Expression of Estrogen Receptors in Relation to Hormone Levels and the Nottingham Prognostic Index

Materials and Methods

Patients' characteristics. At the Capio St. Göran's hospital, pre-menopausal women were invited to participate in a study to determine differences between normal, benign and malignant breast tissue regarding expression of receptors in the estrogen-signaling pathway. In addition to collecting paired samples from the same breast (benign/malignant tumor tissue and the seemingly normal tissue adjacent to the tumor), blood samples were obtained from all women to determine hormone levels and the phase of the menstrual cycle (Table I). The inclusion of the women into the study was performed consecutively during a period of 48 months. This resulted in 13 women with primary invasive ductal breast cancer, 29 women with fibroadenoma and 7 women with other forms of benign breast diseases, bringing the total to 49 women. In the group with benign breast disease, there were 25 women in follicular phase and 11 in luteal phase of the menstrual cycle. In the group of women with malignant disease, 7 were in follicular phase and 6 in luteal phase. Data on patients' characteristics, including age, tumor size and the menstrual phase, are given in Table IIa and b.

Breast tissue sampling. All tissue samples were collected in the clinical setting at the time of surgery, when the tumors were removed for therapeutic purpose. For each woman, tissue samples from the tumor and from adjacent seemingly normal tissue, estimated clinically at the operation by the surgeon, were recovered and frozen at −70°C until analysis. The tissue samples were either extracted for RNA and subsequent polymerase chain reaction (PCR) analyses or embedded in paraffin and cut in 5 μm slices and mounted on glass slides for immunohistochemistry analyses. The tissue slides underwent a re-evaluation by an independent experienced pathologist to confirm the diagnosis of each individual specimen.

RNA preparation and reverse transcription. The frozen breast tissue samples were first transferred to RNAlater-ICE® (Ambion Inc, Austin, TX, USA). Total RNA from the samples was purified with the RNeasy® Lipid Tissue Mini kit (Qiagen GmbH, Hilden, Germany), according to a procedure recommended by the manufacturer for RNA isolation from fatty tissues. Two μg of total RNA from each sample was reverse transcribed at 37°C for 60 min in a final volume of 30 μl with a reaction mixture (Qiagen) containing 1 x RT buffer, dNTP mix (0.5 mM each dNTP), 600 ng random primers (Invitrogen, Paisley, UK), 2 units RNase inhibitor (Qiagen) and 4 units of Omniscript™ reverse transcriptase (Qiagen).

Real time PCR analysis. Real time PCR was performed in an iCycler™ iQ Real Time PCR System (Bio-Rad Laboratories, Inc., Hercules, CA, USA). For PCR, the cDNAs corresponding to 50–100 ng (see Table III) RNA were added to 12.5 μl of iQ™ SYBR® Green Supermix (Bio-Rad Laboratories) and 0.3 μM of each oligonucleotide primer in a final volume of 25 μl. After initial incubation for 3 min at 95°C, the samples were subjected to 40 cycles of 10 s at 95°C, followed by 45 seconds annealing at 55 to 65°C depending upon the genes (see Table III). All reactions were performed in duplicates. The purity of PCR products was confirmed by a melting curve analysis in all experiments (data not shown). The oligonucleotide primers for ERα, ERα36, ERβ, GPER and RPLP0 (housekeeping gene) are presented in Table III, as well as their predicted sizes. All primers were designed to span an intron/exon boundary or to flank an intron, to eliminate amplification of contaminating DNA. Each PCR assay included a negative control containing a RNA sample without reverse transcription. The primers were based on the sequences of the human genes.

Quantification of mRNA. To standardize the quantification method, RPLP0 was selected out of several tested housekeeping genes as an invariable internal control (p=0.482). The PCR amplification rate and the cycle threshold (Ct) values were related to a standard curve using iCycler iQ Optical System Software (Bio-Rad). The values of relative expression of genes of interest were normalized against the RPLP0 product.

Immunohistochemistry. The tissue sections were deparaffinized using xylene, rehydrated in graded ethanol and subjected to microwave treatment for antigen retrieval in 0.01 M sodium citrate buffer (pH 6.0) for 10 min and then allowed to cool for a further 20 min at room temperature (RT).

