Looking for novel breast cancer antigen epitopes is helpful for its treatment, diagnosis, and prevention. brcaa1 gene is mapped at 1q42.1-q43, its whole genome is 93.857 kb, including 18 exons and 17 introns. BRCAA1 protein is composed of 1,214 amino acids with 10 glycosylate sites, and shares 37% amino acid identity and an identical antigen epitope with Rb binding protein 1. The novel antigen epitope, SSKKQKRSHK, was predicted to locate in the region 610 to 619 sites, was synthesized, and its antibody was fabricated. Competent inhibition analysis showed that SSKKQKRSHK is the shortest effective peptide. The antigen epitope was mapped in the cytoplasm of MCF-7 cells. Immunohistochemistry analysis showed that the antigen epitope exhibited positive expression in 65% (39 of 60) breast cancer specimens and negative expression in 60 non-cancerous tissues. Statistical analysis shows that its expression is closely associated with status of ER and PR, with sensitivity of 100% and specificity of 81%, and confidence interval of 85.9% to 96.9%. ELISA analysis showed that the mean absorbance of sera antibody titers from breast cancer patients and healthy donors were 0401 ± 0.163 SD and 0.137 ± 0.121 SD, respectively. Sixty-four percent breast cancer patient sera and 13% healthy donor sera had higher titer than mean titer of healthy donors, and there exists significant difference between breast cancer patients and healthy donors (P < 0.001). In this study, a novel breast cancer antigen epitope, SSKKQKRSHK, is identified. Its expression is associated with characteristics that are themselves associated with prognosis of breast cancer, and its sera antibody level may be helpful for breast cancer diagnosis.

Breast cancer is a common tumor among women. In the United States, for example, one in every eight women will develop breast cancer in life (1). Despite the advances in treating breast cancer, the causal mechanisms underlying this disease have yet to be fully elucidated. Eighty-five percent of breast cancer cases occur sporadically without any known genetic mutation (1). Therefore, looking for its antigen epitopes and fabricating corresponding antibodies is a central objective in developing its specific immunologic diagnosis and immunotherapies. Breast cancer associated antigens can elicit both antibody and cellular immune responses that are active and specific. Therefore, these human antibodies, specific for breast cancer, can be used as probes for the molecular identification of associated breast cancer antigens or genes (2, 3), in favor of its treatment, diagnosis, and prevention.

So far, some useful biomarkers associated with breast cancer have been recognized. For example, HER-2 predicts prognosis of breast cancer and may influence treatment responses (4). Ras expression has been suggested as a marker for tumor aggressiveness of breast cancer, including the degrees of invasion and tumor recurrence (5). γ-Synuclein (SNCG), also referred to as breast cancer-specific gene 1, is highly expressed in human-infiltrating breast carcinomas but not expressed in normal or benign breast tissues. The expression of SNCG was strongly correlated to the stage of breast cancer (6). MUC1 and Met-HGF/SF can be detected in the axillary fluids of patients with breast cancer. The expression of both markers in the axillary drainage is strongly associated with unfavorable tumor features and can be used as a prognostic factor (7, 8). Ki67 antigen is expressed by proliferating cells in the late G1, S, and G2-M phases of the cell cycle. Its expression in breast cancer samples correlates with mitotic activity, recurrence rates after mastectomy, and survival (9). Nuclear matrix protein (NMP) is detected in the blood of women at the early stage of breast cancer, which is absent in the blood of healthy women. NMP66 has been selected as a marker for further developments and clinical trials of a test to be used in the detection and monitoring of women with, or at risk of, breast cancer (10). These tumor markers, such as estrogen receptor (ER), progesterone receptor (PR), p53, and cyclin D1, are also most useful for monitoring response to therapy and detecting early relapse of breast cancer (11-14).

Here we reported the characterization of a novel breast cancer associated antigen BRCAA1 and investigation of its novel antigen epitope and clinical significance. The brcaa1 gene, a breast cancer associated antigen 1 gene (GenBank no. AF208045), was cloned in 1999. So far in GenBank, there are some homology sequences, such as NM_031371, AF214114, NM_053421, AF083249, AL133418, etc. For example, RBP1L1 gene, a gene similar to brcaa1, exhibited up-regulated expression in breast cancer, lung carcinoma, colon cancer, ovary carcinoma and testis, and down-regulated expression in normal tissues (15). Its two antigen epitopes, KASIFLK and IKPSLGSKK were identified by human serum antibodies (16, 17). However, the structure and function of these genes similar to brcaa1 are poorly understood. In the present study, the chromosomal location of brcaa1 was investigated by fluorescence in situ hybridization, and its genomic and protein structure characterization was analyzed. According to antigenic index (AI), a novel breast cancer antigen epitope was predicted and confirmed by a series of experiments, and its clinical significance was further investigated.

PCR Primers and Short Peptides

A pair of PCR primers, with introduction of a COOH-terminal His-tag, was chemically synthesized. The sequences were as follows: primer-C1: 5′-CTT TAA GAA GGA GAT ATA CCA TGA GAG TGA AAG ATG CTC AG-3′, primer-C2: 5′-TGA TGA TGA GAA CCC CCC CCA CTC CAT TTG TAA ACT TTG G-3′. Short peptide SSKKQKRSHK, SKKQKRSHK, and SSKKQKRSH with keyhole limpet hemocyanin (KLH) were chemically synthesized.

Selection Criteria of Breast Cancer Patients and Healthy Donors and Sample Resource

The criteria for the sporadic patients were: primary invasive breast carcinoma less than 5 cm, no axillary metastases, age at diagnosis less than 55 years, and no previous malignancies. The criteria for hereditary patients were: carriers of a germline mutation in BRCA1 or BRCA2, and primary invasive breast carcinoma. ER and PR expressions were determined by immunohistochemical staining.

The criteria for healthy donors were: women without hereditary diseases, malignancies, cardiac vascular diseases and haematological diseases; age ranging from 28 to 55 years.

Sixty pairs of primary breast cancer specimens and non-cancerous breast specimens were collected from patients with breast cancer. Sixty specimens of breast cancer sera were collected from same patients with breast cancer before operation and treatment. Thirty specimens of normal sera were taken from a randomly selected set of healthy female donors. All collected sera were kept in −20°C refrigeration. All these specimens were collected from 1996 to 1999 in Xijing Hospital, China. These breast cancer cases were diagnosed by the Department of Pathology of Xijing Hospital. Tumors and non-cancerous tissues were preserved in liquid nitrogen within 30 minutes after surgery. Non-cancerous tissues were picked up at 5 cm distance away from primary breast cancer sites. Formalin-fixed, paraffin-embedded tumor tissues and lymph nodes were used to evaluate the tumor type, histologic grade, and the presence of metastasis and extensive lymphocytic infiltrate. The data are shown in Table 1. Our study was approved by administration of scientific research of Fourth Military Medical University.

