Sage Journals: Discover world-class research

Abstract

Breast cancer is a major cause of cancer-related mortality in women. There are major discrepancies concerning the usefulness of various antibodies in detecting breast cancer susceptibility gene 1 (BRCA1) protein and its subcellular localization. The aim of the present study was to determine the specificity and sensitivity of immunohistochemistry (IHC) as a screening method for demonstrating BRCA1 expression. BRCA1 gene expression in archival paraffin-embedded breast cancer tissues was studied simultaneously at the protein and mRNA levels, and the two findings were compared. Forty-eight archival paraffin-embedded breast cancer tissues were studied for BRCA1 gene expression at protein level by IHC using four different antibodies against different BRCA1 epitopes and at mRNA level using real-time RT-PCR. BRCA1 mRNA expression was reduced or absent in 79% of the samples, and this finding correlated significantly with loss of BRCA1 protein expression in 83% of breast cancer tissues using one BRCA1 antibody studied (AB-1, against N-terminus epitope). The specificity of this antibody was 91.3%, and its sensitivity was 66.6%. There was no significant correlation between BRCA1 mRNA and protein expression as demonstrated by the remaining three antibodies. Antibody 8F7 had the highest sensitivity of 100%, but its specificity was 30.4% if mRNA levels were considered as the reference standard.

Keywords

breast cancer immunohistochemistry mRNA protein real-time RT-PCR

Breast cancer is a major cause of cancer-related mortality in women (Hill et al. 1997). Although this tumor generally appears in sporadic form, between 5% and 10% of cases can be considered hereditary, attributable to inherited autosomal dominant mutations in several susceptibility genes (Lynch et al. 1994; Hill et al. 1997; Shen et al. 1998). About 40% to 50% of hereditary breast cancers and most hereditary breast and ovarian syndromes are thought to be caused by mutations in breast cancer susceptibility gene 1 (BRCA1) (Easton et al. 1995; Merajver et al. 1995). The BRCA1 gene, identified by positional cloning in 1994, consists of 24 exons, 22 of which encode for a protein of 1863 amino acids (Miki et al. 1994). BRCA1 mRNA is induced at late G₁/early S phase before DNA synthesis (Vaughn et al. 1996), and the expression of the BRCA1 protein closely follows that of its mRNA (Chen et al. 1996; Russell et al. 2000; Vissac et al. 2002; Baldassarre et al. 2003). Although BRCA1 mutation has rarely been detected in sporadic breast cancers (Futreal et al. 1994; Khoo et al. 1999), loss of heterozygocity (Thompson et al. 1995; Khoo et al. 1999), methylation of BRCA1 promoter region (Dobrovic and Simpfendorfer 1997; Mancini et al. 1998), and loss or reduction of BRCA1 mRNA and protein expression have been demonstrated in sporadic breast cancers (Thompson et al. 1995, Scully et al. 1996; Sourvinos and Spandidos 1998).

There are major discrepancies concerning the usefulness of various antibodies in detecting BRCA1 protein expression and localization. Different studies have generated conflicting results related to BRCA1 expression and the subcellular localization of this protein. This controversy is even more pronounced when paraffin-embedded tissues are used (Wilson et al. 1999). Thus, although some researchers conclude that the specificity of some of the BRCA1 antibodies is adequate to consider immunohistochemistry (IHC) as a valuable screening method (Yoshikawa et al. 1999), others believe that commercially available BRCA1 antibodies lack the specificity required to identify the BRCA1 protein (Perez-Valles et al. 2001).

BRCA1 has been claimed to be exclusively a nuclear protein in both normal and cancer cells (Scully et al. 1996; Thomas et al. 1996; Thakur et al. 1997; Wilson et al. 1999), a nuclear protein in normal cells but an aberrantly localized cytoplasmic protein in breast and ovarian tumor cells (Chen et al. 1995; Lee et al. 1999), or a cytoplasmic protein found in tube-like structures that invaginate the nucleus (Coene et al. 1997). The role of BRCA1 in DNA repair is becoming clearer: BRCA1 protein is involved in several DNA repair pathways and also has an effect on global genomic repair, involving transcriptional regulation of nucleotide excision repair genes (Hartman and Ford 2002,2003; Zhang et al. 2004). Also, it has recently been reported that BRCA1 expression influences the choice of chemotherapeutic agents used in the treatment of breast cancer (Egawa et al. 2003; Zhou et al. 2003). Therefore, it is now becoming clear that the knowledge of BRCA1 expression in breast cancer has important clinical ramifications.

This study focused on BRCA1 expression status in paraffin-embedded breast cancer tissues by using RT-PCR and IHC and correlated the results of the two. Such a comparison could be useful in assessing the specificity of commercially available BRCA1 antibodies.

