Pharmacogenetic meta-analysis of baseline risk factors, pharmacodynamic, efficacy and tolerability

23 Pages·2017·1.64 MB·English

RESEARCHARTICLE
Pharmacogenetic meta-analysis of baseline
risk factors, pharmacodynamic, efficacy and
tolerability endpoints from two large global
cardiovascular outcomes trials for darapladib
AstridYeo1,LiLi2¤a,LilingWarren2¤b,JenniferAponte2¤a,DanaFraser2¤a,KarenKing2¤a,
KelleyJohansson2¤a,AllisonBarnes3¤c,ColinMacPhee4,RichardDavies5,
StephanieChissoe2¤d,ElizabethTarka5¤e,MichelleL.O’Donoghue6,HarveyD.White7,
a1111111111
LarsWallentin8,DawnWaterworth9*
a1111111111
a1111111111 1 DepartmentofGenetics,GlaxoSmithKlineMedicinesResearchCentre,Stevenage,Hertfordshire,United
a1111111111 Kingdom,2 DepartmentofGenetics,GlaxoSmithKlineMedicinesResearchCentre,ResearchTrianglePark,
a1111111111 NorthCarolina,UnitedStatesofAmerica,3 ClinicalStatistics,GlaxoSmithKlineMedicinesResearchCentre,
ResearchTrianglePark,NorthCarolina,UnitedStatesofAmerica,4 DepartmentofVascularBiology&
Thrombosis,GlaxoSmithKlineMedicinesResearchCentre,KingofPrussia,Pennsylvania,UnitedStatesof
America,5 MetabolicPathwaysandCardiovascularTherapeuticArea,GlaxoSmithKlineMedicinesResearch
Centre,KingofPrussia,Pennsylvania,UnitedStatesofAmerica,6 TIMIStudyGroup,Cardiovascular
Division,BrighamandWomen’sHospital,Boston,Massachusetts,UnitedStatesofAmerica,7 GreenLane
OPENACCESS CardiovascularService,AucklandCityHospitalandUniversityofAuckland,Auckland,NewZealand,
8 DepartmentofMedicalSciences,Cardiology&UppsalaClinicalResearchCenter,UppsalaUniversity,
Citation:YeoA,LiL,WarrenL,AponteJ,FraserD, Uppsala,Sweden,9 DepartmentofGenetics,GlaxoSmithKlineMedicinesResearchCentre,UpperMerion,
KingK,etal.(2017)Pharmacogeneticmeta- Philadelphia,Pennsylvania,UnitedStatesofAmerica
analysisofbaselineriskfactors,
pharmacodynamic,efficacyandtolerability ¤a Currentaddress:GenomicMedicine,PAREXELInternational,Durham,NorthCarolina,UnitedStatesof
endpointsfromtwolargeglobalcardiovascular America
outcomestrialsfordarapladib.PLoSONE12(7): ¤b Currentaddress:TevaPharmaceuticals,Raleigh-Durham,NorthCarolina,UnitedStatesofAmerica
¤c Currentaddress:Biostatistics,PAREXELInternational,Durham,NorthCarolina,UnitedStatesofAmerica
e0182115.https://doi.org/10.1371/journal.
