Diabetes and Breast Cancer Subtypes

16 Pages·2017·0.99 MB·English

RESEARCHARTICLE
Diabetes and Breast Cancer Subtypes
HeleenK.Bronsveld1,2,VibekeJensen3,PernilleVahl3,MarieL.DeBruin4,
StenCornelissen1,JoyceSanders5,AnssiAuvinen6,JariHaukka7,MortenAndersen8,
PeterVestergaard9,MarjankaK.Schmidt1,2*
1 DivisionofMolecularPathology,TheNetherlandsCancerInstitute,Amsterdam,Netherlands,2 Divisionof
PsychosocialResearchandEpidemiology,TheNetherlandsCancerInstitute,Amsterdam,Netherlands,
3 DepartmentofPathology,AarhusUniversityHospitalTHG,Aarhus,Denmark,4 Divisionof
Pharmacoepidemiology&ClinicalPharmacology,UtrechtUniversity,Utrecht,Netherlands,5 Divisionof
Pathology,TheNetherlandsCancerInstitute,Amsterdam,Netherlands,6 SchoolofPublicHealth,University
ofTampere,Tampere,Finland,7 DepartmentofPublicHealth,UniversityofHelsinki,Helsinki,Finland,
a1111111111 8 DepartmentofMedicine,KarolinskaInstitute,Stockholm,Sweden,9 ClinicalInstitute,AalborgUniversity,
a1111111111 Aalborg,Denmark
a1111111111
*[email protected]
a1111111111
a1111111111
Abstract
OPENACCESS Background
Citation:BronsveldHK,JensenV,VahlP,DeBruin Womenwithdiabeteshaveaworsesurvivalafterbreastcancerdiagnosiscomparedto
ML,CornelissenS,SandersJ,etal.(2017)
womenwithoutdiabetes.Thismaybeduetoadifferentetiologicalprofile,leadingtothe
DiabetesandBreastCancerSubtypes.PLoSONE
developmentofmoreaggressivebreastcancersubtypes.Ouraimwastoinvestigate
12(1):e0170084.doi:10.1371/journal.
pone.0170084 whetherinsulinandnon-insulintreatedwomenwithdiabetesdevelopspecificclinicopatho-
logicalbreastcancersubtypescomparedtowomenwithoutdiabetes.
Editor:AamirAhmad,UniversityofSouthAlabama
MitchellCancerInstitute,UNITEDSTATES
MethodsandFindings
Received:August31,2016
Thiscross-sectionalstudyincludedrandomlyselectedpatientswithinvasivebreastcancer
Accepted:December28,2016
diagnosedin2000–2010.Stratifiedbyageatbreastcancerdiagnosis((cid:20)50and>50years),
Published:January11,2017
womenwithdiabeteswere2:1frequency-matchedonyearofbirthandageatbreastcancer
Copyright:©2017Bronsveldetal.Thisisanopen diagnosis(bothin10-yearcategories)towomenwithoutdiabetes,toselect~300patients
accessarticledistributedunderthetermsofthe
withtumortissueavailable.TumorMicroArrayswerestainedbyimmunohistochemistryfor
CreativeCommonsAttributionLicense,which
permitsunrestricteduse,distribution,and estrogenandprogesteronereceptor(ER,PR),HER2,Ki67,CK5/6,CK14,andp63.A
reproductioninanymedium,providedtheoriginal pathologistscoredallstainsandrevisedmorphologyandgrade.Associationsbetweendia-
authorandsourcearecredited.
betes/insulintreatmentandclinicopathologicalsubtypeswereanalyzedusingmultivariable
DataAvailabilityStatement:Therawdatacannot logisticregression.Morphologyandgradewerenotsignificantlydifferentbetweenwomen
besharedduetorestrictionsasdescribedin withdiabetes(n=211)andwomenwithoutdiabetes(n=101),irrespectiveofmenopausal
DanishLaw(Persondataloven:https://www.
status.PremenopausalwomenwithdiabetestendedtohavemoreoftenPR-negative(OR=
retsinformation.dk/forms/r0710.aspx?id=828).The
anonymizeddatacanonlybeanalyzedonthe 2.44(95%CI:1.07–5.55)),HER2-negative(OR=2.84(95%CI:1.11–7.22)),andbasal-like
serverofStatisticsDenmark.Datarequestsshould (OR=3.14(95%CI:1.03–9.60)tumorsthanthewomenwithoutdiabetes,withnon-signifi-
followtheofficialprocedureofstatisticsDenmark;
cantlyincreasedfrequenciesofER-negative(OR=2.48(95%CI:0.95–6.45))andtripleneg-
seehttp://www.dst.dk/en/TilSalg/Forskningsservice
ative(OR=2.60(95%CI:0.88–7.67)tumors.AfteradjustmentforageandBMI,the
andneedsapprovalfromStatisticsDenmark.
associationsremainedsimilarinsizebutlesssignificant.Weobservednoevidencefor
Funding:Theresearchleadingtotheresultsofthis
associationsofclinicopathologicalsubtypeswithdiabetesinpostmenopausalwomen,or
studyhasreceivedfundingfromtheEuropean
Community’sSeventhFrameworkProgramme withinsulintreatmentingeneral.