Table 1.

Breast cancer samples classification

Sample no.AgeGradeERpPRpBrca1 or Brca2 mutationMetastasisLymphocytic infiltrateDetecting result*
1 43 2 80 80 0 0 0 + 
2 44 1 50 50 0 0 0 + 
3 41 3 10 5 0 0 0 − 
4 41 3 50 70 0 0 1 + 
5 48 3 100 80 0 0 0 + 
6 49 2 80 80 0 0 0 + 
7 46 1 80 50 0 0 0 + 
8 48 3 0 0 0 0 1 − 
9 48 3 60 80 0 0 0 + 
10 38 2 100 10 0 0 0 + 
11 37 3 90 70 0 0 0 + 
12 46 3 0 0 0 0 1 − 
13 41 3 10 5 0 0 0 − 
14 48 1 100 80 0 0 0 + 
15 46 3 30 10 0 0 0 − 
16 49 3 50 50 0 0 0 + 
17 48 2 90 90 0 0 0 + 
18 32 1 40 90 0 0 0 + 
19 48 1 80 30 0 0 0 + 
20 34 3 0 0 0 1 1 − 
21 39 1 50 50 0 0 0 + 
22 45 2 60 80 0 1 1 + 
23 41 2 70 70 0 0 0 + 
24 49 3 0 0 0 0 0 − 
25 45 2 100 100 0 0 0 + 
26 40 3 100 40 0 0 0 − 
27 45 1 80 90 0 0 0 + 
28 40 3 100 0 0 0 0 − 
29 52 2 100 80 0 0 0 + 
30 54 2 100 40 0 0 0 − 
31 52 2 80 90 0 0 0 + 
32 54 2 100 0 0 0 0 − 
33 52 2 100 80 0 0 0 + 
34 54 3 100 40 0 0 0 + 
35 54 3 90 10 0 0 0 + 
36 52 3 0 0 0 0 0 − 
37 54 2 70 30 0 0 0 + 
38 52 2 100 100 0 0 0 + 
39 46 2 50 50 0 0 0 − 
40 53 3 70 10 0 0 0 + 
41 54 3 100 0 0 0 0 − 
42 54 3 50 40 0 0 0 + 
43 54 3 50 5 0 0 0 + 
44 53 1 0 0 0 0 0 − 
45 37 2 100 60 0 0 0 + 
46 44 3 50 100 0 0 0 + 
47 43 3 90 5 0 0 0 + 
48 30 3 0 0 2 1 1 − 
49 39 2 80 100 0 0 0 + 
50 39 3 100 0 0 0 0 − 
51 41 3 80 80 1 0 0 + 
52 45 3 50 50 0 1 1 + 
53 28 3 0 0 0 0 0 − 
54 43 3 90 90 0 0 0 + 
55 41 2 100 100 0 0 0 + 
56 30 2 70 20 0 0 0 − 
57 28 3 0 0 1 1 1 − 
58 38 3 70 90 0 0 0 + 
59 44 3 50 0 2 0 0 − 
60 45 3 70 70 0 0 0 + 
Sample no.AgeGradeERpPRpBrca1 or Brca2 mutationMetastasisLymphocytic infiltrateDetecting result*
1 43 2 80 80 0 0 0 + 
2 44 1 50 50 0 0 0 + 
3 41 3 10 5 0 0 0 − 
4 41 3 50 70 0 0 1 + 
5 48 3 100 80 0 0 0 + 
6 49 2 80 80 0 0 0 + 
7 46 1 80 50 0 0 0 + 
8 48 3 0 0 0 0 1 − 
9 48 3 60 80 0 0 0 + 
10 38 2 100 10 0 0 0 + 
11 37 3 90 70 0 0 0 + 
12 46 3 0 0 0 0 1 − 
13 41 3 10 5 0 0 0 − 
14 48 1 100 80 0 0 0 + 
15 46 3 30 10 0 0 0 − 
16 49 3 50 50 0 0 0 + 
17 48 2 90 90 0 0 0 + 
18 32 1 40 90 0 0 0 + 
19 48 1 80 30 0 0 0 + 
20 34 3 0 0 0 1 1 − 
21 39 1 50 50 0 0 0 + 
22 45 2 60 80 0 1 1 + 
23 41 2 70 70 0 0 0 + 
24 49 3 0 0 0 0 0 − 
25 45 2 100 100 0 0 0 + 
26 40 3 100 40 0 0 0 − 
27 45 1 80 90 0 0 0 + 
28 40 3 100 0 0 0 0 − 
29 52 2 100 80 0 0 0 + 
30 54 2 100 40 0 0 0 − 
31 52 2 80 90 0 0 0 + 
32 54 2 100 0 0 0 0 − 
33 52 2 100 80 0 0 0 + 
34 54 3 100 40 0 0 0 + 
35 54 3 90 10 0 0 0 + 
36 52 3 0 0 0 0 0 − 
37 54 2 70 30 0 0 0 + 
38 52 2 100 100 0 0 0 + 
39 46 2 50 50 0 0 0 − 
40 53 3 70 10 0 0 0 + 
41 54 3 100 0 0 0 0 − 
42 54 3 50 40 0 0 0 + 
43 54 3 50 5 0 0 0 + 
44 53 1 0 0 0 0 0 − 
45 37 2 100 60 0 0 0 + 
46 44 3 50 100 0 0 0 + 
47 43 3 90 5 0 0 0 + 
48 30 3 0 0 2 1 1 − 
49 39 2 80 100 0 0 0 + 
50 39 3 100 0 0 0 0 − 
51 41 3 80 80 1 0 0 + 
52 45 3 50 50 0 1 1 + 
53 28 3 0 0 0 0 0 − 
54 43 3 90 90 0 0 0 + 
55 41 2 100 100 0 0 0 + 
56 30 2 70 20 0 0 0 − 
57 28 3 0 0 1 1 1 − 
58 38 3 70 90 0 0 0 + 
59 44 3 50 0 2 0 0 − 
60 45 3 70 70 0 0 0 + 
*

Detection of expression of novel antigen epitope in breast cancer tissues by immunohistochemistry.

