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Expression profile of BRCA1 and BRCA2 genes in premenopausal Mexican women with breast cancer: clinical and immunohistochemical correlates
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Expression profile of BRCA1 and BRCA2 genes in premenopausal Mexican women with breast cancer: clinical and immunohistochemical correlates

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Gloria Loredo-Pozos, Erwin Chiquete, …

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.

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  • 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. References 1. Ganz PA, Greendale GA, Petersen L, Kahn B, Bower JE. Breast cancer in younger women: reproductive and late health effects of treatment. 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