Materials and Methods

Forty-eight archival formalin-fixed paraffin-embedded breast cancer tissues of female patients (aged 29–65 years) were randomly chosen and used for the study. The genetic background of the population studied was unknown. Sections of 5-μm thickness were cut from each tissue. One section was stained with hematoxylin and eosin to confirm the diagnosis, type, and grading of the tumor. The remaining sections were stored for use in IHC and RNA extraction. Histological grading of the carcinomas was done according to the system of Scarff-Bloom-Richardson (Dalton et al. 1994). The MCF-7 cell line, grown in RPMI medium (Gibco BRL; Carlsbad, CA) and known to express BRCA1 (Scully et al. 1996), was used as positive control for IHC. In addition, RNA extracted from this cell line was used as positive control RNA in the real-time RT-PCR method.

Immunohistochemistry

To study BRCA1 protein expression, four antibodies were applied to formalin-fixed paraffin-embedded breast cancer sections (Table 1). Breast cancer sections were deparaffinized, then rehydrated through three different concentrations of alcohol and 0.3% H₂O₂ for 30 min to block endogenous peroxidase. Epitope retrieval was carried out in 0.1 M citrate buffer at 95C in water bath for 20 min. Nonspecific binding was blocked using rabbit anti-mouse serum before monoclonal antibodies (AB-1, AB-8F7), and swine anti-rabbit serum before polyclonal antibodies (AB-D20, AB-C-terminus) for 30 min. After overnight incubation, biotinylated antibody (link antibody) was added, followed by streptavidin (dilution 1:500). Diaminobenzene was used as chromogen. Sections from MCF-7 breast cancer cell line paraffin blocks were used as positive control. For negative controls, the antibodies were substituted with the corresponding serum. It is worthy of note that several buffers and antigen retrieval methods were tested before this optimal protocol was achieved. These methods include microwave treatment at 90C for 10 min and at 95C for 20 min.

Subcellular localization of staining was categorized as cytoplasmic, nuclear, or both cytoplasmic and nuclear.

RNA Extraction and Real-time RT-PCR

Of the 48 breast cancer tissues studied using IHC, 29 samples had enough tissue for successful RNA extraction and RT-PCR amplification of GAPDH. To control for RNA degradation, GAPDH RNA had to be amplified in RNA samples extracted from breast tissue sections before BRCA1 expression in that tissue was deemed negative. Macrodissection of tumor tissue from several paraffin sections was used to minimize the influence of surrounding normal tissues. In these samples, 70% or more of the tissue sections contained tumor. The sections were rehydrated and scraped off the glass slide, using a needle, into an Eppendorf tube containing RNase inhibitor, proteinase K buffer (pH 8.3), 1% Tween surfactant, and proteinase K (10 mg/ml). The tubes were kept at 60C in water bath overnight, then mixed with 400 μl Trizol and chloroform to precipitate the RNA. There was no need to treat the samples with Dnase, because the BRCA1 probe and GAPDH primers spanned an exon–exon junction. RT-PCR master mixes were prepared according to the Invitrogen ThermoScript Platinum Quantitative One-Step RT-PCR System (Invitrogen; Carlsbad, CA) BRCA1 primers (Egawa et al. 2001) (forward, 5'-ACAGCTGTGTGGTGCTTCTGTG-3', reverse 5'-CATTGTCCTCTGTCCAGGCATC-3') and BRCA1 probe labeled with FAM (Egawa et al. 2001) (5'-CATCATTCACCCTTGGCACAGGTGT-3') were used to amplify and detect BRCA1 mRNA. GAPDH primers (forward 5'-TCATTGACCTCAACTACATGGTTT-3', reverse 5'-GAAGATGGTGATGGGATTTC-3') and GAPDH probes labeled with JOE [TaqMan] (JOE-CAAGCTTCCCGTTCTCAGCC-TAMRA) were used as internal control for RT-PCR. Samples for no-RT and no-template were also included in each test to detect any DNA or RNA contamination. The Applied BioSystems ABI7000 real-time sequence detection system (Applied Biosystems, Foster City, CA) was used to detect amplifications. Amplification conditions were 60C for 60 min for RT, followed by 95C for 5 min, 45 PCR cycles at 95C for 15 sec, and 60C for 1 min. The cut-off value for BRCA1 mRNA negativity was when there was no specific or significant reduction in BRCA1 signals detected even after 45 cycles of amplification. Each sample was assayed in triplicate in independent reactions.

Table 1

Details of four different anti-BRCA1 antibodies

Anti-BRCA1 antibody	Mono/polyclonal	Epitope	Dilution	Source
AB-1 (MS110)	Monoclonal	N-terminus (exon 1–10)	1:50	Oncogene Research Products (CA)
AB-D20	Polyclonal	N-terminus	1:500	Santa Cruz Biotechnology (CA)
AB-8F7	Monoclonal	Exon 11	1:10	Genetex (TX)
AB-C-terminus (hBRCA1)	Polyclonal	C-terminus (exon 12–24)	1:100	Pharminogen (CA)

Table 2

Histological grade, BRCA1 mRNA and protein expression, and BRCA1 protein subcellular localization of breast cancer tissues studied

