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CIRCULATINGDBPLEVELANDPROGNOSISINOPERATEDLUNG
CANCER:ANEXPLORATIONOFPATHOPHYSIOLOGY
AMTurner1,LMcGowan1,AMillen2,PRajesh2,CWebster2,GLangman2,GRock2,ITachibana3,
MGTomlinson4,FBerditchevski5andBNaidu6
1SchoolofClinicalandExperimentalMedicine,UniversityofBirmingham,B152WB,UK
2BirminghamHeartlandsHospital,HeartofEnglandNHSFoundationTrust,B95SS,UK
3DepartmentofRespiratoryMedicine,AllergyandRheumaticDiseases,OsakaUniversity
GraduateSchoolofMedicine,2‐2Yamada‐oka,Suita,Osaka,Japan
4SchoolofBiosciences,UniversityofBirmingham,B152TT,UK
5SchoolofCancerSciences,UniversityofBirmingham,B152TT,UK
6WarwickMedicalSchool,UniversityofWarwick,Coventry,CV47AL,UK
Correspondingauthor:BNaidu,addressasabove,[email protected]
Telephone:02476523523 Fax:01214242200
Keywords:Lungcancer;vitaminD;epidemiology;prognosis
Wordcount=3353(excludingtablesandlegends)
. Published on May 3, 2012 as doi: 10.1183/09031936.00002912ERJ Express
Copyright 2012 by the European Respiratory Society.
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ABSTRACTBackground:VitaminDstimulatestranscriptionofanti‐angiogenicandapoptoticfactorsthat
maysuppresstumours,whilstvitaminDbindingprotein(DBP)maybeabiomarkerinmurine
lungcancermodels.WesoughttoascertainifthevitaminDaxisisalteredinlungcanceror
influencesprognosis.
Methods:148lungcancerpatients,68otherintrathoraciccancerpatientsand33non‐cancer
controlswerestudiedforupto5years.CirculatingDBPandvitaminDlevelswerecompared
betweengroupsandtheireffectonsurvivalassessedbyCoxregressionanalysis.Expressionof
DBPandvitaminDreceptor(VDR)wasexaminedinlungcancercelllinesandinnormaland
tumourlungtissuebyWesternblotandimmunohistochemistry.
Results:LowserumDBPlevelspredictedlungcancerspecificdeath(p=0.04),andDBPwas
poorlyexpressedinlungcancercellsonWesternblotandimmunohistochemistry.VitaminD
didnotpredictcancersurvival,andVDRexpressionwasvariableintumours.
Conclusions:PreservationofserumDBPisasignificantindependentfactorassociatedwith
bettercanceroutcomeinoperatedlungcancerpatients.GiventheestablishedroleofDBPin
macrophageactivationandclearanceofabnormalcellsfurtherstudyonitsinvolvementinlung
cancerismerited.
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INTRODUCTIONVitaminDisafatsolublevitaminbestknownforitsroleincalciumandphosphate
homeostasis.ItisalsoincreasinglyapparentthatVitaminDhasbeneficialhealtheffectsbeyond
theskeletalsystem.Serumconcentrationofcholecalciferol(vitaminD3;25OHD3)isthebest
indicatorofvitaminDstatusasitreflectscutaneousproductionaswellasthatintakeinfoods
andsupplements,whereas1,25‐Dihydroxycholecalciferol(1,25(OH)2D3)hasashorthalflife
andserumconcentrationsaretightlyregulated[1].VitaminDstatushasbeenreportedto
correlatewithcancerrisk,andplayaroleinthepreventionofcolon,prostateandbreast
cancers[2],althoughlessisknownaboutitsinfluenceonlungcancer.Noneoftheavailable
epidemiologicalworkhasbeenabletodeterminethelevelofriskconferredbyvitaminD
deficiency,becauseofconfounderssuchasobesityandamountofsunlightexposure.
