Prognostic role of clusterin in resected adenocarcinomas of the lung

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ContentslistsavailableatSciVerseScienceDirect

Lung

Cancer

j o ur na l ho me p ag e :w w w . e l s e v i e r . c o m / l o c a t e / l u n g c a n

Prognostic

role

of

clusterin

in

resected

adenocarcinomas

of

the

lung

Francesca

Panico

a

_{, Christian}

_Casali

b

_{, Giulio}

_Rossi

c

_,

_Federica

_Rizzi

d,e

_,

_Uliano

_Morandi

b

_,

Saverio

Bettuzzi

d,e

_,

_Pierpaola

_Davalli

f

_,

_Lorenzo

_Corbetta

g

_,

_Erica

_Susanna

_Storelli

b

_,

Arnaldo

Corti

f

_,

_Leonardo

_M.

_Fabbri

a,∗

_,

_Serenella

_Astancolle

f,1

_,

_Fabrizio

_Luppi

a,1 a_Section_of_Respiratory_Diseases,_Department_of_Oncology,_Haematology_&_Pulmonology,_University_of_Modena_and_Reggio_Emilia,_Modena,_Italy b_Division_of_Thoracic_Surgery,_Department_of_General_Surgery_and_Surgical_{Specialities,}_University_of_Modena_and_Reggio_Emilia,_Modena,_Italy c_Section_of_Pathologic_Anatomy,_University_of_Modena_and_Reggio_Emilia,_Modena,_Italy

d_Department_of_Experimental_Medicine_and_Centre_for_Molecular_and_{Translational}_Oncology_(COMT),_University_of_Parma,_Parma,_Italy e_National_Institute_of_{Biostructures}_and_Biosystems_(INBB),_Rome,_Italy

f_Department_of_Biomedical_Sciences,_University_of_Modena_and_Reggio_Emilia,_Modena,_Italy g_Functional_Unit_of_Respiratory_Medicine,_University_of_Florence,_Florence,_Italy

a

r

t

i

c

l

e

i

n

f

o

Articlehistory:

Received24September2012 Receivedinrevisedform 27November2012 Accepted28November2012

Keywords: Biomarkers

Bronchioloalveolarcarcinoma Non-smallcelllungcarcinoma Immunohistochemistry Relapse

Survival

a

b

s

t

r

a

c

t

Rationale:Clusterinexpressionmaychangein varioushuman malignancies,including lungcancer.

Patientswithresectablenon-smallcelllungcancer(NSCLC),includingadenocarcinoma,haveapoor

prognosis,witharelapserateof30–50%within5years.NuclearfactorkB(Nf-kB)isanintracellular

proteininvolvedintheinitiationandprogressionofseveralhumancancers,includingthelung.

Objectives:WeinvestigatetheroleofclusterinandNf-kBexpressioninpredictingtheprognosisofpatients

withearly-stagesurgicallyresectedadenocarcinomaofthelung.

Findings:Thelevelofclusteringraduallydecreasedfromwell-differentiatedtopoorlydifferentiated

adenocarcinomas.Clusterinexpressionwassigniﬁcantlyhigherinpatientswithlow-grade

adenocar-cinoma,inearly-stagediseaseandinwomen.Clusterinexpressionwasinverselyrelatedtorelapseand

survivalinbothunivariateandmultivariateanalyses.Finally,weobservedaninversecorrelationbetween

Nf-kBandclusterin.

Conclusions:Clusterinexpressionrepresentsanindependentprognosticfactorinsurgicallyresectedlung

adenocarcinomaandwasproventobeausefulbiomarkerforfewerrelapsesandlongersurvivalin

patientsintheearlystageofdisease.TheinversecorrelationbetweenNf-kBandclusterinexpression

conﬁrmthepreviouslyreportedroleofclusterinaspotentdownregulatorofNf-kB.

1. Introduction

Lungcanceristheleadingcauseofcancer-related mortality. Despitemajoradvancesintreatment, theprognosisforpatients withlungcancerhasnotsigniﬁcantlyimprovedinthelast2decades [1].Whenidentiﬁedatanearlystage,lungadenocarcinomais pri-marilytreatedbysurgicalresection,whichispotentiallycurative. However,30–60%ofpatientswithstageIBtoIIIAnon-smallcell lung cancer(NSCLC)diewithin5 years aftersurgery, primarily fromrecurrenceofthetumor[2].Therefore,althoughTNM stag-ingistheestablishedprognosticindicator,betterparametersare

∗ Correspondingauthorat:SectionofRespiratoryDiseases,Departmentof Oncol-ogy,Haematology&Pulmonology,UniversityofModenaandReggioEmilia,Largo delPozzo71,41124Modena,Italy.Tel.:+390594222198;fax:+390594224231.

