SERBP1 protein expression in breast cancer patients was assessed using two independent breast cancer cohorts on TMAs. The first tumour cohort has been previously described [14, 15] and consisted of 193 breast cancer specimens and 48 normal breast tissue specimens. The TMA contained one tissue core from non-selected, formalin-fixed and paraffin-embedded primary breast cancer specimens diagnosed between 1994 and 2002 at the Institute of Pathology, University of Regensburg, Germany. Patients’ age ranged from 25 to 82 years with a median age of 56 years. An experienced surgical pathologist (A.H.) evaluated H&E-stained slides of all specimens prior to construction of the TMA in order to identify representative tumour areas. Histologically, all tumours were graded according to Elston and Ellis . Clinical follow-up data, provided by the Central Tumour Registry, Regensburg, Germany were available for all 193 breast cancer patients with a median follow-up period of 78 months (range 0-148 months).
The second TMA for validation consisted of 967 breast specimens of which 605 breast carcinomas were analysable. The second TMA was constructed from a cohort of breast cancer patients who were participants of a case control trial for the assessment of breast cancer susceptibility markers and prognostic factors, the Bavarian Breast Cancer Cases and Controls Study (BBCC), which has been described previously, including methods for the data collection [17, 18]. Briefly, database closure for this analysis was December 31, 2008 with a median follow up of 5.3 years. Of 967 BBCC patients a paraffin embedded tissue was available. A 0.6 mm punch was retrieved from the breast cancer tumour after a pathologist (A.D.) evaluated H&E-stained slides of all specimens. Data concerning the hormone receptor status were obtained from original pathological reports of these specimens; the other data were collected from the patients’ medical records and an epidemiological questionnaire which was completed in a personal interview.
All patients included in this study gave informed consent for further analysis of their tissue for research purposes and publication of the data. The Instructional Review Board of the participating centre approved the study (Ethics Committee of the Medical Faculty of the Friedrich-Alexander University of Erlangen, reference number: 2700; Ethics Committee of the Medical Faculty of the University of Aachen (reference number: 206/09) and Regensburg).
Benign human tissue mRNAs
For RT-PCR analysis commercially available mRNAs (Clontech, Germany) derived from different benign human organs (n = 25) were used. Tissue included in this selection were skeletal muscle, heart, spinal cord, brain, thymus, kidney, colon, bone marrow, placenta, adrenal gland, prostate, stomach, trachea, small intestine, uterus, thyroid, lymph node, pituitary gland, mammary gland, spleen, pancreas, cervix, salivary gland, testis and liver.
Cryoconserved human breast cancer samples
For Western blot analysis we used lysates of cryoconserved human breast cancer tissues with low (n = 8) and high (n = 8) levels of PAI-1 protein. The PAI-concentrations were measured by application of a commercially available ELISA-test (FEMTELLE®). As established in clinical routine, a PAI-1 level <14 ng/mg total protein was defined as low and >14 ng/mg total protein as high [10, 11]. Additionally we analysed cryoconserved matched pairs of normal (n = 7) and cancerous (n = 7) breast tissue samples for mRNA expression analysis.
The human mammary epithelial cell lines MCF12A and MCF10A as well as the breast cancer cell lines ZR75-1, BT20, HS578T, SKBR3, MDA-MB468 and MDA-MB231 were obtained from the ATCC (Rockville, MD, USA) and cultured as previously described .
RNA extraction and reverse transcription
Total RNA was isolated by use of TRIzol reagent (Invitrogen, Carlsbad, CA, USA) according to the manufacturers’ recommendations. Of the obtained RNA, 1 μg was reverse transcribed using the Reverse Transcription System (Promega, Madison, WI, USA). In order to improve transcription rate we mixed oligo-dT and pdN6 primers 1:1.
Semiquantitative realtime PCR
Semiquantitative PCR was performed using the LightCycler system together with the LightCycler DNA Master SYBR Green I Kit (Roche Diagnostics, Mannheim, Germany) . To ensure experiment accuracy, all reactions were performed in triplicate. Primer sequences were: SERBP1 sense 5′- CCA CCT CGT GAA CGA AGA TT -3′ and antisense 5′- ACC ACC ACG ACC TCG AAT AG -3′; GAPDH sense 5′- GAA GGT GAA GGT CGG AGT CA -3′ and antisense 5′- AAT GAA GGG CTC ATT GAT GG -3′. Annealing temperatures for both genes were set to 60°C. Reaction specificity was controlled by post-amplification melting curve analyses as well as by gel electrophoresis of the obtained products.
