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Nuclear detection of Y- box protein-1 (YB-1) closely associates with progesterone receptor negativity… Dahl, Edgar; En-Nia, Abdelaziz; Wiesmann, Frank; Krings, Renate; Djudjaj, Sonja; Breuer, Elisabeth; Fuchs, Thomas; Wild, Peter J; Hartmann, Arndt; Dunn, Sandra E; Mertens, Peter R Nov 24, 2009

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ralssBioMed CentBMC CancerOpen AcceResearch articleNuclear detection of Y-box protein-1 (YB-1) closely associates with progesterone receptor negativity and is a strong adverse survival factor in human breast cancerEdgar Dahl*1, Abdelaziz En-Nia2, Frank Wiesmann1, Renate Krings3, Sonja Djudjaj2, Elisabeth Breuer1, Thomas Fuchs4, Peter J Wild5, Arndt Hartmann6, Sandra E Dunn7 and Peter R Mertens*2Address: 1Molecular Oncology Group, Institute of Pathology, Medical Faculty, RWTH Aachen University, Aachen, Germany, 2Department of Nephrology and Hypertension, Otto-von-Guericke-University Magdeburg, Magdeburg, Germany, 3Department of Nephrology and Immunology, Medical Faculty, RWTH Aachen University, Aachen, Germany, 4Department of Computer Science, ETH Zurich, Zurich, Switzerland, 5Institute of Surgical Pathology, University Hospital Zurich, Zurich, Switzerland, 6Department of Pathology, University of Erlangen, Germany and 7Laboratory for Oncogenomic Research, Department of Pediatrics, Child and Family Research Institute, University of British Columbia, Vancouver, British Columbia, CanadaEmail: Edgar Dahl* - edahl@ukaachen.de; Abdelaziz En-Nia - aen-nia@ukaachen.de; Frank Wiesmann - fwiesmann@ukaachen.de; Renate Krings - rkrings@ukaachen.de; Sonja Djudjaj - sonja.djudjaj@med.ovgu.dE; Elisabeth Breuer - ebreuer@ukaachen.de; Thomas Fuchs - thomas.fuchs@inf.ethz.ch; Peter J Wild - peter.wild@cell.biol.ethz.ch; Arndt Hartmann - arndt.hartmann@uk-erlangen.de; Sandra E Dunn - sedunn@interchange.ubc.ca; Peter R Mertens* - peter.mertens@med.ovgu.de* Corresponding authors    AbstractBackground: Y-box binding protein-1 (YB-1) is the prototypic member of the cold shock proteinfamily that fulfills numerous cellular functions. In the nucleus YB-1 protein orchestratestranscription of proliferation-related genes, whereas in the cytoplasm it associates with mRNA anddirects translation. In human tumor entities, such as breast, lung and prostate cancer, cellular YB-1 expression indicates poor clinical outcome, suggesting that YB-1 is an attractive marker to predictpatients' prognosis and, potentially, is suitable to individualize treatment protocols. Given thesepredictive qualities of YB-1 detection we sought to establish a highly specific monoclonal antibody(Mab) for diagnostic testing and its characterization towards outcome prediction (relapse-free andoverall survival).Methods: Hybridoma cell generation was carried out with recombinant YB-1 protein asimmunogen and Mab characterization was performed using immunoblotting and ELISA withrecombinant and tagged YB-1 proteins, as well as immunohistochemistry of healthy and breastcancer specimens. Breast tumor tissue array staining results were analyzed for correlations withreceptor expression and outcome parameters.Results: YB-1-specific Mab F-E2G5 associates with conformational binding epitopes mapping totwo domains within the N-terminal half of the protein and detects nuclear YB-1 protein byimmunohistochemistry in paraffin-embedded breast cancer tissues. Prognostic evaluation of Mab F-E2G5 was performed by immunohistochemistry of a human breast cancer tissue microarrayPublished: 24 November 2009BMC Cancer 2009, 9:410 doi:10.1186/1471-2407-9-410Received: 28 February 2009Accepted: 24 November 2009This article is available from: http://www.biomedcentral.com/1471-2407/9/410© 2009 Dahl et al; licensee BioMed Central Ltd. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.Page 1 of 17(page number not for citation purposes)comprising 179 invasive breast cancers, 8 ductal carcinoma in situ and 37 normal breast tissuesamples. Nuclear YB-1 detection in human breast cancer cells was associated with poor overallBMC Cancer 2009, 9:410 http://www.biomedcentral.com/1471-2407/9/410survival (p = 0.0046). We observed a close correlation between nuclear YB-1 detection andabsence of progesterone receptor expression (p = 0.002), indicating that nuclear YB-1 detectionmarks a specific subgroup of breast cancer. Likely due to limitation of sample size Cox regressionmodels failed to demonstrate significance for nuclear YB-1 detection as independent prognosticmarker.Conclusion: Monoclonal YB-1 antibody F-E2G5 should be of great value for prospective studiesto validate YB-1 as a novel biomarker suitable to optimize breast cancer treatment.BackgroundSince the seminal description of Janz et al. [1] that highYB-1 expression levels in breast cancer and surroundingtissues are indicative for poor outcome, follow-up studieshave extended these findings to larger cohorts and othercancer entities, including non-small cell lung cancer [2],ovarian cancer [3], prostate cancer [4], and synovial sar-coma [5]. A recent study confirmed the unfavorable out-come of patients with YB-1 expression in breast cancertissue with a large cohort of 4049 cases over an observa-tion period of 20 years, which reached statistical signifi-cance in nearly all subgroups [6]. Furthermore, Bargou etal. [7] reported that nuclear localization of YB-1 was asso-ciated with P-glycoprotein expression in human primarybreast cancers, other studies have shown a co-expressionof YB-1 and P-glycoprotein in osteo- and synovial sar-coma as well as breast, ovarian and prostate cancer [3,5,8-11].Cold-shock proteins like the Y-box binding (YB) proteinfulfill pleiotropic functions, e.g. regulation of target genes,(pre-)mRNA splicing and translation [12-16]. YB-1 is amultifunctional protein with fascinating roles in cell biol-ogy. First and utmost, YB-1 serves multiple roles in genetranscription with numerous target genes involved inDNA replication and proliferation [17-22]. Overexpres-sion of YB-1 via a transgene in a mouse model induced thedevelopment of breast cancers of many histological types[23], suggesting that YB-1 is oncogenic. Reports confirmthe relevance of YB-1 expression for EGF-independentbreast cancer cell growth [24]. Following its original clon-ing as binding