E-mailaddress:leonardo.fabbri@unimore.it(L.M.Fabbri).

1 _These_authors_contributed_equally_as_senior_authors_to_this_work.

urgentlyneededformoreaccuratepredictionoftheoutcomeand morepreciseindicationoftheefﬁcacyoftreatment.

Clusterin(CLU,alsoknownasApoJ)isaheterodimeric glycopro-tein,expressedinalltissuesandfoundinallhumanﬂuids,including thelung,suggestingthatthisproteinperformsfunctionsof funda-mentalimportancebothinsideandoutsidethecell.Indeed,CLUhas beenfoundtobeinvolvedinvariousphysiologicprocesses impor-tantforcarcinogenesisandtumorgrowth,includingapoptoticcell death,cellcycleregulation,DNArepair,tissue remodeling,lipid transportation,membranerecycling,andimmunesystem regula-tion[3–5].Severalstudieshaveexaminedtheprognosticvalueof CLUinvariousmalignancies,withconﬂictingresults[6–9].Only onestudyinvestigatedtheprognosticroleofCLUina heteroge-neousseriesofresectedNSCLC,andtheresultssuggestedthatCLU expressioncouldbeapositiveprognosticindicatorforNSCLC[10]. Nf-kB is a transcription factor associated withthe initiation andprogressionofseveralhumantumors,includinglungcancer [11]andisconsideredapotentiallyoncogenictranscription fac-torimportantforproliferationandsurvivalofneoplasticcells[12].

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Several in vitro studies demonstrated that Nf-kB activation is inhibitedbyCLU[13,14]andthatthedeletionofCLUinduces acti-vationofNf-kB[15]suggestinganinterrelationshipbetweenthese 2proteins.

The aim of the present study was to investigate the level ofexpression and theprognostic role of CLU,in terms of both overallsurvivalanddisease-freesurvival,inawell-deﬁned pop-ulationof patients withearly-stage (stages Iand II), surgically resectedadenocarcinomaofthelung.Furthermore,weanalyzed therelationshipbetweenCLUand Nf-kBexpressionandvarious clinical–pathologicalfeaturesinthesamecohortofpatients.

2. Methods 2.1. Patients

Files from the archives of the Section of Pathology and of the Division of Thoracic Surgery of the University of Modena and Reggio Emilia were retrospectively reviewed to retrieve all patients with a definitive diagnosis of non-small cell lung carcinoma(NSCLC) who underwent a radical surgicalresection betweenJanuary2000and December2004.Allthepathological slides werereviewed by one pathologist(G.R.) and reclassified accordingtothecriteriasetbythe2004WHOlungtumors clas-sification.Infact,thisstudywasperformedpriorthepublicationof thecurrentconsensuspaper[16].

Onlycaucasianpatientswithdeﬁnitivediagnosisofprimitive adenocarcinomaofthelungandacompleteresection(R0resection) wereincluded;patientswithotherknownprimarytumorsanda minororincompleteresection(R1orR2resection)wereexcluded fromthestudy.Onlyearlystagesofdisease(StageIandStageII accordingtothe1997IASLC/UICC TNMstagingsystemforlung tumors)wereconsidered.Eighty-threepatientsmetallcriteriaand wereenrolledinthestudy.

Thepercentageofbronchioloalveolarcomponent(BAC) (asa non-replacingtypeadenocarcinomaaccordingtothe2004-WHO classification)wasassessedin allpatients. BACpercentage was measuredbycountingtheBACratioforeach slideunderocular fieldwithgrid(100gridsperfield)andaveragingtheBAC compo-nentacrossslides.Theprocesswasrepeatedtwiceforeachslide, andthefinalaveragewasusedasthepercentageofBACforagiven tumorcase.

