:171–83. doi:10.1385/MO:23:2:171. relation to p53 expression in breast cancer among young women.
12. Acharya CR, Hsu DS, Anders CK, Anguiano A, Salter KH, Cancer Epidemiol Biomarkers Prev. 1999;8:255–63.
Walters KS, et al. Gene expression signatures, clinicopathologi- 29. Gerson R, Alban F, Villalobos A, Serrano A. Recurrence and
cal features, and individualized therapy in breast cancer. JAMA. survival rates among early breast cancer cases with triple nega-
2008;299:1574–87. doi:10.1001/jama.299.13.1574. tive immunophenotype. Gac Med Mex. 2008;144:27–34.
13. Kimura M, Koida T, Yanagita Y. A study on telomerase activity 30. Crabtree B, Neme Y, Rivera S, Olivares G. Hormone Receptors,
and prognosis in breast cancer. Med Oncol. 2003;20:117–26. doi: HER2/neu and p53 in 1027 Mexican patients with breast cancer.
10.1385/MO:20:2:117. J Clin Oncol. 2005;23:S903.