The present study was conducted on 72 patients diagnosed with breast cancer at the Oncology Center of Mansoura University (Mansoura, Egypt) between September 2013 and December 2013. Prior to commencing treatment, relevant information was obtained from each patient regarding age, menopausal status, number of children, lactation history and family history of breast cancer. Patients with chronic diseases (such as diabetes mellitus, liver dysfunction or rheumatoid arthritis) were excluded from the study. All pathological data were recorded following surgery (Table I). The control group included 100 healthy female volunteers. Each study participant provided written informed consent. The study was approved by the ethics committee of the Faculty of Medicine of Mansoura University.

For the assessment of GSTP1, TLR2 and TLR9 genetic polymorphisms, 5 ml blood was drawn from each subject enrolled in the study following an overnight fast, and was collected in EDTA tubes (BD Vacutainer Systems, Plymouth, UK) for DNA extraction. DNA was extracted from EDTA-anticoagulated blood using a Gentra Puregene Blood Kit for DNA purification (Qiagen, Inc., Valencia, CA, USA).

Tumor evaluation and grading were assessed histopathologically, as previously described (23). Tumor size, regional lymph nodes and distant metastasis were also evaluated and staged according to the American Joint Committee on Cancer Staging Manual (6th edition) (24). For immunohistochemical staining, samples were fixed with 10% neutral-buffered formalin and routinely processed to paraffin blocks. Next, 4-µm sections were cut from the blocks and mounted on poly-L-lysine pre-coated glass slides. Antigen retrieval was performed in a pressure cooker for 20 min at 120°C followed by incubation with blocking 6% H₂O₂ for 3 min to block endogenous peroxidase activity. The sections were then incubated with the Lab Vision™ Avidin Biotin Blocking Solution (cat. no. TA-015-BB (Thermo Fisher Scientific, Pittsburgh, PA, USA) to block nonspecific protein binding. Following antigen retrieval, the sections were incubated for 1 h at room temperature with primary antibodies, followed by horseradish peroxidase-conjugated secondary antibodies (Dako, Glostrup, Denmark) for 30 min at room temperature. The primary antibodies used were as follows: Monoclonal rabbit anti-human estrogen receptor (ER) antibody (clone SP1; cat. no. MA1-39540; 1:200; Thermo Fisher Scientific, Waltham, MA, USA), monoclonal mouse anti-human progesterone receptor (PR) antibody (clone PgR 636; 1:50; cat. no. M3569; Dako) and monoclonal mouse anti-human human epidermal growth factor receptor 2 (HER2) antibody (clone CB11; 1:10; cat. no. 18-7107; Thermo Fisher Scientific). The secondary antibodies used were as follows: Polyclonal goat anti-rabbit IgG antibody (1:200; cat. no. P0448; Dako) or polyclonal rabbit anti-mouse IgG antibody (1:200; cat. no. P0260; Dako). 3,3′-diaminobenzidine (Thermo Fisher Scientific) was then added as a chromogen for color development. The expression of the ER and PR were assessed immunohistochemically, as described previously (25). Scoring for both ER and PR expression was based on the proportion of cells in a given tumor specimen exhibiting distinct nuclear immunopositivity, as well as the intensity of staining (0, 5, 10, 20, 30, 40, 50, 60, 70, 80, 90 or 100%). Positive immunostaining for ER/PR expression was defined as >10% of cells with positively stained nuclei of any intensity. Samples exhibiting >10% immunoexpression were considered positive. HER 2-positivity was defined immunohistochemically, according to the Food and Drug Administration-approved criteria, as uniform and intense circumferential membrane staining in >10% of invasive tumor cells (26).

The GSTP1 105 Ile/Val polymorphism was investigated using a polymerase chain reaction (PCR)-restriction fragment length polymorphism (RFLP) method with the following primers (27): p105 forward, 5′-ACCCCAGGGCTCTATGGGAA-3′; and p105 reverse, 5′-TGAGGGCACAAGAAGCCCCT-3′ (Biosearch Technologies, Petaluma, CA, USA). PCR was performed using DreamTaq Green PCR Master Mix (Thermo Fisher Scientific). The procedure was conducted in a total reaction volume of 40 µl. containing 1.5 mM MgCl₂, 1 unit of Taq DNA polymerase, 200 ng of each primer and 10 µl DNA as template. PCR cycling conditions were as follows: Initial denaturation at 95°C for 5 min; 30 cycles of denaturation at 95°C for 30 sec, primer annealing at 55°C for 30 sec and extension at 72°C for 1 min; and a final polymerization step of 72°C for 5 min to complete the elongation processes. The PCR products (20 µl) were then digested using 5 units of Alw261 restriction enzyme (Thermo Fisher Scientific) in a total volume of 25 µl, and subsequently separated on a 3.5% agarose gel (Bio Basic Inc., Markham, ON, Canada) prior to staining with ethidium bromide (10 mg/ml; Bio Basic Inc.) to visualize the bands (Fig. 1). Individuals with the wild genotype (Ile/Ile) exhibit a single band (176 bp), whereas those with the mutant homozygote (Val/Val) exhibit two bands (85 and 91 bp). Individuals with the variant allele (Ile/Val) exhibit three bands (176 bp corresponding to Ile; and 91 and 85 bp corresponding to Val).