2.2. Immunostainingcorrelations

Ineachcase,3-␮mthicksectionswereobtainedfroma rep-resentativeblock.Sectionswereair-driedovernightat37◦C,then deparafﬁnizedinxyleneandrehydratedthroughadecreasing con-centration of alcohol towater. Endogenous peroxidaseactivity was blocked by immersion for 10min in 3% hydrogen per-oxide (H2O2) in methanol. Incubation with primary antibody

(CLU,clone41D,UpstateBiotechnologies,LakePlacid,NY;1.200 dilution; microwave antigen retrieval) was accomplished with a modiﬁed streptavidin–biotin–peroxidase technique using an automatedimmunostainer(Benchmark,Ventana,Tucson,AZ);3 -3-diaminobenzidine was used as the chromogene and Harris’s hematoxylinasthecounterstain.Negativeandpositivecontrols wereincludedineachbatch.Phospho-Nf-kBp65Ser536,(93H1) (CellSignaling1:200)wasusedforNf-kBstaining,underthesame conditions.Percentageofpositivecellsandstainingintensity (neg-ative=0, weak=1, moderate=2, strong=3) were evaluated and multipliedtorenderthestainingscore(CLUstainingindexand Nf-kBstainingindex).InordertoﬁndthevalueofCLUstainingindex withthebestaccuracyinpredictingprognosis,weperformeda

ReceiverOperatingCharacteristic(ROC)analysisfortheCLU stain-ingindexinpredictingrecurrenceaftersurgicalresection(Fig.1).

Thevalueof40wasassociatedtothecut-offvalue.Infurther survivalanalysisandCLUcorrelation,weconsideredaCLUstaining indexmorethan40asdeﬁnitivelypositiveandavalueofNf-kB stainingindexhigherthan0asdeﬁnitivelypositive.

2.3. Follow-up

MostpatientswerefolloweddirectlyattheDivisionofThoracic SurgeryandDivisionofRespiratoryDiseasesoftheUniversityof ModenaandReggioEmiliawithperiodicofﬁcevisits.Information abouttheremaining patientswasobtainedfromthe Oncologic Institutes treating them or by telephone interviews with the patient and/or his/her relatives. Data regarding long-term sur-vival,presenceandtypeofrecurrencewererecorded.Weassigned apatientto“recurrentdisease”onlyafterhistological conﬁrma-tion,exceptforobviousclinicalsituations(widespreadmultiple lesions).

2.4. Statisticalanalysis

Thedescriptiveanalysiswasexpressedintermsoffrequency, mean,medianandstandarddeviation.Frequencieswerecompared withthechi-squaretestforcategoricalvariables;Fischer’sexact testwasusedforsmallsamples.T-testandANOVAwereperformed whencomparingcontinuousvariables.CorrelationbetweenCLU stainingindexandotherquantitativevariables wasinvestigated withthePearson’scorrelationcoefﬁcient;thegoodness-of-ﬁt mea-sureofeachlinearmodel(R2₎_were_reported._Correlation_between

CLUstainingindexandNf-kBstainingindexwasinvestigatedwith theSpearman’srankcorrelationcoefficient.Overallsurvival(OS) wascalculatedfromthedateofsurgicalresectiontothedateof deathorlastfollow-up.Disease-freesurvival(DFS)wascalculated fromthedateofsurgicalresectiontothedateofrecurrence.TheOS andDFSwerecalculatedaccordingtotheKaplan–Meiermethod. OverallSurvival(OS)andDFSratesat5yearswerechosenasend pointsfor evaluating theprognosis. Univariateanalysisfor any potentialprognosticfactorwasperformedusingtheLog-Ranktest. ACoxproportionalhazardsmodelwasfittedtoestimatehazard ratiosandconfidenceintervalsforthoseprognosticfactors signifi-cantinunivariateanalysis.Aprobabilityvalue(P)wasconsidered statisticallysignificantwhen<0.05;allPvalueswerebasedon two-sidedtests.

3. Results

Theresectedtumorsof83caucasianpatientswithpulmonary adenocarcinomawhounderwentradicalresectionwereanalyzed forCLUandNf-kBexpression.

Theclinicopathologiccharacteristicsofthepatientsarereported in Table 1. Mean age for all patients was 66.0±9.1 years (range 36–84). The mean smoking index was 20.2 pack/years (range0–60).