activity of the EGF receptor promoter [25],Dunn et al. recently reported that YB-1 is a strong trans-stimulator of EGF receptor expression in breast cancercells [26].A predominant nuclear localization of YB-1 protein goesalong with intrinsic expression of putative oncogenes[1,4,24,27], response to oxidative stress and coordinationof DNA excision repair [28]. On the other hand, there isevidence for a prominent role of YB-1 as RNA-bindingmolecule and constituent of cytoplasmic major messengerribonucleoprotein particles (in this context denoted p50)protein modifications have shed some light on underlyingmechanisms. The transcription of YB-1 is upregulated bybasic helix-loop-helix transcription factor Twist [31].Twist itself is an immediate target of signal tranducers andactivator of transcription (STAT)-3, which may be activatedby epidermal growth factor receptor signaling [32]. Knock-down of YB-1 completely abrogated the proliferative effectof Twist, emphasizing the fundamental role that YB-1plays for the EGF receptor axis. Furthermore, serine 102 ofYB-1 protein is a direct target of protein kinases B (AKT)and RSK [33], both of which are signaling cascadesinvolved in cell transformation, proliferation and anchor-age-independent growth [17,27,34].A general evaluation of YB-1 expression in breast cancerpatients is limited due to lack of a suitable antibody ofunrestricted quantity and defined quality, preferablymonoclonal. This could potentially allow to diversifytheir relative risk profile and chemotherapy sensitivity[7,35]. Furthermore, most polyclonal antibodies, like theones used for breast cancer tissue by Janz et al. [1] andHabibi et al. [6], predominantly detect YB-1 in the cyto-plasm, whereas YB-1 protein activities relating to chromo-somal instability and gene regulation must take placewithin the nuclear compartment [36].In the following we present data on a newly establishedmonoclonal YB-1 antibody that is characterized as suita-ble for immunohistochemistry in paraffin-embedded tis-sue. The expression of YB-1 by means of this antibody wasanalyzed by immunohistochemistry in a cohort of breastcancer specimens. For all tumour specimens analyzed, fullhistopathological and clinical follow-up data were availa-ble, allowing uni- and multivariate analyses of nuclear YB-1 expression in correlation to well-established factors ofbreast cancer prognosis (grade, nodal status, HER2, estro-gen receptor (ER) status, progesterone receptor (PR) sta-tus). Thereby we characterized the Mab as a novel toolthat can be used in breast cancer prognosis and therapystratification.MethodsMab generationPage 2 of 17(page number not for citation purposes)[29,30]. Studies addressing the question what regulatesYB-1 expression, its subcellular localization and potentialMouse work was in full compliance with the guidelinesfor animal care and was approved by the animal care com-BMC Cancer 2009, 9:410 http://www.biomedcentral.com/1471-2407/9/410mittee from the government. Female BALB/c mice wereimmunized by intraperitoneal and intravenous injectionof 100 μg recombinant YB-1 full-length protein emulsi-fied in equal volume of Freund's adjuvant (Gibco-BRL lifetechnologies, Karlsruhe, Germany) followed by a boosterinjection six weeks later containing 10 μg of antigen inFreund's adjuvant. The response was assessed by dot blotassay with recombinant YB-1 protein. Three days after thelast booster injection, spleen cells were obtained andfused with X63-Ag/653 (BALB/c) mouse myeloma cellsand propagated according to standard procedures. Thefused cells were resuspended in DMEM medium (GibcoBRL life technologies) supplemented with 10% bovinecalf serum, 100 U/ml penicillin, 100 μg/ml streptomycin,50 μM hypoxanthine, 160 μM thymidine, and 400 nMaminopterin. Aliquots of the cell suspension (100 μl)were dispensed into 96-well plates and incubated at37°C/5% CO2. Every week the medium was replaced withfresh selection medium supplemented with recombinantinterleukin-6 (100 U/ml). After two weeks the mediumwas replaced by HT-medium (DMEM medium (GibcoBRL life technologies) supplemented with 10% bovinecalf serum, 100 U/ml penicillin, 100 μg/ml streptomycin,100 μM hypoxanthine and 160 μM thymidine andanother week later cells were cultured in standard DMEMmedium (4500 mg/l D-Glucose supplemented withGlutaMax (Gibco BRL life technologies) with 10% bovinecalf serum, 100 U/ml penicillin and 100 μg/ml streptomy-cin. Hybridoma supernatants from 96-well plates werescreened by dot blot assay with recombinant YB-1 proteinfor specific antibody synthesis. The positive-tested hybrid-oma cells were single-cell cloned twice by limited dilutionin standard medium as described [37]. Immunoglobulinsin the hybridoma cell culture supernatants were purifiedusing protein A sepharose or protein G sepharose col-umns according to the immunoglobulin isotype(ÄKTAFPLC systems, Amersham Biosciences, Freiburg, Ger-many). F-E2G5 was of IgG2b isotype. Purified monoclonalantibodies were conjugated to biotin as described [38].Plasmids, cell line and transfectionPlasmids encoding for GFP and YB-1-GFP fusion proteins(pcDNA6/V5-His-YB-1-GFP) have been described previ-ously [20]. HEK293T cells were cultured in DME-mediumcontaining 10% fetal bovine serum, 100 U/ml penicillin,100 μg/ml streptomycin and 2 mM L-glutamine. Tran-sient transfections of HEK293T cells with vectors were per-formed by means of calcium phosphate precipitationmethodology using purified endotoxin-free plasmid DNApreparations. HEK293T cells expressing the respectiveproteins were harvested 48 h after transfection and cyto-plasmic and nuclear fractions were prepared by cell lysisin buffer A (10 mM HEPES [pH 7.9], 1.5 mM MgCl2, 102.000 rpm for 1 minute. The supernatant corresponds tosoluble cytoplasmic proteins and contains GFP and YB-1-GFP proteins.Western BlottingSDS-PAG electrophoresis was performed with proteinextracts containing GFP or YB-1-GFP protein under reduc-ing and non-reducing conditions. Proteins were separatedon 12% SDS-PAG and transferred to nitrocellulose mem-branes (Schleicher-Schuell, Dassel, Germany). Mem-branes were blocked in TTBS (10 mM Tris-HCl [pH 8.0],150 mM NaCl, 0.2% Tween-20) containing 5% non-fatdry milk for 1 h at room temperature. After three washingsteps in TTBS, blots were incubated with biotin-labeledYB-1 