Garcia-Casado et al. BMC Cancer 2010, 10:36 http://www.biomedcentral.com/1471-2407/10/36 endogenous peroxidase was inhibited with 3% hydrogen peroxide for 30 min. Immunoreactivity was enhanced with antigen retrieval treatment by heating the slides in a microwave oven for 10 min (700 W) in 10 mM sodium citrate buffer pH 7, followed by cooling for 20 min at room temperature. Sections were blocked with 20% horse serum in phosphate-buffer saline (PBS) and incubated with primary antibody for 1 h at room tem- perature. The incubation time for the secondary anti- body and avidin-biotin complexes was 30 min at room temperature. Sections were extensively washed and the immunoreactions developed using DAB (0.05% 3’3’ dia- mino-benzidine in 0.1% hydrogen peroxide). Negative controls included substitution of the primary antibody by mouse ascites or PBS. Slides were counterstained in Mayer hematoxylin, dehydrated, and mounted. ER and PgR expression in primary tumor was immu- nohistochemically evaluated using the anti-ER (clone 6F11) and anti-PgR (clone 1A6) antibodies (Novocastra Laboratories, Newcastle upon Tyne, England) at a dilu- tion of 1:40 and 1:30 respectively. Ki67 expression was also determined using the anti-Ki67 (clone MIB1) anti- body (Dako Corp., Carpinteria, CA) at a 1:50 dilution. HER2 protein expression was immunohistochemically evaluated using the HercepTest Kit (Dako Corp., Car- pinteria, CA) according to the manufacturer’s protocol. The scoring system was as follows: 0, tumors with no or weak staining in less than 10% of the cells; +, tumors with a faint or barely perceptible membrane staining in more than 10% of cells or with noncircumferential stain- ing; ++, moderate circumferential membrane staining; + ++, strong circumferential membrane staining. Scores 0 and + were considered as negative; scores ++ and +++ were considered as positive for HER2 overexpression [31]. DNA isolation Genomic DNA used for genotyping studies was isolated from peripheral blood mononucleated cells, previously separated by centrifugation with Histopaque®-1077 (SIGMA-ALDRICH, LTD Irvine, UK), using the Ultra- Clean DNA Blood Isolation Kit (MO BIO, Carlsbad, CA, USA). DNA integrity was evaluated by the A260/ A280 absorbance ratio with a Nanodrop-1000 (Thermo Scientific) spectrophotometer. CYP19A1 Genotyping The single nucleotide polymorphisms (SNPs) rs10046 (A/G) and rs4646 (C/A) located at the 3’-untranslated region (3’-UTR) of CYP19A1, previously associated with letrozole efficacy in advanced BC [29], and the rs700519 (C/T) in the first base of codon 264 (Arg264Cys), related with low levels of aromatase activity [32], were evalu- ated. The rs700519 was analyzed by PCR amplification Page 3 of 11 and direct sequencing. Briefly, amplification was carried out in a final volume of 25 μl containing 100 ng geno- mic DNA, 0.3 μM of forward (5’-CATGAAGTG- TAGGGTC-TATGTAAT-3’) and reverse (5’- GATCTTTACACACCTCTACACAGT-3’) primers, 0.2 mM dNTPs (deoxyribonucleoside triphosphates), 2 mM MgCl2, 1× _Buffer II (Applied Biosystems, New Jersey, USA) and 1 unit AmpliTaq Gold (Applied Biosystems, New Jersey, USA). PCR conditions were 94°C for 10 minutes; 35 cycles with denaturation at 94°C for 30 sec- onds, annealing at 52°C for 30 seconds, and elongation at 72°C for 30 seconds; and a final extension step at 72° C for 10 minutes. PCR products were visualized on 1.5% agarose gels containing ethidium bromide and after UV radiation. Purified PCR products were then sequenced using BigDye Terminator v3.1 Cycle Sequencing Kit (Applied Biosystems, Warrington WA1 4SR, UK) and electrophoresed on a ABI 3130xl Genetic Analyzer (Applied Biosystems). Sequencing analysis was per- formed with the Sequencing Analysis 5.2 Software (Applied Biosystems). Genotyping of the rs10046 and rs4646 polymorphisms were carried out by allelic discrimination using specific TaqMan SNP Genotyping Assays and following the manufacturer’s instructions. In brief, 20 μl PCR reac- tions were carried out containing 10.0 μl TaqMan Uni- versal PCR Master Mix (Applied Biosystems, New Jersey, USA), 20.0 ng DNA template and 1.0 μL Taq- Man SNP Genotyping Assays (Applied Biosystems, Fos- ter City CA 94404). The references of selected assays were: C_8234731_1 (rs10046) and C_8234730_1 (rs4646). All assays were performed in 96-well plates including non-template controls. PCR reactions were read on a 7500 Fast Real Time PCR System in end- point mode using the Allelic Discrimination Sequence Detector software (Applied Biosystems). Statistical analysis Statistical analysis was carried out using the SPSS statis- tical software package (version 15.0.1, SPSS Inc., Chi- cago, Illinois, USA). A total of 95 patients were included in the analysis. Data on radiological response at the fourth month of induction therapy, progression and sur- vival status, immunohistochemical markers, histological grade, stage and genotype were available for these patients. For the statistical analysis we used binary variables reflecting the positivity status of the measures (yes or no) as well as the genotypic status of each polymorphism (homozygous reference or heterozygous/homozygous less frequent). Association with histopathological parameters, all categorical, was also assessed using a chi-square test to determine homogeneity or linear trend for ordinal vari- ables. The significance level was set at 5%.