|Year : 2015 | Volume
| Issue : 1 | Page : 158-163
Expression of BRCA1 and BRCA2 proteins and their correlation with clinical staging in breast cancer
Suresh Hedau1, Madhu Batra2, Usha Rani Singh2, Alok C Bharti1, Amitabha Ray3, Bhudev C Das4
1 Division of Molecular Oncology, Institute of Cytology and Preventive Oncology (ICMR), Noida, Uttar Pradesh, India
2 Department of Pathology, Guru Teg Bahadur Hospital, Sahadara, India
3 Faculty of Basic Sciences, Saint James School of Medicine, Anguilla, West Indies
4 Department of Molecular Oncology, Dr. B. R. Ambedkar Centre for Biomedical Research, University of Delhi, New Delhi, India
|Date of Web Publication||16-Apr-2015|
Division of Molecular Oncology, Institute of Cytology and Preventive Oncology (ICMR), I-7, Sector - 39, Noida - 201 301
Source of Support: Institutional, Institute of Cytology and Preventive Oncology, Conflict of Interest: None
Objectives: The purpose of this study was to evaluate the level of expression of the BRCA1 and BRCA2 proteins in sporadic breast cancer cases to determine the functional role of these genes in breast carcinogenesis.
Materials and Methods: Paraffin embedded histologically proven invasive breast tissue sections that were obtained from 40 patients and the adjacent normal breast tissue sections used as controls to determine breast carcinoma specific changes in the expression of BRCA1 and BRCA2 by immunohistochemistry (IHC).
Results: Majority of the cases express either low or no detectable level of BRCA1 expression in tumor tissues in comparison with control; the decline in BRCA1 expression was found to be more prominent in advanced grade 3 disease. On the other hand, the expression of BRCA2 protein was moderate or low in breast cancer cases and its overall distribution did not show significant difference when compared with controls. Interestingly, those breast cancer cases, which were found to express low or no BRCA1 expression, demonstrated a higher protein level of BRCA2. The inverse correlation of BRCA1 and BRCA2 expression was more prominent in post-menopausal patients.
Conclusions: Our results demonstrate in a subset of cases that decline in BRCA1 expression that may be associated with potentially compensatory increase in BRCA2 protein, which may depend on tumor grade as well as menopausal status.
Keywords: BRCA1 gene, BRCA2 gene, immunohistochemistry, sporadic breast cancer
|How to cite this article:|
Hedau S, Batra M, Singh UR, Bharti AC, Ray A, Das BC. Expression of BRCA1 and BRCA2 proteins and their correlation with clinical staging in breast cancer. J Can Res Ther 2015;11:158-63
|How to cite this URL:|
Hedau S, Batra M, Singh UR, Bharti AC, Ray A, Das BC. Expression of BRCA1 and BRCA2 proteins and their correlation with clinical staging in breast cancer. J Can Res Ther [serial online] 2015 [cited 2019 May 26];11:158-63. Available from: http://www.cancerjournal.net/text.asp?2015/11/1/158/140985
| > Introduction|| |
Breast cancer is the most commonly diagnosed cancer and the leading cause of cancer death in females worldwide.  In India, breast cancer is the second most predominant cancer in women, showing a fast rising trend, especially in urban India. Among Indian women, breast cancer and uterine cervix cancer together account for about 50% of all the other cancers. According to the recent nationwide figures, there were about 80,000 new cases and approximately 35,000 deaths due to breast cancer.  The development of breast cancer is associated with several genetic and non-genetic factors. The non-genetic factors include reproductive risk factors and lifestyle changes.  For instance, prolonged exposure to estrogen due to early age of menarche, late menopause, use of oral contraceptives, high fat diet, and sedentary lifestyle have been shown to play an important role in the development of breast cancer. 