				BRCA1 protein expression
Breast cancer patient	Histological grade	BRCA1 mRNA expression	AB-1	AB-8F7	AB-D20	AB-C-terminus
Case 1	High	—	NC	NC	C	NC
Case 2	High	—	—	—	—	—
Case 3	High	^∗	—	NC	C	C
Case 4	High	—	—	C	C	—
Case 5	High	—	—	C	C	—
Case 6	High	+	C	NC	—	NC
Case 7	Low	+	C	NC	C	—
Case 8	High	—	—	—	C	—
Case 9	High	^∗	C	—	—	—
Case 10	High	^∗	—	NC	—	—
Case 11	High	^∗	—	NC	—	—
Case 12	Low	—	—	NC	C	NC
Case 13	High	^∗	—	—	C	—
Case 14	High	^∗	—	—	—	—
Case 15	High	—	—	NC	C	—
Case 16	High	—	—	C	C	—
Case 17	Low	^∗	—	—	—	—
Case 18	High	^∗	—	—	—	—
Case 19	High	^∗	—	NC	C	—
Case 20	High	+	—	NC	C	—
Case 21	High	^∗	—	—	—	—
Case 22	High	—	—	NC	C	NC
Case 23	High	—	—	C	C	—
Case 24	Low	—	—	—	C	—
Case 25	High	—	—	—	C	—
Case 26	High	^∗	—	—	—	—
Case 27	Low	+	—	C	—	—
Case 28	High	—	—	C	C	—
Case 29	High	^∗	—	C	—	—
Case 30	Low	—	—	C	—	—
Case 31	High	+	C	C	C	—
Case 32	Low	+	C	C	C	C
Case 33	Low	—	C	C	C	C
Case 34	High	^∗	C	—	—	—
Case 35	High	^∗	—	—	—	C
Case 36	High	—	—	—	C	C
Case 37	High	—	—	C	—	C
Case 38	Low	—	—	NC	C	C
Case 39	High	^∗	—	C	—	C
Case 40	High	—	—	C	C	—
Case 41	High	—	—	C	C	C
Case 42	High	^∗	—	NC	C	C
Case 43	High	^∗	—	—	—	—
Case 44	High	—	—	—	C	—
Case 45	Low	^∗	—	NC	C	NC
Case 46	High	—	—	—	C	—
Case 47	High	^∗	—	NC	C	C
Case 48	Low	—	—	NC	C	—

C, cytoplasmic staining; NC, nuclear and cytoplasmic staining; +, positive; -, negative, ^∗ tissue not sufficient for RNA extraction.

Statistical Analysis

The data were analyzed using the Statistical Package for Social Sciences (SPSS version 11.01) software. The association between BRCA1 mRNA and protein expression was analyzed using the χ² test. A p-value less than 0.05 was considered significant.

Results

Breast cancers were categorized according to their histological type. Five main histological types were present in this study: infiltrative ductal carcinoma (35 cases), intraductal carcinoma (1 case), medullary carcinoma (7 cases), infiltrative lobular carcinoma (3 cases), and tubulo-lobular carcinoma (2 cases). The most common histological type was infiltrative ductal carcinoma, which accounted for 72.8% of cases. In this study, cancers with total score of 3 to 5 were considered low grade; and those with scores of 6 to 9 were considered high grade. Of the 48 breast cancer cases, 37 (77%) were of high histological grade (Table 2).

Immunohistochemistry

Table 3 summarizes the BRCA1 protein expression and its subcellular localization in 48 breast cancer tissues using the four different antibodies.

Monoclonal Antibody against N-terminus, AB-1. IHC using monoclonal antibody against N-terminus, AB-1 showed positive staining for BRCA1 protein in only 16.7% of cases. In 87.5% of these cases, staining was localized in the cytoplasm. The remaining 12.5% showed staining in both the cytoplasm and nuclei (Figure 1A).

Table 3

BRCA1 protein expression and subcellular localization in breast cancer tissue using four different antibodies

			Subcellular localization of positive BRCA1 protein
BRCA1 antibodies	Negative BRCA1 expression (%)	Positive BRCA1 expression (%)	Cytoplasmic only (%)	Nuclear + cytoplasmic (%)
AB-1	40 (83.3)	8 (16.7)	7 (87.5)	1 (12.5)
AB-8F7	17 (35.4)	31 (64.6)	15 (48.4)	16 (51.6)
AB-D20	18 (37.5)	30 (62.5)	30 (100)	0 (0)
AB-C-terminus	32 (66.7)	16 (33.3)	11 (69)	5 (31)

Figure 1.

BRCA1 IHC using four different antibodies. (A) Positive BRCA1 IHC using monoclonal AB-1. (B) Positive BRCA1 IHC using AB-D20 antibody. (C) Positive BRCA1 IHC using AB-8F7. (D) Immunostaining of breast cancer tissue using antibody AB-C-terminus. (E) Negative control.

Monoclonal Antibody against Exon-11, AB-8F7. Monoclonal antibody against exon-11, AB-8F7 showed positive BRCA1 protein staining in 64.6% of cases. In half of these cases, staining was nuclear and cytoplasmic. In the remaining half, staining was cytoplasmic only (Figure 1B).