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Mapping of brcaa1 on Human Chromosome by Fluorescence In situ Hybridization

Chromosome slides were made using peripheral blood lymphocytes (PBL) and stained with G strip according to standard methods (18). brcaa1 fragments (5.3 kb) were labeled with fluorescence dye according to the manual of the labeling reagent kit. Chromosome slides were prehybridized overnight, and then hybridized with denatured probes for 24 hours. The slides were subsequently washed twice in solutions of 2× SSC + 0.2% SDS, 0.1× SSC + 0.2% SDS, and 0.1× SSC, 10 minutes each time, then dried at room temperature, observed under fluorescence microscope, and photographed for mapping analysis (19). According to hybridization results and searching against Human Genomic Resource, the brcaa1's concrete position on human chromosome was determined.

Characterization of Genomic Structure and Protein Structure

In accordance with brcaa1 gene sequence, its exons and introns were identified by searching against Human Genomic Resource, and its amino acids sequence was derived. Its hydrophilicity, surface probability, flexibility, antigen index, secondary structure, and glycosyl site were analyzed by using GCG software (Genetics Computer Group, Madison, WI) (20-22).

Expression of Short Peptide A via Rapid Translation System

DNA fragment matching with 600 to 700 amino acid sites in Fig. 3 was selected. A pair of primers with introduction of a COOH-terminal His-tag, primer-C1 and primer -C2, was designed and chemically synthesized. PCR reaction 1 was used to obtain specific fragments: 10× PCR buffer 3 μL, 2.5 mmol/L deoxynucleotide triphosphate 3 μL, primer-C1 1 μL, primer-C2 1 μL, brcaa1 vector template 2 μL, 25 mmol/L MgCl2 3 μL, and Taq enzyme 1 μL. Sterilized minipure water was added until the total volume was up to 30 μL. PCR reaction condition: pre-denature at 94°C for 4 minutes, then 94°C for 1 minute, 55°C for 1 minute, 72°C for 1 minute, 25 cycles, finally extended at 72°C for 7 minutes. The PCR product was purified with a PCR purification kit. PCR reaction 2 was used to obtain the products with regulatory elements and His-tag by using rapid translation system (RTS) E. coli Linear Template Generation Set and His-tag kit (Roche Diagnostics GmbH, Penzberg, Germany). The PCR products were purified by PCR purification kit. The purified products were used for expressing corresponding proteins by RTS 100 E. coli kit (Roche Diagnostics) (23). The protein products were rapidly purified using Vivapure S Spin column. The expressed protein, named as short peptide A, was confirmed by Western blotting using anti-His antibody as first antibody.

Rabbit IgG Antibody against the Peptide SSKKQKRSHK

A rabbit antibody against SSKKQKRSHK was developed. Five hundred micrograms of SSKKQKRSHK were mixed with Freund's complete adjuvant and injected around the lymph nodes of two rabbits. Twenty days later, the rabbits received an i.m. injection of 250 μg of SSKKQKRSHK in Freund's incomplete adjuvant, followed by three immunizations with the same dose every 20 days. Ten days later, the rabbits were bled, sera were collected, and serum IgG antibody titers were tested for SSKKQKRSHK using ELISA. The anti-SSKKQKRSHK IgG antibody was purified from the serum using an affinity column with the synthetic peptide.

Labeling of Rabbit IgG Antibody and Determination of Antigen Epitope Specificity

Rabbit IgG antibody was purified after the precipitation by standard affinity chromatography on protein G-Sepharose 4 Fast Flow. Coupling of purified antibody to peroxidase was made in accordance with the method of Nakane and recommendations of Boehringer Mannheim Biochemica (24). For a determination of epitope specificity, a 96-well plate was coated with peptide A (1 μg/mL) in 100 μL of 0.1 mol/L sodium bicarbonate buffer (pH 9.6). Nonspecific binding sites of the wells were then blocked with a solution of 1% bovine serum albumin in PBS for 1 hour at 37°C. After washing with PBS containing 0.1% Tween 20 (PBS-T), 100 μL of rabbit IgG antibody were added and incubated for 1 hour at 37°C. Then, the plate was washed 5 times with PBS-T and 100 μL of a 5,000-fold diluted solution of goat anti-rabbit IgG antibody labeled with peroxidase were added to each well. After incubation for 1 hour at room temperature and washing with PBS-T, 100 μL of the mixture of o-phenylenediamine (0.43 mg/mL in 0.1 mol/L citrate buffer, pH 5.0) and H2O2 (0.002%) were added in each well. Then, the mixture was incubated for 30 minutes at room temperature in darkness. The reaction was stopped by adding 50 μL of 1 mol/L HCl. The absorbance of the product was measured at 490 nm using Bio-Rad 680 Microplate Reader.

Screening and Purification of Positive Sera to Bind Peptide A

Purified peptide A in 5 μg/mL PBS solution was coated onto 96-well polystyrene plates and incubated overnight at 4°C. The wells were then incubated with blocking buffer for 2 hours. One hundred microliters of 1:500 diluted individual human sera were added in triplicate to the peptide A–coated plates and incubated for 2 hours at room temperature. Controls included 1:500 diluted individual human sera plus the second antibody added to the wells without peptide A. The remaining steps followed standard ELISA procedure. A specific absorbance was obtained by subtracting background absorbance from experimental absorbance. Sixty sera from breast cancer patients and 30 sera from healthy women were screened for their antibody reactivity to expressed peptide A. The positive sera from breast cancer patients were measured the titer of IgG antibody to peptide A by ELISA. The immunoglobulin fraction of those positive sera was precipitated by 40% saturation ammonium sulfate, resuspended in PBS (pH 7.4), and dialyzed at 4°C against PBS. The IgG was purified by passing through a protein G column. The bound IgG was eluted with 0.3 mol/L acid glycine buffer (pH 3.0), neutralized with 1 mol/L Tris (pH 8.0), and dialyzed against PBS (25).