CLUimmunostainingwas consideredpositive in 63 patients (76%).ThemeanCLUstainingindexwas170±91.8(median140, range 50–270) for patient with BAC, 97.64±77.9 (median 70, range0–270)for patientswithadenocarcinomawithavariable BACcomponentand41.09±62.79(median9,range 0–270). Nf-kBimmunostainingwasconsideredpositivein48patients(57.8%). ThemeanNf-kBstainingindexwas3±6.7(median0,range0–15) forpatientwithBAC,13.52_±38.85(median0,range 0–135)for patientswithadenocarcinomawithavariableBACcomponentand 36.43±44.29(median20,range0–210).

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Fig.1.Receiveroperatingcharacteristic(ROC)analysisandinteractivedotdiagramfortheCLUstainingindexinrelationtorecurrence.Thevalueof40istheidealcutoff pointtodifferentiatepatientswhoexperiencerecurrenceaftersurgicalresection(value1)frompatientswhoremainfreeofdisease(value0).

3.1. CLUimmunostainingcorrelations

Thecorrelationsbetweenimmunohistochemicalexpressionof CLUandNf-kBwithclinical–pathologicalvariablesarereportedin Table2.

Asignificantassociation wasnotedbetweenCLUexpression andsex(P=0.024),histologicalgrading(P<0.001),presenceofBAC component(P=0.002) andpathologicstage(P=0.016).CLUwas expressedsignificantlymoreofteninwomen,althoughsmoking habitandagewerenotstatisticallysignificantbetweenmalesand females,inlow-gradeadenocarcinomaandinearlystagesof can-cerprogression. In particular,allpureBAC tumors significantly expressedCLU, whereas poorly differentiated adenocarcinomas frequentlydidnotexpressCLU.

Nf-kBwassigniﬁcantlymoreexpressedinhigh-grade(P=0.034) andinvasiveadenocarcinomaswithoutBACcomponent(P=0.028). Therefore, a highly signiﬁcant inverse correlation was found

Table1 Patients’characteristics. Variable No.(%) Sex Male 62(74) Female 21(26) Smokinghistory Yes 66(79) No 17(21) Typeofresection Lobectomy 77(93) Bilobectomy 2(2) Pneumonectomy 4(5) Pathologicstage IA 26(31) IB 42(50) IIA 3(4) IIB 12(15) Grading G1 12(14) G2 20(24) G3 51(62) Adenocarcinomasubtype Acinar 38(46) Mixed

WithBACcomponent 13(18)

Othermixed 4(4)

Papillar 8(10)

Solidwithmucinproduction 8(10)

Mucinous“colloid 3(3)

Signetring 2(2)

Fetaltype 2(2)

Clearcell 1(1)

PureBAC 4(4)

betweenCLUandNf-kBexpression:highlevelsofCLUexpression

wereassociatedwithlowlevelsofNf-kBexpression(Spearman’s

rho=−0.4282,P=0.0001)(Fig.2).

Tobetterclarifywhich clinical-pathologicparameters signif-icantly correlated with CLU expression, we also performed a bivariatestatisticalanalysis.TumorsthatexpressedaBAC compo-nent(pureBAC,andmixedadenocarcinomawithBACcomponent) were correlated with CLUstaining index, and a highly signifi-cantlinearcorrelationwiththepercentageofBACcomponentwas found.TheCLUstainingindexincreasedsignificantlywiththe per-centageofBACcomponent(Pearsoncorrelationcoefficient0.726; R2₌_0.527;_P₌_0.001).

Inthewholeseries,aninverselinearcorrelationbetweenCLU andsmokingindexapproachedsigniﬁcance(Pearsoncorrelation coefﬁcient −0.215; R2₌_0.046; _P₌_0.067). _Conversely, _no

signiﬁ-cantcorrelationwasfoundbetweentheCLUstainingindexand age(Pearsoncorrelationcoefﬁcient−0.086;R2₌_0.007;_P₌_0.440)

ortumorsize(Pearsoncorrelationcoefﬁcient−0.025;R2₌_0.001;

P=0.823).

3.2. Analysisofsurvival

Follow-upwasdiscontinuedforallpatientsinJuly2008.Median follow-upwas44.5months(3.7years)(range1–97months). Over-all5-yearsurvivalratewas50%withamediansurvivaltimeof61 months.Eightdeathswerenotcancerrelated;33patientsdiedof thedisease.TherateofDFSwas48%withamediandisease-free sur-vivaltimeof55months.Thetumorsrecurredin38patients(47.4%): 13patientswithintrathoracicrecurrenceand25patientswith sys-temicdissemination.Sixpatientswerealivewithrecurrentdisease attheendoffollow-up.Asigniﬁcantcorrelationwasfoundbetween thecruderecurrencerateandtheCLUstainingindex:29.4%for positiveCLUexpressionand59.2%for negativeCLUexpression; P=0.008.