PLOSONE|DOI:10.1371/journal.pone.0170084 January11,2017 1/16
DiabetesandBreastCancerSubtypes
(FP-7)undergrantagreementnumber282526,the Conclusions
CARINGproject.Thefundingsourcehadnorolein
Wefoundnocompellingevidencethatwomenwithdiabetes,treatedwithorwithoutinsulin,
studydesign,datacollection,dataanalysis,data
interpretationorwritingofthereport. developdifferentbreastcancersubtypesthanwomenwithoutdiabetes.However,premeno-
pausalwomenwithdiabetestendedtodevelopbreasttumorsthatdonotexpresshormonal
CompetingInterests:Theauthorshadno
competinginterests. receptors,whicharetypicallyassociatedwithpoorprognosis.
Introduction
Diabetesmellitusandbreastcancerarechronicdiseaseswithincreasingincidenceinmany
countries[1,2].Recentestimatesindicatethatdiabetesprevalenceis9.1%amongwomenin
Europe[1]andlife-timeriskforbreastcanceris9.7%[3].Mostpatientswithdiabetes(~90%)
havetype2disease,characterizedbyreducedinsulinsecretionandinsulinresistancewith
diagnosisinlateadulthood,whilepatientswithtype1diabetesareinsulindeficient[4].
Severalstudieshaveinvestigatedwhetherdiabetesand/orinsulin(analogue)treatment
increasebreastcancerrisk[5–10]oraffectprognosis[11–18],becauseoftheirpotentialimpact
ontumorprogressionthroughe.g.theinsulin-likegrowthreceptorpathway[5,19].Women
withdiabeteshavea15–20%increasedriskofbreastcancercomparedtowomenwithoutdia-
betes[6–9],butnoimpactofinsulinanaloguetreatmenthasbeenshown[5].Breastcancerin
womenwithdiabetesisoftendiagnosedatanadvancedstagecomparedtowomenwithout
diabetes[13,14,20–22].Moreover,overallmortalityafterbreastcancerdiagnosishasbeen
showntobe30–60%higherinwomenwithdiabetescomparedtowomenwithoutdiabetes
[11–16],evenafteradjustmentfortumorstage[13,14,16].However,studiesthatinvestigated
theassociationbetweenbreastcancer-specificmortalityanddiabetesshowinconsistentresults
[11,23–27].
Diabetesitselfmighthaveadirecteffectonbreastcancerprognosisduetophysiological
effectsofhyperglycemia,orhyperinsulinemia,whichisahallmarkofinsulinresistancecom-
monlyobservedinpatientswithtype2diabetes[28,29].Ithasbeenshownthatcancer-specific
survivalwasdecreasedforwomenwithabnormalglycemicstatus[25,27]andthatfastinginsu-
linlevelsareassociatedwithworseoutcome(distantrecurrenceanddeath),independentof
BodyMassIndex(BMI)[30].However,diabetesitselfanditscomplicationsmayalsoincrease
riskofoverallmortality[4]andsharedcancer-promotingfactorsinpatientswithdiabetes,
suchasobesityandasedentarylifestyle,increasesalsotheriskofdeathfromcompetingcauses
(metabolic/cardiovasculardiseases).
Anotherreasonfortheworsebreastcancersurvivalmaybethatwomenwithdiabetes
developamoreaggressiveorlesstreatment-responsivetumorsubtype.Ithasalreadybeen
shownthathormone-relatedbreastcanceranddiabetesriskfactors,suchasobesity,areassoci-
atedwiththedevelopmentofER-negativebreastcancersubtypes[31,32].Insulininteracts
withestrogens;thereisexperimentalsupportthatinsulinmayenhanceestrogenproduction,
stimulatingthedevelopmentofER-positivebreastcancer[19].Furthermore,thepromotionof
tumorcellgrowthuponinsulinexposuremaydifferbybreastcancersubtype;weknowfrom
invitrostudiesthatmitogenicpotentialofinsulinsdependsonthetypeofbreastcancercell
line[5,33].Althoughbreastcancersubtypeshavebeenextensivelystudiedinthegeneralpopu-
lation[31],fewstudieshaveassessedbreastcancersubtypesinwomenwithdiabetes.
Theaimofthisstudyistodeterminewhetherbreastcancerpatientswithdiabeteshavea
specificclinicopathologicaltumorsubtypecomparedtothosewithoutdiabetes,andwhether
theuseofinsulinisrelatedtothis.