Non-radioisotopic in situ hybridisation
Non-radioisotopic RNA in situ hybridisation [20, 21] was used to analyse the mRNA expression of SERBP1. In short, cRNA sense and antisense probes were transcribed from linearised SERBP1 cDNA clone AA164643 using an in vitro transcription kit (Roche Diagnostics, Mannheim, Germany) according to the instructions of the manufacturer. The size of the in vitro transcribed RNA was confirmed by agarose gel electrophoresis. Paraffin embedded tissue sections were deparaffinised, rehydrated and fixed prior to hybridisation. Hybridisation of sections was performed in hybridisation buffer containing 2.5 ng/μl Digoxigenin-labeled RNA probes for 12 h at 65°C. After stringent washes to remove excess probe, the slides were incubated with the anti-DIG-AP antibody (Roche Diagnostics, Mannheim, Germany) for 12 h at 4°C in order to visualise the hybridised transcripts. The slides were then rinsed for 2–4 days in washing buffer. For detection of hybridisation signals, slides were incubated in 1 ml BM-Purple (Roche Diagnostics, Mannheim, Germany) containing 2 mM Levamisol and 0.1% Tween-20. Sections were counterstained with Texas Fast Red (Sigma, Munich, Germany).
Immunohistochemical (IHC) studies for the expression of HER2 utilised an avidin-biotin peroxidase method with a 3,3′-diaminobenzidine (DAB) chromatogen. After antigen retrieval (microwave oven for 30 min at 200 W) IHC was carried out in a NEXES immunostainer (Ventana, Tucson, AZ, USA) following the manufacturer’s instructions. The following primary antibodies were used: anti-HER2 (DAKO, Hamburg, Germany; 1:400), anti-ER and anti-PR (Novocastra, Newcastle Upon Tyne, UK; 1:20). For target proteins the ChemMate detection kit (DAKO) was used. A surgical pathologist (A.H.) performed a blinded evaluation of the TMA slides without knowledge of clinical data. Causes of non-interpretable results included lack of tumour tissue and presence of necrosis or crush artefacts. HER2 expression was scored according to the DAKO HercepTest. For the evaluation of ER and PR presence, a semiquantitative immunoreactivity score (IRS), as described by Remmele and Stegner , was used considering staining intensity and percentage of positive cell nuclei. The staining intensity was described by scores between 0 and 3 (0 = no reaction, 1 = low, 2 = moderate, 3 = strong). Accordingly, the number of positive cell nuclei was counted and scored between 0 and 4 (0 = no positive cell nuclei, 1 = < 10% positive cell nuclei, 2 = 10-50% positive cell nuclei, 3 = 51-80% positive cell nuclei, 4 = > 80% positive cell nuclei). The product of staining intensity and percentage of positive cell nuclei resulted in a score (IRS) between 0 and 12. Each sample was categorised by this rating score. Immunohistochemical evaluation of nuclear and cytoplasmic staining for SERBP1 was done equivalently .
Generation of a polyclonal SERBP1 antibody
As we started our investigations commercial polyclonal SERBP1 antibodies which were applicable for immunohistochemistry on paraffin embedded tissues were not available. Therefore we commissioned Eurogentec Inc. (Liège, Belgium) to produce a polyclonal SERBP1 antibody. Eurogentec gained the SERBP1 antibody by immunisation of rabbits with the corresponding peptide (peptide sequence: CKKEGIRRVGRR). Specificity was proven by a peptide competition experiment using Western blot analysis (Additional file 1: Figure S1).