VitaminDmaysuppresstumourprogressionbyreducingcellproliferation,invasivenessand
angiogenesis,andstimulatingapoptosis[2‐4].Italsoprotectsagainstmetastasesinvarious
tumourmodels,includingthelung[2‐4].InorderforvitaminDtoexertitsintracellulareffectsit
mustentercellsbydiffusionorbyendocytosiswhenboundtoitsmaincarrierprotein,vitamin
Dbindingprotein(DBP).Whenintracellular,vitaminDisdissociatedfromDBPandthen
undergoesaseriesofreactionsthatenableinteractionwiththevitaminDreceptor(VDR)–a
processillustratedinourpreviouswork[5].ThereissomesuggestionthatVDRexpressionis
reducedinlungcancer[6],implyingthatvitaminDwillbelessabletoexertitsanti‐tumour
effects,suchthatotheraspectsofthevitaminDaxiscouldbemoreimportant.
DBPisaglycosylatedalphaglobulin,partofthealbuminsuperfamily,beingabout58kDain
size,producedintheliverandlocatedpredominantlyinserum.Itisdividedinto2large
domains(IandII)andashorterdomainattheCOOHterminus(domainIII)[7],andisexpressed
inmanytissues[8]andbyneutrophils[9].Itcontributestomacrophageactivation[10],
augmentsmonocyteandneutrophilchemotaxistoC5‐derivedpeptidesandactsasanactin
scavengerprotein,asdiscussedinourrecentreview[5].Itmayplayaroleinmalignancy
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becauseofitseffectonmacrophages,whichareimportantbecauseoftheirpotentialtoclear
abnormaltissue[11].Indeedinlungcancer,thenumberofcytotoxicmacrophageswithinthe
tumourpredictssurvival[12].ThevitaminDaxismayoptimiseanti‐tumouractionsof
macrophagesintwoways.Firstly,DBPcanbeconvertedbyde‐glycosylationtoapotent
macrophageactivatingfactor(DBP‐MAF)[10].ThusintissuewhereDBPispoorlyexpressed,or
poorlyconvertedtoDBP‐MAF,macrophageactivationwillbesub‐optimal.Littleisknownabout
DBPexpressioninlungtissue,althoughwehavedemonstratedpreviouslythatDBPispresentin
airwaysecretions[13].Secondly,macrophagescanconvert25OHD3to1,25(OH)2D3[14],thus
optimizingdownstreameffectsonanti‐tumourgenetranscriptioninthesecells.
WehypothesizedthatthevitaminDaxismaybealteredinlungcancerandrelateadverselyto
prognosis:thismaybeduetoeithervitaminDdeficiency,inabilityoftumourtissuetorespond
tovitaminD,orreducedmacrophageactivationbyDBP‐MAFinandaroundtumours.
METHODSPrognosticeffectofserummarkersofthevitaminDaxis
PatientswererecruitedconsecutivelyfromthoracicsurgerylistsatHeartofEnglandNHS
FoundationTrust(HEFT)between2006and2009.Serumsamplesweretakenatseveraltime
points,aspartoftheCLUBstudy,aprospectivestudyofpotentiallungcancerbiomarkerswhich
isdescribedpreviously[15].Thecurrentprojectwasasub‐study,andonlythosewithpre
operativesamplesremainingwereselected.Thisgaveatotalof148lungcancerpatients,68
otherintrathoracictumoursand33non‐cancercontrols.Demographicfeatures,tumour
histologyandpathologicalstage,surgerytype,resectionmargins,smokeexposureandco‐
morbiditywererecorded.Pathologicalstagingwastakentobethegoldstandardandhasbeen
updatedtoreflectthelateststagingguidancefornon‐smallcelllungcancer(NSCLC)[16].Lung
cancerpatientswerefollowedforupto5yearsandsurvivalassessedusingCancerIntelligence
data.Thestudywasapprovedbythelocalethicscommitteeandallpatientsgaveinformed
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consent.VitaminD(25OHD3)wasmeasuredbytandemmassspectrometryatHEFT.DBPwas
measuredbyspecificELISA(Immunodiagnostik).