Among the genes that have been associated with the genesis of breast cancer, the BRCA1 and BRCA2 genes are strongly implicated in the pathogenesis; both genes have been now cloned and fully characterized. , BRCA1 and BRCA2 are putative tumor suppressor genes located on chromosome 17q21 and 13q12, respectively. BRCA1 encodes a protein of 220 kDa consisting of 1863 amino acids; whereas BRCA2 produces a 384 kDa protein that has 3418 amino acids. Many tumors with germ line BRCA1 mutations show loss of heterozygosity at these loci, which uniformly involves loss of wild-type BRCA1 allele, indicating the role of the BRCA1 gene as a tumor suppressor gene.  Although somatic mutations have not been well characterized, loss of heterozygosity, decreased levels of BRCA1 mRNA and protein expression and methylation of the BRCA1 promoter region have been shown in some sporadic breast carcinoma, indicating the involvement of BRCA1 even in sporadic form. , Studies have indicated that the promoter region of BRCA1 gene has been found to be hypermethylated in most of the sporadic cases of breast cancer. This is indicative of interference with the expression of the gene leading to the abnormal expression of the protein. 
Although the prevalence of BRCA1 and BRCA2 mutations may vary according to geographical location, perhaps BRCA2 is not frequently mutated in sporadic breast cancers in comparison with BRCA1.  However, studies have pointed to a considerable role of these two genes in the genesis of sporadic breast cancer. , BRCA2 is found either in the nucleus or in perinuclear compartments, such as endoplasmic reticulum and Golgi vesicles. Little is known about BRCA2 gene expression, except that BRCA2 mRNA has a tissue-specific expression pattern similar to that of BRCA1 mRNA and that these two genes are coordinately regulated during mammary epithelial proliferation and differentiation, despite the fact that they display no homology.  Overall, the extent to which expression of these two genes are modulated a significant role in sporadic cancer is still not clear.
The BRCA1 protein is localized exclusively in the nuclei of normal and malignant breast tissue. Several investigators , have reported that BRCA1 protein expression is reduced or absent in familial and sporadic breast cancer by immunohistochemical analysis. Mechanisms other than direct mutation of BRCA1 gene, such as allelic loss or methylations of the BRCA1 promoter region  may be involved in its altered protein expression. Our data also suggest that reduced expression of BRCA1 and BRCA2 protein may play an important role in mammary carcinogenesis in Indian sporadic cases and that mechanisms other than mutation may be involved in the reduced expression of BRCA1 and BRCA2 protein. Therefore, the present study has been designed to analyze BRCA1 and BRCA2 protein expression in sporadic breast cancer cases and correlate the expression of these proteins with various clinico-pathological variables.
| > Materials and methods|| |
Paraffin embedded tissue sections were obtained from 40 sporadic breast cancer patients, who underwent surgery between the years 2003-2005. All the cases had histological evidence of invasive breast carcinoma, and none had a family history in first-degree relatives as determined at the time of surgery. Diagnosis of all the patients was confirmed by taking fine needle aspiration cytology (FNAC)/Trucut biopsy of the lump for histopathology. The patients had undergone modified radical mastectomy and the specimen was sent for histopathological examination in the pathology department. The histological typing and Bloom Richardson's grading was done by Pathologist. Same number of control samples (n = 40) were taken from the normal tissue adjacent to the cancerous growth.
For IHC studies, 3 μm thin sections were cut from the paraffin-embedded tissues. The immunostaining was done by avidin-biotin complex method. Briefly, the method involved fining paraffin sections on poly-L-lysine coated slide, which were de-waxed with xylene and rehydrated through a graded alcohol series. Endogenous peroxidase activity was blocked in phosphate buffered saline (PBS) solution containing 3% hydrogen peroxide for 30 minutes and the slides were washed in PBS. For antigen retrieval, slides were immersed in 1 mM citrate-phosphate buffer, pH 6.0, and microwaved at 100 watts for about 15 minutes. After the buffer had been cooled, normal horse-serum was reacted with the slides for 1 hour to eliminate non-specific immunostaining. The slides were incubated with BRCA1 and BRCA2 primary antibodies overnight at 4 o C in a humidified chamber. The details of the primary antibodies used in this study were as follows: Monoclonal antibody (mAb) Ab-1 for BRCA1 (MS110) with dilution of 1:50 (Oncogene Research, UK), mAb D0-1 for BRCA2 protein with a dilution of 1:50 (Santa Cruz Biotechnology). After proper washing with PBS, sections were treated with mouse biotinylated secondary antibody. Again, after PBS washings, avidin-biotinylated horseradish peroxidase (HRP) was used. (Santa Cruz Biotechnology). Subsequently, diaminobenzidine was used as a chromogenic substrate. The slides were then rinsed in tap water, counterstained in Mayer's hematoxylin, dehydrated through alcohol grades, cleared in xylene and mounted in DPX.