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DiabetesandBreastCancerSubtypes
Methods
ThestudyprotocolwasapprovedbytheScienceEthicsCommitteeoftheRegionMidtjylland
inDenmark.TheScienceEthicsCommitteeoftheRegionMidtjyllandinDenmarkapproved
thatinformedconsentforthisstudywasnotobtained;however,allwomenhadthepossibility
toopt-outfromresearchthroughthenation-wideregistry.Tumortissueofthewomenhad
beencollectedfordiagnosticortherapeuticpurposesaroundthetimeofbreastcancerdiagno-
sis.Thistissueisstoredinbiobanksandmaybeusedforresearch(‘secondaryuse’)aslongas
codedandanonymoustotheresearcher.Notissuewasusedagainstthewillofthepatients
(womenwhoopt-outwithregardtotissueuseforfuturescientificpurposeswereexcluded
(http://sundhedsdatastyrelsen.dk/da/registre-og-services/vaevsanvendelsesregisteret);norisk
wasposedtothewomenasthetissuehadalreadybeenremoved;andtumortissueanddata
wereanonymousfortheresearcher.
Studydesignandpatientselection
ThestudypopulationconsistsofCaucasianwomenwithandwithoutdiabetes,diagnosedwith
primarybreastcancerbetween2000and2010.Thebreastcancerpatientswereselectedfroma
previouslyestablishednation-widehospital-basedcohort,bytheDanishBreastCancerCoop-
erativeGroup(DBCG)[34].ThiscohortwaslinkedtotheNationalPatientRegisterinDen-
marktoidentifywomenwithandwithoutdiabetes,coveringtheyearssince1977.Intotal,
43,701womenwerediagnosedwithincidentbreastcancerin2000–2010intheDBCG,of
whom3,047haddiabetes(7.0%).Weusedacross-sectionalstudydesignwitharandomly
selectedtargetpopulationof300breastcancerpatients.Theselectedwomenincludedbreast
cancerpatientswithdiabetes(exposed)andwithoutdiabetes(non-exposed)sampledasfol-
lows:arandomsampleofwomenwithdiabetesinstrataofage(cid:20)50and>50years(1:1)at
breastcancerdiagnosis(stratificationbyagetoincreasethenumberofyoungwomen)fre-
quencymatchedwithwomenwithoutdiabetesfromthesamedatabase(1:2)byyearofbirth
andageatdiagnosis(bothin10-yearcategories)(Fig1).Twiceasmanywomenwithdiabetes
wereincludedaswomenwithoutdiabetestoallowanalysesbyinsulintreatment.Patientswith
ahistoryofothercancers,non-invasiveormetastasizedbreastcancer,thosetreatedwithneo-
adjuvanttherapy,patientswithdiabetesdiagnosed(cid:20)1yearpriortotheirbreastcancerdiagno-
sis,andpatientswithnoorinsufficienttumortissuewereexcluded.
Datacollection
Age,menopausalstatus,yearofbreastcancerdiagnosisandinformationontumorandtumor
treatmentwereobtainedfromtheDBCGdatabankandthepathologyregisterofthewomen.
Onlyage,yearofbreastcancerdiagnosis,anddiabetesstatuswereavailableatthetimeof
patientidentification.Diabetesstatus,diabetestype(1or2),andageatdiabetesdiagnosis,as
wellasdataonsocioeconomicstatuswerecollectedbylinkagewiththeNationalPatientRegis-
ter(whichincludedallmedicaldiagnosesfrom1977onwards)inDenmark.Dataonmedica-
tionuse,availablefrom1995onwards,wasobtainedbylinkagewiththeDanishRegisterof
MedicinalProductsStatistics.Alllinkagesweredoneusingcodeswhichrenderthedataanon-
ymoustotheresearcherswhodonothavedirectaccesstothesesourcedatabases.Women
weredefinedasoralcontraceptiveorhormonereplacementusersifatleast2prescriptionsof
thedrugwereprescribedcumulativelyintheperioduptooneyearpriortobreastcancerdiag-
nosis.Additionalinformationonheight,weight,BodyMassIndex(BMI),smoking,alcohol
use,andHbA Clevels(measureofaverageglucoselevels)priortobreastcancerdiagnosis
1
wereretrievedfromelectronicpatientfilesandanonymizedbeforeinclusioninthedatabase
fortheresearchers.Formalin-fixed,paraffin-embeddedtissuesamplesoftheprimarytumors
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DiabetesandBreastCancerSubtypes
Fig1.Flowchartofpatientidentificationandselection.Stratifiedbyageatbreastcancerdiagnosis((cid:20)50and>50years),womenwithdiabeteswere
2:1frequency-matchedonyearofbirthandageatbreastcancerdiagnosis(bothin10-yearcategories)towomenwithoutdiabetes,toselect~300patients
withtumortissueavailable.ǂExactnumbers<5cannotbeshownaccordingtoregulationsofStatisticsDenmark.
doi:10.1371/journal.pone.0170084.g001
wereretrievedfromdifferentDepartmentsofPathologyinDenmark,forcentralpathology
reviewandimmunohistochemical(IHC)analyses.
TumorreviewandIHCanalyses
Allformalin-fixed,paraffin-embeddedtumorsblocksoftheprimarytumorofeachpatient
werecollectedandwholeslideswerestainedwithHematoxylinandEosin.Themostrepresen-
tativetumorblockwasselectedfortheanalyses.HematoxylinandEosinslideswerereviewed
byabreastpathologistformorphologyandgrade(VJ).Gradewasscoredfollowingthemodi-
fiedBloom-Richardsonsystem.