Mab F-D2G5 diluted 1:2,000 and StreptABComplexdiluted 1:50 (DAKO Cytomation, Glostrup, Denmark)was added. Following two more washes in TTBS the per-oxidase reaction was visualized by ECL system (Amer-sham, Freiburg, Germany). Immunoprecipitationexperiments were performed with the indicated antibod-ies that were incubated with pre-cleared pansorbin.ELISARecombinant YB-1 protein was expressed in E. coli andaffinity purified as previously described [39]. For ELISA96-well polystyrene plates were incubated with 200 μl ofrecombinant YB-1 protein solution (3 ng/μl) in coatingbuffer (15 mM Na2CO3, 35 mM NaHCO3) overnight at4°C. After four washing steps with phosphate-bufferedsaline with 0.05% Tween-20 (50 mM sodium phosphate,3 mM potassium phosphate, 150 mM sodium chloride[pH 7.4]), blocking with 2.5% low fat milk powder in PBSwas performed at 37°C for 1 h. After four additionalwashing steps Mabs dissolved in washing buffer at 1:50were added to each well (200 μl) and incubated anotherhour at 37°C and washed four times. Bound Mabs weredetected either by incubation with 100 μl HRP-conju-gated polyclonal anti-mouse IgG diluted 1:1,000 in PBS/Tween (non-biotin-labelled Mabs) or by incubation with100 μl StrepABComplex diluted 1:50 (DAKO Cytoma-tion, Glostrup, Denmark) with biotin-labelled Mabs for 1h at 37°C and developed with ABTS (2,2'-azino-di-3-ethylbenzthiazoline-6-sulfonic acid)/H2O2. One row wasused as blank containing immobilised YB-1 protein butwith no addition of Mabs.ImmunohistochemistryImmunohistochemistry was performed as describedrecently [40] using an established breast tissue microarray[41]. The breast tissue samples of this tissue microarraywere obtained from patients treated by primary surgeryfor breast cancer at the Department of Gynecology, Uni-versity Hospital Regensburg, Germany, with institutionalPage 3 of 17(page number not for citation purposes)mM KCl, 0.2 mM PMSF, 1 mM sodium vanadate, 0.5 mMDTT) for 10 min on ice, followed by centrifugation atreview board approval. All patients gave informed consentto the study for retention and analysis of their tissue forBMC Cancer 2009, 9:410 http://www.biomedcentral.com/1471-2407/9/410research purposes. Briefly, tissue sections were deparaffin-ized and rehydrated, and the endogenous peroxidaseactivity was quenched by treatment with 3% H2O2. Anti-gen retrieval was performed by pretreatment in citratebuffer [pH 6.0] in a microwave oven (three times for 8minutes at 600 W). Slides were incubated with a 1:25dilution of monoclonal biotin-labeled YB-1 antibody in2% milk powder dissolved in PBS in a humidified cham-ber overnight at 4°C. After another wash Vectastain avi-din-biotin complex (Vector Laboratories, California,USA) was added for 10 minutes. Immunostaining was vis-ualized using 3,3'-diaminobenzidine tetrahydrochloride(DAB, Sigma, Deisenhofen, Germany) and hydrogen per-oxide for 5 minutes. Finally, sections were counterstainedwith Mayer's hematoxylin. The primary antibody wasomitted for negative controls. Tissue microarray slideswere analyzed by a pathologist without knowledge ofclinicopathological parameters of the tumors. Nuclear YB-1 staining was simply scored as positive (1) or negative(0), depending on visual inspection of tumor nucleiunder high magnification (400×). The decision to scorethe staining pattern according to a dichotomous crite-rium, nuclear versus non-nuclear staining seemed justi-fied as the pattern was evident throughout all specimens.There was no tissue with restricted focal nuclear staining.Cytoplasmic YB-1 staining was scored according to theimmunoreactivity score (IRS) system developed by Rem-mele and Stegner [42], subsequently tumors were groupedinto low expressers (IRS 0-3) and high expressers (IRS 4-12). For comparison of the F-E2G5 tissue staining patternwith peptide-derived affinity-purified YB-1 antibodies,the following antibodies were utilized: YB-1#1 has beengenerated by immunization and purification with a pep-tide corresponding to an N-terminal YB-1 epitope and waspurchased from antibodies-online; YB-1#2 has beendescribed by Janz et al. [1])Statistical analyses of tissue microarray dataStatistical analyses were performed with SPSS version 17.0(SPSS, Chicago, IL). Differences were considered statisti-cally significant when p < 0.05. Contingency table analy-ses and two-sided Fisher's exact tests were used to studythe statistical association between clinicopathologic andimmunohistochemical variables. Recurrence-free and dis-ease-specific survival curves comparing patients with orwithout any of the factors were calculated using the Kap-lan-Meier method, with significance evaluated by two-sided log-rank statistics. Disease-specific survival andrecurrence-free survival were measured from time of sur-gery. For the analysis of recurrence, patients were censoredat the time of their last tumor-free clinical follow-upappointment. For disease-specific survival analysis,patients were censored at the time of their last tumor-freeregression model was adjusted, testing the independentprognostic relevance of nuclear YB-1 immunoreactivity.The limit for reverse selection procedures was p = 0.01.The proportionality assumption for all variables wasassessed with log-negative-log survival distribution func-tions.Multiple logistic regression modelRegression analyses were conducted with R 2.8 (r-project.org). The binary response variable y is regressed onthe explanatory variables, x1, x2, ..., xq. The model for nobservations is defined by ln(p/(1-p)) = Xβ + ε, where εcontains the residual error terms and p = Pr(y = 1). Theparameters are estimated by maximum likelihood estima-tion. The regression coefficients β1, β2, ..., βq give thechange in the response variable corresponding to a unitchange in the appropriate explanatory variable, condi-tional on the other variables remaining constant. Theintercept corresponds to the expected value of theresponse variable y when all the explanatory variables arezero. Significance tests of whether the coefficients take thevalue zero can be derived on the assumption that for agiven set of values of the explanatory variables, y has anormal distribution with constant variance. The data wascentered and scaled previous to applying logistic regres-sion. Centering is performed by subtracting the columnmeans (omitting missing