The staining intensity of the tissue sections was evaluated under a microscope and was scored in the absence of any clinical data. Similarly, another observer evaluated all the sections and the mean of the two observations were recorded in each case. The sections were examined for the presence of a brown colored end-product, at the site of the target antigen. The absence of specific staining in the negative control specimens was checked to confirm the specificity of the primary antibody. The immunoreactivity was observed and recorded as cytoplasmic, membranous or nuclear.
Evaluation of nuclear staining
For nuclear staining, the percentage of the positive cells was determined by scoring 100-200 cells in the field at ×400 magnification from 10 fields (1000-2000), in randomly selected areas. Each tumor was assigned one of following scores according to the ratio of the cancer cells; 0 = score 1, 1-25% = score 2, 26-50% = score 3, 51-75% = score 4 and >75% = score 5.
The data was analyzed by using standard statistical Chi-squared test or Fisher's exact test.
| > Results|| |
In the present study, paraffin embedded breast cancer and normal adjacent tissues from 40 breast carcinoma patients were collected for BRCA1 and BRCA2 IHC. Out of 40 patients, 23 patients (57.5%) were post-menopausal; and 31 patients (77.5%) had lymph node metastasis at the time of surgery. Out of these 40 cases, 6 (15.0%) were evaluated as histological grade I, 23 (57.5%) as grade II and 11 (27.5%) as grade III. The age of the patients ranged from 20-60 years with the mean age group was 39.6 years (±11.9 Standard Deviation (SD). The majority of the cases were in the age group of 40-50 years [Table 1].
|Table 1: Clinicopathological characteristic of 40 sporadic breast cancers evaluated for expressions of BRCA1 and BRCA2|
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To determine the expression pattern of BRCA1 and BRCA2 proteins in breast cancer, IHC distribution of these proteins was checked in cancer tissue sections and was compared with the expression of respective proteins in normal adjacent tissue, which were used as controls. Analysis showed a high expression of BRCA1 protein in majority of the normal tissue sections (12/40; 30.0%) irrespective of the grade of disease or the menopausal status [Figure 1]. BRCA1 protein was expressed in the nuclei of normal epithelial and myoepithelial cells of the ductal and lobular region. In contrast, majority of breast cancer tissues showed moderate (26/40; 65.0%) or no expression (2/40) of BRCA1. In breast carcinoma, BRCA1 expression was heterogeneous and frequently less intense than in normal adjacent tissue. Out of the 40 breast cancer cases that were compared to their respective normal adjacent controls, 12 cases (30.0%) showed a decreased BRCA1 protein expression compared to their respective normal adjacent controls. In these cases, immunoreactivity was complete absent in some tumor tissues, whereas in others the number of immunoreactive cells were very few, while 28 (70.0%) cases revealed only a minor decline in the expression of BRCA1 in tumor tissue. The significant association was not observed in BRCA1 expression normal tissue and breast cancer cases (P < 0.15) in [Table 2].
|Figure 1: Immunohistochemical analysis of BRCA1 in breast carcinomas. The photomicrographs show (a) normal breast tissue showing both cytoplasmic and nuclear immunostaining; and (b) negative control showing no detectable BRCA1 immunoreactivity in which BRCA1 antibody has been replaced with isotype specific IgG (c) invasive ductal breast carcinoma showing less BRCA1 immunolocalization in cytoplasm and nucleus of tumor cells; (A-C, original magnification ×200)|
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|Table 2: Immunochemical staining pattern of BRCA1 and BRCA2 proteins in breast cancer patients and normal controls|
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All samples that were tested for BRCA1 were subsequently tested for BRCA2 expression by IHC. The results showed a similar pattern of BRCA2 protein expression in normal tissues, but less number of cases exhibited higher BRCA2 expression (24/40 for BRCA2 versus 26/40 cases for BRCA1). Moreover, degree of expression of BRCA2 did not correlate with the BRCA1 expression.