Results of univariate analysisregarding OS and DFSfor the clinicopathologicprognosticfactorsareshowninTable3.Stage, grading,andCLUstainingindexpredictedbothOSandDFS,while sexpredictedonlyoverallsurvival.Nf-kBstainingindexdidnot predictOSneitherDFS.RegardingtheCLUstainingindex, univari-atesurvivalanalysisshoweda5-yearOSrateof72%forpatients withpositiveCLUexpressionand35%forpatientswithnegative CLUexpression(logrank9.92;P=0.001)anda5-yearDFSrateof 67%forpatientswithpositiveCLUexpressionand34%forpatients withnegativeCLUexpression(logrank8.49;P=0.003).

Whenwestratiﬁedtheanalysisofsurvivalbypathologicstage, theCLUstainingindex remaineda signiﬁcantprognosticfactor even at early stages (IA and IB): a 5-year OS rate of 73% for

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Fig.2.Hematoxylin–eosinstaining,clusterinstainingandNf-kBstaininginapuremucinous-typebronchioloalveolarcarcinoma(A,DandG,respectively),inapapillary adenocarcinoma(B,EandH,respectively)andinpartialcolonizationofanormalbronchioleby2fociofwell-differentiatedadenocarcinoma(seearrows)(C,FandI, respectively).

CLU-positivepatientsvs.42%forCLU-negativepatients(P=0.016), anda5-yearDFSrateof66%forCLU-positivepatientsvs.39%for CLU-negativepatients(P=0.021).

ToevaluatetheindependentprognosticvalueofCLUexpression, weperformedamultivariateanalysisusingtheCoxproportional hazardsmodelwiththevariablessignificantlyrelatedtosurvival byunivariateanalysis(Table4).TheCLUstainingindexwasfound tobetheonlyindependentfactorpredictingrecurrence,whereas pathologicstagewastheonlyprognosticfactorsignificantly associ-atedwithOS.ApositiveCLUstainingindexscorewassignificantly associated withlongerDFS (P=0.048);the associationwith OS approached statistical significance (P=0.052). Adenocarcinomas witha negativeCLUstaining indexscore had a relative risk of

recurrenceof2.18(95%confidenceinterval1.00–4.75)comparedto CLU-positivetumors;patientswithadenocarcinomahadarelative riskofdeathof2.39(95%confidenceinterval0.99–5.77). Early-stagedisease(IAandIB)predictedgreatersurvivalthanstageII disease(P=0.02).Neithertumordifferentiationnorsexhadany significantassociationwithdeath orrecurrence inmultivariate analysis.

4. Discussion

Toourknowledge,thisistheﬁrststudyexploringinvivothe existenceofacorrelationbetweenNf-kBandCLUexpressionin lungcancer.

Table2

AssociationbetweenclusterinstainingindexandNf-kBstainingindexandclinicopathologicvariables.

Variable Clusterinstaining P Nf-kBstaining P

Positivea _Negativea _Positivea _Negativea

Sex Male 21(34%) 41(66%) 0.024 37(60%) 25(40%) 0.615 Female 13(62%) 8(38%) 11(52%) 10(48%) Grading G1 10(83%) 2(17%) 0.001 4(%) 8(%) 0.034 G2 11(55%) 9(45%) 9(%) 11(%) G3 13(25%) 38(75%) 35(%) 16(%) Histologicalsubtype BAC 4(100%) 0 0.002 1(25%) 3(75%) 0.028 ADK-BAC 9(69%) 4(31%) 4(31%) 9(69%) Othersubtypes 21(32%) 45(68%) 43(65%) 23(35%) Pathologicstage IA+IB 32(47%) 36(53%) 0.016 40(83%) 8(17%) 0.776 IIA+IIB 2(13%) 13(87%) 8(53%) 7(47%)

Abbreviation:ADK-BAC,adenocarcinomawithBACcomponent.

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Table3

Resultsofunivariateanalysisofoverallsurvivalanddisease-freesurvival.