The TLR2 −196 to −174 del/ins polymorphism was assessed using an allele-specific PCR method with the following primers (28): forward, 5′-CACGGAGGCAGCGAGAAA-3′; and reverse, 5′-CTGGGCCGTGCAAAGAAG-3′ (Biosearch Technologies). The procedure was conducted in a total reaction volume of 25 µl, containing 2.5 µl 10X PCR buffer, 2 µl dNTPs (1.25 µmol/l), 0.5 µl MgCl₂ (25 mmol/l), 1.25 µl of each primer (25 mmol/l; Sigma-Aldrich, St. Louis, MO, USA), 15.3 µl dH₂O, 2 µl DNA (100 ng/µl), and 0.2 µl Taq DNA polymerase (5 U/µl; Invitrogen Life Technologies, Carlsbad, CA, USA). PCR cycling conditions were as follows: Initial denaturation step at 95°C for 5 min; amplification at 95°C for 30 sec, 60°C for 40 sec and 72°C for 40 sec, for 35 cycles; and a final elongation step at 72°C for 7 min. Fig. 2 shows the resulting homozygous (ins/ins; band at 286 bp) and heterozygous (ins/del; bands at 286 and 264 bp) genotypes following 3.5% agarose gel electrophoresis and ethidium bromide staining of the PCR products.

Genotyping of TLR9 was conducted using PCR-RFLP, as previously described (29). PCR reactions consisted of an initial step of 95°C for 5 min, followed by 35 cycles at 95°C 30 sec, 60°C for 30 sec and 72°C for 30 sec, and a final incubation at 72°C for 5 min. Briefly, PCR mixtures (30 µl final volume) contained 20 ng of genomic DNA, 20 pmol of each primer (25 pmol/ml; Intergrated DNA Technologies, Coralville, IA, USA), 3 µl ml of dNTPs (2.5 nM; Thermo Fisher Scientific), 3 µl MgCl₂ (25 mM; Thermo Fisher Scientific), 1 ml of NH₄ buffer and 0.2 µl of Taq polymerase (Thermo Fisher Scientific). The following primers for TLR9-rs187084 (1237T/C) were used: forward, 5′-ACTATCGAGCCTGCCTGCCATGATACC-3′; and reverse, 5′-ATCCAGCCTTCTTACAAACCTCCCACCC-3′ (Biosearch Technologies). A 15-µl aliquot of each product was digested with 0.5 units of BspTI (Thermo Fisher Scientific). PCR products were subjected to electrophoresis on a 2% agarose gel and visualized by ethidium bromide staining (Fig. 3). The presence of a wild type allele (C allele) for TLR9 resulted in an intact 423-bp band, whereas the RFLP profile of the T variant was characterized by two bands of 172 and 251 bp.

The χ² test was employed to examine differences in genotypic and allelic distribution between breast cancer patients and controls. The odds ratio (OR) and 95% confidence intervals (CI) were calculated. P<0.05 was considered to indicate statistically significant differences. Statistical tests were performed with GraphPad Prism software version 5.0 (GraphPad Software, Inc, La Jolla, CA, USA).

The breast cancer group (n=72) had a median age of 46.2 years (range, 27–76 years), with postmenopausal status in 61.1%, positive lactation history in 95.8%, positive family history of breast cancer in 8.3% and metastasis in 20.8%. The staging and other clinical and pathological characteristics are shown in Table I.

The distribution of the different genotypes of GSTP1 is presented in Table II. A significantly lower percentage of the GSTP1 Ile/Val (heterozygous) genotype was observed in women with breast cancer compared with healthy individuals (26.4 vs. 41%, respectively; P=0.0297; OR=2.112). There was also a significant decrease in the combined number of Val/Val and Ile/Val genotypes frequencies in breast cancer patients compared with controls, at rates of 36.1 and 51%, respectively (P=0.0283; OR=1.995). Furthermore, the GSTP1 Val allele frequency was decreased in breast cancer patients relative to controls, at rates of 22.9 and 32.5%, respectively.

The genotype and allele frequencies of TLR2 did not differ significantly between breast cancer patients and controls (Table III). However, the frequency of the TLR9 CC and CT genotypes were significantly lower in women with breast cancer compared with healthy individuals [CC, 12.5% vs. 20%, respectively (P=0.0016; OR=0.316); CT, 47.2% vs. 63%, respectively (P=0.0069; OR=0.264)]. A significant decrease was also observed in the overall TLR9 C allele frequency in breast cancer patients compared with controls, at rates of 36.1 and 51.5%, respectively (P=0.0047; OR=0.532).