Study of TLR3, TLR4 and TLR9 in breast carcinomas and their association with metastasis

This study included 74 women with histologically confirmed early breast cancer confirmed and treated between 1990 and 2003. We selected patients with the following inclusion criteria: invasive ductal carcinoma, in cases of non-recurrence, patients had been followed-up for a minimum of 5 years of follow-up. The exclusion criteria were the following: metastatic disease at presentation, prior history of any kind of malignant tumor, bilateral breast cancer at presentation, any type of neoadjuvant therapy, development of loco-regional recurrence during the follow-up period, development of a second primary cancer, and absence of sufficient tissue in paraffin blocks. From a total of 1,264 patients fulfilling these criteria, we randomly selected a sample size of 74 patients in accordance divided them into two different groups of similar size and stratified each group with regard to the development of metastasis. Patients and tumor characteristics are listed in Table 1. Patients were treated according to approved guidelines at our institution. The study adhered to national regulations and was approved by our institutions Ethics and Investigation Committee. Tissue samples were obtained prior informed consent from the patients. All patients were followed for distant metastasis status by clinical and biological studies every 3 months for the first 2 years and then yearly. Radiological studies were performed yearly, or when considered necessary. The end-point was distant metastatic relapse. The median follow-up period was 85 months in patients without metastases, and 46 months in patients with metastases.

Breast carcinoma tissue samples were obtained at the time of surgery. All specimens were routinely fixed in 10% neutral buffered formalin and stored in paraffin at room temperature for a period of four months to five years before further testing. Histopathological representative tumor areas were defined on haematoxylin and eosin-stained sections and marked on the slide. Tumour tissue array (TA) blocks were obtained by punching a tissue cylinder (core) with a diameter of 1.5 mm through a histological representative area of each 'donor' tumor block, which was then inserted into an empty 'recipient' TA paraffin block using a manual tissue arrayer (Beecher Instruments, Sun Prairie, Winconsin, USA) as described elsewhere [15]. Collection of tissue cores was carried out under highly controlled conditions. Two cores were employed for each case.

Four composite high-density TA blocks were designed, and serial 5-μm sections were consecutively cut with a microtome (Leica Microsystems GmbH, Wetzlar, Germany) and transferred to adhesive-coated slides. One section from each TA block was stained with haematoxylin and eosin, and these slides were then reviewed to confirm that the sample was representative of the original tumor. Immunohistochemistry was done on these sections of TA fixed in 10% buffered formalin and embedded in paraffin using a TechMate TM50 autostainer (Dako, Glostrup, Denmark). Monoclonal antibodies for TLR3 (TLR3.7; ref: sc-32232), TLR4 (H-80; sc-10741), and TLR9 (H-100; sc-25468) were obtained from Santa Cruz Biotechnology (California, USA). The dilution for each antibody was established based on negative and positive controls (1/50 for TLR3, 1/100 for TLR4 and TLR9).

Tissue sections were deparaffinized in xylene, and then rehydrated in graded concentrations of ethyl alcohol (100%, 96%, 80%, 70% and water). To enhance antigen retrieval for the three antibodies, TA sections were microwave-treated (H2800 Microwave Processor, EBSciences, East Granby, Connecticut, USA) in citrate buffer, (Target Retrieval Solution, Dako), with high pH (pH9) for TLR3 and low pH (pH6) for TLR4 and 9, at 99°C for 16 min. Endogenous peroxidase activity was blocked by incubating the slides in peroxidase-blocking solution (Dako) for 5 min. The EnVision Detection Kit (Dako) was used as the staining detection system. Sections were counterstained with haematoxylin, dehydrated with ethanol, and permanently coverslipped.