Like BRCA1, we also checked the expression pattern of BRCA2 protein in breast cancer patients and compared it with the expression in adjacent normal tissues. Out of 40 cases, 12 cases (30.0%) showed decreased level of BRCA2 protein expression as compared to normal subject, whereas 12 cases (30.0%) showed higher BRCA2 protein expression while the remaining samples revealed similar level of expression [Figure 2]. Like BRCA1, BRCA2 expression was significantly different between cases and control (P < 0.05) in [Table 3].
|Figure 2: Immunohistochemical analysis of BRCA2 in breast carcinomas. The photomicrographs show (a) normal breast tissue showing less both cytoplasmic and nuclear immunostaining; and (b) negative control showing no detectable BRCA2 immunoreactivity in which BRCA2 antibody has been replaced with isotype specific IgG (c) invasive ductal breast carcinoma depicting BRCA2 immunolocalization in cytoplasm and nucleus of tumor cells; (A-C, original magnification ×200)|
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|Table 3: Expression of BRCA1 and BRCA2 in normal and cancerous breast tissues|
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Further comparison of decreased levels of BRCA1 protein expression in 12 cases with the expression pattern of BRCA2 showed a corresponding increase in BRCA2 protein expression in 10 cases (83.3%); whereas 1 case (0.83%) showed no detectable difference and 1 case (0.83%) had decreased level of BRCA2 protein expression.
Similarly, we checked the level of BRCA1 expression dynamics in cases that showed increased BRCA2 expression. Out of these, 12 cases showed higher expression in tumors; 6 cases (50.0%) exhibited a corresponding decreased level of BRCA1 protein expression.
A further analysis of the results following re-grouping of cases based on their menopausal status showed a significantly decreased expression of BRCA1 and correspondingly increased BRCA2 expression in 13 cases (56.5%). Out of 23 post-menopausal women, these cases indicate an inverse correlation of BRCA1 and BRCA2 expression dynamics.
| > Discussion|| |
Though mutational inactivation of BRCA1 tumor suppressor gene has been implicated in the predisposition of familial breast cancer, , the contribution of BRCA1 and BRCA2 gene mutations in sporadic breast cancer perhaps is nominal. , Our earlier studies for BRCA1 and BRCA2 mutations in breast cancer patients showed somatic missense mutation in BRCA1 gene patients, whereas no mutation was detected in BRCA2 gene.  In this report, considering the potential overlapping tumor suppressor role of BRCA1 and BRCA2 genes  and lack of any germline mutations in these genes during sporadic breast cancer, we analyzed an in situ expression of BRCA1 and BRCA2 proteins and investigated the correlation, if any, among the expression dynamics of the two proteins. Our data showed a tumor tissue-specific decline in the expression of BRCA1 and in a subset of such cases, a corresponding increase in BRCA2 protein expression was observed. This phenomenon was found to be more common in patients with post-menopausal status. The loss of BRCA1 expression was found to be most prominent in advanced grade 3 diseases.
In the present study, we observed a tumor tissue-specific decline in the protein expression of BRCA1 in 50.0% of the cases investigated. A subsequent comparative analysis with respect to adjacent normal tissue revealed a significant decline of BRCA1 expression in 30.0% of total breast cancer biopsies. Similar observation indicating loss of BRCA1 immunoreactivity has been reported earlier by several groups, which ranged from 30-83%. , In a comprehensive analysis using a panel of 19 antibodies of different epitope specificities, Wilson et al., showed loss of BRCA1 expression in 82% of high grade lesions of invasive ductal carcinoma (IDC); whereas staining was found more intense in normal and low grade tumor tissues.  Though the samples size of IDC in our study was small, the results revealed a significant decline of BRCA1 expression in 57% cases (i.e. 4/7), while the other cases showed some decrease but was not significantly different from corresponding adjacent control tissues. Similar studies in 108 Japanese and 51 Chinese women in sporadic breast cancer showed decline of BRCA1 in 28% and 49% of cases, respectively.  Furthermore, Amirrad et al. showed no detectable BRCA1 expression in about 79% of tissues sections by using IHC and reverse transcriptase-polymerase chain reaction (RT-PCR).  On the other hand, in population of southern region of India, 54% of breast cancer patients showed low or undetectable levels of BRCA1 expression.  Lack of BRCA1 expression observed in most of the above studies may not necessarily reflect the tumor-specific decline of BRCA1 expression. Expression of the BRCA1 in these patients can also be attributable to overall low BRCA1 expression that may predispose the risk in such women. Our study too showed a similar increased rate (up to 50.0%) of low or undetectable levels of BRCA1; however, when compared to adjacent tissue it was evident that 30.0% of the total cases truly reflected tumor-specific decline in BRCA1 levels. Moreover, involvement of BRCA1 has been reported to be higher in pre-menopausal women with age less than 35 years. Our study indicated that the expression of BRCA1 was proportionately reduced in cases with post-menopausal status (57.5%).