values) of x from their corre-sponding columns, and scaling is done by dividing the(centered) columns of x by their root-mean-square.Explanatory variables were considered statistically signifi-cant when P < 0.05.ResultsCharacterization of monoclonal YB-1-specific antibody F-E2G5In a first approach the F-E2G5 antibody was examined forits substrate specificity by western blot analysis using cellextracts from HEK293 cells expressing GFP, YB-1-GFP orYB-1(21-147)-GFP proteins. GFP and YB-1-GFP fusionprotein expression was ascertained by detection withmonoclonal GFP antibody (Figure 1A, lanes 1-3). Immu-noblotting with biotinylated F-E2G5 Mab was performedfollowing addition of non-reducing (lanes 4-6) or reduc-ing buffer (lanes 7-8) to protein samples. Mab F-E2G5detects full-length YB-1-GFP fusion protein and also thetruncated protein encompassing amino acids 21-147.However, detection succeeds only with non-reducingbuffer and not under reducing buffer conditions withinclusion of mercaptoethanol (Figure 1A, compare lanes5 and 7). Endogenous YB-1 protein with a relative molec-ular weight of 50 kDa was not detected by immunoblot-ting using F-E2G5 antibody, neither in denaturing nor innon-denaturing gels (data not shown). These results indi-Page 4 of 17(page number not for citation purposes)clinical follow-up appointment or at their date of deathnot related to the tumor. A stepwise multivariable Coxcate that (i) the epitope(s) recognized by F-E2G5 resideswithin the YB-1 protein domains aa21-147 and (ii) detec-BMC Cancer 2009, 9:410 http://www.biomedcentral.com/1471-2407/9/410tion is highly susceptible to the chosen conditions ofwestern blotting.means of Mab F-E2G5, as the endogenous YB-1 proteinfrom cell lysates (~50 kDa) was not detected by westernblotting (only minute amounts were immunopositive fol-Detection of YB-1-GFP and YB-1(21-147)-GFP by immunoblot and recombinant YB-1 by ELISAFigure 1Detection of YB-1-GFP and YB-1(21-147)-GFP by immunoblot and recombinant YB-1 by ELISA. (A) HEK293T cell extracts containing GFP (lanes 1 and 4), YB-1-GFP (lane 2, 5 and 7) or YB-1(21-147)-GFP proteins (lanes 3, 6 and 8) were separated in denaturing gels and following transfer to nitrocellulose probed with GFP antibody (lanes 1 to 3) or Mab F-E2G5 (lanes 4 to 8). Relative mobilities of expressed proteins are indicated by arrowheads. Notably, reducing sample buffer prevents detection of tagged YB-1 proteins by immunoblot (lanes 7 and 8). (B) ELISA was performed by coating with recombinant affin-ity purified hexahistidin-tagged YB-1 protein. Biotin-labeled Mab F-E2G5 yields an even stronger signal than non-biotinylated Mab. Controls included omission of antibody (Con1) or antigen (Con2).recomb. YB-1*Page 5 of 17(page number not for citation purposes)One may conclude that the GFP-tag stabilizes the YB-1protein conformation and permits successful detection bylowing prolonged exposure). To ensure that Mab F-E2G5successfully binds YB-1 protein under non-reducing andBMC Cancer 2009, 9:410 http://www.biomedcentral.com/1471-2407/9/410non-denaturing conditions, we thereafter established adirect ELISA with recombinant, affinity-purified YB-1 pro-tein expressed in E. coli. Biotinylated as well as non-bioti-nylated Mabs F-E2G5 were tested. Under both conditionsrecombinant YB-1 protein was detected (Figure 1B), evenbetter with biotinylated Mab F-E2G5. Controls includedomission of antibody (Con1) or antigen (Con2) andaddition of irrelevant IgG2/irrelevant biotinylated IgG2antibody (not shown), all yielding negative results. Thus,Mab F-E2G5 binds epitope(s) that reside within YB-1 pro-tein domains aa21 to 147. In addition, a sandwich ELISAwas established with a series of GFP-tagged fusion pro-teins that include the following domains of YB-1 protein:aa21-147, aa146-317, aa146-225, aa146-172, aa260-317,aa146-262, aa21-262. Biotinylated Mab F-E2G5 was ableto detect all fusion proteins that encompassed aa146-172.ELISA results were scored positive with an optical densityof >0.4, whereas background values were below 0.05.These results support the notion that two distinct confor-mational epitopes are recognized by the Mab, one withinthe N-terminal (aa21-147) and one within the centrallylocalized domains (aa146-172).To further evaluate the propensity of Mab F-E2G5 to asso-ciate with YB-1-GFP or endogenous YB-1 proteins Mab F-E2G5 was evaluated in immunoprecipitation studies (Fig-ures 2A and 2B). Mab F-E2G5 successfully pulled downYB-1-GFP, that was subsequently detected by monoclonalanti-GFP-tag antibody (Figure 2A). Furthermore Mab F-E2G5 immunoprecipitated endogenous YB-1 proteinfrom HEK293 whole cell extracts, with immunoprecipi-tated protein being detected by polyclonal peptide-derived anti-YB-1 antibody directed against the protein C-terminus (denoted YB-1(C-term), Figure 2B, lane 2).Immunoprecipitation studiesFigure 2Immunoprecipitation studies. (A) Control IgG1, F-E2G5 and anti-GFP-tag antibodies were added to binding reactions with YB-1-GFP protein. Immunoblotting (IB) was carried out with anti-GFP antibody. (B) HEK293 cell protein extracts containing endogenous YB-1 protein were incubated with control IgG1, F-E2G5 and polyclonal anti-YB-1(C-term) antibodies and immu-noprecipitated using pansorbin. Detection of protein pull-down was accomplished by immunoblotting with anti-YB-1(C-term) antibody. Sample buffers utilized were reducing (left) or non-reducing (right). Recombinant YB-1 protein was run aside w/o A BGFP YB-1(C-term) YB-1(C-term)T7-tagIB T7-tagIPrYB-1kDa97-66-45-30-- - -1 2 3 1 2 3 4 5 6 7 8 9IBkDa97-66-45-30-12-YB-1-GFP cell extract inputkDa97-66-45-30-12-**rYB-1cell extract reducing buffer non-reducing bufferIPinputPage 6 of 17(page number not for citation purposes)immunoprecipitation to visualize relative mobilities. "*" indicated mobilities of immunoglobulin heavy and light chains.BMC Cancer 2009, 9:410 http://www.biomedcentral.com/1471-2407/9/410Notably, when endogenous YB-1 protein was immuno-precipitated by polyclonal anti-YB-1(C-term) immunob-lotting was unsuccessful under reducing conditions(Figure 2B, lane 3).Next immunoprecipitated proteins were loaded on gels innon-reducing buffer (omission of mercaptoethanol andEDTA). Under these conditions immunoprecipitatedendogenous YB-1 pulled down by monoclonal F-E2G5antibody was no longer