FortheIHCanalysis,tissuemicroarrayswith2coresof2mmpertissueblockwerecon-
structed.Tissuemicroarrays3μsliceswereplacedonsuperfrost+glassslides,andstainedand
scoredforER,PR,HER2,Ki67,CK5/6,CK14,andp63.HER22+tumorswereevaluatedusing
SISH(SilverInSituHybridization).ScoringoftheIHCstainingwasperformedbyabreast
pathologist(VJ).A10%cut-offwasusedtodefineapositivestainingforallmarkers,except
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Ki67:lowif(cid:20)14%andhighif>14%accordingtotheStGallenguidelinesof2013[35],and
HER2:negativeif0/1+andpositiveif2+(SISHconfirmed)/3+.Tumorsweredefinedasbasal-
likeifatleastoneoutofthreebasalmarkers(CK14,CK5/6,P63)werepositive.Wealsoclassi-
fiedthetumorsusingtheStGallenguidelinesof2013usingER,PR,HER2,andKi67[35,36].
Diabetestreatmentclassification
DiabetesstatuswasdeterminedbasedonmedicaldiagnosisfromtheNationalPatientRegister.
Diabetesdurationwasdefinedastimefromageofdiabetesdiagnosistillageofbreastcancer
diagnosis.Womenwithdiabeteswereclassifiedasinsulinusersifatleast2prescriptionsof
insulinwereprescribedcumulativelyintheperioduptooneyearpriortobreastcancerdiag-
nosis.Exposuretimewasdefinedastimefromageofstartofinsulintillageofbreastcancer
diagnosis.Forwomentreatedwithothernon-insulinantidiabeticdrugs,thesamemethodwas
used.Womenwithdiabetestreatedwithinsulinonlywereconsideredpatientswithtype1dia-
betes,iftheyhadarecordeddiagnosisoftype1diabetes(n=21),oramedicalcodewasmiss-
ingbuttheywereunderage30yearsatdiabetesdiagnosis(n=4).Allotherwomenwith
diabeteswereconsideredtype2.
Imputation
Forwomenwithunknownmenopausalstatus(n=5),ageover52years[37]wasusedasa
proxyforpostmenopausalstatus.MissingvaluesforBMI(n=51inwomenwithdiabetes,
n=42inwomenwithoutdiabetes)wereimputedusingMultivariateImputationsbyChained
Equations[38]inRstudiowithapredictivemeanmatchingregressionmodelforeachana-
lyzeddataset,imputingvariableswithascendingnumberofmissingvalues;numberofimputa-
tions=10,numberofiterations=25;see(S1Table).Weassumedthatdatawasmissingat
randomandcouldbeimputedbecauseofcorrelationswithothervariables(S2andS3Tables).
VariablesderivedfromtheDBCG,i.e.,ageofbreastcancerdiagnosis,yearofbreastcancer
diagnosis,menopausalstatus(foranalysesinallwomen),breastcancertreatment;theelec-
tronicpatientfiles,i.e.,smoking,alcohol,height,weight,HbA Clevels;theNationalPatient
1
Registry,i.e.,diabetestype,diabetesduration,cardiovasculardisease,microvasculardisease,
income,education;theDanishRegisterofMedicinalProductsStatistics,i.e.,diabetesmedica-
tion,hormonereplacementtreatmentandoralcontraceptionuse;anddataonbreastcancer
characteristicsandclinicopathologicalsubtypes.Inthesubsequentanalyses,weonlyincluded
thevariablesrelevantforthepredictionofclinicopathologicalsubtype,i.e.age,menopausal
status,smoking,alcohol,BMI,HbA C,diabetesduration,oralcontraceptionuseandhormone
1
replacementtreatment.
Statisticalanalyses
Patientandbreastcancercharacteristicsatdiagnosiswerecomparedbetweenbreastcancer
patientswithandwithoutdiabetesusingchi-squaretests.Multivariablelogisticregression
modelswereusedtoestimatetheassociationbetweendiabetesstatusorinsulintreatmentwith
primarybreastcancerclinicopathologicalsubtypes.Weconstructedseparatelogisticregres-
sionmodelsforeachexposure(diabetesorinsulin)toevaluatetumorsubtype(variousdefini-
tions)asmodel-specificoutcomes.Multinomiallogisticregressionmodelswereusedfor
tumorsubtypeswhichconsistedof>2categories.Wetestedforheterogeneitybetweeninsulin
andnon-insulinusersinanalysisrestrictedtodiabetespatientsonly.Intheanalysescompar-
ingwomenwithandwithoutdiabetes,potentialcovariateswereaddedinaonebyone-step-
wisemanner;however,noneofthecovariateschangedthebeta-estimatefordiabeteswith
>10%foranyofthesubtypeclassifications,exceptforBMIintheanalysisofPRstatusand
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ER-/PR-inpremenopausalwomen.Nonetheless,wearealsoshowingadjustedmodelswith
breastcancersubtypesforageandBMI,becausepreviousliteraturehasshownassociations
betweenage,BMIandbreastcancersubtypes[31].Modelsforgradewereadjustedforage
only.