Polyclonal Antibody against N-terminus, AB-D20. BRCA1 protein staining with polyclonal antibody against N-terminus, AB-D20 was positive in 62.5% of cases, and staining was localized in the cytoplasm in all these cases (Figure 1C).

Polyclonal Antibody against C-terminus, AB-C-terminus. Polyclonal antibody against AB-C-terminus showed positive staining for BRCA1 protein in 33.3% of cases. In 69% of these cases, staining was localized only in the cytoplasm. Staining in the remaining 31% was localized in both the nucleus and cytoplasm (Figure 1D).

Cytoplasmic staining appeared to be the most common pattern, with or without nuclear staining, and none of the antibodies demonstrated nuclear staining only (Tables 2 and 3).

Real-time RT-PCR

Given the generally poor quality of RNA extracted from formalin-fixed paraffin-embedded tissues, several steps were implemented to ensure the accuracy of the results. First, only samples that had successful amplification of GAPDH, a housekeeping gene, were considered for subsequent BRCA1 amplifications. This step ensured that samples with only high-quality RNA were considered and represented a control for RNA degradation. Second, to ensure amplification of the less common transcript, namely, BRCA1, the number of amplification cycles was increased to 45. Third, the MCF7 cell line and a paraffin-embedded tissue known to express BRCA1 were used in all reactions as positive controls. Finally, to ensure reproducibility, three independent RT-PCR reactions were performed for each sample. Such stringent criteria reduced our analyzable tissues to 29 of the 48 for which IHC was successful. Of 29 breast cancer tissues analyzed for BRCA1 mRNA expression using real-time RT-PCR, only 6 (21%), showed detectable BRCA1 mRNA. Twenty-three breast cancer tissues (79.3%) showed no BRCA1 mRNA amplification, even after 45 cycles. The amplification curves of RNA extracted from the breast cancer MCF-7 cell line and paraffin-embedded breast cancer tissues using BRCA1 primers are demonstrated in Figure 2.

Correlation between BRCA1 mRNA and Protein Expression

Table 4 compares BRCA1 mRNA expression with corresponding protein expression. BRCA1 mRNA expression in breast cancer tissues showed a significant relationship with protein expression by only one of the antibodies, AB-1 (p = 0.002). Thus, 72% of breast cancers, which also did not demonstrate BRCA1 at mRNA level, did not show BRCA1 protein expression by IHC using AB-1. No significant relationship could be detected between BRCA1 mRNA and BRCA1 protein expression using the remaining three antibodies (AB-8F7, AB-D20, and AB-C-terminus).

Specificity and Sensitivity of the Four Different BRCA1 Antibodies

To evaluate the specificity and sensitivity of the four anti-BRCA1 antibodies, the RT-PCR results were considered as standard. AB-1 has the best combined specificity (91.3%) and sensitivity (66.6%) in detecting BRCA1 protein (Table 5). Although AB-8F7 had 100% sensitivity, its specificity was low (30.4%).

Discussion

The present study shows lack of both BRCA1 mRNA and protein expression in the majority of breast cancers studied. This finding is consistent with other reports, which have shown that BRCA1 mRNA and protein are decreased in mammary tumors compared with matched normal breast tissue (Thompson et al. 1995; Rio et al. 1999). The negative BRCA1 mRNA and protein expression in this study was associated with infiltrative ductal carcinoma, although this relationship was not statistically significant. On the other hand, there was a significant, albeit marginal, relationship between BRCA1 mRNA expression and high histological grade of breast cancer (p = 0.05). This finding is consistent with the observation that the majority of high-grade breast cancers show weak or no BRCA1 expression (Jacquemier et al. 1995; Wilson et al. 1999; Lakhani et al. 2000), suggesting that absence of BRCA1 may contribute to the pathogenesis of a significant percentage of breast cancers. Seventy-seven percent of our patients had a high histological grade of breast cancer; although this could indicate a selection bias, this represents a consistent observation in Kuwaiti patients (unpublished data). It follows that our low BRCA1 detection rate, although consistent with the study of Wilson et al. (1999), could be attributed to this selection bias.

Figure 2.

Amplification curves of RNA extracted from breast cancer MCF7 cell line (red arrow) and paraffin-embedded breast cancer tissues from several patients (blue arrows) using BRCA1 primers. The x-axis represents the PCR cycle number at which the cDNA started the amplification; the y-axis represents Delta-Rn, which is the normalized reporter signal minus the baseline signal. Blue arrow corresponds to the BRCA1 mRNA amplification curve of the breast cancer tissues shown in Figure 1. The last two curves are samples considered negative for BRCA1 expression (black arrows).