¤d Currentaddress:GlaxoSmithKline,ElliottAvenue,Seattle,Washington,UnitedStatesofAmerica
pone.0182115
¤e Currentaddress:JanssenPharmaceuticals,SpringHouse,Pennsylvania,UnitedStatesofAmerica
Editor:GiuseppeDaniloNorata,Universitadegli *[email protected]
StudidiMilano,ITALY
Received:November1,2016
Abstract
Accepted:July11,2017
Published:July28,2017 Darapladib,alipoprotein-associatedphospholipaseA2(Lp-PLA2)inhibitor,failedtodemon-
strateefficacyfortheprimaryendpointsintwolargephaseIIIcardiovascularoutcomestri-
Copyright:©2017Yeoetal.Thisisanopenaccess
articledistributedunderthetermsoftheCreative als,oneinstablecoronaryheartdiseasepatients(STABILITY)andoneinacutecoronary
CommonsAttributionLicense,whichpermits syndrome(SOLID-TIMI52).Nomajorsafetysignalswereobservedbuttolerabilityissuesof
unrestricteduse,distribution,andreproductionin
diarrheaandodorwerecommon(upto13%).Wehypothesizedthatgeneticvariantsassoci-
anymedium,providedtheoriginalauthorand
atedwithLp-PLA activitymayinfluenceefficacyandtolerabilityandthereforeperformeda
sourcearecredited. 2
comprehensivepharmacogeneticanalysisofbothtrials.Wegenotypedpatientswithinthe
DataAvailabilityStatement:Duetolimitations
STABILITYandSOLID-TIMI52trialswhoprovidedaDNAsampleandconsent(n=13,577
detailedintheinformedconsentprovidedbythe
subjectsenrolledintotheSTABILITYandSOLID- and10,404respectively,representing86%and82%ofthetrialparticipants)usinggenome-
TIMI52clinicaltrials,fullaccesstopatient-level widearrayswithexomecontentandperformedimputationusinga1000Genomesreference
datacannotbeprovidedpublicly,inorderto
panel.WeinvestigatedbaselineandchangefrombaselineinLp-PLA activity,twoefficacy
protecttheprivacyofpatientsandindividuals 2
endpoints(majorcoronaryeventsandmyocardialinfarction)aswellastolerabilityparame-
involvedinourstudies.However,allanalysis
results(summarystatistics)andanonymised tersatgenome-wideandcandidategenelevelusingameta-analyticapproach.Werepli-
patient-leveldatacanberequestedbyqualified catedassociationsofpublishedlocionbaselineLp-PLA activity(APOE,CELSR2,LPA,
2
independentresearchers,subjecttoreviewbyan
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Pharmacogeneticanalysisofdarapladibclinicaltrials
independentpanel,accordingtodetailsprovided PLA2G7,LDLRandSCARB1)andidentifiedthreenovelloci(TOMM5,FRMD5andLPL)
onwww.clinicalstudydatarequest.com. usingtheGWAS-significancethresholdP(cid:20)5E-08.ReviewofthePLA2G7gene(encoding
Funding:Theworkdescribedinthismanuscript, Lp-PLA )withinthesedatasetsidentifiedV279Fnullallelecarriersaswellasthreeother
2
includingthedarapladibSTABILITYandSOLID- rareexonicnullalleleswithinvariousethnicgroups,howevernoneofthesevariantsnorany
TIMI52clinicaltrials,wasfundedby
otherlociassociatedwithLp-PLA activityatbaselinewereassociatedwithanyofthedrug
GlaxoSmithKline. 2
responseendpoints.Theanalysisofdarapladibefficacyendpoints,despitelowpower,iden-
Competinginterests:Ihavereadthejournal’s
tifiedsixlowfrequencylociwithmaingenotypeeffect(thoughwithborderlineimputation
policyandtheauthorsofthismanuscripthavethe
followingcompetinginterests:AstridYeo,Colin scores)andonecommonlocus(minorallelefrequency0.24)withgenotypebytreatment
MacPhee,RichardDavies,StephanieChissoe, interactioneffectpassingtheGWAS-significancethreshold.Thislocusconferredriskinpla-
DawnWaterworth:Employmentbyandstock
cebosubjects,hazardratio(HR)1.22with95%confidenceinterval(CI)1.11–1.33,butwas
ownershipofGlaxoSmithKline.LiLi,Jennifer
protectiveindarapladibsubjects,HR0.79(95%CI0.71–0.88).Nomajorlocifortolerability
Aponte,DanaFraser,KarenKing,Kelley
Johansson,AllisonBarnes:Employeesof werefound.Thus,geneticanalysisconfirmedandextendedtheinfluenceoflipoproteinloci
GlaxoSmithKline(GSK)duringtheconductofthe onLp-PLA levels,identifiedsomenovelnullallelesinthePLA2G7gene,andonlyidentified
2
study;currentlyemployeesofPAREXEL
onepotentiallyefficacioussubgroupwithinthesetwolargeclinicaltrials.
International.LilingWarren:Employeeof
GlaxoSmithKline(GSK)duringtheconductofthe
study;currentlyemployeeofAcclarogenLtd.
ElizabethTarka:EmployeeofGlaxoSmithKline
(GSK)duringtheconductofthestudy;currently
employeeofJanssenPharmaceuticals.M.L.