The location of immunoreactivity, percentage of stained cells, and intensity were determined for each antibody preparation. All the cases were semiquantified for each protein-stained area. An image analysis system using the Olympus BX51 microscope and analysis soft (analySIS^®, Soft imaging system, Münster, Germany) was employed as follows: tumor sections were stained with antibodies according to the method explained above and counterstained with haematoxylin. There were different optical thresholds for both stains. Each core was scanned with a 400× power objective in two fields per core. Fields were selected on the basis of protein-stained areas. The computer program selects and traces a line around antibody-stained areas (red spots) for higher optical threshold. The remaining non-stained areas (haematoxylin-stained tissue with lower optical threshold) appear as a blue background. Each field has an area ratio of stained (red) versus non-stained areas (blue). A final area ratio was obtained after averaging two fields. To evaluate immunostaining intensity we used a numeric score ranging from 0 to 3, reflecting the intensity as follows: 0, no staining; 1, weak staining; 2, moderate staining; and 3, intense staining. Using an Excel spreadsheet, the mean score was obtained by multiplying the intensity score (I) by the percentage of stained cells [16] and the results were added together (total score: I × PC). This overall score was then averaged with the number of cores that were done for each patient. If there was no tumor in a particular core, then no score was given. In addition, for each tumor, the mean score of two core biopsies was calculated.

Samples were separated by SDS-PAGE using 10% polyacrylamide gels and run at constant 120 V (Mini-Protean^® Tetra Electrophoresis System, Bio-Rad, Hercules, USA). The tetraprotean transference kit was used to electrotransfer proteins to nitrocellulose membranes at 160 mA for 1 h in transfer buffer (0.248 M Tris pH 8.8, 1.92 M glycine and 20% methanol). The nitrocellulose filters containing the transferred proteins were blocked by rocking for 1 h in Tris-buffered saline (TBS) contained 1% skimmed milk and rinsed 3 tines in TBS. The filters were incubated for 2 h at room temperature with one of these monoclonal antibodies: anti-TLR3 (ref: sc-32232), anti-TLR4 (sc-10741), anti-TLR9 (sc-25468) (Santa Cruz Biotechnology, California, USA) diluted in TBS containing 1% skimmed milk. The blots were then washed with TBS, incubated with protein A peroxidase and the reactive protein bands were visualized by chemiluminiscence (Pierce ECL Western Blotting Substrate, Rockford, USA).

Total RNA was isolated from breast tissue using the RNeasy Mini kit (Quiagen, Hilden, Germany), including DNase treatment. The integrity of the eluted total RNA was checked by agarose gel electrophoresis and the RNA concentration was determined spectrophotometrically. First strand cDNA was made using the High Capacity cDNA Reverse Transcrition kit (Applied Byosystems, Cheshire, UK) following the manufacturer's instructions. The reverse transcription step was carried using the following program: 25°C for 10 min, 37°C for 120 min and 85°C for 5 sec. The expression levels of TLR3, TLR4, TLR9 and β-actin were assessed by real-time PCR using ABI Prism 7900 HT thermocycler (Applied Biosystems, Cheshire, UK) and the Fast SYBR Green Master Mix (Applied Biosystems, Cheshire, UK) with the following cycling conditions: 95°C for 20 sec, 40 cycles of 95°C for 1 sec and 60°C for 20 sec. The primers used were 5'-TAGCAGTCATCCAACAGAATCAT-3' (forward) and 5'-AATCTTCTGAGTTGATTATGGGTAA-3' (reverse) for TLR3, 5'-ACTCCCTCCAGGTTCTTGATTAC-3' (forward) and 5'-CGGGAATAAAGTCTCTGTAGTGA-3' (reverse) for TLR4, 5'-CTTCCCTGTAGCTGCTGTCC-3' (forward) and 5'-CCTGCACCAGGAGAGACAG-3' (reverse) for TLR9 and 5'-GGCACCCAGCACAATGAAG-3' (forward) and 5'-CCGATCCACACGGAGTACTTG-3' (reverse) for β-actin. PCR products were separated on 2% agarose gels containing ethidium bromide (0.5 μg/ml).

Differences in percentages were calculated with the chi-square test. Immunostaining score values for each protein were expressed as median (range). A comparison of group immunostaining values was made with the Mann-Whitney or Kruskal-Wallis tests. For metastasis-free survival analysis we used the Cox's univariate method. Cox's regression model was used to examine interactions of different prognostic factors in a multivariate analysis. The SPSS 17.0 program was used for all calculations.