Immunoprecipitation and Western Blotting Analysis

All steps of the immunoprecipitation were carried out on ice. MCF-7 cells were lysed in an NP40 isotonic lysis buffer with freshly added protease inhibitors [142.5 mmol/L KCl, 5 mmol/L MgCl2, 10 mmol/L HEPES (pH 7.2), 1 mmol/L EGTA, 0.2% NP40, 0.2 mmol/L phenylmethylsulfonyl fluoride, 0.1% aprotinin, 0.7% μg/mL pepstatin, and 1 μg/mL leupeptin] by shaking for 30 minutes. Nuclei and unlysed cellular debris were removed by centrifugation at 15,000 × g for 10 minutes. Purified human IgG antibody and anti-SSKKQKRSHK antibody were added for 90 minutes, and immunoprecipitates were captured with 10% (v/v) protein G-Sepharose for 60 minutes. Immunoprecipitates were washed 3 times in lysis buffer. Immunoprecipitates were solubilized with SDS-PAGE sample buffer and electrophoresed through 15% SDS-polyacrylamide gels. For Western blotting, proteins were electrotransferred during 1 hour (400 mA) on polyvinylidene difluoride membranes. Filters were blocked for 2 hours with PBS-T containing 5% nonfat milk. All immunostaining steps were done in PBS-T at room temperature. Filters were incubated with human IgG antibody or anti-SSKKQKRSHK IgG antibody for 2 hours, respectively. After washing, secondary antibodies labeled with peroxidase were added for 2 hours. Then filters were washed in PBS-T and developed by the enhanced chemiluminescense system (26).

ELISA Competition Inhibition Assays with Synthetic Peptide Antigens

For ELISA competition inhibition assays, 100 μL of human antibody in 5 μg/mL PBS were added to 96-well polystyrene plates, incubated overnight at 4°C, and then blocked with 1% bovine serum albumin. Specific IgG antibody (1.7 × 10−9 mol/L) was incubated overnight at 4°C with peptides SSKKQKRSHK, SKKQKRSHK, and SSKKQKRSH at concentrations of 1.7 × 10−9, 1.7 × 10−8, 1.7 × 10−7, 1.7 × 10−6, 1.7 × 10−5, and 1.7 × 10−4 mol/L. Pre- and post-inhibited antibodies were then added in triplicate to peptide-coated plates and incubated for 3 hours at room temperature. Peroxidase-conjugated goat anti-human IgG was added for 1 hour at room temperature, followed by the addition of o-phenylenediamine dihydrochloride peroxide substrate solution. Reactivity was measured by absorbance at 490 nm.

Immunohistochemical Analysis with Human Specific Antibody

Sixty pairs of breast cancer specimens and non-cancerous tissues were analyzed by immunohistochemistry. The avidin-biotin complex (ABC) technique was used for immunohistochemical staining of these tissues. Specific IgG antibody (10 μg/mL) and PBS were added to tissue slides that previously had been blocked for 5 hours with goat serum and incubated overnight. After washing with PBS, the slides were incubated with a goat anti-human IgG conjugated to biotin at room temperature for 1 hour. Alkaline phosphatase substrate was then added for color development. The slides were counterstained with H&E (27).

Statistical Analysis

Paired and unpaired t test and χ2 analysis were used to explore the correlation of the expression of novel antigen epitope and sera antibody levels with patient ages, breast cancer stages, ERp, and PRp. Values of P ≤ 0.05 were considered to be statistically significant. The model based on the threshold on ERp and PRp was established to explain the data, and was obtained after applying dimensionality reduction with a stepwise forward selection, using χ2 to rank the worth of each variable with respect to the outcome. The final predictor was chosen in the form of classification trees, with informational loss function (28). A logistic regression with stepwise forward selection (29) was found to be less accurate than a classification tree. Neither PCA dimensionality reduction nor Bayesian modeling (in which ERp and PRp were assumed Gaussian distributions) did not show any clear advantage compared with classification trees. All statistical analyses were done with statistical Package for the social science software (SPSS, Chicago, IL), and S-plus language.

Human Chromosomal Location

Fluorescence in situ hybridization result showed that brcaa1 was mapped at 1q42.1-q43 as shown in Fig. 1. Searching against Human Genome Resource, NT_004836.14/Hs1_4993 clone on human No. 1 chromosome was found to include the whole brcaa1 cDNA sequence. Further analysis showed that the cDNA of brcaa1 is located between 167428 and 73571 sites, that is, 1q42.1-q43. Therefore, brcaa1 is confirmed to locate 1q42.1-q43. Final result was submitted to GenBank (AF208045).

Figure 1.
Figure 1. Human chromosome location of brcaa1 by fluorescence in situ hybridization. A. Fluorescence in situ hybridization map; B. Concrete location of brcaa1 gene on No. 1 chromosome with G band; C. Schematic of concrete location of brcaa1 on No. 1 chromosome with G band.
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Human chromosome location of brcaa1 by fluorescence in situ hybridization. A. Fluorescence in situ hybridization map; B. Concrete location of brcaa1 gene on No. 1 chromosome with G band; C. Schematic of concrete location of brcaa1 on No. 1 chromosome with G band.

Figure 1.
Figure 1. Human chromosome location of brcaa1 by fluorescence in situ hybridization. A. Fluorescence in situ hybridization map; B. Concrete location of brcaa1 gene on No. 1 chromosome with G band; C. Schematic of concrete location of brcaa1 on No. 1 chromosome with G band.
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Human chromosome location of brcaa1 by fluorescence in situ hybridization. A. Fluorescence in situ hybridization map; B. Concrete location of brcaa1 gene on No. 1 chromosome with G band; C. Schematic of concrete location of brcaa1 on No. 1 chromosome with G band.

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Characterization of Genomic Structure and Protein Structure

Searching against Genome Resource, the region of 167428 to 73571 nucleotide sites in NT_004836.14/Hs1_4993 clone was found to include the full-length cDNA sequence of brcaa1. The region of 167428 to 73571 nucleotide sites in NT_004836.14/Hs1_4993 is genomic region of brcaa1. The whole length of this region is 93.857 kb, including 18 exons and 17 introns, as shown in Fig. 2.

Figure 2.
Figure 2. Plotstructure graph of BRCAA1 protein created by the PLOTSTRUCTURE command of GCG software.
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Plotstructure graph of BRCAA1 protein created by the PLOTSTRUCTURE command of GCG software.

Figure 2.
Figure 2. Plotstructure graph of BRCAA1 protein created by the PLOTSTRUCTURE command of GCG software.
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Plotstructure graph of BRCAA1 protein created by the PLOTSTRUCTURE command of GCG software.

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Compared with GenBank Database, BRCAA1 protein has 37% amino acid identity compared with RB binding protein1. BRCAA1 protein includes two known antigen epitopes, such as KASIFLK (sites 250 to 256, RBP1) and IKPSLGSKK (sites 34 to 42, BRCAA1, Fig. 2). Its peptide is predicted to include 10 glycosylate sites. The region 740 to 750 at NH2 terminus is very hydrophilic, indicating that this region may be exposed to solvent, and it may form α helix. Its probability, flexibility, AI, and secondary structure are shown in Fig. 3.