Modificationsoftheassociationsbetweendiabetesstatusandbreastcancersubtypesby
menopausalstatus,BMI,anddiabetestypewereassessedusinginteractionsterms.Although
wefoundnostatisticallysignificantinteractionsbetweenmenopausalstatusanddiabetessta-
tus(thelowestp-valuewas0.07intheanalysesofPR),weshowresultsforpre-andpostmeno-
pausalwomenseparatelybasedonpreviousevidencefordifferentriskprofiles[31].To
excludepotentialbiasbytheinclusionsofwomenwithtype1diabetesweperformedasensi-
tivityanalysisexcludingwomenwithtype1diabetes.Moreover,explorativeanalyseswereper-
formedwithinwomenwithtype1andtype2diabetes.SASEnterpriseguide4.2forWindows
wasusedforstatisticalanalyses.
Results
Thiscross-sectionalstudyconsistedof211womenwithdiabetesand101womenwithoutdia-
betes,alldiagnosedwithbreastcancerandwithtumortissueavailable(Fig1).Breastcancer
patientswithdiabeteshadasimilardistributionofmenopausalstatus(asaresultoftheage-
stratifiedselection),butweremoreoftenobese(BMI(cid:21)30)(p<0.0001),comparedtothose
withoutdiabetes(Table1).Themajorityofwomenwithdiabetes(88.2%)werediagnosedwith
type2diabetesandthemeandiabetesdurationwas8.9years(S3Table).Twenty-fivepercent
(n=53)ofthewomenwithdiabetesweretreatedwithinsulin;including18combinedwith
non-insulinantidiabeticdrugs.Thenon-insulinusersweretreatedwithnon-insulinantidia-
beticdrugs(35%)ordiabeteswascontrolledbydietandexerciseonly(40%)(S3Table).The
meandurationofinsulinusewas8.4years(S3Table).Insulinusers(47%type1diabetes
women)weremoreoftenpremenopausalcomparedtonon-insulinusers(p=0.04);andinsu-
linuserswithpremenopausalbreastcancerhadlowerBMIcomparedtothosenottreatedwith
insulin(p=0.0003)(S4Table).
Associationbetweendiabetesandclinicopathologicalbreastcancer
subtypes
Breastcancerpatientswithdiabeteshadasimilardistributionofmorphology,tumorsize,and
numberofpositivelymphnodescomparedtothosewithoutdiabetes(Table1);alsoifstratified
formenopausalstatus(S5Table).
PremenopausalbreastcancerpatientswithdiabeteshadmoreoftenPR-negative
(OR=2.44(95%CI:1.07–5.55),p=0.03),HER2-negative(OR=2.84(95%CI:1.11–7.21),
p=0.03),andbasal-like(OR=3.14(95%CI:1.03–9.60),p=0.05)tumorsthanthosewithout
diabetes,withnon-staticallysignificantincreasedfrequenciesofER-negative(OR=2.48(95%
CI:0.95–6.45))andtriplenegative(OR=2.60(95%CI:0.88–7.67)tumors(Table2andS6
Table).AfteradjustmentforageandBMI,theassociationsremainedsimilarinsizebutlesssta-
tisticallysignificant.Wefoundnostatisticallysignificantassociationsbetweendiabetesstatus
andgradeorKi67,norusingthemorerefinedSt.Gallensubtyping(Table2andS6Table).We
foundnomodificationofbreastcancersubtypebyBMIordiabetestype.Sensitivityanalyses,
inwhichwomenwithtype1diabeteswereexcluded,resultedinhazardratiosofthesame
directionandsimilarsize(S7Table).Wedidnotfindanassociationbetweenanyoftheclini-
copathologicalbreastcancersubtypesanddiabetesinpostmenopausalwomen(Table2).In
analysesincludingallwomen,weonlyfoundstatisticallysignificantmorebasal-liketumorsin
womenwithdiabetescomparedtothosewithout(OR=2.39(95%CI:1.07–5.35),p=0.03).
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DiabetesandBreastCancerSubtypes
Table1. Characteristicsofbreastcancerpatientswithandwithoutdiabetes.