O’Donoghue:ResearchGrantfromEisai,Merck, Introduction
Janssen,GlaxoSmithKlineandAstraZeneca.Lars
Wallentin:Institutionalresearchgrants, Lipoprotein-associatedphospholipaseA2(Lp-PLA2)istheproductofactivatedinflammatory
consultancyfees,lecturefees,andtravelsupport cellswithcirculatingLp-PLA predominantlyboundtolow-densitylipoprotein(LDL)choles-
2
fromBristol-MyersSquibb/Pfizer,AstraZeneca, terol[1].ElevatedplasmalevelsofLp-PLA areassociatedwithanincreasedriskofcardiovas-
2
GlaxoSmithKline,BoehringerIngelheim;
cular(CV)events,suchascoronaryheartdisease(CHD)death,myocardialinfarction(MI)
institutionalresearchgrantsfromMerck&Co,
andstroke[2,3].HighexpressionofLp-PLA hasbeennotedinhumanatheroscleroticlesions,
Roche;consultancyfeesfromAbbott;holdstwo 2
particularlyinthincapfibroatheromaorrupturedplaques[4,5].Inthesehighrisklesionsthat
patentsinvolvingGDF-15.HarveyDWhite:
Researchgrantsandpersonalfeesfrom oftenleadtosuddendeathorMI,theenzymeishighlylocalizedtoactivatedmacrophages
GlaxoSmithKline,duringtheconductofthestudy; undergoingapoptosisinthelipidnecroticcoreandfibrouscapsuggestingapotentialrolein
researchgrantsandconsultancyfeesfromDaiichi- promotingplaqueinstability[5].Therefore,ithadbeenproposedthatdirectinhibitionofthe
SankyoPharmaDevelopment;researchgrantsand
pro-inflammatoryLp-PLA enzymemayreducevascularinflammationandpromotestability
advisoryboardmemberforAstraZeneca;research 2
ofvulnerableplaques.TheIBIS-2studydemonstrated(usingintravascularultrasoundvirtual
grantsfromSanofi-Aventis,EliLilly,National
InstituteofHealth,MerckSharp&Dohme.There histology)thatdarapladibcouldhaltthegrowthoftheplaquenecroticcorevolume(asecond-
arenofurtherpatents,productsindevelopmentor aryendpoint)over12monthsinCHDpatients,furthersupportingthishypothesis,though
marketedproductstodeclare.Thisdoesnotalter therewerenosignificantdifferencesintheprimaryendpointsofplaquedeformabilityor
ouradherencetoallthePLOSONEpolicieson
plasmahigh-sensitivityC-reactiveprotein[6].Areductionofplaquenecroticcoreareaand
sharingdataandmaterials,asdetailedonlineinthe
reductioninmedialdestructionhadalsopreviouslybeenobservedinhypercholesteremic
guideforauthors.
swinetreatedwithdarapladib[7].Fewhumangeneticdatawasavailabletosupporttargetvali-
dationofLp-PLA priortothePhaseIIItrialscommencing.