Figure 3.
Figure 3. Schematic of genomic structure of brcaa1. Black column, exon; short line between black columns, intron; white column, 5′ and 3′ untranslated regions.
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Schematic of genomic structure of brcaa1. Black column, exon; short line between black columns, intron; white column, 5′ and 3′ untranslated regions.

Figure 3.
Figure 3. Schematic of genomic structure of brcaa1. Black column, exon; short line between black columns, intron; white column, 5′ and 3′ untranslated regions.
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Schematic of genomic structure of brcaa1. Black column, exon; short line between black columns, intron; white column, 5′ and 3′ untranslated regions.

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In the region 580 to 620 sites, the AI values are higher than average; therefore, potential antigen epitope likely exists in this region. Especially in the region 610 to 619 sites, SSKKQKRSHK, their AI values are the highest, suggesting that the peptide SSKKQKRSHK possibly is a potential antigen epitope.

Expression and Confirmation of Peptide A by Using RTS and Western Blotting

Western blotting result, as shown in Fig. 4, shows that peptide A, composed of 100 amino acids (600 to 700 sites, Fig. 2), including predicated antigen epitope with His-tag, was successfully expressed by RTS.

Figure 4.
Figure 4. Western blotting analysis of peptide A by using anti-his antibody as first antibody. A. Protein marker; B-D hybridization bands of peptide A with His-tag expressed by RTS.
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Western blotting analysis of peptide A by using anti-his antibody as first antibody. A. Protein marker; B-D hybridization bands of peptide A with His-tag expressed by RTS.

Figure 4.
Figure 4. Western blotting analysis of peptide A by using anti-his antibody as first antibody. A. Protein marker; B-D hybridization bands of peptide A with His-tag expressed by RTS.
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Western blotting analysis of peptide A by using anti-his antibody as first antibody. A. Protein marker; B-D hybridization bands of peptide A with His-tag expressed by RTS.

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Screening Positive Sera with Peptide A

ELISA results are shown in Fig. 5; some statistical analyses are shown in Tables 2 and 3. No statistical difference is found between the age distribution of breast cancer patients and healthy donors as shown in Table 2. The mean absorbance of serum IgG antibody from patients and healthy donors were 0.401 ± 0.163 SD and 0.137 ± 0.121 SD, respectively. Sixty-five percent (39 of 60) of breast cancer patients and 13% (4 of 30) of healthy donors had a specific absorbance of IgG antibody against peptide A greater than the mean absorbance of healthy donors; there exists a statistically significant difference between serum antibody levels from patients and healthy donors (t test, P < 0.001). This result strongly indicates that the antigen expressed by breast cancer cells may be responsible for the higher level of sera antibody in breast cancer patients.

Figure 5.
Figure 5. ELISA analysis of peptide A in serum from 60 patients with breast cancer and 30 healthy female donors. A. Scatter plot of the titers of peptide A in breast cancer patients sera; B. Scatter plot of the titers of peptide A in healthy donors.
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ELISA analysis of peptide A in serum from 60 patients with breast cancer and 30 healthy female donors. A. Scatter plot of the titers of peptide A in breast cancer patients sera; B. Scatter plot of the titers of peptide A in healthy donors.

Figure 5.
Figure 5. ELISA analysis of peptide A in serum from 60 patients with breast cancer and 30 healthy female donors. A. Scatter plot of the titers of peptide A in breast cancer patients sera; B. Scatter plot of the titers of peptide A in healthy donors.
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ELISA analysis of peptide A in serum from 60 patients with breast cancer and 30 healthy female donors. A. Scatter plot of the titers of peptide A in breast cancer patients sera; B. Scatter plot of the titers of peptide A in healthy donors.

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Table 2.

Comparison of ages and sera antibody levels between patients and donors

Mean ± SD
Percentage of difference*P from unpaired t test
PatientsDonors
Age (y) 44.63 ± 7.05 43.89 ± 6.09 1.6 0.96 
Sera level 0.401 ± 0.163 0.137 ± 0.121 192.7 <0.001 
Mean ± SD
Percentage of difference*P from unpaired t test
PatientsDonors
Age (y) 44.63 ± 7.05 43.89 ± 6.09 1.6 0.96 
Sera level 0.401 ± 0.163 0.137 ± 0.121 192.7 <0.001 
*

Expressed as (meanpatients − meandonors)/meandonors.

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Table 3.

Comparison of sera antibody levels between breast cancer stage and menopause status

Pre-menopause
Post-menopause
P value from t test
N (mean ± SD) 95% CIsN (mean ± SD ) 95% CIs
Stage I 5 (0.446 ± 0.230) (0.220-0.672) 3 (0.460 ± 0.147) (0.168-0.752) 0.929 
Stage II 10 (0.432 ± 0.175) (0.328-0.536) 9 (0.362 ± 0.133) (0.252-0.472) 0.346 
Stage III 18 (0.327 ± 0.125) (0.256-0.398) 15 (0.462 ± 0.173) (0.384-0.540) 0.014 
Pre-menopause
Post-menopause
P value from t test
N (mean ± SD) 95% CIsN (mean ± SD ) 95% CIs
Stage I 5 (0.446 ± 0.230) (0.220-0.672) 3 (0.460 ± 0.147) (0.168-0.752) 0.929 
Stage II 10 (0.432 ± 0.175) (0.328-0.536) 9 (0.362 ± 0.133) (0.252-0.472) 0.346 
Stage III 18 (0.327 ± 0.125) (0.256-0.398) 15 (0.462 ± 0.173) (0.384-0.540) 0.014 
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Further analysis showed that sera antibody levels in 33 cases of stage III is likely associated with menopause status. Sera antibody levels of patients before menopause are significantly higher than sera level of patients after menopause, as shown in Table 3. However, no difference of sera antibody levels was found between patients before and after menopause in the cases of breast cancer stage I or II. Similarly, no difference of sera antibody levels was found between healthy donors before menopause (0.102 ± 0.117 SD) and after menopause (0.182 ± 0.114SD) (P = 0.071).

Determination of Antigen Epitope Specificity

ELISA competition inhibition assay results (Fig. 6) show that labeled anti-SSKKQKRSHK antibody was completely inhibited by the unlabeled anti-SSKKQKRSHK antibody and human IgG antibody from positive sera. Results also showed that anti-SSKKQKRSHK antibody is specific to an antigen epitope on peptide A.