detected with polyclonal anti-YB-1(C-term) antibody. Successful immunoprecipiation byanti-YB-1(C-term) antibody was however possible (Figure2B, lane 8). Heavy and light chains from immunoglobu-lins, that are contaminants from the immunoprecipita-tion procedure, were detected in samples loaded undernon-reducing buffer conditions (indicated by "*" in Fig-ure 2B).From these results it is concluded that monoclonal F-E2G5 antibody has the propensity to immunoprecipitateYB-1-GFP as well as endogenous YB-1 protein, however,detection by means of immunoblotting with the utilizedpolyclonal anti-YB-1 antibody is susceptible to the chosenbuffer conditions.Mab F-E2G5 is suitable for YB-1 immunodetection in formalin-fixed paraffin-embedded breast cancer tissueFrom our previous studies it was clear that the fixationprocedure markedly affects detection of YB-1 in rat tissue[43]. In the following we performed immunohistochem-istry of sequential tissue sections from patients diagnosedwith invasive breast cancer. Biotinylated Mab F-E2G5 andtwo polyclonal peptide-derived YB-1 antibodies generatedagainst distinct epitopes within the protein N-terminuswere utilized. Notably, one of the polyclonal antibodiesused (YB-1#2) has been previously applied by Janz et al.[1], the other is commercially available (YB-1#1; see Mate-rials). Sequential sections were stained for Ki67 to identifycycling cells and exclude the possibility that biotinylatedMab F-E2G5 merely detects such cells. As can be seen inFigure 3 there was strong cytoplasmatic staining of cancer-ous cells with both polyclonal antibodies (#1 and #2).The overall pattern of immunopositive cells was similarwith the Mab, however, a considerable fraction of cellsexhibited strong nuclear staining. Ki67 positivity wasobserved in about 10 to 25% of all tumor cells, which wasconsiderably lower than YB-1 positive cells detected byMab F-E2G5. This finding excludes the possibility that theMab only detects cycling cells.From these results we conclude that the established MabF-E2G5 is suitable for immunohistochemistry with for-malin-fixed paraffin-embedded tissue, as routinely per-pattern with Mab and polyclonal antibodies is similar.However, Mab F-E2G5 preferentially detects nuclear YB-1.Nuclear YB-1 staining pattern in breast cancer tissueImmunohistochemical analysis was used to investigateYB-1 protein expression in normal breast tissue and breastcancers. We analyzed a tissue microarray with 224 breasttissue samples, i.e. 179 invasive ductal carcinomas, eightductal carcinoma in situ (DCIS) and 37 normal breast tis-sue samples. To our knowledge, the latter group is largerthan all other control groups analyzed by immunohisto-chemistry for YB-1 expression so far. Nuclear YB-1 immu-nohistochemical staining was only detectable in invasiveductal carcinomas, but not in DCIS and normal breast tis-sue. Figure 4 shows representative images of YB-1 expres-sion in normal and malignant breast tissues. YB-1 stainingwas not detectable in normal breast tissue (Figures 4A and4B). In DCIS (Figures 4C and 4D) cytoplasmic YB-1expression was present in 75% (6/8) of cases, whilenuclear YB-1 expression was not present. Invasive ductalcarcinomas exhibited nuclear staining in 24% (43/179) ofcases (Figures 4E and 4F).YB-1 expression, clinico-pathological parameters and patient survivalClinicopathologic and immunohistochemical characteris-tics were correlated with nuclear and cytoplasmic YB-1staining for descriptive data analysis (Table 1). Cytoplas-mic YB-1 expression detected with the Mab F-E2G5 wasnot significantly correlated to overall (p = 0.134) andrecurrence-free survival (p = 0.39).However, nuclear YB-1 expression detected by antibody F-E2G5 was associated with shorter overall survival (OS; p =0.0046) and exhibited a strong trend towards associationwith shorter recurrence-free survival (RFS; p = 0.09). Thesewere compared by Kaplan Meier analysis between inva-sive breast tumours with nuclear YB-1 expression versus allother invasive breast tumours exhibiting no nuclearexpression (Figure 5). Patients with nuclear YB-1 expres-sion in the tumor had an estimated mean OS of 90months (95% confidence interval (CI): 72-109 months)compared to 117 months (95% CI: 108-126 months) inpatients with absent nuclear YB-1 immunoreactivity.Nuclear YB-1 detection also correlated with tumor stage(p = 0.004), higher (G2/G3) histological grade (p =0.011) and negativity of progesterone receptor status (p =0.002) (Table 1). These strong correlations are demon-strated in Figure 6. In a stratified univariate analysis, theprognostic value of nuclear YB-1 detection became evenmore pronounced in the clinically important subgroup ofstage pT1/T2 tumors, representing ~80% of all diagnosedcarcinomas, and breast tumors with negative progester-Page 7 of 17(page number not for citation purposes)formed for histological analyses. The overall staining one receptor status (Figures 6 and 7). While pT1 tumoursexhibit no prominent nuclear YB-1 staining (Figure 7A),BMC Cancer 2009, 9:410 http://www.biomedcentral.com/1471-2407/9/410distinct nuclear YB-1 staining was detectable in pT4tumours (B). In a similar comparison nuclear YB-1 pro-tein was not detectable in most progesterone receptor pos-itive breast tumours (Figure 7C), while there wasprominent nuclear YB-1 staining in most tumors withnegative progesterone receptor status (D).Cox regression models including factors possibly influ-encing tumor-related survival and recurrence-free survivalin relation to nuclear YB-1 expression were applied. Ascould be expected due to the size of our cohort, this anal-ysis failed to demonstrate significance for nuclear YB1detection as an independent prognostic marker (Table 2).Subsequently we analyzed nuclear YB-1 expression withmodel was calculated with nuclear YB-1 expression as tar-get variable and CK5/6, ER and HER2 as covariates (Table3). None of the tested covariates remained significant; i.e.,none of the covariates was significantly correlated withnuclear YB-1 expression, arguing that YB-1 is not selec-tively expressed in a specific molecular subtype of breastcancer.DiscussionMajor challenges in cancer diagnostics relate to earlydetection of malignant tissue and the possibility to predictthe rate of disease progression and sensitivity to chemo-therapy. Regarding all these