Variable Overallsurvival Disease-freesurvival

5years(%) Pvalue 5years(%) Pvalue Sex Male 42 0.013 42 0.118 Female 71 62 Grading G1 83 0.026 90 0.016 G2 58 54 G3 37 35 Histologicalsubtype BAC+ADK-BAC 57 0.158 61 0.159 Othersubtypes 45 45 Pathologicstage 0.001 76 IA 79 36 0.024 IB 42 21 IIA+IIB 28

CLUstainingindex

Positive 72 0.001 67 0.003

Negative 35 34

NFKBstainingindex

Positive 45 0.990 42 0.662

Negative 52 55

Abbreviation:ADK-BAC,adenocarcinomawithBACcomponent.

OurdatasuggestthatCLUexpressionisanindependent

pro-gnosticfactorinsurgicallyresectedadenocarcinomaofthelung.

MultivariateanalysisshowedthatlowCLUexpressionis

signiﬁ-cantlyrelatedtoahighrateofrecurrenceanddeathaftersurgical

resection.Giventheestablishedprognosticroleofpathologicstage

inlungcancer,wealsoperformedasubgroupanalysisstratiﬁed

fordiseasestage:patientswithstageIdiseasewhohadhigh

lev-els of CLU expression showed signiﬁcantly higher OS and DFS

withrespecttopatientswiththesamestagewithlowlevelsof

CLU.

ThehighlysigniﬁcantinversecorrelationfoundbetweenCLU

andNf-kBexpressionis consistentwithwhat isalreadyknown

abouttheinhibitoryactivityofCLUonNf-kBactivityandexpression

[15].However,althoughNf-kBisknowntobeinvolvedininitiation andcancerprogression,therearenostudiesshowingacorrelation betweenNf-kBandcancerOSandDFS.

Inarecentstudy,Dimitrakopoulosandcolleaguesshowedthat cytoplasmicimmunoreactivityofNF-␬B2andRelBwascorrelated withtumorstage.Inaddition,cytoplasmicNF-␬B2levelswerealso relatedtotumorgradeandexpressionofRelBin thecytoplasm wastumorhistologictype-speciﬁc,withsquamouscellcarcinomas havingthehighestproteinlevels[17].Incontrast,inourstudyCLU servesasabiomarkerforOSandDFS.

Similarﬁndings werereportedbyAlbert et al.,who investi-gatedCLUexpressionlevelsinpatientswithdifferenthistologic typesofNSCLC[10].Incontrast,wefocusedourstudyon early-stage(stagesIandII)adenocarcinomainordertoinvestigatethe possibleinvolvementofCLUinearlystagesofcancerprogression.

Inaddition,thisistheonlyhistologictypeoflungcancerwhose incidenceisincreasing,accordingtoepidemiologicalstudies[18]. Moreover,ourseriesincludedaselectgroupofpatientswhowere raciallyhomogeneous,beingallcaucasian,becauseofthedifferent characteristicsofadenocarcinomas[19].

Theprognostic andpredictive[20] value ofCLUhasalready beenevaluatedinvariousmalignancies,butconflictingresultshave beenreported[6–9,21–24].ConflictingdataonthelevelsofCLUin lungcancerhavealsobeenfound.Invitroandinvivostudieshave reportedthepresenceofhighlevelsofCLUinlungcancercells [25].However,dataavailableontheOncominedatabaseshowed thatCLUwasdownregulatedintumorsascomparedtonormallung (www.oncomine.org).DownregulationofCLU,andafurther reduc-tionofitsexpressionduringcancerprogression,canalsobegleaned fromtheresultsofthepresentstudy.CLUexpressionsignificantly decreasedfromwell-differentiatedtopoorlydifferentiated adeno-carcinoma,andfromstageItostageII.Thisdataandthesurvival analysesconfirmthepositiveprognosticroleofCLUinlung adeno-carcinoma.TheearlierfindingsofhighlevelsofCLUinlungcancer cells[25]isnotcontradictorywithourexperimentaldata,because CLUwasindeedpositivelyexpressedin41%ofadenocarcinomas fromourselectpopulation.