Table 4

Comparison between BRCA1 mRNA and protein expression in breast cancer tissues

	BRCA1 protein expression (IHC)
	AB-1		AB-8F7		AB-D20		AB-C-terminus
BRCA1 mRNA expression (RT-PCR)	Negative	Positive	Negative	Positive	Negative	Positive	Negative	Positive
Positive (6)	2	4	0	6	2	4	4	2
Negative (23)	21	2	7	16	3	20	15	8
Correlation	p = 0.002		p = 0.11		p = 0.59		p = 0.33

One of the major tasks of this study was to choose the appropriate and reliable antibody for BRCA1 protein IHC. There is a major controversy about the usefulness and specificity of different BRCA1 antibodies. This controversy is even more pronounced when paraffin-embedded tissues are used. In the present study, four anti-BRCA1 antibodies against different BRCA1 epitopes were used. The results obtained were compared to determine which antibody is most reliable for detecting BRCA1 protein on paraffin-embedded breast cancers. To evaluate the specificity and sensitivity of the four anti-BRCA1 antibodies, the RT-PCR results were considered as a standard. Our results demonstrate a significant relationship between BRCA1 mRNA and BRCA1 protein expression with only one of the antibodies, monoclonal AB-1. Almost all breast cancers that had no or significantly reduced BRCA1 mRNA expression in this study were also negative for BRCA1 protein expression with AB-1 (21/23, 91%; p = 0.002). Accordingly, AB-1 has the best combined specificity (91.3%) and sensitivity (66.6%) in detecting BRCA1 protein (Table 5). In a study using four commercially available anti-BRCA1 antibodies on paraffin-embedded breast cancers (including AB-1 and AB-D20), Perez-Valles et al. (2001) reported that available BRCA1 antibodies lack the specificity required to identify the BRCA1 protein. Others, however, have used AB-1 for immunolocalization of BRCA1 on breast cancer and have reported that this antibody is the most reliable antibody for detecting BRCA1 protein (Lee et al. 1999; Wilson et al. 1999; Niwa et al. 2000). Our data are consistent with this finding. IHC results using the other three antibodies have shown no statistically significant relationship with BRCA1 mRNA. Of all the antibodies, AB-8F7 had the best sensitivity, but its specificity was low (30.4%). Results reported by Yoshikawa et al. (1999) suggest that N-terminus AB-1 could be useful in prescreening tumors for BRCA1 mutations because of a high detection rate (7 of 19, 37%) of alterations in the BRCA1 gene product in breast cancer. Our data are also consistent with the findings that BRCA1 loss is at mRNA level (Russell et al. 2000; Baldassarre et al. 2003). Interestingly, we have found that some of the breast cancers with positive BRCA1 mRNA expression had no BRCA1 protein expression with AB-1. This finding supports the hypothesis that there might be other mechanisms by which BRCA1 expression is controlled at translational or posttranslational levels (Miyamoto et al. 2002; Sobczak and Krzyzosiak 2002). Nevertheless, loss of both mRNA and BRCA1 protein could indicate that the genetic control of BRCA1 expression in the breast cancer analyzed is at transcriptional level.

Table 5

Sensitivity and specificity of various antibodies used

Antibody	Specificity (%)	Sensitivity (%)
AB-1	91.3	66.6
AB-8F7	30.4	100
AB-D20	13	66.6
AB-C-terminus	62.5	33.3

The subcellular localization of BRCA1 has been also been controversial. BRCA1 has been claimed to be an exclusively nuclear protein in both normal and cancer cells (Scully et al. 1996; Thomas et al. 1996; Thakur et al. 1997; Wilson et al. 1999), a nuclear protein in normal cells but an aberrantly localized cytoplasmic protein in breast and ovarian tumor cells (Chen et al. 1995; Lee et al. 1999), and a cytoplasmic protein found in tubelike structures that invaginate the nucleus (Coene et al. 1997). The variation in the subcellular localization of BRCA1 might be attributable to several causes, including the specificity of the antibodies used to localize the protein, antibody cross-reactivity (Smith et al. 1996; Bernard-Gallon et al. 1997; Wilson et al. 1999), and the presence of splice variant isoforms (Thakur et al. 1997; Wilson et al. 1997). The present study confirms that BRCA1 is expressed as both nuclear and cytoplasmic antigen in breast cancer tissues. Cytoplasmic staining was a consistent feature in the breast cancers positive for BRCA1, but the nuclear expression of BRCA1 protein ranged from 37% to 83% of breast cancers (depending on the type of antibody used) and was absent in a subset of them. AB-1 monoclonal N-terminus antibody, which showed a nuclear dot pattern both in normal and cancer cell lines in other studies (Scully et al. 1996,1997; Wilson et al. 1999; Hsu et al. 2001), resulted in only 16.7% positive staining for BRCA1. In 87.5% of these cases, staining was located in the cytoplasm only; in the remaining 12.5%, staining was in both the cytoplasm and nuclei. Differences in subcellular localization of the BRCA1 protein can affect the interpretation of the IHC results (Chen et al. 1995). Wilson et al. (1997) compared 19 BRCA1 antibodies and detected nuclear foci in both a normal breast epithelial cell line and a breast malignancy–derived cell line. In accord with Wilson's study are the studies of Thomas et al. (1996), Ruffner and Verma (1997), and Zhang et al. (1997), which have also reported the nuclear subcellular localization of BRCA1 protein on cell lines. On the other hand, Chen et al. (1995) have reported that BRCA1 is a nuclear protein in normal cells and becomes aberrantly localized in cytoplasm of breast and ovarian tumor cells. In the present study, none of the breast cancer tissues showed BRCA1 nuclear localization alone. It can be argued that breast cancer cell lines, not archival paraffin-embedded breast cancer tissues, were used in the majority of these studies. Subcellular localization of proteins in cell lines cannot be compared with formalin-fixed, paraffin-embedded tissue, because several factors affect the protein localization in the tissue. The staining pattern in the paraffin-embedded tissues could be altered by changes in tissue fixation conditions and antigen retrieval methods; differences in immunostaining methodology, antibody concentrations, specificity of the antibodies used to localize the protein, and antibody cross-reactions (Scully et al. 1996; Smith et al. 1996; Bernard-Gallon et al. 1997; Wilson et al. 1997,1999); the presence of splice variant isoforms (Lu et al. 1996; Wang et al. 1997; Orban and Olah 2001; Fabbro et al. 2002); and the problems of working with preserved archival tissue (Scully et al. 1996; Thomas et al. 1996; Bernard-Gallon et al. 1997; Wilson et al. 1999). According to Yoshikawa et al. (1999), results obtained by using C-terminus antibody should be evaluated carefully because of false-positive immunostaining demonstrated. BRCA1 protein localization is a complex issue, but the antibodies used in this study were chosen against different BRCA1 protein epitopes (N-terminus, exon 11, and C-terminus), which enabled us to recognize different protein isoforms. In addition, the realtime probe used for BRCA1 detects all known mRNA variants (because it hybridizes to the junction of exon 22–exon 23).