issues the Y-box protein-1may gain a prominent role, given that (i) it has an onco-Immunohistochemical comparison of different YB-1 antibodies in human invasive breast cancerFigure 3Immunohistochemical comparison of different YB-1 antibodies in human invasive breast cancer. While the pro-liferation marker Ki67 is strongly expressed in a defined fraction of breast tumor cells there is no concordance to the expres-sion pattern of YB-1. The three YB-1 antibodies present very similar expression patterns. However, while the two established polyclonal YB-1 antibodies (#1 is against an N-terminal epitope and available from antibodies-online; #2 has been established by Janz et al. [1]) predominantly detect cytoplasmic YB-1 expression, the newly characterized monoclonal YB-1 antibody Mab F-E2G5 is able to detect nuclear YB-1 besides less abundant expression in the cytoplasm.patient 2patient 1Ki67YB-1 Ab #1 (polycl.)YB-1 Ab #2 (polycl.)YB-1 Mab F-E2G5 100x 300x 100x 300xPage 8 of 17(page number not for citation purposes)respect to the molecular breast cancer subtypes defined byPerou and Sorlie [44,45]. A multiple logistic regressiongenic property with induction of breast tumors in 100%of transgenic animals overexpressing YB-1 in the mam-BMC Cancer 2009, 9:410 http://www.biomedcentral.com/1471-2407/9/410Page 9 of 17(page number not for citation purposes)Expression of YB-1 in normal breast tissue, non-invasive and invasive breast cancerFigure 4Expression of YB-1 in normal breast tissue, non-invasive and invasive breast cancer. (A) Only weak cytoplasmic YB-1 staining is detectable in normal breast tissue. (B) Scale-up of specimen shown in A. The magnification demonstrates weak YB-1 expression in luminal-epithelial breast cells. (C) Moderate YB-1 expression in a ductal carcinoma in situ (DCIS) of the breast. (D) Scale-up of specimen shown in C. (E) Invasive ductal breast cancer with abundant nuclear YB-1 expression. (F) Scale-up of specimen shown in E. Magnifications: A, C, E: 100×; B, D, F: 400×.AFEDBC DBMC Cancer 2009, 9:410 http://www.biomedcentral.com/1471-2407/9/410mary gland [23], (ii) analyses of dysplasia-associatedlesions in colitis ulcerosa patients reveal that lesions withincreased cancer risk are distinguishable due to their YB-1expression pattern [46], (iii) several reports on breast can-cer indicate that YB-1 expression levels are strongly predic-tive for relapse rates and negatively correlate with diseasefree survival [6,34,36,47-50], and (iv) YB-1 upregulated P-glycoprotein expression. To date, however, no clear-cutstandard for the assessment of YB-1 expression levels iset al. even included the YB-1 expression within the adja-cent tissue for analyses, which profoundly improved thepredictive value on relapse-free survival [1].One of the major obstacles towards a unifying scoring sys-tem in cancer tissue is the lack of a common expressionpattern observed with diverse antibodies directed againstYB-1. Most antibodies provide a highly specific stainingpattern that is confined to the cytoplasm with completeTable 1: Clinico-pathological and immunohistochemical parameters in relation to YB-1 immunoreactivity, and univariate analysis of factors regarding tumor-related and recurrence-free surivalVariable Categorization Nuclear YB-1 immunoreactivity Tumor-related death Tumor recurrencen analyzable neg. pos. p* n events p† n events p†Clinico-pathological data:Age at diagnosis<50 years 51 43 8 0.036 51 9 0.0064 51 16 0.1895≥ 50 years 108 74 34 108 45 102 42Tumor stagepT1 40 35 5 0.004 40 4 <0.0001 39 5 0.0001pT2 83 63 20 83 28 81 34pT3 10 4 6 10 4 9 4pT4 26 15 11 26 18 24 15Lymph node statuspN0 75 60 15 0.101 75 11 <0.0001 74 13 <0.0001pN1-3 80 54 26 80 41 76 42Histologic gradeG1 17 16 1 0.011 17 4 0.0047 17 4 <0.0001G2 69 55 14 69 17 65 16G3 73 46 27 73 33 71 38Multifocalityunifocal tumor 141 104 37 1.000 141 45 0.0903 136 49 0.0683multifocal tumor 18 13 5 18 9 17 9Histologic typeductal 125 93 32 0.710 125 43 0.9302 123 49 0.5730lobular 16 11 5 16 5 14 4other 15 10 5 15 5 13 4Immunohistochemistry (IHC):Estrogen receptor statusnegative 42 30 12 1.000 42 19 0.0129 42 24 0.0026positive 96 69 27 96 25 92 27Progesterone receptor statusnegative 98 63 35 0.002 98 40 0.0045 93 44 0.0019positive 46 41 5 46 8 46 9Cytokeratin 5/6 IHCnegative (0) 118 85 33 0.540 118 34 0.0100 114 38 0.0349positive (1+-3+) 41 32 9 41 20 39 20TP53 IHC<10% 85 62 23 1.000 85 28 0.3823 82 28 0.4422≥ 10% 48 31 17 48 18 46 19HER2 IHCnegative (0-1+) 108 83 25 1.000 108 28 0.0081 102 31 0.0008positive (2+-3+) 35 21 14 35 18 35 20* Fisher's exact test (two-sided), bold face representing significant data.† Log rank test (two-sided), bold face representing significant data.Page 10 of 17(page number not for citation purposes)available. In most immunohistochemical analyses overallexpression levels have been scored in cancer tissues. Janzabsence within the nuclear compartment, as also reportedin the most recent and largest study performed hithertoBMC Cancer 2009, 9:410 http://www.biomedcentral.com/1471-2407/9/410[6]. On the other hand, most - if not even all - activities ofYB-1, that relate to oncogenic transformation, cell prolif-eration and drug resistance, must take place within thenuclear compartment. These are accompanied by alteredgene transcription as well as chromosomal instability[18,20,23]. Thus, it is conceivable that a sensitive detec-tion system and valid predictive testing must include suchinformation, which is also reiterated by the observation ofnuclear YB-1 being associated with P-glycoprotein expres-sion and drug resistance [8,11]. It becomes even morecomplex when one envisions that YB-1 is a quite abun-dant cytoplasmic protein and has been detected in thenucleus in non-transformed cells as well, when tissue isfixed in Carnoys fixative [43]. The question is therefore,how these inconsistencies may be reconciled. One answermay be that YB-1 in cancer tissue has a different antigenic-ity due to post-translational modifications, e.g. phospho-rylation of serine 102 [27]. In line with this interpretationis the observation of nuclear as well as cytoplasmic YB-1in most cells using immunoblotting and senescence ofcells with YB-1 knock-out [18,51]. Alternatively, cleavage ofYB-1 within the protein C-terminus, as proposed beforevia the 20S proteasome [52,53], may