Immunostaining of ERα, ERβ and GPER was performed on 5-μm thick sections using a standard immunohistochemical technique (avidin-biotin-peroxidase) as previously described (11). Before applying the primary antibody, all slides were blocked with normal horse serum diluted in either phosphate-buffered saline or Tris-buffered saline. Thereafter, the primary and secondary antibodies were applied, using dilutions and incubation times as presented in Table IV. After incubation for 30 min with a horseradish peroxidase-avidin biotin complex (Vectastain Elite, Vector, CA, USA), the bound enzyme was visualized by the application of 3,3’-diaminobenzidine (DAKO Cytomation, Carpinteria, CA, USA). The sections were counterstained with hematoxylin and dehydrated before mounted with Pertex®. We have not found any commercial antibody that works for immunohistological analysis of ERα36.

Image analysis. To assess the ERα immunostaining quantitatively, a Leica microscope was connected to a computer using a Colorvision software (Leica Imaging system Ltd. Cambridge UK). Ten fields were randomly selected from the glandular tissue in a systematic way for quantification of the area of positively immunostained nuclei. The stromal tissue was actively excluded from the measurements. In a few samples, there was not possible to obtain measurements from ten separate sites due to lack of tissue. In those few cases, all glandular tissue was measured. Using color discrimination software, the total area of positively stained nuclei (brown reaction product) was measured and expressed as a ratio of the total area of cell nuclei (brown reaction product + blue hematoxylin).

Manual scoring. ERβ and GPER immunostainings were assessed by manual scoring due to their character of staining, i.e. not only nuclear. The scoring was performed by two independent observers on a four-point scale from negative (0), (+) faint, (++) moderate to (+++) strong immunostaining. The results by this method, from two independent observers, show a good consistency between investigators (11).

Statistical analysis. Statistical analysis was performed by ANOVA on ranks (Kruskal-Wallis test) and significances were evaluated by the Dunn's test. When two samples were compared, the Mann Whitney Rank Sum Test was used. Paired analyses were run by the Wilcoxon's Signed rank test. Correlations were evaluated by the Spearman's test. Values were considered significantly different when p<0.05.

Hormone analyses. The hormone levels were established using venous blood samples obtained at the time of surgery. Serum concentrations of sex hormone-binding globulin (SHBG) and E2 were determined by chemiluminiscence enzyme immunoassay using commercial kits obtained from Siemens (Immulite® SHBG and Estradiol; Diagnostic Products Corp., Los Angeles, CA, USA). The serum concentration of IGF-1 was determined by an enzyme-labeled chemiluminescent immunocentric assay using a commercial kit obtained from Siemens (Immulite® IGF-1; Diagnostic Products Corp.). Progesterone (P) was determined by sequential competitive immunoassay, using a commercial kit obtained from Siemens (Immulite® Progesterone Diagnostic Products Corp.). Total levels of testosterone were determined by a radioimmunoassay (RIA) using a commercial kit obtained from Orion (Spectria® Testosterone RIA; Orion Diagnostica OY, Esbo, Finland). All analyses were performed according the manuals from the manufacturers. The concentration of free testosterone (fT) was calculated from the value of total testosterone, SHBG and a fixed albumin concentration of 40 g/l by successive approximation using a computer program based upon an equation system derived from the law of mass action (12). Detection limits and coefficients of variation for within and between assays were: 0.1 U/l, 5.7% and 12.3% for luteinizing hormone (LH); 0.1 U/l, 3.0% and 5.9% for follicle-stimulating hormone (FSH); 0.2 nmol/l, 6.5% and 8.7% for SHBG; 55 pmol/l, 9.3% and 10.6% for E2; 20 ng/ml, 3.6% and 6.6% for IGF-1; 0.11 nmol/l, 5.3% and 5.4% for testosterone; 0.6 nmol/l, 9.6% and 9.9% for progesterone, respectively.

Ethics and consent. The study was approved by the Local Ethics Committee at Karolinska Institutet, Stockholm, Sweden (98-173). All women gave their written consent to participate in the study.