2
Darapladib(SB-480848)isanovel,selective,reversible,orallyactiveinhibitorofLp-PLA
2
activitythatwasindevelopmentbyGlaxoSmithKline(GSK)forCVriskreduction.GSKcon-
ductedtwophaseIIIoutcomestudiesinvestigatingtheclinicalefficacyoflong-termtreatment
withdarapladibentericcoated(EC)tablets,160mg(oraloncedailydose)ascomparedtopla-
cebowhenaddedtostandardofcare.ItwashypothesizedthatdirectinhibitionofLp-PLA
2
activitywithdarapladibmayreduceresidualcardiovasculareventsinhighriskindividuals
whengivenasanadjuncttostandardofcare,includinglipid-loweringandanti-plateletthera-
pies[1;3;8–10].Eachtrialwasrandomized,double-blind,placebo-controlled,multicenter,and
event-drivenwithapproximately1,500majoradversecardiovascularevents(MACE:first
occurrenceofnon-fatalstroke,non-fatalMIorCVdeath).ThefirststudywasSTABILITY
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Pharmacogeneticanalysisofdarapladibclinicaltrials
(STabilisationofAtheroscleroticplaqueByInitiationofdarapLadIbTherapY;NCT00799903),
whichfocusedondarapladib’seffectontheincidenceofMACEin15,828subjectswithchro-
niccoronaryheartdisease(CHD)[11].TheSOLID-TIMI52(TheStabilizationOfpLaques
usIngDarapladib-ThrombolysisInMyocardialInfarction52)trial,evaluatedtheeffectsofdar-
apladibontheincidenceofmajorcoronaryevents(MCE:CHDdeath,non-fatalMIorurgent
revascularizationformyocardialischemia)in13,026subjectswithin30daysofhospitalization
withanacutecoronarysyndrome(ACS)(NCT01000727)[12].Theprimaryendpointofthe
SOLID-TIMI52trialwaschangedfromMACEtoMCEpriortostudyunblinding,following
theavailabilityoftheresultsoftheSTABILITYtrial.Neitherofthetrialsshowedstatistically
significantresultsfortheprimaryefficacyendpoints[13;14].However,therewasapotential
signalofefficacyintheSTABILITYtrialwherenominallysignificantreductionswereobserved
fortheMCEHR0.90;95%CI,0.82to1.00;p=0.045)andtotalcoronaryeventsendpoints
(CHDdeath,non-fatalMI,hospitalizationforunstableangina,oranycoronaryrevasculariza-
tionprocedure;HR0.91;95%CI,0.84to0.98;p=0.02)[14],butnotintheSOLID-TIMI52
trial[13].Bothtrialsobservedahigherpercentageofpatientsexperiencingdiarrhea(darapla-
dibtreatedvsplacebo:12%vs6%inSTABILITYand11%vs6%inSOLID-TIMI52),odor
(darapladibtreatedvsplacebo:13%vs2%inSTABILITYand12%vs2%inSOLID-TIMI52)
butnoconsistent,seriousadverseeventswereobserved.
AnaturaldeficiencyinLp-PLA ispresentinEastAsianpopulationsduetoageneticnull
2
variant(V279F;rs76863441)[15]andasaresultsubjectswhocarrytwocopiesofthisgenetic
nullvarianthaveabsentorverylowlevelsofenzymeactivityinplasma.Thereforesubjects
withbothparentsofJapanese,ChineseorKoreanancestrywerescreenedandthosewithLp-
PLA activitylevels(cid:20)20nmol/min/mL(indicativeof279FFsubjects)wereexcludedfrom
2
enrollmentintotheSTABILITYandSOLID-TIMI52studiesastherewouldbenoclearratio-
naleforexposingsuchsubjectstodarapladib.AttemptstousethisvarianttopredictCHDrisk
haveprovidedmixedresults,withsomemeta-analysessuggestingnorelationship[16,17]and
astudyinKoreanssuggestingriskreduction,butonlyinmenwithMIorangiographically
defineddisease[18].AmorerecentstudyinChinaprovidedrobustevidenceofalackofasso-
ciationwithstroke(concordantwithresultsfrombothdarapladibtrials)thoughitwasunder-
poweredforcoronaryheartdisease[19].Nullallelesarerareinotherethnicgroupsasevi-
dencedbyaveryrecentmeta-analysisinamulti-ethnicstudy,designedtotesttheassociation
ofPLA2G7variantsandCHD[20].Inover180,000subjectsofEuropeanorSouthAsian
descent,lessthan40carriersofnullalleleswerefound.NosignificantassociationswithCHD
wereobservedwitheitherthesenullvariantsortheV279FinanEastAsiansample(~10K
casesand~15Kcontrols),withpointestimatesbroadlyconcordantwiththeclinicaltrial
results.
Severalgenome-wideassociationstudies(GWAS)ofLp-PLA activitylevelsandmasswere
2
performed(FraminghamHeartStudy[FHS][21];CohortsforHeartandAgingResearchin
GenomicEpidemiology[CHARGE][22];JustificationfortheUseofstatinsinPrimarypre-
vention:anInterventionTrialEvaluatingRosuvastatin[JUPITER][23]).Themajorityofthe
locithatshowedgenome-wideassociationcontainedgeneticvariantsthatareknowntobe
involvedinlipidmetabolismandhavebeenobservedtostronglyinfluencecirculatinglevelsof
LDL-Cand/orHDL-C[22].