AssessmentofVDRandDBPinlungcancersandnormallung
Tumourandnon‐tumourlungtissuewasobtainedfrom25patientsundergoingresectionat
HEFTbetween2009and2010.Afterresectionlungsweretakenimmediatelytothepathology
departmentforinflationwith10%formalinatconstant25cmH2Opressureviacannulationof
themajorairway,andonceinflated,wereimmersedinformalinfor24hours.Representative
blocksofnormallungdistantfromthetumourandtumourblockswereselectedbyasingle
pathologistforsubsequentstaining.BothVDRandDBPwerestainedusingtheVentana
BenchmarkXTsystemwithultraviewtechnology.Theprimarymousemonoclonalantibodies
usedwereanti‐VDR(D‐6):SC‐13133(SantaCruzBiotechnologyInc,USA),andanti‐DBPA0021
(Dako,UK).Stainingwithbothantibodiesinvolveda30minuteCC1antibodyretrievalstep,
followedby32minutesantibodyincubation.TheVDRantibodywasdiluted1:100andDBP
1:10000.Anadditional4minutehaematoxylincounterstainwasusedintheanti‐DBPprotocol.
TheVDRprotocolwasadaptedfromthatpublishedforlungtissue[6]andtheDBPprotocol
fromthatpublishedforkidneytissue[17].Positivecontrolswerekidneytissue(VDR)andliver
(DBP)respectively;positivestainingwasdeterminedbyapathologistusingstandardsemi‐
quantitativetechniqueswhichgradeintensityofstaining[18].
AssessmentofVDRandDBPinlungcancercelllinesandnormallungtissue
Cancercelllinesweredescribedinourpreviousworkandculturedasindicatedtherein[19].
NCI‐231wasoriginallygiftedtoITbyDrYShimosataoftheNationalCancerResearchInstitute
inJapanin2003.A549s,NCI‐H292andNCI‐H69werepurchasedfromtheAmericanType
CultureCollection,USA,andauthenticatedatsourcein2003.Lu65andLu99werepurchased
fromtheRikenBioresourceCellCenterJapan,andauthenticatedatsourcein2003.HARAwere
purchasedfromtheHealthSciencesResearchResourceCenter,Japan,againin2003.Allcells
weretestedpriortotheexperimentshereinforN‐CAMexpressionbyflowcytometry(either
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positiveornegative–datanotshown)andMycoplasma(allnegative),asdescribedinour
previouswork[19].
Cellswerelysedinabuffercontaining1%TritonX‐100,0.1%SDS,1mMEDTA,10mM
Tris/HCl,pH7.5,150mMNaCl,0.01%sodiumazideandaproteaseinhibitorcocktail(Sigma).
TheproteinconcentrationsoflysatesweredeterminedusingtheDetergentCompatibleProtein
Assay(Bio‐Rad),and45µgreducingsampleswereseparatedonSDS‐polyacrylamidegels.
ProteinwastransferredtoPVDFmembranesandprobedwithchickenanti‐DBP,rabbitanti‐
VDRormouseanti‐tubulinantibodies(allSigma).ForDBPandtubulinblots,secondary
antibodieswereIRDye800CW‐conjugatedforvisualizationusingtheOdysseyInfraredImaging
System(LI‐COR).ForVDRblots,thesecondaryantibodywashorse‐radishperoxidase‐
conjugated(ThermoScientific)forvisualizationusingPierceECLchemiluminescencereagents
(ThermoScientific)andHyperfilm(AmershamBiosciences),whichwasdevelopedusingafilm
processor(AGFACurix60).
Statisticalanalysis
AllanalyseswerecarriedoutinSPSSversion16.0(Chicago,USA).Clinicaldatanormalitywas
assessedusingtheKolmogorov‐Smirnovtest(normal,p>0.05);parametricdataisreportedas
mean(SEM)andnon‐parametricdataasmedian(range).Thet‐testwasusedtocomparemeans
ofparametricdataandtheMannWhitneyorKruskalWallisfornon‐parametricdatabetween
groups.FrequencyvariableswerecomparedusingtheChisquaredtest.Bonferronicorrection
formultipletestswasusedfortheseanalysesmeaningthatunadjustedoverallpforsignificance
was0.01.AmultivariateCoxregressionanalysiswascarriedoutforsurvivalofthoseNSCLC
caseswithclearresectionmarginsusingage,gender,smokeexposure,histologicaltypeand
cancerstage,plusDBPorvitaminDlevelaspredictors.DBPwasassessedinquartiles(4
groups),ratherthanasacontinuousvariable.AllcomparisonsofvitaminDtookintoaccount
seasonofcollection,asdescribedinourpreviouswork[13].Statisticalsignificancewas
assumedatp<0.05intheabsenceofBonferronicorrection.