Womenwithbreastcancer
Diabetes(n=211) NoDiabetes(n=101) Pd
Age,median(IQrange)a,b
(cid:20)50years 47.0(43.0–50.0) 47.0(43.0–50.0)
>50years 67.0(60.0–75.0) 67.0(62.0–73.0)
%(n) %(n)
Yearofbreastcancerdiagnosesa
2000–2002 12.8(27) 6.9(7)
2003–2004 15.6(33) 16.8(17)
2005–2006 17.5(37) 33.7(34)
2007–2008 27.5(58) 18.8(19)
2009–2010 26.6(56) 23.8(24)
Menopausalstatusb 0.57
Pre 52.1(110) 48.5(49)
Post 47.9(101) 51.5(52)
BMIinkg/m2c
Premenopausalwomen 0.0002
<25(normal) 30.3(27) 46.7(14)
(cid:21)25(overweight) 24.7(22) 50.0(15)
(cid:21)30(obese) 44.9(40) <5(<5)ǂ
Postmenopausalwomen 0.005
<25(normal) 22.5(16) 55.2(16)
(cid:21)25(overweight) 38.0(27) 31.0(9)
(cid:21)30(obese) 39.4(28) <14(<5)ǂ
Morphology 0.54
Ductal 75.8(160) 70.3(71)
Lobular 7.6(16) 10.9(11)
Other 16.6(35) 18.8(19)
Tumoursizeinmm
(cid:20)20 57.8(122) 57.4(58) 0.54
21–50 36.5(77) 39.6(40)
>50 5.7(12) <5(<5)ǂ
Numberofpositivelymphnodes 0.50
0 50.3(102) 54.0(54)
1–3 32.5(66) 26.0(26)
>3 17.2(35) 20.0(20)
Grade 0.03
Grade1 20.3(41) 19.0(19)
Grade2 35.6(72) 51.0(51)
Grade3 44.1(89) 30.0(30)
ER 0.08
Positive 77.6(163) 86.1(87)
Negative 22.4(47) 13.9(14)
PR 0.17
Positive 64.4(136) 72.3(73)
Negative 35.6(75) 27.7(28)
HER2 0.07
Positive 10.5(22) 17.8(18)
(Continued)
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Table1. (Continued)
Womenwithbreastcancer
Diabetes(n=211) NoDiabetes(n=101) Pd
Negative 89.5(187) 82.2(83)
aMatchingvariable,
batbreastcancerdiagnosis,
cclosestmeasurepriortobreastcancerdiagnosis,
dChi-squaretest.Missingvaluesarenotshown,thereforethesumofthecategoriesdoesnotadduptothetotalnumberofpatientsforBMI,positivelymph
nodes,grade,ERandHER2.
ǂExactnumbers<5withpercentagescannotbeshownaccordingtoregulationsofStatisticsDenmark.
IQ=interquartilerange,BMI=BodyMassIndex.
doi:10.1371/journal.pone.0170084.t001
Associationbetweeninsulintreatmentandclinicopathologicalbreast
cancersubtypes
Tumormorphology,tumorsizeandnumberofpositivelymphnodesdidnotdifferbetween
womenwithdiabetestreatedwithorwithoutinsulin(S4Table);similarresultswerefoundin
analysesstratifiedformenopausalstatus(datanotshown).
Weobservednostatisticallysignificantevidenceforthedevelopmentofpoorprognosis
tumorsamonginsulinusers(Table3andS8Table).Premenopausalwomenwithdiabetesnot
usinginsulinweremorelikelytodevelopER-negative(OR=3.06(95%CI:1.30–7.20),p=0.01)
andPR-negative(OR=2.98(95%CI:1.11–8.00),p=0.03)comparedtowomenwithoutdiabe-
tes,whileORsforERandPR-negativetumorsininsulinuserscomparedtowomenwithout
diabeteswereonlyslightlyincreased(Table3andS8Table).Weperformedexplorativeanaly-
sesseparatelyintype1andtype2insulin-treatedpremenopausalwomenwithdiabetestrying
tounderstandthesedifferencesbetweeninsulinandnon-insulinusers.Theassociations
betweendiabetesandtumorsubtypesamongtype1diabetesinsulinusersweremoreinline
withthefindingsinthenon-insulinusers(e.g.poorprognosistumors),whileweobserveda
suggestionthattype2diabetesinsulinusershadbetterprognosistumors(S8andS9Tables).
However,overall,therewasnoevidenceforastatisticallysignificantheterogeneitybetween
insulinandnon-insulinusersforanyoftheclinicopathologicalsubtypesintheanalyses
restrictedtobreastcancerpatientswithdiabetes(Table3).Inaddition,adjustmentforageand
BMIdidnotmateriallychangetheeffectestimatesortheir95%confidenceintervals(S8and
S10Tables).Inpostmenopausalwomen,weobservednoassociationofinsulin,withbreast
cancersubtypes(Table3).Wedidnothaveenoughpowertoincludesubtypesusingthemore
refinedStGallencriteriaintheanalysesstratifiedbymenopausalstatus.Inanalysesincluding
allwomen,wefoundsignificantlymorebasal-liketumors(OR=2.5(95%CI:1.09–5.74),
p=0.03)andER-/PR-negativetumors(OR=1.99(95%CI:1.00–3.95),p=0.05)innon-insulin
userscomparedtowomenwithoutdiabetes.
Discussion
Wefoundnocompellingevidencethatwomenwithdiabetesdevelopdifferentclinicopatho-
logicalsubtypescomparedtowomenwithoutdiabetes.However,premenopausalbreastcancer
patientswithdiabetestendtodevelopbreasttumorsthatdonotexpresshormonalreceptors
andbasal-liketumors,whicharetypicallyassociatedwithpoorprognosis.Themajorityofthe
womeninourpopulationhadtype2diabetesmellitus,sotheresultsaremostapplicablefor
thesepatients.Wealsofoundnostrongevidencethatinsulintreatmentisassociatedwith
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Table2. Crudeandadjustedoddsratiosforbreastcancerclinicopathologicalsubtypesofwomenwithdiabetescomparedtowomenwithoutdia-
betesinsubgroupsofmenopausalstatususing(multinomial)logisticregression.