Wehypothesizedthatgeneticvariantsmayalsoinfluenceresponsetodarapladib.Asthe
STABILITYandSOLID-TIMI52trialsweresolarge(>24,000patientscombined),wecon-
ductedgenotypingaheadoftimesothatthedatawouldbeavailablecontemporaneouslywith
theclinicaldataupontrialcompletion.Inordertomaximizethepowerweperformedmeta-
analysesoftheSTABILITYandSOLID-TIMI52trialsforefficacy,pharmacodynamicandtol-
erabilityendpoints.TheefficacyanalysiswasfocusedonMCEandMIendpoints,asthose
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Pharmacogeneticanalysisofdarapladibclinicaltrials
phenotypeshadgreaterefficacywithinSTABILITYascomparedtoothers.Wealsoinvesti-
gatedbaselineandchangefrombaselineLp-PLA activitylevels.BaselineLp-PLA activitylev-
2 2
elswereassociatedwithprimaryandsecondaryendpointsinSTABILITY[24],butnotin
SOLID-TIMI52.Weusedbothagenome-wideandcandidategeneapproach,withthecandi-
dategenesincludingthoseknowntoinfluenceLp-PLA activitylevelsanddarapladibabsorp-
2
tion,distribution,metabolismandexcretion(ADME).Theresultsreportedhererepresentthe
oneoflargestpharmacogenetic(PGx)studiesofcardiovasculartreatmentendpointsreported
todateandalsocontainexomechipvariantswhichhavenotbeenincludedinmanyPGxeval-
uationstodate.WefoundgeneticvariationinfluencingbaselineLp-PLA activitylevels,but
2
thosevariantsdidnotinfluenceoutcomes.Despitethelargesizeoftheclinicaltrialsandavail-
abilityofagoodpharmacodynamicmarker,wefoundjustonecommonlocusthatappearedto
influencecardiovascularoutcomes.
Results
Samplesize,demographics,andlaboratorycharacteristicsofeachendpointbystudyarepre-
sentedinTable1.AlthoughmostcharacteristicsweresimilarfortheSTABILITYandSOLID-
TIMI52trials,therewereconsiderabledifferencesintheuseofP2Y12inhibitors(34%vs89%
respectively)andtheprevalenceofchronickidneydisease(30%vs17%respectively),which
maybeexplained,atleastinpart,bythedifferentstudydesignwithregardtodiseasecharac-
teristics(chronicvsacute).
Table1. CharacteristicsofPGxstudysubjects(n=23,981).
STABILITY SOLID-TIMI52
Placebo Darapladib160mgdaily Placebo Darapladib160mgdaily
N* 6863 6714 5201 5203
Age(years) 64.3(9.3) 64.5(9.2) 64.3(9.4) 64.0(9.4)
Male,% 81.2 82.0 74.0 74.8
Currentsmoking,% 19.0 17.5 18.9 18.9
BMI(kg/m2) 29.0(4.9) 29.1(5.09) 28.5(5.10) 28.6(5.11)
Waist/hipratio 0.97(0.08) 0.97(0.08) 0.97(0.09) 0.98(0.09)
Systolicbloodpressure(mmHg) 132.0(16.4) 132.0(16.4) 126.0(16.3) 126.0(16.2)
Diastolicbloodpressure(mmHg) 78.8(10.2) 78.7(10.4) 74.6(10.1) 74.9(9.9)
Totalcholesterol(mg/dL) 4.2(1.0) 4.2(1.1) 4.0(1.1) 4.0(1.1)
HDL-C(mmol/L) 1.2(0.3) 1.2(0.3) 1.1(0.3) 1.1(0.3)
LDL-C(mmol/L) 2.2(0.8) 2.2(0.9) 2.1(0.8) 2.1(0.9)
Triglycerides(mg/dL) 1.8(1.3) 1.8(1.4) 1.8(1.1) 1.8(1.1)
hsCRP(mg/L) 3.0(6.6) 2.9(5.8) 12.4(21.9) 12.6(24.4)
Lp-PLA activitylevels(nmol/min/ml) 176(47.2) 175(47.6) 178(48.2) 178(49.1)
2
Statin,% 97.2 97.5 95.2 94.6
Aspirin,% 93.4 92.1 96.4 96.4
Betablockers,% 79.8 79.2 88.0 87.9
P2Y12inhibitors,% 33.8 33.5 88.5 88.7
ACEinhibitororARB,% 77.7 77.6 83.1 83.2
Chronickidneydisease,% 29.7 30.2 16.7 16.0
Diabetes,% 38.0 38.9 33.5 33.9
Mean(SD)forcontinuous,percentforcategoricalvariables
*numberofsubjectsthatpassedgenotypingQC
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OurPGxanalysisincludedvariousclinicalendpointsandanalysispopulations,asoutlined
inTable2.Furtherdetailontherationaleanddefinitionoftheendpointsandanalysispopula-
tionscanbefoundinthetablefootnotesandundertheMethodssection.