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RESULTSPrognosticeffectofserummarkersofthevitaminDaxis
Characteristicsofthepatientsareshownintable1.NoneweretakingprescribedvitaminD
supplementswhenadmittedforsurgery.Therewerenosignificantdifferencesbetweenthetwo
cancergroups(allp>0.05).Non‐cancercontrolswereyounger,morelikelytobemaleandhad
beenfollowedupforfeweryears(p=0.03,<0.01and0.02respectively).
Lungcancer
N=148
Othercancer
N=68
Non‐cancercontrols
N=33
Age 69.7(1.5) 66.6(54‐80) 54.5(33‐88)
Malegender 57(38.5) 22(32.4) 24(72.7)
Packyearssmoked 50.0(5‐120) 60.0(30‐100) 43.3(0‐70)
Currentsmoker 57(38.5) 9(13.2) 1(3.0)
Neversmoked 7(4.7) 3(4.4) 4(12.1)
Cancerdeath 33(22.3) ‐ ‐
Otherdeath 23(15.5) ‐ ‐
Yearsoffollowup 4.3(1.5‐5.3) 4.5(1.8‐5.7) 2.38(1.4‐5.0)
DBP(mg/dl) 33.7(1.5) 35.9(2.4) 45.5(5.1)
Cholecalciferol(ng/ml) 38.5(1.6) 15.7(2.0) 30.8(3.0)
Albumin(g/l) 37.6(3.8) 33.4(27.2‐39.6) 41.3(2.7)
Table1:Characteristicsofthepatients
Frequencydataisshowninboldtypesandislistedn(%).Forthequantitativedata,whereit
wasnormallydistributedmean(SE)isshownandfornon‐normaldatamedian(range).
Thehistologyofthelungtumours,andpathologyoftheotherpatientgroupsisshowninFigure
1.Squamouscellcarcinomaswerethemostfrequentlungtumour,whilstoesophagealcancers
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formedthemajorityoftheothercancers.6patientshadsmallcelllungcancerandwere
excludedfromfurtheranalyses.AmongsttheNSCLCcasespathologicalstagesweredistributed
asfollows:Ia=27patients;Ib=45;IIa=11;IIb=10;IIIa=22;IIIb=7.In20casesnewstagingcould
notbedeterminedfromthepathologyreportduetothelevelofdetailgiven.Amongsttheother
cancersalloesophagealpatientswerestageIIaorIIb,allmesotheliomapatientswerestageII
andofthe2lymphomapatientsonewasstageIIandonestageIII.
Cholecalciferolvariedwithseasonofcollection,asexpected,althoughthisdifferencewas
marginalandunlikelytobeofclinicalsignificance(Supplementarydata).Afteradjustmentfor
thisittendedtobehigherinlungcancerthannon‐cancerouslungdisease,althoughthiswasnot
statisticallysignificant(38.5v30.8ng/ml;p=0.06).InothercancersvitaminDwaslower
(15.7ng/ml;p<0.01).ThisisshowninFigure2a.Frequenciesofthe3usualclassesofvitaminD
levelinthelungcancerpatientsareshowninFigure2b.DBPwaslowerinlungcancerpatients
thannon‐cancerouslungdisease(33.7v45.5mg/dl;p=0.02)butdidnotdifferfromother
cancers(35.9mg/dl;p=0.72).ThisisalsoshowninFigure2a.DBPandcholecalciferoldidnot
correlatewithoneanother(p=0.62).Albumindidnotvarysignificantlybetweengroups(both
p>0.32);therewasnosignificantcorrelationbetweenthisandcholecalciferol(p=0.72)orDBP
(p=0.24).