Hereweinvestigatedforthefirsttimethecorrelationbetween CLUexpressionandadenocarcinomasubtypes,inparticularthose thatexpressaBACcomponent.WefoundBACinonlyfourpatients (4.8%).However,upto20%ofinvasiveadenocarcinomasofthelung showedamixedpattern,rangingfrompredominantBACwitha smallfocusofinvasion,toinvasiveadenocarcinomawithan iso-latedgroupofcellswithBACfeatures[18,26].Inourseries,mixed adenocarcinomaswithaBACcomponentaccountedfor16%ofall adenocarcinomas.IncaseswithpureBAC,CLUwasexpressedat highlevels,whereasonly30%ofcasesofinvasiveadenocarcinoma expressedhighlevelsofCLU.Inmixedadenocarcinomas,witha variableamountofBACcomponent,CLUexpressionwas interme-diate,beingpresentin69%ofcases;furthermore,CLUexpression wasdirectlyrelatedtotheamountofBACcomponent,decreasing withtheincreasedinvasivenessofthelesions.Themolecularevents thatregulatethemultistepcarcinogenesis–hypothesizedinlung adenocarcinoma–arecurrentlyunknown,butmultiplepathways andgeneticchangesarecertainlyimplicated[27–31].The progres-sivedecreaseof CLUexpressionisclearly oneof themolecular changesimplicatedintheprogressionfrompureBACtoinvasive adenocarcinoma,andconfirmthepossibleroleofCLUasatumor suppressorfactoragainstinfiltrationorinvasiveness.Infact,CLU hasbeenalreadyfoundasanimportantregulatorofcellfate, neg-ativelycontrollingfurtherprogressionandinvasiveness[12,32].

Tangetal.[11]showedthatNf-kBexpressionisanearlyand fre-quentfeatureoflungcancer.Furthermore,Nf-kBexpressionlevel wassigniﬁcantlyhigherinadvancedadenocarcinomasofthelung comparedtoearlierstages[12].

InverselinearcorrelationofCLUandNf-kBexpressionis consis-tentwithinvitrostudiesdemonstratingandimportantregulatory

Table4

Resultsofmultivariateanalysis(CoxproportionalHazardModels).

Variable Death Recurrence

HR(95%CI) Pvalue HR(95%CI) Pvalue

CLU Negativevs.positive 2.39(0.99–5.77) 0.052 2.18(1.00–4.75) 0.048 Grading G3vs.G1+G2 1.40(0.64–3.07) 0.393 1.59(0.73–3.48) 0.237 Pathologicstage IIA+IIBvs.IA+IB 2.26(1.10–4.65) 0.026 1.19(0.52–2.68) 0.675 Sex Malevs.female 2.02(0.92–4.40) 0.077 1.62(0.74–3.58) 0.221

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loopbetweenthesetwogenes,i.e.whileCLUisoneofthemost

highlyregulatedgenebyNf-kB,CLUalsoregulatesNf-kBactivity

inanegativemannerbystabilizingI-kBs.Thesedataledtothe

hypothesisthatCLUparticipatesinanegativeloopinwhich

trans-criptionalactivationof CLUis evokedtodampenNf-kBactivity

[14].

Inconclusion,CLUexpressionisanindependentpositive pro-gnosticfactorinsurgicallyresectedadenocarcinomaofthelung. CLUexpressioncouldthereforebeusedasanovelbiomarkerfor identifyingpatientswithstageIB adenocarcinomawho require chemotherapeutic treatment after surgical resection. The pro-gressivedecrease inCLUexpression fromwell-differentiatedto poorlydifferentiatedadenocarcinomas,alongwiththeoncogenetic progressionfrompureBAC,tomixedadenocarcinomawithBAC component,toinvasivecarcinoma,confirmapositiveprognostic role forCLU inpatients withlung adenocarcinoma.Finally, we observedaninversecorrelationbetweenNf-kBandCLU,thus pro-vidingnewinsightsintothepotentialroleinpathogenesisofthese proteinsduringlungcancerprogressionandfinaloutcome. Conflictofinterest

Nonedeclared. Acknowledgments

We acknowledge Paul Wegener (Epitome Pharmaceuticals Limited,Halifax,NovaScotia,Canada)forhelpfulsuggestions,Elisa Veratelli(UniversityofModenaandReggioEmilia,Modena,Italy) andMaryMcKenney(Menominee, MI49858, USA)for editorial assistancewiththepreparationofthemanuscript.

SupportedbyunrestrictedgrantsfromRegioneEmiliaRomagna (Italy);AssociazioneperloStudiodeiTumoriedelleMalattie Pol-monari(ASTMP,Padova,Italy); ConsorzioFerraraRicerche(CFR, Ferrara,Italy);andAssociazioneperlaRicercaelaCuradell’Asma (A.R.C.A.,Padova,Italy).

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