In conclusion, the clinical benefits of establishing BRCA1 expression status and its effects on breast cancer treatment, prophylaxis, and prognosis are obvious (Lafarge et al. 2001; Egawa et al. 2003; Zhou et al. 2003). Thus, IHC can be a valuable preliminary test for detecting the reduction in BRCA1 protein expression. Among the different available BRCA1 antibodies, we consider AB-1 to be the best anti-BRCA1 antibody that can be applied on formalin-fixed paraffin-embedded tissues, with 91.3% specificity and 66.6% sensitivity. Nevertheless, AB-8F7 detected six of six tumors positive for BRCA1 mRNA, making it highly sensitive. The antibody, however, also produced signals in another 16 tumors that were negative for BRCA1 mRNA, which indicates low specificity if one considers, as we did, RT-PCR to be the reference standard. We acknowledge that the small number of positive RT-PCR values constitutes a limitation of the study. A further, larger study focusing on identifying various BRCA1 splice variants and their correlation with a range of antibodies raised against a larger number of epitopes is now warranted.

Footnotes

Acknowledgements

This study was funded by the College of Graduate Studies of Kuwait University and the Kuwait Institute for the Advancement of Sciences, project number 990707 (FA-M), and Shared Facility Grant, number GM/0101.

We wish to thank Dr. Josley George, Dr. Shirley George, Mrs. Bency John, and Mrs. Tessy Saji for their technical support.

References

Baldassarre

Battista

Belletti

Thakur

Pentimalli

Trapasso

Fedele

(2003) Negative regulation of BRCA1 gene expression by HMGA1 proteins accounts for the reduced BRCA1 protein levels in sporadic breast carcinoma. Mol Cell Biol 23: 2225–2238

Bernard-Gallon

Crespin

Maurizis

Bignon

(1997) Cross-reaction between antibodies raised against the last 20 C-terminal amino acids of BRCA 1 (C-20) and human EGF and EGF-R in MCF 10a human mammary epithelial cell line. Int J Cancer 71: 123–126

Chen

Sharp

Lee

(1996) The nuclear localization sequences of the BRCA1 protein interact with the importin-alpha subunit of the nuclear transport signal receptor. J Biol Chem 271: 32863–32868

Chen

Riley

Allred

Chen

Von Hoff

Osborne

(1995) Aberrant subcellular localization of BRCA1 in breast cancer. Science 270: 789–791

Coene

Van Oostveldt

Willems

van Emmelo

De Potter

(1997) BRCA1 is localized in cytoplasmic tube-like invaginations in the nucleus. Nat Genet 16: 122–124

Dalton

Page

Dupont

(1994) Histologic grading of breast carcinoma. A reproducibility study. Cancer 73: 2765–2770

Dobrovic

Simpfendorfer

(1997) Methylation of the BRCA1 gene in sporadic breast cancer. Cancer Res 57: 3347–3350

Easton

Ford

Bishop

(1995) Breast and ovarian cancer incidence in BRCA1-mutation carriers. Breast Cancer Linkage Consortium. Am J Hum Genet 56: 265–271

Egawa

Miyoshi

Taguchi

Tamaki

Noguchi

(2001) Quantitative analysis of BRCA1 and BRCA2 mRNA expression in sporadic breast carcinomas and its relationship with clinicopathological characteristics. Jpn J Cancer Res 92: 624–630

10.