Allpatientshadundergoneatleast12monthsoffollowupatthetimeofdataanalysis.One
yearsurvivalwas79.1%.Ofthosethatdiedduringtheirfollowupperiodthemediantimeto
deathwas0.93years(range0‐3.54years);whenonlycancerrelateddeathswereselectedmean
timetodeathwas1.04years(range0‐3.45years).Survivalatthemeanofcovariates,excluding
DBPandvitaminD,isshowninFigure3a,sub‐stratifiedforquartilesofDBPinFigure3b.Stage,
ageandpackyearssmokedwereallsignificantpredictorsofdeath(p=0.039,0.005and0.009
respectively),whilstgenderwasnot(p=0.96).IntheallcausemortalityanalysisneitherDBP
nor25OHD3predicteddeath(bothp>0.30).Whenonlydeathssecondarytolungcancerwere
consideredDBPbecameapredictor(p=0.041),theoddsratioofdeathfallingto0.95(0.91‐0.98)
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foreachunitgainedinDBP.Toputthisintocontext,alungcancerpatientexhibitingDBPlevel
equivalenttothatofourhealthycohortwouldhaveanORofdeathof0.59comparedtoa
patientwiththemeanDBPlevelseeninourcohort.FurtherdetailsoftheDBPanalysesare
shownintable2;thewideCIsreflectthesmallnumbersofdeaths.
DBPquartile DBPrange(mg/dl) Deaths(n/total) OR(95%CI) p
4 >43.02 3/32 ‐ ‐
3 33.25‐43.01 9/35 5.49(0.62‐48.52) 0.125
2 19.95‐33.24 7/31 5.77(0.59‐56.64) 0.133
1 <19.94 7/34 10.4(1.03‐125.42) 0.044
Table2:RelationshipofquartilesofDBPtolungcancerspecificdeathintheNSCLCcases
ThetableshowstherangeofDBPvaluesineachquartile(numberedindescendingorder,such
that4isthehighest),thenumberineachquartilewhodiedalungcancerspecific
death(n)/numberofindividualsinthatquartile,andtheoddsratioformortalitycomparedto
thehighestquartile.
AlbuminwasalsoassessedasapredictorinordertoascertainiftheDBPeffectwasspecific;
albuminwasnotsignificant(p=0.38).Cholecalciferoldidnotpredictlungcancerdeath(p=0.52).
AssessmentofVDRandDBPinlungcancersandnormallung
InnormallungVDRwasexpressedmoststronglyinbronchialepithelium,withlesserstainingin
pneumocytes(Figure4a).OnlyonetumourexhibitedabsentVDRexpression,howeverhalf
exhibitedlessintensestainingthanthenormallungtissuefromthatindividual(Figure4b).In
normallungDBPwaspresentpredominantlyinbloodandairwaysecretionswithlessintense
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staininginmacrophages(Figure4c).IngeneraltumourtissueonlystainedpositiveforDBPin
necroticareasandassociatedmacrophages,elsewhereitexhibitedintensityhalfthatofairway
secretions(Figure4d).16%oftumoursshowednoDBPexpression.Consistentwithrelatively
lowexpressionofVDRandDBPinlungtumours,eightlungcancercelllinesexhibitedlowor
absentexpressionoftheseproteinsbywesternblotting,comparedwithpositivecontrolblotsof
fournormallungsamples(Figure5).Normallungwasusedapositivecontrolbecauseofthe
detectionoftheseproteinsinlungsections(Figure4).