We observed that majority of the cancer tissue biopsies demonstrated a reduction in the expression of BRCA1 rather than changes into its subcellular localization suggesting a post-transcriptional/post-translation suppression rather than affecting the upstream signaling of BRCA1 and functional inactivation. Lack of somatic mutations in sporadic breast carcinomas , suggest that BRCA1 expression might be down regulated by mechanisms other than point mutations. One non-mutational mechanism of BRCA1 inactivation that has been observed in sporadic breast carcinoma is methylation.  Hypermethylation of CpG-rich areas located within the promoter of genes may be a common mechanism of silencing tumor suppressor genes.  The decreased BRCA1 expression also might be secondary effect caused by changes in upstream regulatory pathways, controlling BRCA1 expression. In addition, environmental exposures may alter BRCA1 expression levels. Polycyclic aromatic hydrocarbons have been reported to be capable of reducing BRCA1 mRNA expression in human breast carcinoma cells. 
On the other hand, analysis of BRCA2 protein expression in breast cancer tissue showed low or undetectable level in 52.5% of cases indicating a higher expression level of this protein in breast cancer as compared to BRCA1. Present situation is in contrast to the condition in normal mammary gland development where BRCA1 expression was shown to be much higher than BRCA2.  Interestingly, our study on a subset showed lack of BRCA1 expression whereas a reciprocal increase in BRCA2 levels (4/7 cases), though the sample size in these cases was small. Therefore, the current study indicates an inverse kinetics of these proteins during breast carcinogenesis. Moreover, like the present study, studies on BRCA2 showed a similar BRCA2 expression pattern in breast cancer.  Overexpressed BRCA2 might play a role in the aggressiveness of breast tumors. Likewise, reports also documented the upregulation of BRCA2 expression in rapidly proliferating cells.  Nonetheless, the authors did not correlate their finding with BRCA1 expression. It is important to note that the promoter of BRCA2 showed absence of methylation of CpG dinucleotides in breast tumors,  suggesting that this alternative mechanism of tumor-suppressor gene inactivation by transcriptional regulation of mRNA expression is unlikely to explain the differences in BRCA2 mRNA expression observed in this study.
BRCA2, like BRCA1 is not frequently mutated in sporadic breast cancers.  However, high frequencies of loss of heterozygosity on 17q12-21 and 13q12-13; , point to a significant role of these two genes in the pathogenesis of sporadic breast cancer. In the present study, we observed a tumor tissue-specific decline in the protein expression of BRCA2 in 72% cases and 28% cases exhibited overexpression. A similar observation has been reported earlier by several authors. , Bieche et al. showed weak BRCA2 expression in 14/127 (11%) cases and its overexpression in 25/127 (20%) cases and the expression levels correlated significantly with grade III  , suggesting that overexpression of BRCA2 may influence the aggressiveness of breast tumors.
The clinical relevance of our finding is that the BRCA1 expression decreases upon tumor grade increase and severity of disease. Women who go through menopause (when regular menstrual periods stop) at a later age have a higher risk of breast cancer. This is because a late menopause exposes a woman's body to greater amounts of the hormone estrogen over her lifetime. These higher levels of estrogen increase the risk that breast cells will become cancerous. The early detection of breast cancer improves the prognosis, survival of patients and management practices of the disease. This pilot study very well lays the foundation that the BRCA1 and BRCA2 expression pattern analyses in early stage or non-symptomatic menopausal women may help in identifying the risk of breast cancer. The study with larger number sample size may establish BRCA1, BRCA2 to be used as a potential marker in population based screening for risk assessment.
| > Conclusions|| |
The study shows downregulation of BRCA1 protein during breast carcinogenesis. Our results also demonstrate that in a subset of cases, the decline in BRCA1 expression may be associated with potentially compensatory increase in BRCA2 protein, which may depend on grade of tumor as well as menopausal status of the patients.
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[Figure 1], [Figure 2]
[Table 1], [Table 2], [Table 3]