Figure 6.
Figure 6. Competition inhibition analysis using human IgG antibody and unlabeled anti-SSKKQKRSHK antibody as inhibitors. Points, average of three determinations.
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Competition inhibition analysis using human IgG antibody and unlabeled anti-SSKKQKRSHK antibody as inhibitors. Points, average of three determinations.

Figure 6.
Figure 6. Competition inhibition analysis using human IgG antibody and unlabeled anti-SSKKQKRSHK antibody as inhibitors. Points, average of three determinations.
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Competition inhibition analysis using human IgG antibody and unlabeled anti-SSKKQKRSHK antibody as inhibitors. Points, average of three determinations.

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ELISA Competition Inhibition Assays with Synthetic Peptides

ELISA competition inhibition assay results (Fig. 7) show that SSKKQKRSH was the shortest peptide with optimal inhibition effect among synthetic peptides SSKKQKRSHK, SKKQKRSHK, and SSKKQKRSH.

Figure 7.
Figure 7. Competition inhibition assays of synthetic peptides by ELISA. Synthetic peptides SSKKQKRSHK, SKKQKRSHK, and SSKKQKRSH were used for competition inhibition assays at concentrations of 1.7 × 10−9, 1.7 × 10−8, 1.7 × 10−7, 1.7 × 10−6, 1.7 × 10−5, and 1.7 × 10−4 mol/L. Three inhibition curves were drawn. SSKKQKRSH is the shortest peptide with optimal inhibition effect.
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Competition inhibition assays of synthetic peptides by ELISA. Synthetic peptides SSKKQKRSHK, SKKQKRSHK, and SSKKQKRSH were used for competition inhibition assays at concentrations of 1.7 × 10−9, 1.7 × 10−8, 1.7 × 10−7, 1.7 × 10−6, 1.7 × 10−5, and 1.7 × 10−4 mol/L. Three inhibition curves were drawn. SSKKQKRSH is the shortest peptide with optimal inhibition effect.

Figure 7.
Figure 7. Competition inhibition assays of synthetic peptides by ELISA. Synthetic peptides SSKKQKRSHK, SKKQKRSHK, and SSKKQKRSH were used for competition inhibition assays at concentrations of 1.7 × 10−9, 1.7 × 10−8, 1.7 × 10−7, 1.7 × 10−6, 1.7 × 10−5, and 1.7 × 10−4 mol/L. Three inhibition curves were drawn. SSKKQKRSH is the shortest peptide with optimal inhibition effect.
View largeDownload slide

Competition inhibition assays of synthetic peptides by ELISA. Synthetic peptides SSKKQKRSHK, SKKQKRSHK, and SSKKQKRSH were used for competition inhibition assays at concentrations of 1.7 × 10−9, 1.7 × 10−8, 1.7 × 10−7, 1.7 × 10−6, 1.7 × 10−5, and 1.7 × 10−4 mol/L. Three inhibition curves were drawn. SSKKQKRSH is the shortest peptide with optimal inhibition effect.

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Immunoprecipitation and Western Blotting Analysis

Immunoprecipitation and Western blotting results (Fig. 8) show that the IgG antibody reactivity to peptide A was completely absorbed by the soluble cytoplasmic fraction of MCF-7 cells; there was no absorption by cell membrane or the nuclear fractions. The results also show that SSKKQKRSHK peptide epitope is in the cytoplasm of breast cancer cells.

Figure 8.
Figure 8. Western blotting comparing the absorption of specific IgG antibody with a control using preabsorbed antibody (A), membrane protein (B), cytoplasmic protein (C), and nuclear protein obtained from MCF-7 cells (D, E). M, protein marker.
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Western blotting comparing the absorption of specific IgG antibody with a control using preabsorbed antibody (A), membrane protein (B), cytoplasmic protein (C), and nuclear protein obtained from MCF-7 cells (D, E). M, protein marker.

Figure 8.
Figure 8. Western blotting comparing the absorption of specific IgG antibody with a control using preabsorbed antibody (A), membrane protein (B), cytoplasmic protein (C), and nuclear protein obtained from MCF-7 cells (D, E). M, protein marker.
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Western blotting comparing the absorption of specific IgG antibody with a control using preabsorbed antibody (A), membrane protein (B), cytoplasmic protein (C), and nuclear protein obtained from MCF-7 cells (D, E). M, protein marker.

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Immunohistochemical Staining and Statistical Analysis

Immunohistochemical staining results are shown in Table 1,Table 1. Thirty-nine of 60 primary breast cancer tissues showed a strong positive staining, which is only in the cytoplasm, as shown in Fig. 9A. No positive staining was found in the non-cancerous tissues. There is a statistically significant difference between expression levels of novel antigen epitope in breast cancer tissues and in non-cancerous tissues (P < 0.01), this result indicates that the expression of novel antigen epitope is associated with breast cancer status. Positive rate of novel antigen epitope in patients before menopause and after menopause are 56.4% (22 of 39) and 43.59% (17 of 39), respectively. χ2 analysis showed that the expression of novel antigen epitope is uncorrelated with patient ages, even if they are classified as pre- and post-menopause.

Figure 9.
Figure 9. Immunohistochemical analysis using specific IgG antibody to SSKKQKRSHK of human breast cancer tissues and normal breast tissues. The cytoplasmic protein of breast cancer tissues stained intensive positive (A, magnification, ×400). No positive cytoplasmic staining existed in normal breast tissues (B, magnification, ×400).
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Immunohistochemical analysis using specific IgG antibody to SSKKQKRSHK of human breast cancer tissues and normal breast tissues. The cytoplasmic protein of breast cancer tissues stained intensive positive (A, magnification, ×400). No positive cytoplasmic staining existed in normal breast tissues (B, magnification, ×400).

Figure 9.
Figure 9. Immunohistochemical analysis using specific IgG antibody to SSKKQKRSHK of human breast cancer tissues and normal breast tissues. The cytoplasmic protein of breast cancer tissues stained intensive positive (A, magnification, ×400). No positive cytoplasmic staining existed in normal breast tissues (B, magnification, ×400).
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Immunohistochemical analysis using specific IgG antibody to SSKKQKRSHK of human breast cancer tissues and normal breast tissues. The cytoplasmic protein of breast cancer tissues stained intensive positive (A, magnification, ×400). No positive cytoplasmic staining existed in normal breast tissues (B, magnification, ×400).

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Further statistical analysis showed that novel antigen epitope expression is closely associated with the values of ERp and PRp in breast cancer tissues, as shown in Table 4.

Table 4.