DISCUSSIONWehaveshownthatlowserumDBPbeforesurgerymaybeapredictorofsubsequentdeath
fromlungcancer,andthatexpressionofDBPiseitherloworabsentinlungcancertissue.This
supportsapathogenicroleforDBPinlungcancer,whichismostlikelytocentreonitsroleasa
precursorforDBP‐MAF,basedonitslocationonmacrophagesinnormallung,andinnecrotic
areasintumours.ItseemslikelythatDBPisnotproducedextensivelybylungtissue,but
diffusesfrombloodtoairwaysecretionsandtissuefluidsgiventhatlittlestainingwasobserved
inanyprimarypulmonarycellsinthenormallungsamples,andtheWesternblotsfromcell
linesshowednoexpression.Thismayexplainwhyaserummarkerwascapableofpredictinga
lungspecificoutcome.SmallamountsofDBPexpressionbynormallungandtumoursremainsa
possibility.PrognosticmarkersinlungcancerincludeERCC1,EGFR,RRM1andKRAS,although
mostofthesearebettervalidatedasmarkersintumourthancirculatingblood[20].Arecent
reviewoflungcancerbiomarkersnotedthatmanyofthestudieslookingatsuchbiomarkers
andsurvivalhavebeenconductedretrospectivelyonsamplescollectedduringclinicaltrials,
suchthattheirroleinpredictingresponsetotherapyratherthanoutcomeperseisbetter
known[20].ThehazardratioforthelowestquartileofDBPwassimilartothatconferredby
highlevelsofcirculatingcancercellsinarecentstudyof101patientswithstageIIIorIVNSCLC
[21].
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ThelinkbetweenDBPandlungcancerhasnotbeenstudiedindetail;onestudyofcirculating
DBPlevelsshowednodifferencebetweencancerandhealthyindividuals[22].Howeverthe
techniquesformeasuringDBPusedinthisstudyweremuchlesssensitivethanthecurrent
ELISA,andthestudyitselfwasnotspecifictolungcancer,comprisingatotalof100cases,split
betweenlung,prostateandgastrointestinalmalignancies. Morerecently,proteomicworkina
mousemodeloflungcancersuggestedthatDBPactsasadiseasebiomarker[23].DBPis
regulatedatatranscriptionallevelbypro‐inflammatorycytokinesandsteroids[24],andcould
potentiallyrelatetonutritionandcatabolicstates,ratherlikealbumin,sinceitisinthesame
familyofproteins.Wedidnotshowanyrelationshipofsurvivaltoalbuminlevels,butcannot
excludeanepiphenomenonlinkingDBPtoanotherunmeasuredpoorprognosticfactor
influencingourDBPmortalityanalyses.
PreviousworkhasshownthatconversionofDBPtoDBP‐MAFmaybereducedinmalignancy
duetotheactionofα‐N‐acetylgalactosaminidase[25].Duringtumourinvasionvariouscellsin
canceroustissuesproduceexo‐andendoglycosidases[26]andifthelatterenterthe
bloodstreamtheyarecapableofdeglycosylatingcirculatingDBP,aprocesswhichappearsto
relatedirectlytotumourburdeninamurinemodel[27].OurdatashowsthatDBPislowinthe
bloodoflungcancerpatients.ThusevenifDBPdeglycosylationisnotinvolved,macrophage
activationmaybelower,adverselyaffectingprognosis.AugmentationofDBP‐MAFhasbeen
proposedasadjuvanttherapyinsurgicallyresectedcancersforthesereasons;indeedincolonic
andprostatecancersDBP‐MAFimmunotherapyusedinthiswaywassafeandwelltoleratedin
earlyphasetrials[28‐29].DBP‐MAFhasalsoshownbeneficialeffectsonbreastcancercellsin
vitro[30].TheseconceptsrequirefurtherfollowupbeforetrialsofDBP‐MAFwouldbe
appropriateinlungcancer,butprovideanexcitingnewavenueforresearch.Specifically,amore
extensiveanalysisontheexpressionofDBP‐MAFandthemechanismsofdeglycosylationinlung
tissuewouldberequiredinthefuture.
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Cholecalciferoldidnotpredictoutcomeinoursurvivalanalysis.Fewstudieshavebeendone
examiningvitaminDstatusspecificallyinlungcancer.Zhouetal.investigatedtheassociation
betweensurgeryseasonandvitaminDintakewithrecurrence‐freesurvival(RFS)andoverall
survival(OS)in456early‐stageNSCLCpatients.Theyconcludedthatthejointeffectofseason
andintakeareassociatedandhigher25OHD3levelscorrelatedwithimprovedOSandRFS[31].
