Eleven histologically confirmed primary breast tumors were obtained from the Tissue Bank of the Fondazione IRCCS Istituto Nazionale Tumori between October 2006 and March 2007. For each sample a written informed consent signed by the patient was available authorizing the use of leftover material for research purposes. The study was approved by Independent Ethics Committee and Institutional Review Board. Samples used in the study were selected based on the size, carefully avoiding necrotic areas, fat and normal tissue. An adjacent section was stained and used for defining the percentage of tumor cells. Only specimens with more than 70% of tumor cells were included in the study. Each tissue sample was divided into 4 aliquots. One was immediately frozen while the remaining three were frozen after 2, 6 and 24 hours at room temperature.
RNA isolation and expression profiling
Tissue was pulverized using a Mikrodismembrator (Braun Biotech International, Germany). Total RNA was extracted with the Trizol reagent (Invitrogen, Carlsbad, CA) according to manufacture instructions and an additional DNase digestion was performed using the RNeasy kit (Qiagen, Valencia, CA). After each extraction a small fraction of RNA was used for quality and yield assessment. RNA total concentration and purity were determined by UV spectrometry. Total RNA electrophoretic profile was analysed by the Agilent RNA 6000 NanoLabChip kit on the Agilent 2100 Bioanalyzer (Agilent Technologies, Palo Alto, CA) using the software provided by the manufacturer for determination of RIN (RNA integrity number).
Probe labelling and hybridization were performed as previously described . The samples and a reference RNA (Universal Human Reference RNA, Stratagene, La Jolla, CA) were labelled directly with Cy3-dCTP (reference RNA) or Cy5-dCTP (sample RNA) (Amersham Biosciences, Little Chalfont, UK) and indirectly with 3DNA Submicro Expression Array Detection kit (Genisphere, Montvala, NJ). Hybridization was carried out in a hybridization station (Genomic Solutions, Ann Arbour, MI), slides were scanned using the GenePix 4000B microarray scanner and quantified using GenePix Pro 18.104.22.168 (Axon Instruments/Molecular Devices). The RNAs were hybridized on two different cDNA microarrays containing a total of 17172 unique clones selected from the Human sequence-verified I.M.A.G.E. clone collection (Research Genetics/Invitrogen, Carlsbad, CA) and spotted in triplicate. Raw gene expression data have been deposited at the European Bioinformatics Institute (EBI) ArrayExpress and are accessible through accession no. E-MEXP-2035.
Quantitative real-time PCR
Total RNA isolated for the microarray analysis was used to verify the quantity of specific messengers by real-time PCR for 4 biologically relevant differentially expressed genes (i.e., FGFR4, ESR1, ERBB2, FBLN2). RNA was reverse transcribed using the High-Capacity cDNA Archive Kit (Applied Biosystems, Foster City, CA). Samples were amplified in multiplex PCR reactions using one of the assays of interest labelled with FAM. PPIA was used as housekeeping gene. Reactions were performed in a final reaction volume of 20 μl containing cDNA template, 10 μl 2× TaqMan Gene Expression Assay (Applied Biosystems, Foster City, CA) and thermal cycling was performed on an ABI PRISM 7700 Sequencer Detector (Applied Biosystems, Foster City, CA).
Protein Profile Analysis
Following tissue pulverization, tumor samples were processed as described in . Briefly, samples were lysed in an ice-cold buffer containing 50 mM HEPES (pH 7.6), 150 mM NaCl, 10% glycerol, 1% Triton X-100, 1.5 mM MgCl2, 1 mM EGTA, 10 mM Na4P2O7, 100 mM NaF, protease inhibitors and in presence or absence of 1 mM sodium orthovanadate. After 30 min incubation with gentle rocking at 4°C, lysates were cleared by centrifugation (10 min at 13,000 rpm). Supernatants were collected and, after protein concentration determination, tested for the presence of specific proteins by Western blotting. For Western blotting, cell lysates were resolved by 4-12% SDS-PAGE (precast gel NuPAGE, Invitrogen). Proteins were transferred to nitrocellulose membranes (Hybond-C Super, Amersham Bioscience, Little Chalfont, UK), checked for equal sample loading by Red Ponceau S staining, and probed with the appropriate antibodies. Immunoreactive bands were visualized using horseradish peroxidase-linked secondary antiserum (Amersham Biosciences, Little Chalfont, UK) and detected using an enhanced ECL system (Amersham Biosciences, Little Chalfont, UK). For Western blotting, the following antibodies were used: anti-p-Tyr 4G10 (Upstate Biotechnology, Millipore Corporation, Billerica, MA); p-Neu (Tyr 1248)-R (Santa Cruz, Biothecnology, Santa Cruz, CA) for ERBB2 and anti-FAK (Transduction Laboratories, BD, Franklin Lakes, NJ).
Data analysis and Statistics
Raw microarray images and quantifications were stored and processed in Bio Array Software Environment (BASE, Lund Sweden) . Poor signal quality of background-corrected Cy3 and Cy5 intensities were flagged and a lowess normalization  was applied to each slide. Replicated spots were averaged and their log (base2) expression ratios (tumor/reference) were downloaded from BASE and imported into BRB-ArrayTools version 3.4.0. (Bethesda, MD) developed by Richard Simon and Amy Peng Lam . ANOVA analysis was performed following their instructions. All I.M.A.G.E clones annotations were updated with the latest release of NCBI Unigene (Build No. 194) using SOURCE. In order to find the overlap between our list of genes and genes included in public available signatures, we employed the Merge function of the web-application MatchMiner . Hierarchical clustering was performed using centered correlation and average linkage method.
For gene function analyses, a score obtained calculating the -Log(p-value) of the Fisher Exact Test indicates the probability that a function is obtained by chance; scores equal or greater than 3 give a 99.9% confidence level of not being generated by chance.
The genes identified by microarray analyses were challenged for gene function. After having imported their accession numbers into the Ingenuity Pathway Analysis (IPA) software (Ingenuity Systems, Mountain View, CA), they were categorized according to the gene functions present in the database.
Data analysis for real-time PCR was done using the Sequence Detector version 1.9 software. Relative log-expression of the genes (-ΔCt) was obtained subtracting the number of cycle threshold observed for the 18S gene from that observed for the gene of interest. After being exported further data analysis was performed in a BRB_ArrayToolv3.4.0.
The association between gene expression evaluated by cDNA microarry and real-time PCR was evaluated by linear regression analysis.