render epitopes ofstrated that the cleaved N-terminal fragment itself mayshuttle to the nucleus and predict chemoresistance,whereas the overall full-length YB-1 expression level wasunchanged in chemotherapy resistant versus chemosensi-tive cells. Attempts to explain the hidden secret beyondthe different detection patterns observed already hint atthe difficulties that the design and establishment of amonoclonal antibody to detect YB-1 faces.In this report, we describe the generation of a monoclonalYB-1 antibody, termed F-E2G5, which detects YB-1 in for-maldehyde-fixed paraffin-embedded tissues. Specificity ofYB-1 detection by this antibody was confirmed by immu-noblotting with YB-1-GFP fusion protein and ELISA. Onedetected epitope must reside within the central domainsencompassing amino acids 21-147, a second one withindomains aa146-172. Notably, using this antibody endog-enous YB-1 protein is not detected by immunoblotting,likely due to its "misfolding" under the given conditions.Likely, that the GFP-tag within the protein C-terminus sta-bilizes the recognized epitope(s) for detection by westernblotting. A non-specific band detected under some immu-noblotting conditions does not seem to be of relevance forA and B: Distribution of time (months) to tumor-related death (A) and tumor recurrence (B) among breast cancer patients with negative and positive YB-1 immunoreactivity as estimated by the meth d of Kaplan and MeierFigur  5A and B: Distribution of time (months) to tumor-related death (A) and tumor recurrence (B) among breast cancer patients with negative and positive YB-1 immunoreactivity as estimated by the method of Kaplan and Meier.OS (months) 140 120 100 80 60 40 20 0 1.0 .8 .6 .4 .2 0.0 P=0.0046positive nuclear YB-1 immunoreactivity(N=42, events=22)negative nuclear YB-1 immunoreactivity(N=117, events=32)Tumor-related survival (months) RFS (months) 140 120 100 80 60 40 20 0 1.0 .8 .6 .4 .2 0.0 P=0.0927positive nuclear YB-1 immunoreactivity(N=39, events=18)negative nuclear YB-1 immunoreactivity (N=114, events=40)Recurrence-free survival (months)A BPage 11 of 17(page number not for citation purposes)YB-1 accessible for antibodies and result in positiveimmunodetection. Sorokin et al. have nicely demon-the performed immunohistochemistry, given that tissuespecimens from healthy breast tissue do not demonstrateBMC Cancer 2009, 9:410 http://www.biomedcentral.com/1471-2407/9/410Page 12 of 17(page number not for citation purposes)A -- D: Distribution of time (months) to tumor-related death among the various subgroups (A, pT1-2; B, pT3-4; C, negative progesterone recept r status; D; posi ive pr gesterone receptor status) of breast cancer patients with negative and positive YB 1 immu oreactivity as stimated by the method f Kaplan nd MeierFigure 6A -- D: Distribution of time (months) to tumor-related death among the various subgroups (A, pT1-2; B, pT3-4; C, negative progesterone receptor status; D; positive progesterone receptor status) of breast cancer patients with negative and positive YB-1 immunoreactivity as estimated by the method of Kaplan and Meier.OS (months) 140 120 100 80 60 40 20 0 1.0 .9 .8 .7 .6 .5 .4 .3 .2 .1 0.0 Primary invasive unilateral breast cancern=123, events 32 P=0.0043Apositive YB-1 immunoreactivitynegative YB-1 immunoreactivityTumor-related survival (months)Stage pT1-2: OS (months) 140 120 100 80 60 40 20 0 1.0 .9 .8 .7 .6 .5 .4 .3 .2 .1 0.0 Bpositive YB-1 immunoreactivitynegative YB1 immunoreactivity Primary invasive unilateral breast cancern=36, events 22 P=0.6609Tumor-related survival (months)Stage pT3-4:OS (months) 140 120 100 80 60 40 20 0 1.0 .9 .8 .7 .6 .5 .4 .3 .2 .1 0.0 Primary invasive unilateral breast cancern=98, events 40 P=0.01Cpositive YB-1 immunoreactivitynegative YB-1 immunoreactivityTumor-related survival (months)Negative PR status:. OS (months) 140 120 100 80 60 40 20 0 1.0 .9 .8 .7 .6 .5 .4 .3 .2 .1 0.0 Primary invasive unilateral breast cancern=46, events 8 P=0.3011Dpositive YB-1 immunoreactivitynegative YB-1 immunoreactivityPositive PR status:Tumor-related survival (months)BMC Cancer 2009, 9:410 http://www.biomedcentral.com/1471-2407/9/410immunopositivity with Mab F-E2G5 and correlationresults indicate that the outcome correlates with a nuclearstaining pattern.We have used this antibody to analyze YB-1 expression incessfully used to define novel breast tumor markers [40].Unlike Abba et al. [54] who described an absence of YB-1mRNA expression in DCIS compared to normal tissues,we found an increased cytoplasmic concentration of YB-1protein in six of eight analyzed DCIS compared to normalRepresentative tissue microarray staining results illustrating YB-1 expression in relation to tumor stage (A and B) and proges-terone receptor tatus (C and D)Figure 7Representative tissue microarray staining results illustrating YB-1 expression in relation to tumor stage (A and B) and progesterone receptor status (C and D). A and B: While YB-1 staining in small (pT1) tumors is predomi-nantly cytoplasmic (A), large (pT4) tumors preferentially show a distinct condensation of YB-1 signal in the nuclei of tumor cells (B). C and D: In breast tumors with positive progesterone receptor status (C) YB-1 expression is predominantly cytoplas-mic, while tumors that lost progesterone receptor expression (D) exhibit prominent nuclear YB-1 expression.A BDCpT1 pT4PR + PR -Page 13 of 17(page number not for citation purposes)a large and clinically well characterized collection ofbreast cancer specimens [41] that has previously been suc-tissue. In our study, nuclear YB-1 expression was restrictedto invasive ductal carcinomas. Conversely, tissue sectionsBMC Cancer 2009, 9:410 http://www.biomedcentral.com/1471-2407/9/410representing DCIS or normal breast tissue did not shownuclear expression of YB-1. Concordantly with theseobservations, nuclear expression of YB-1 in invasive breastcancer was significantly associated with overall survival (p= 0.0046). In our cohort this correlation was more predic-tive as observed by Janz et al. [1], who found a less tightassociation between YB-1-linked tumor aggressivenessand poor clinical outcome (p = 0.011).So far we are the first group that describes a correlationbetween nuclear YB-1 expression and increased tumorgrading (p = 0.011) and tumor stage (p = 0.004) in