Comparison of ERp and PRp association with expression of novel antigen epitope

Positive novel antigen epitope
Negative novel antigen epitope
P from unpaired t test
N (mean ± SD) (95% CIs )N (mean ± SD) (95% CIs)
ERp 39 (76.9 ± 19.4) (67.21 to 86.63) 21 (39 ± 44.1) (25.82 to 52.28) <0.001 
PRp 39 (64.9 ± 29.5) (56.67 to 73.08) 21 (8.10 ± 15.6) (−3.085 to −19.28) <0.001 
Positive novel antigen epitope
Negative novel antigen epitope
P from unpaired t test
N (mean ± SD) (95% CIs )N (mean ± SD) (95% CIs)
ERp 39 (76.9 ± 19.4) (67.21 to 86.63) 21 (39 ± 44.1) (25.82 to 52.28) <0.001 
PRp 39 (64.9 ± 29.5) (56.67 to 73.08) 21 (8.10 ± 15.6) (−3.085 to −19.28) <0.001 
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As shown in Table 5, ERp is associated with either metastasis or infiltrate of breast cancer, whereas PRp is not associated with either metastasis or infiltrate of breast cancer.

Table 5.

Comparison of ERp and PRp association with breast cancer metastasis and infiltrates

Metastasis or infiltrate
No metastasis or No infiltrate
P from t test
N (mean ± SD) 95% CIsN (mean ± SD) 95% CIs
ERp 8 (20 ± 27.8) (−1.832 to 41.83) 52 (70.4 ± 31.2) (61.82 to 78.95) <0.001 
PRp 8 (25 ± 35.5) (−1.005 to 51) 52 (48.1 ± 36.9) (37.88 to 58.28) 0.104 
Metastasis or infiltrate
No metastasis or No infiltrate
P from t test
N (mean ± SD) 95% CIsN (mean ± SD) 95% CIs
ERp 8 (20 ± 27.8) (−1.832 to 41.83) 52 (70.4 ± 31.2) (61.82 to 78.95) <0.001 
PRp 8 (25 ± 35.5) (−1.005 to 51) 52 (48.1 ± 36.9) (37.88 to 58.28) 0.104 
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Clinical data showed that among 39 positive specimens of novel antigen epitope, 35 positive specimens were with non-metastasis and non-lymphocytic infiltrates, and with ERp > 30 and PRp > 30; the remaining 4 positive specimens were respectively found in 1 of 4 specimens of hereditary breast cancer, 1 of 5 metastasis specimens, and 2 of 6 specimens with lymphocytic infiltrates; these specimens all exhibited ERp < 30 and PRp < 30. Further statistical analysis showed that ERp was associated with metastasis or infiltrate in the cases of breast cancer with positive novel antigen epitope; PRp was not associated with metastasis or infiltrate (PER = 0.044, and PPR = 0.619). Therefore, positive expression of novel antigen epitope, simultaneously with ERp > 30 and PRp > 0, possibly predicts good prognosis of breast cancer patients, such as no-metastasis and no-lymphocytic infiltrate. Conversely, the positive expression of novel antigen epitope, ERp ≤ 30 and null PRp, possibly predicts bad prognosis of breast cancer, such as metastasis and lymphocytic infiltrate.

Positive ratio of novel antigen epitope in breast cancer tissues with I, II, III grade were 87% (57.20% to 98.84%), 79% (60.12% to 90.56%), and 45% (29.58% to 53.29%), respectively; all CI with a 90% of confidence, P = 0.0989. This result indicates that the expression of novel antigen epitope has no difference with histologic grade I, II, and III.

According to the abovementioned results, a simple prognosis model based on PR and ER thresholds is established to explain the data. A prognosis based on PR and ER thresholds may be reasoned as a Bernoulli process, in which each patient is an independent event that either has expression of novel antigen epitope or not. Therefore, confidence limits can be estimated for with a two-tailed probability Pr[−z ≤ x ≤ z] = 90%, z = 1.65, N = 60. Thus, the probability of issuing a successful prognosis lies between 85.9% and 96.9%.

The model we found is depicted in Fig. 10A, and it implies that a good prognosis (positive novel antigen epitope) is associated to ERp > 30 and PRp > 0; meanwhile the bad prognosis (negative novel antigen epitope) sets two cases: (i) ERp ≤ 30 and null PRp, and (ii) when PRp is null. The classification result with the model is shown in Fig. 10B. Its sensitivity is 100% and its specificity is 81%; false positives or misclassifications were found in four cases, such as No. 26, 30, 39, 56 samples, which actually exhibited negative expression of novel antigen epitope and positive expression of ERp > 30 and PRp > 0.

Figure 10.
Figure 10. Model based on ERp and PRp and classification result with model. A. Resulting prognosis model found by stepwise forward selection. This tree shows how the co-expression between PRp and ERp is related to novel antigen epitope. N, negative novel antigen epitope; P, positive novel antigen epitope. B. Classification result with the model based on PR and ER thresholds. Correct positive prognosis, specimen with positive expression of novel antigen epitope. Correct negative prognosis, specimen with negative expression of novel antigen epitope. Misclassification, specimen with mistaken positive or negative expression of novel antigen epitope.
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Model based on ERp and PRp and classification result with model. A. Resulting prognosis model found by stepwise forward selection. This tree shows how the co-expression between PRp and ERp is related to novel antigen epitope. N, negative novel antigen epitope; P, positive novel antigen epitope. B. Classification result with the model based on PR and ER thresholds. Correct positive prognosis, specimen with positive expression of novel antigen epitope. Correct negative prognosis, specimen with negative expression of novel antigen epitope. Misclassification, specimen with mistaken positive or negative expression of novel antigen epitope.

Figure 10.
Figure 10. Model based on ERp and PRp and classification result with model. A. Resulting prognosis model found by stepwise forward selection. This tree shows how the co-expression between PRp and ERp is related to novel antigen epitope. N, negative novel antigen epitope; P, positive novel antigen epitope. B. Classification result with the model based on PR and ER thresholds. Correct positive prognosis, specimen with positive expression of novel antigen epitope. Correct negative prognosis, specimen with negative expression of novel antigen epitope. Misclassification, specimen with mistaken positive or negative expression of novel antigen epitope.
View largeDownload slide

Model based on ERp and PRp and classification result with model. A. Resulting prognosis model found by stepwise forward selection. This tree shows how the co-expression between PRp and ERp is related to novel antigen epitope. N, negative novel antigen epitope; P, positive novel antigen epitope. B. Classification result with the model based on PR and ER thresholds. Correct positive prognosis, specimen with positive expression of novel antigen epitope. Correct negative prognosis, specimen with negative expression of novel antigen epitope. Misclassification, specimen with mistaken positive or negative expression of novel antigen epitope.