Premenopausalwomenwithbreastcancer
Dependentvariable Independentvariableofexposure
Diabetesvs.NoDiabetes Diabetesvs.NoDiabetes
crudeOR(95%CI) P adjustedOR*(95%CI) P
Grade2(vs.grade1) 0.56(0.22–1.42) 0.22 0.56(0.22–1.42) 0.22
Grade3(vs.grade1) 1.02(0.40–2.61) 0.97 1.08(0.41–2.86) 0.88
ER-(vs.ER+) 2.48(0.95–6.45) 0.06 2.32(0.86–6.31) 0.10
PR-(vs.PR+) 2.44(1.07–5.55) 0.03 2.18(0.92–5.17) 0.07
HER2-(vs.HER2+) 2.84(1.11–7.22) 0.03 2.94(1.08–8.02) 0.04
Highki67(vs.lowki67) 1.23(0.62–2.42) 0.55 1.17(0.53–2.58) 0.70
Basal-likea(vs.non-basal-like) 3.14(1.03–9.60) 0.05 3.11(0.98–9.86) 0.05
ER+/PR-(vs.ER+/PR+) 2.10(0.55–7.96) 0.28 1.77(0.43–7.18) 0.42
ER-/PR-(vs.ER+/PR+) 2.67(1.02–7.00) 0.05 2.46(0.90–6.75) 0.08
LuminalB-like,HER2-c(vs.luminalA-likeb) 1.15(0.47–2.82) 0.76 1.05(0.40–2.73) 0.92
HER2+d(vs.luminalA-like) 0.46(0.17–1.23) 0.12 0.41(0.14–1.20) 0.10
Triple-negativee(vs.luminalA-like) 2.60(0.88–7.67) 0.08 2.21(0.71–6.69) 0.17
Postmenopausalwomenwithbreastcancer
Dependentvariable Independentvariableofexposure
Diabetesvs.NoDiabetes Diabetesvs.NoDiabetes
crudeOR(95%CI) P adjustedOR*(95%CI) P
Grade2(vs.grade1) 0.80(0.32–2.04) 0.65 0.80(0.31–2.03) 0.64
Grade3(vs.grade1) 1.97(0.72–5.39) 0.19 1.97(0.72–5.39) 0.19
ER-(vs.ER+) 1.27(0.52–3.14) 0.60 1.33(0.52–3.40) 0.55
PR-(vs.PR+) 0.96(0.48–1.93) 0.92 1.06(0.51–2.19) 0.88
HER2-(vs.HER2+) 1.15(0.43–3.13) 0.78 1.20(0.40–3.59) 0.75
Highki67(vs.lowki67) 1.11(0.56–2.22) 0.77 1.06(0.52–2.18) 0.87
Basal-likea(vs.non-basal-like) 1.62(0.50–5.29) 0.43 1.73(0.51–5.91) 0.38
ER+/PR-(vs.ER+/PR+) 0.79(0.33–1.87) 0.59 0.89(0.36–2.19) 0.79
ER-/PR-(vs.ER+/PR+) 1.20(0.48–3.04) 0.69 1.29(0.49–3.39) 0.60
LuminalB-like,HER2-c(vs.luminalA-likeb) 0.65(0.29–1.44) 0.29 0.58(0.25–1.35) 0.21
HER2+d(vs.luminalA-like) 0.79(0.28–2.26) 0.66 0.88(0.28–2.71) 0.82
Triple-negativee(vs.luminalA-like) 1.29(0.41–4.00) 0.66 1.30(0.40–4.20) 0.67
Logisticregressionfortumorsubtypeswith2categoriesandmultinomiallogisticregressionfortumorsubtypewith>2categoriesasthedependentvariable.
aPositivefor(cid:21)1ofthebasalmarkersCK56,CK14,andP63,
bER+,PR+,HER2-,lowKi67,
cER+,PR-,HER2-withhighKi67,
dER+orER-,PR+orPR-,HER2+,
eER-,PR-,HER2-.
*AdjustedforageandBMI(continuous),exceptforgrade,whichisadjustedforageonly.
OR=OddsRatio,CI=ConfidenceInterval.
doi:10.1371/journal.pone.0170084.t002
clinicopathologicalsubtypes;thoughthepoor-prognosistumorsweremoreoftenoccurringin
premenopausalwomenwithdiabetesnotusinginsulinandintype1diabetesinsulinusers.