Inourstudylevelswerehigherinsummer,althoughthedifferenceswereunlikelytobe
clinicallysignificant;aspecificsurvivalanalysisaccordingtoseasonofsurgerywasnotcarried
outforthisreason.AsthemainsourceofvitaminDissynthesisintheskinfollowingsun
exposure,severalstudieshaveinvestigatedseasonalandgeographicalvariationincancerrisk
andsurvival[32‐33].Onesuchstudyinvestigatedtheimpactofseasonofdiagnosisand
residentialregionontheriskofdeathfromlungcancerinNorwegianlungcancerpatients[34].
ResultssuggestedthatvitaminDstatusatlungcancerdiagnosisisofprognosticvalueandthat
cancermortalitydecreaseswithincreasingsunexposure[34].Ourresultsareindirectcontrast
tothesestudies,perhapsbecauseofdifferencesinthestudycohorts.Firstlylessthan20%of
patientsweredeficientinvitaminD(Figure2b).Secondlyweshowedthatmosttumours
exhibitedlowerVDRexpressionthannormalepithelialtissue.Thismeansthatthetumours
wouldbelessresponsivetovitaminD,thuspreventingitsanti‐tumouractivities.Our
immunohistochemistryresultsconcurwithalargerstudyontheexpressionofVDRinnormal,
premalignantandmalignantbronchialtissue[6].Furthermore,theyarealsoconsistentwith
geneticepidemiologyworkwhichshowsthatVDRpolymorphismswhichleadtolessVDR
function,areassociatedwithmalignancyingeneral[35].Thisobservationechoessmallerlung
cancerstudieswhichhaveshownthattheVDRFokIpolymorphismisassociatedwithworse
survivalinNSCLC[36‐37],whilsttheTaqIpolymorphisminfluenceslungcancerrisk,itseffect
beingmodifiedbyage,genderandsmokinghabit[38].Itisalsopossiblethatunmeasured
confounders,suchasbodyweight,couldhavehadaninfluenceonourresults.
Ourstudyislimitedtosurgicallyresectedcases,whichledtorelativelysmallnumbersforthe
survivalanalyses;neverthelessthecohortremainscompetitiveinthefieldforitssizeand
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degreeofcharacterization.Theproportionoffemalepatientsishigherthantheaverage,and
manycaseswerequiteadvancedonpathologicalstaging(stageIIIaorb)whichmayreducethe
abilityoftheresultstobegeneralizedtootherpatientcohorts.Wedidnotformallyaccountfor
adjuvanttherapyuseinouranalyses,sinceonly3patientsreceivedit–giventhelownumbers
wefeltitwouldbeuninformativetodoso,butacknowledgethatthereisasmallchancethis
couldaffectresults.ThestudyisalsothefirsttoreportDBPimmunohistochemistryinthelung.
WewereunabletoconfirmthelocationofDBPonmacrophagesbyco‐localisationofDBPand
CD68stains,duetoahighlevelofbackgroundstainingintheduallystainedimages[datanot
shown,availableonrequest],althoughmanyoftheslidesshowmorphologicallythatthe
stainingisonthiscelltype.Wecorrectedouranalysesformultipletests,andacknowledgethat
itisonlytheunadjustedpvalueforDBPthatreachessignificance,since4quartilesweretested.
However,giventhemarkeddifferenceinsurvivalinthisgroup,andthefunctionaldatawe
presenttosupportourfindingsthereremainspotentialforclinicalsignificance.
InsummarywehaveshownthatlowcirculatingDBPmaypredictpoorprognosisinNSCLC,
whichwehypothesiseisbecauseofitsroleasaprecursortoDBP‐MAF.Theresultsrequire
independentreplicationandassessmentinlargercohortsbeforewecanbecertainofthe
validityofDBPasaprognosticmarker.Ifourresultsarevalidatedbyothergroupsfurther
researchtodetermineifDBP‐MAFmaybeausefultherapyinthefuturecouldbewarranted.