Egawa

Motomura

Miyoshi

Takamura

Taguchi

Tamaki

Inaji

(2003) Increased expression of BRCA1 mRNA predicts favorable response to anthracycline-containing chemotherapy in breast cancers. Breast Cancer Res Treat 78: 45–50

11.

Fabbro

Rodriguez

Baer

Henderson

(2002) BARD1 induces BRCA1 intranuclear foci formation by increasing RING-dependent BRCA1 nuclear import and inhibiting BRCA1 nuclear export. J Biol Chem 277: 21315–21324

12.

Futreal

Liu

Shattuck-Eidens

Cochran

Harshman

Tavtigian

Bennett

(1994) BRCA1 mutations in primary breast and ovarian carcinomas. Science 266: 120–122

13.

Hartman

Ford

(2002) BRCA1 induces DNA damage recognition factors and enhances nucleotide excision repair. Nat Genet 32: 180–184

14.

Hartman

Ford

(2003) BRCA1 and p53: compensatory roles in DNA repair. J Mol Med 81: 700–707

15.

Hill

Doyle

McDermott

O'Higgins

(1997) Hereditary breast cancer. Br J Surg 84: 1334–1339

16.

Hsu

Doan

White

(2001) Identification of a gamma-tubulin-binding domain in BRCA1. Cancer Res 61: 7713–7718

17.

Jacquemier

Eisinger

Birnbaum

Sobol

(1995) Histoprognostic grade in BRCA1-associated breast cancer. Lancet 345: 1503

18.

Khoo

Ozcelik

Cheung

Chow

Ngan

Done

Liang

(1999) Somatic mutations in the BRCA1 gene in Chinese sporadic breast and ovarian cancer. Oncogene 18: 4643–4646

19.

Lafarge

Sylvain

Ferrara

Bignon

(2001) Inhibition of BRCA1 leads to increased chemoresistance to microtubule-interfering agents, an effect that involves the JNK pathway. Oncogene 20: 6597–6606

20.

Lakhani

Gusterson

Jacquemier

Sloane

Anderson

van de Vijver

Venter

(2000) The pathology of familial breast cancer: histological features of cancers in families not attributable to mutations in BRCA1 or BRCA2. Clin Cancer Res 6: 782–789

21.

Lee

Jin

Chang

Lin

(1999) Immunolocalization of BRCA1 protein in normal breast tissue and sporadic invasive ductal carcinomas: a correlation with other biological parameters. Histopathology 34: 106–112

22.

Conzen

Cole

Arrick

(1996) Characterization of functional messenger RNA splice variants of BRCA1 expressed in nonmalignant and tumor-derived breast cells. Cancer Res 56: 4578–4581

23.

Lynch

Conway

Watson

Feunteun

Lenoir

Narod

(1994) Hereditary breast cancer and family cancer syndromes. World J Surg 18: 21–31

24.

Mancini

Rodenhiser

Ainsworth

O'Malley

Singh

Xing

Archer

(1998) CpG methylation within the 5' regulatory region of the BRCA1 gene is tumor specific and includes a putative CREB binding site. Oncogene 16: 1161–1169

25.

Merajver

Pham

Caduff

Chen

Poy

Cooney

Weber

(1995) Somatic mutations in the BRCA1 gene in sporadic ovarian tumours. Nat Genet 9: 439–443

26.

Miki

Swensen

Shattuck-Eidens

Futreal

Harshman

Tavtigian

Liu

(1994) A strong candidate for the breast and ovarian cancer susceptibility gene BRCA1. Science 266: 66–71

27.

Miyamoto

Fukutomi

Asada

Wakazono

Tsuda

Asahara

Sugimura

(2002) Promoter hypermethylation and post-transcriptional mechanisms for reduced BRCA1 immunoreactivity in sporadic human breast cancers. Jpn J Clin Oncol 32: 79–84

28.

Niwa

Oyama

Nakajima

(2000) BRCA1 expression status in relation to DNA methylation of the BRCA1 promoter region in sporadic breast cancers. Jpn J Cancer Res 91: 519–526

29.

Orban

Olah

(2001) Expression profiles of BRCA1 splice variants in asynchronous and in G1/S synchronized tumor cell lines. Biochem Biophys Res Commun 280: 32–38

30.

Perez-Valles

Martorell-Cebollada

Nogueira-Vazquez

Garcia-Garcia

Fuster-Diana

(2001) The usefulness of antibodies to the BRCA1 protein in detecting the mutated BRCA1 gene. An immunohistochemical study. J Clin Pathol 54: 476–480

31.

Rio

Maurizis

Peffault de Latour

Bignon

Bernard-Gallon

(1999) Quantification of BRCA1 protein in sporadic breast carcinoma with or without loss of heterozygosity of the BRCA1 gene. Int J Cancer 80: 823–826

32.