The tissue microarrays – both, the evaluation set and the validation set - were subjected to immunostaining using the Advance Kit (DAKO K4068) following the manufacturer’s instructions. Paraffin-embedded breast carcinomas served as positive controls. After deparaffinisation and rehydration the tissue samples were boiled in a microwave oven for 30 min at 200 W in 10 mM sodium citrate buffer (pH 7.2). Endogenous peroxidase was blocked by peroxidase blocking solution (DAKO S2023) for 5 min. The polyclonal primary antibody SERBP1 (Eurogentec, EP060994, rabbit, Liège, Belgium) was applied (1:40 dilution) for 30 min at room temperature. In negative controls the primary antibody was omitted. For signal detection 3,3′-diaminobenzidine (DAB) was used. Slides were counterstained with hematoxylin and after dehydration mounted in Vitro-Clud (Langenbrinck, Emmendingen, Germany). Two experienced pathologists (N.B.S., A.H.) scored the immunohistochemical staining intensity according to the scoring system suggested by Remmele and Stegner .
SERBP1 Western blot analysis
Cell lines were lysed in NP40 buffer (1% Nonidet P-40, 150 mM NaCl, 25 mM Tris-HCl pH 7.6, 1 mM DTT, 1 mM EDTA, 1 mM Sodium Orthovanadat, proteinase inhibitor (Roche, Lewes, UK)), sonicated for 30 sec and finally centrifuged for 30 min at 13.000 rpm. 20 μl of the supernatant (approximately 15-20 μg protein) was then mixed with 4x NuPage buffer (Invitrogen) and run on a 4-12% Bis-Tris Gel (Invitrogen).
Lysis of frozen tissue samples was processed according to the recommendations of the Femtelle Kit (FEMTELLE®). Approximately 100-150 mg cryoconserved tissue was homogenised with mortar and pestle. Next lysis was performed through addition of 2 ml lysis buffer (1,8 ml TBS (pH 8,5) + 0,2 ml 10% Triton-X-100) and centrifugation for 1 h at 100.000 g. 25 μl of the supernatant (approximately 15-20 μg protein) was then mixed with 4x NuPage buffer (Invitrogen) and run on a 4-12% Bis-Tris Gel (Invitrogen).
After SDS-PAGE (sodium dodecylsulfate polyacrylamide gel electrophoresis) the proteins were transferred to nitrocellulose. Blocking was performed with 5% nonfat dry milk overnight at 4°C. The primary antibody (SERBP1, Eurogentec) was then incubated for 1 h at room temperature at a dilution of 1:2000 for the cell line-lysates and 1:1000 for the tissue-lysates. Western blots were processed using a horseradish peroxidase goat anti-rabbit IgG (1:2000; Dako). The Pierce ECL Western Blotting Substrate was used for visualisation (Amersham Biosciences, Amersham, UK).
The same membrane was then used for development with a β-actin antibody (from mouse; Sigma) in a dilution of 1:2000. As secondary antibody a horseradish peroxidase goat anti- mouse IgG (1:2000; Dako) was used. As positive controls, tissue-lysates of liver and placenta were used. As a negative control rabbit IgG was used in place of the SERBP1 antibody.
The intensity ratio of SERBP1 protein expression was calculated densitometrically using a STORM-860 phosphoimager (Molecular Dynamics, Sunnyvale, CA, USA) and normalised against the β-actin expression.
For statistical evaluation the SPSS version 17.0 (SPSS Inc, Chicago, IL) was used. Differences were considered statistically significant when p-values were < 0.05. A non-parametrically two-tailed Mann-Whitney U-test was employed to analyse differences in expression levels. A statistical association between clinicopathological and molecular parameters was tested, using two-sided Fisher’s exact test. Recurrence-free (RFS) and overall survival (OS) were calculated according to the Kaplan-Meier equation.
For both TMAs, OS was defined as tumour-related death and RFS as any local recurrence or distant recurrence whatever occurred first. Simple Cox proportional hazard models were used with the biomarkers under investigation (nuclear and cytoplasmic IRS score of SERBP1) to analyse the prognostic effect with the biomarker as an ordinal variable. Kaplan-Meier estimates were used to display the survival curves and log rank test was used to compare patients with high vs. low SERBP1 expression. For multivariate analyses a Cox proportional hazard model was constructed for OS and RFS including tumour size (pT), nodal status (pN), Grading (G), ER status, PgR status and HER2/neu status. After analysing both datasets independently a joint analysis of the data was done for the adjusted hazard ratio with the same covariates