breastcancer. Although this does not necessarily reflect a causalrelationship, it emphasizes the role of YB-1 as predictivemarker. In non-small lung cancer and ovarian cancer andby applying polyclonal antibodies, other groupsdescribed an apparent link between YB-1 positivity in thenucleus and tumor staging. However, they did not findany correlation between YB-1 positivity and tumor gradein these entities [3,55,56].A further finding of our study is the highly significant cor-relation between nuclear YB-1 expression and negativityof the progesterone receptor status (p = 0.002), which hasnot been described so far. Since YB-1 has been directlylinked to intrinsic or acquired resistance to chemotherapyand thus might be responsible for failure of current treat-ment regimens, these findings implicate an interestingopportunity for the selection of chemotherapy resistantbreast cancer patients out of the whole cohort of patients.ER-positive/PR-negative breast cancers are known torespond less well to selective ER modulator (SERM) ther-apy than ER-positive/PR-positive tumors [57]. ThereforeYB-1 expressing breast cancer may have a general ten-dency to be hormone refractory, their relative neoplasticpotential thus requiring chemotherapy, even if the tumorsTable 2: Multivariate Cox regression analysis of factors possibly influencing tumor-related survival and recurrence-free survivalTumor-related survival Recurrence-free survivalName Variables Categorisation Global Stepwise reverse selection† Global Stepwise reverse selection†p* Hazard ratio95% Confidence intervalp p* Hazard ratio95% Confidence intervalpModel with dichotomous covariablesT Tumor stage 0 pT1-2 0.402 - 0.153 -1 pT3-4N Lymp node status0 pN0 0.002 5.050 2.264-11.267 <0.001 0.038 2.934 1.303-6.607 0.0091 pN1-3G Histologic grade0 G1-2 0.136 1.875 0.946-3.715 0.072 0.007 3.188 1.442-7.052 0.0041 G3ER Estrogen receptor status0 negative 0.168 0.420 0.213-0.829 0.012 0.047 0.357 0.173-0.733 0.0051 positiveHER2 HER2 IHC 0 negative (0-1+) 0.386 - 0.045 2.164 1.019-4.594 0.0451 positive (2+-3+)CK5/6 Cytokeratin 5/6 IHC0 negative (0) 0.085 - 0.305 -1 positive (1+-3+)TP53 TP53 IHC 0 <10% 0.607 - 0.043 0.529 0.256-1.092 0.0851 ≥ 10%YB1 YB1 IHC 0 negative (0) 0.530 - 0.738 -1 positive (1+-3+)Table 3: Multiple logistic regression model. Estimate Std. Error t value Pr(>| t|)(Intercept) -1.187 0.4558 -2.603 0.009CK56 0.032 0.2460 0.131 0.896ER -0.009 0.0452 -0.207 0.836HER2 0.226 0.1577 1.434 0.152Null Deviance: 170.1; Residual Deviance: 167.8; AIC: 175.8; Degrees of Freedom: 146The estimate for the regression coefficients show the change in YB-1 corresponding to a unit change in the corresponding marker (ER, Page 14 of 17(page number not for citation purposes)are of small size, low grade or estrogen receptor positive.HER2, CK5/6), conditional on the other markers remaining constant. No significant correlation can be seen between YB-1 and one of the other markers.BMC Cancer 2009, 9:410 http://www.biomedcentral.com/1471-2407/9/410Our data are not in line with two reports, where YB-1 ismore commonly found in ER negative breast tumors[6,26]. One may explain this with differences in antibodydetection sensitivity, altered epitopes of YB-1 in thenuclear compartment, with the latter being the morelikely reason.In our stratified univariate analysis the prognostic value ofnuclear YB-1 expression became even more pronouncedin the clinically important subgroup of stage pT1/T2tumors and breast tumors with negative progesteronereceptor status, respectively. Routine mammographyscreening for breast cancer in women aged 51-70 isbecoming a standard in the European Community. Thus,the number and frequency of small sized stage pT1/T2breast tumors detected is steadily increasing. A YB-1immunohistochemistry-based diagnostic test could helpto identify young patients with small tumors but signifi-cant risk of relapse. We are confident that this novel mon-oclonal YB-1 antibody is of great value in performingprospective studies to establish YB-1 as a tumor marker inthe management of human breast cancer. For this purposelarge confirmative meta-analyses on independent breastcancer cohorts will be necessary. Therefore we would liketo encourage other academic groups to validate our dataon their own tumor collections.ConclusionThe newly established monoclonal anti-YB-1 antibody(clone F-E2G5) is suited to detect nuclear YB-1 expressionand may be of great value for prospective studies to vali-date YB-1 as a prognostic marker and to adjust treatmentin breast cancer patients.AbbreviationsAKT: protein kinase B; EGF: epidermal growth factor;ELISA: enzyme-linked immunosorbant assay; ER: estro-gen receptor; GFP: green fluorescent protein; Mab: mono-clonal antibody; PR: progesterone receptor; YB-1: Y-boxbinding protein-1.Competing interestsPRM is co-founder of antibodies-online and filed a patentrelating to monoclonal anti-YB-1 antibody clone F-E2G5.The other authors declare that there are no competingfinancial or non-financial interests.Authors' contributionsED and PRM designed the study, performed data analysesand drafted the manuscript, AE and RK carried out mono-clonal antibody generation and immunoassays, FW andEB were involved in scoring of the tissue array and datainterpretation, TF performed statistical analyses and waspretation, SD was involved in data interpretation anddrafting of the manuscript, UK participated in the designof the study. PRM originally conceived the study. Allauthors read and approved the final manuscript.AcknowledgementsWe are grateful to Ellen Krott for performing the immunohistochemistry. The study has been funded by grants from the Deutsche Forschungsge-meinschaft (SFB542 TP A11, C4 and C12 to P.R.M.).References1. Janz M, Harbeck N, Dettmar P, Berger U, Schmidt A, Jurchott K, Sch-mitt M, Royer HD: Y-box factor YB-1 predicts drug resistanceand patient outcome in breast cancer independent of clini-cally relevant tumor biologic factors HER2, uPA and PAI-1.Int J Cancer 2002, 97(3):278-282.2. Shibahara K, Sugio K, Osaki T, Uchiumi T, Maehara Y, Kohno K, Yas-umoto K, Sugimachi K, Kuwano M: Nuclear expression of the Y-box binding protein, YB-1, as a novel marker of disease pro-gression in non-small cell lung cancer.  Clin Cancer Res 2001,7(10):3151-3155.3. 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