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Looking for novel antigen epitopes of breast cancer helps us to clarify its mechanism in favor of its diagnosis, treatment, and prevention (30, 31). The brcaa1 gene (breast cancer associated antigen 1 gene, AF208045), which we previously cloned, exhibits differential expression between breast cancer and normal breast tissues. Its genomic characteristics, such as location at 1q42.1-43 on human chromosome, full-length sequence of 93.857 kb, including 18 exons and 17 introns, are identified. Its protein structure characteristic also has been analyzed as shown in Fig. 3. For example, its second structure includes 10 glycosylate sites; the region 740 to 750 sites at NH2 terminus is very hydrophilic, indicating that this region may form α helix (32, 33).

Because BRCAA1 protein includes the same antigen epitope IKPSLGSKK as RB binding protein 1, BRCAA1 protein may be one member of RB binding protein family. Rb is an important antioncogene; RB protein regulates transcriptional events, which are important for cell proliferation. A major target of pRB is the E2F family of transcription factors that controls expression of many genes required for DNA synthesis and cell cycle progression. Binding of pRB to E2F species inhibits expression of E2F-regulated genes, resulting in withdrawal from the cell cycle. pRB and related pocket proteins use multiple mechanisms to elicit this effect (34-37). RB family members are able to recruit the mSIN3-HDAC complex via a pocket-dependent association with RBP1 to actively repress transcription. RBP1 is the first molecule described that seems to contribute to two separated classes of transcriptional repression activities, which are important for transcriptional repression by RB family members, both by recruiting the mSIN3-HDAC complex via R2 and via an as yet unidentified repression mechanism using the R1 domain. These dual repression activities of RBP1 may account for the ability of pRB to repress transcription in a variety of promoters, including those that have been reported to be insensitive or only partially sensitive to the HDAC inhibitor tissue specific antigen (38, 39). Therefore, BRCAA1 may be associated with regulating cell cycle. Further clarification of its concrete function is under way.

Besides the known antigen epitopes, another novel antigen epitope inside BRCAA1 protein has been predicted by AI, and identified by series of experiments (40, 41). The novel antigen epitope is in the region 610 to 619 sites; its shortest sequence is SSKKQKRSHK. The antigen epitope is in the cytoplasm of breast cancer cells by immunohistochemical staining. In our experiments, the peptide A, including predicted antigen epitope, was expressed by using RTS system. This RTS system is very useful, which can produce 50 to 100 mg target proteins with natural activity in 3 to 5 hours, and the target protein is easily purified and kept active (23, 42). Our experiments also fully demonstrate that AI can be used to predict the potential antigen epitope.

In our study, the clinical significance of novel antigen epitope was investigated. Its expression levels in breast cancer and non-cancerous tissues were analyzed via immunohistochemistry. Our initial exploration showed that the expression of novel antigen epitope seems to be associated with breast cancer, but not associated with histologic grade of breast cancer, uncorrelated with patient ages and menopause status. Later, further statistical analysis also shows that it is closely associated with the status of ER and PR in breast cancer tissues. A simple model to explain the data is established based on two variables, ERp and PRp. The model is described in the previous section. This analysis unravels an astonishing model with 100% of sensitivity and 81% of specificity. Unfortunately, this model is unable to capture four cases, such as four patients of No. 26, 30, 39, and 56, who actually exhibited negative expression of novel antigen epitope, but positive expression of ERp > 30 and PRp > 0. Confidence limits show that the probability of issuing a successful prognosis lies between 85.9% and 96.9%. The model also fully supports that the expression of novel antigen epitope is closely associated with the status of ER and PR in breast cancer.

Previous studies have shown that ER- and PR-positive breast cancers compared with ER- and PR-negative ones have a less aggressive, more differentiated phenotype, and a more favorable prognosis (43-45). In this study, the expression level of ER was found to be associated with prognosis of breast cancer, such as metastasis or infiltrate. Our result also corresponds with some reports (9, 46, 47). Conversely, the expression level of PR was not found to be associated with prognosis, such as metastasis or infiltrate of breast cancer (P = 0.104). This result conflicts with some reports (45, 48-50). This may be because our analysis is based on a small data set. Increasing the sample number may result in more precise results.

The status of ER and PR in breast cancer is an important reference factor for breast cancer therapy. Muss et al. reported that tamoxifen therapy can significantly decrease the risk of recurrence and improve survival in women of all ages having ER- or PR-positive invasive breast cancer (50, 51). Therefore, the status of novel antigen epitope in breast cancer tissues is also possibly an important reference factor for breast cancer therapy. Investigation for its significance on breast cancer therapy is under way.

In our study, the sera antibody levels of novel antigen epitope in breast cancer patients and healthy female donors were investigated via ELISA. The results showed that titer levels of sera antibody from breast cancer patients are dramatically higher than healthy donors, highly indicating that sera antibody level of novel antigen epitope is helpful for breast cancer diagnosis, although the result is based on a small sample. Statistical analysis also shows that sera antibody levels of breast cancer stage III are associated with menopause status; the sea antibody level of patients before menopause is significantly higher than ones of patients after menopause. However, no difference of sera antibody levels was found between patients before and after menopause in breast cancer stage I or II. Similarly, no difference was found between healthy female donors before menopause and after menopause. So far the critical threshold of sera antibody for distinguishing the breast cancer patients from non-breast cancer patients still needs to be further determined by screening a lot of breast cancer sera.

In conclusion, a breast cancer associated antigen 1 gene, brcaa1, genomic characteristics were obtained, such as location in 1q42.1-43 and full length of 93.857 kb, including 18 exons and 17 introns. BRCAA1 protein structure is characterized as being composed of 1,214 amino acids, including 10 glycosylate sites and two known antigen epitopes, such as KASIFLK and IKPSLGSKK, the region 740 to 750 sites at NH2 terminus being very hydrophilic, indicating that this region may form α helix. Its novel antigen epitope, SSKKQKRSHK, was identified and found in the cytoplasm of breast cells. The expression of novel antigen epitope in breast cancer tissues is closely associated with the status of ER and PR in breast cancer tissues. Its sera antibody level is helpful for early diagnosis of breast cancer. Because our result is based on a small sample, BRCAA1 function and its novel antigen epitope potential significance still need to be further investigated.

Grant support: National Natural Scientific Fund of China grants 39970725 and 39470683.

The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

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