Onlyafewstudieshaveinvestigatedbreastcancercharacteristicsamongwomenwithdia-
betes[20,22,39,40].Twopreviousstudiesstratifiedtheresultsformenopausalstatusandthey
alsofoundthatpremenopausalwomendevelopedmoreoftentumorsthatwerehormone
receptornegative[22,39],aftermultivariableadjustment[39].Overallresultswereconsistent
PLOSONE|DOI:10.1371/journal.pone.0170084 January11,2017 9/16
DiabetesandBreastCancerSubtypes
Table3. Crudeandadjustedoddsratiosforbreastcancerclinicopathologicalsubtypesofwomenwithdiabetestreatedwithorwithoutinsulin
comparedtowomenwithoutdiabetesinsubgroupsofmenopausalstatususing(multinomial)logisticregression.
Premenopausalwomenwithbreastcancer
Dependentvariable Independentvariableofexposure
Insulin*vs.NoDiabetes NoInsulin†vs.NoDiabetes Diabetesonly
Insulinvs.NoInsulin
crudeOR(95%CI) P crudeOR(95%CI) P P
Grade2(vs.grade1) 0.55(0.18–1.68) 0.29 0.57(0.21–1.58) 0.28 0.93
Grade3(vs.grade1) 0.53(0.16–1.74) 0.30 1.34(0.49–3.67) 0.57 0.09
ER-(vs.ER+) 1.54(0.45–5.24) 0.49 2.98(1.11–8.00) 0.03 0.20
PR-(vs.ER+) 1.37(0.47–4.00) 0.57 3.06(1.30–7.20) 0.01 0.08
HER2-(vs.ER+) 8.97(1.10–73.36) 0.04 2.16(0.82–5.67) 0.12 0.19
Highki67(vs.lowki67) 0.80(0.32–1.96) 0.62 1.48(0.72–3.05) 0.29 0.15
Postmenopausalwomenwithbreastcancer
Dependentvariable Independentvariableofexposure
Insulin*vs.NoDiabetes NoInsulin†vs.NoDiabetes Diabetesonly
Insulinvs.NoInsulin
crudeOR(95%CI) P crudeOR(95%CI) P P
Grade2(vs.grade1) 0.60(0.12–2.96) 0.53 0.85(0.32–2.25) 0.75 0.66
Grade3(vs.grade1) 2.05(0.43–9.78) 0.37 1.95(0.69–5.55) 0.21 0.95
ER-(vs.ER+) 1.47(0.39–5.58) 0.57 1.23(0.38–3.15) 0.66 0.78
PR-(vs.ER+) 1.01(0.34–3.01) 0.98 0.95(0.46–1.96) 0.89 0.90
HER2-(vs.ER+) 0.83(0.19–3.60) 0.80 1.26(0.44–3.63) 0.67 0.56
Highki67(vs.lowki67) 0.80(0.26–2.46) 0.70 1.19(0.58–2.45) 0.63 0.46
Logisticregressionfortumorsubtypeswith2categoriesandmultinomiallogisticregressionfortumorsubtypewith>2categoriesasthedependentvariable.
*Womenwithdiabetestreatedwithinsulin(analogues)regardlesstheuseofconcomitantnon-insulinantidiabeticdrugs,
†womenwithdiabetestreatedonlywithdietandexerciseandusersofnon-insulinantidiabeticdrugsonly.
OR=OddsRatio,CI=ConfidenceInterval.
doi:10.1371/journal.pone.0170084.t003
withours,showingmoreER-negative,PR-negativeandHER2-negativetumorsinwomen
withdiabetes,withrelativefrequenciesof1.5to2.5,butmostdifferenceswerenotstatistically
significant,exceptforPR[20,22]andER,evenafteradjustmentforBMI[40].Afewstudies
thatreportedtumormarkers(ER,PR,andsomeHER2status)amongwomenwithdiabetes
[11–13,26,41]compared(breastcancer)mortalityordisease-freesurvivalamongwomenwith
andwithoutdiabetesastheirprimaryobjective.Therefore,onlycrudeestimatesofassociations
betweendiabetesandtumorsubtypewerereportedandnotstratifiedformenopausalstatus.
Womenincludedinthesestudiesweremainlypostmenopausalandnosignificantassociations
werefoundbetweentumormarkersanddiabetesstatus.
Studiesontheassociationbetweendiabetestreatmentandbreastcancersubtypeareeven
morescarce.Nodifferenceintumorstageandtumorsubtypeamongglargineversusnon-glar-
gineuserswaspreviouslydescribed[42,43].Studiesthatcomparedmetforminuserstowomen
withdiabetestreatedwithsulphonylureaorinsulin(non-metformin)showednodifferencein
ERstatus[20,44],butsulphonylureaorinsulinuserspresentedmorePR-negativetumors
(63.0%versus26.7%,p=0.041)[44]andmoreHER-2positive(29.5%versus21%,p=0.002)
[20]thaninthemetformin-treatedsubgroup.
Ourstudywasbasedonthecomprehensivebiobanks(archivaltumortissuefromaran-
domlyselectedgroupofwomen),anddatabasesavailableinDenmark,andincludedmedica-
tionhistoryatleastfiveyearspriortobreastcancerdiagnosisfromprescriptionrecords,
PLOSONE|DOI:10.1371/journal.pone.0170084 January11,2017 10/16