Acknowledgements
TheauthorswouldliketothankstaffinthedepartmentofthoracicsurgeryatHeartofEngland
NHSTrustwhocontributedtotissuecollection.WearealsogratefultoJingYangandVera
Novitskayafortheirhelpwithcultureoflungcelllines.
Funding
14
AMTissupportedbytheWestMidlandsChestFundandCancerResearchUK.LMissupported
bytheSocietyforEndocrinology.MGTissupportedbyaSeniorFellowshipfromtheBritish
HeartFoundation.FBissupportedbyCancerResearchUK.BNissupportedbyTheHealth
FoundationandtheMidlandsLungTissueCollaborative.Nofunderhadanycontributiontothe
design,conductoranalysisofthework.
Statementofcontribution
AMTconceivedthestudy,collectedthetissueforimmunohistochemistry,performedstatistical
analysesanddraftedthemanuscript.AMperformedlaboratoryworkandfollowupdata
collection.LM,CW,GR,IT,YJandMTperformedlaboratorywork.SRandBNcollectedserum
samplesandbaselinepatientdata.GLpreparedalllungsamplesbyinflationandanalysedall
immunohistochemistry.FB,MGTandBNalsoreviewedthemanuscript,andBNsupervisedthe
work.
Conflictofintereststatement
Theauthorshavenorealorperceivedconflictsofinteresttodeclare.
15
FIGURELEGENDSFigure1:Pathologicalfindingsinthepatients
Thebarchartshowsthepathologicalfindingsinthethreepatientgroups.Themajorityofthe
lungcancercaseswereeithersquamousoradenocarcinomas,withsmallernumbersofsmall
cell,largecell,bronchoalveolarcellandmixedcellularitytumours.Themajorityoftheother
intrathoraciccancerswereoesophageal,whilstthebulkofthenon‐cancercaseswerebenign
nodules.
Figure2:ComponentsofthevitaminDaxisincancerandnon‐cancerpatients
(a) Thebarchartshowsmean(SEM)vitaminDandDBPlevelsinthe3groups.VitaminD
didnotdifferbetweenlungcancerandnon‐cancerpatients(p=0.06),butwas
significantlylowerintheotherintra‐thoracicmalignancies(p<0.01).DBPwaslowerin
16
lungcancerthannon‐cancerpatients(p=0.02)butdidnotdifferfromothercancers
p=0.72).
(b) ThepiechartshowsclinicalcategoriesofvitaminDlevelinthelungcancerpatients.The
majorityoflungcancerpatientsweresufficientinvitaminD.
Figure3:Survivalinthelungcancerpatients
(a) ShowssurvivalfromtheCoxregressionanalysesatthemeanofallcovariates,beforethe
additionofDBPorvitaminDtothemodel.
(b) Showssurvivalatthemeanofcovariates,sub‐stratifiedbyDBPlevel.Thetopline(4.00)
showsthehighestquartileofDBP,whilst3,2and1representdescendingquartilesof
DBP,withcorrespondinglylowersurvival.
17
Figure4:VDRandDBPexpressioninlungtissue
(a) VDRstainsstronglyinbronchialepithelium,seenat10xmagnification(top)andmore
stronglythanadjacenttumourtissuewhenseenat40x(bottom).
(b) VDRgenerallyexhibitedlessintensestainingintumourtissue;2tumoursareshownat
20xand40x(bottom)magnification.
(c) DBPisseeninblood(top)andairwaysecretions(bottom);10xmagnification.
(d) DBPisseenonmacrophages(top)andatlowintensityinanadenocarcinoma(bottom);
both40xmagnification
18
Figure5:DBPandVDRexpressionbyWesternblottinginlungcancercelllinesandlung
lysates
TheblotsshowDBP,VDRandtubulin(control)expressioninlungcancercelllines(A549to
NCI‐N231)andnormallung(lung1‐4).ThescaleontheleftisinkDa.DBPandVDRwere
universallyexpressedbywholelunglysates.InthecancercelllinesDBPwasexpressedvery
weaklyinsomelines,andabsentinmost.,whilstVDRwasnotexpressed.
19
20
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