Ruffner

Verma

(1997) BRCA1 is a cell cycle-regulated nuclear phosphoprotein. Proc Natl Acad Sci USA 94: 7138–7143

33.

Russell

Pharoah

De Foy

Ramus

Symmonds

Wilson

Scott

(2000) Frequent loss of BRCA1 mRNA and protein expression in sporadic ovarian cancers. Int J Cancer 87: 317–321

34.

Scully

Chen

Ochs

Keegan

Hoekstra

Feunteun

Livingston

(1997) Dynamic changes of BRCA1 subnuclear location and phosphorylation state are initiated by DNA damage. Cell 90: 425–435

35.

Scully

Ganesan

Brown

De Caprio

Cannistra

Feunteun

Schnitt

(1996) Location of BRCA1 in human breast and ovarian cancer cells. Science 272: 123–126

36.

Shen

Weaver

Weinstein

Chen

Guan

(1998) A targeted disruption of the murine Brca1 gene causes gamma-irradiation hypersensitivity and genetic instability. Oncogene 17: 3115–3124

37.

Smith

Lee

Szabo

Jerome

McEuen

Taylor

Hood

(1996) Complete genomic sequence and analysis of 117 kb of human DNA containing the gene BRCA1. Genome Res 6: 1029–1049

38.

Sobczak

Krzyzosiak

(2002) Structural determinants of BRCA1 translational regulation. J Biol Chem 277: 17349–17358

39.

Sourvinos

Spandidos

(1998) Decreased BRCA1 expression levels may arrest the cell cycle through activation of p53 checkpoint in human sporadic breast tumors. Biochem Biophys Res Commun 245: 75–80

40.

Thakur

Zhang

Peng

Carroll

Ward

Yao

(1997) Localization of BRCA1 and a splice variant identifies the nuclear localization signal. Mol Cell Biol 17: 444–452

41.

Thomas

Smith

Rubinfeld

Gutowski

Beckmann

Polakis

(1996) Subcellular localization and analysis of apparent 180-kDa and 220-kDa proteins of the breast cancer susceptibility gene, BRCA1. J Biol Chem 271: 28630–28635

42.

Thompson

Jensen

Obermiller

Page

Holt

(1995) Decreased expression of BRCA1 accelerates growth and is often present during sporadic breast cancer progression. Nat Genet 9: 444–450

43.

Vaughn

Davis

Jarboe

Huper

Evans

Wiseman

Berchuck

(1996) BRCA1 expression is induced before DNA synthesis in both normal and tumor-derived breast cells. Cell Growth Differ 7: 711–715

44.

Vissac

Peffault De Latour

Communal

Bignon

Bernard-Gallon

(2002) Expression of BRCA1 and BRCA2 in different tumor cell lines with various growth status. Clin Chim Acta 320: 101–110

45.

Wang

Shao

Ding

Cui

Reddy

Rao

(1997) BRCA1 proteins are transported to the nucleus in the absence of serum and splice variants BRCA1a, BRCA1b are tyrosine phosphoproteins that associate with E2F, cyclins and cyclin dependent kinases. Oncogene 15: 143–157

46.

Wilson

Payton

Elliott

Buaas

Cajulis

Grosshans

Ramos

(1997) Differential subcellular localization, expression and biological toxicity of BRCA1 and the splice variant BRCA1-delta11b. Oncogene 14: 1–16

47.

Wilson

Ramos

Villasenor

Anders

Press

Clarke

Karlan

(1999) Localization of human BRCA1 and its loss in high-grade, non-inherited breast carcinomas. Nat Genet 21: 236–240

48.

Yoshikawa

Honda

Inamoto

Shinohara

Yamauchi

Suga

Okuyama

(1999) Reduction of BRCA1 protein expression in Japanese sporadic breast carcinomas and its frequent loss in BRCA1-associated cases. Clin Cancer Res 5: 1249–1261

49.

Zhang

Zhao

Kajino

Weber

Davis

Wang

(1997) Relationship of p215BRCA1 to tyrosine kinase signaling pathways and the cell cycle in normal and transformed cells. Oncogene 14: 2863–2869

50.

Zhang

Willers

Feng

Ghosh

Kim

Weaver

Chung

(2004) Chk2 phosphorylation of BRCA1 regulates DNA double-strand break repair. Mol Cell Biol 24: 708–718

51.

Zhou

Smith

Liu

(2003) Role of BRCA1 in cellular resistance to paclitaxel and ionizing radiation in an ovarian cancer cell line carrying a defective BRCA1. Oncogene 22: 2396–2404

BRCA1 Gene Expression in Breast Cancer: A Correlative Study between Real-time RT-PCR and Immunohistochemistry

Abstract

Keywords

Materials and Methods

Immunohistochemistry

RNA Extraction and Real-time RT-PCR

Statistical Analysis

Results

Immunohistochemistry

Real-time RT-PCR

Correlation between BRCA1 mRNA and Protein Expression

Specificity and Sensitivity of the Four Different BRCA1 Antibodies

Discussion

Footnotes

Acknowledgements

References