WhenweassessedwhetherthePGxpopulationwasrepresentativeoftheoverallclinical
studyinSTABILITYstudy,abiaswasidentifiedinthePGxpopulationresultinginslightly
lowerhazardratiosintheefficacyendpoint(MCE:PGxsubjectsHR0.87[CI95%0.78,0.97]
vsallsubjectsHR0.90[CI95%0.82,1.00])andthetolerabilityendpoints(diarrhea:PGxsub-
jectsHR1.9[CI95%1.7,2.1]vsallsubjectsHR2.0[95%CI1.8,2.3];anyodor:PGxsubjects
HR5.9[CI95%4.9,6.9]vsallsubjectsHR6.19[95%CI5.3,7.3]).ThisdifferenceinSTABIL-
ITYmaybeexplainedbythecollectionofPGxsamplesatthemonth3studyvisits,ratherthan
atrandomization,therebymissingcollectionofsamplesfromsubjectswithearlyMCEevents
andthosewhowithdrewfromthestudyduetotolerabilityissues.PGxsamplecollectionwas
alsodelayedinsomecountriesduetolateapprovalofthePGxcomponenttothestudy.The
SOLID-TIMI52study,wherePGxsampleswerecollectedatbaseline,didnotshowthisbias.
Genome-wideassociationoflipoprotein-associatedphospholipaseA2
activityatbaselineandchangefrombaseline
WefirstperformedasinglevariantassociationanalysisforLp-PLA activityatbaselinefor
2
imputedvariantswithimputationr2(cid:21)0.5usingalinearregressionmodelthatincludedthe
top10principlecomponent(PC)scoresascovariatesinSTABILITY(n=12,640)andSOLID-
TIMI52(n=9,516)separately.Meta-analysisofthe~13.6millioncommon,lowfrequency
Table2. Overviewofpharmacogeneticanalysispopulationsbyendpoint.
Outcome Endpoint AnalysisPopulation MAF* STABILITY(n.event/ SOLID-TIMI52(n. Meta-Analysis#(n.
Variable cut-off n.total) event/n.total) event/n.total)
Lp-PLA Baseline PGxsubjectsinplaceboarmand >0% 12640 9516 22156
2
enzyme darapladibtreatedarm
activity
Changefrom PGxsubjectsindarapladibtreated >0.5% 6170 4704 10874
baseline arm
Efficacy MCE PGxsubjectsinplaceboarmand >0.5% Placebo:688/6863; Placebo:752/5201; Placebo:1440/12064;
darapladibtreatedarm Darapladib:585/6714 Darapladib:733/5203 Darapladib:1318/11917
MI(fatal+non- PGxsubjectsinplaceboarmand >0.5% Placebo:358/6863; Placebo:475/5201; Placebo:833/12064;
fatal) darapladibtreatedarm Darapladib:292/6714 Darapladib:448/5203 Darapladib:740/11917
Tolerability Diarrhea§ WhitePGxsubjectswithdiarrhea >0.5% Darapladib:235/3405 Darapladib:246/3155 Darapladib:481/6560
eventwithin90daysofreceiving
darapladib,excludingsubjectson
Metformin
MSdiarrhea§- WhitePGxsubjectswithdiarrhea >0.5% Darapladib:97/3267 Darapladib:115/3024 Darapladib:212/6291
Moderateand eventwithin90daysofreceiving
severe darapladib,excludingsubjectson
Metformin
Odor— PGxsubjectsindarapladibtreated >0.5% Darapladib:735/6568 Darapladib:548/5071 Darapladib:1283/11639
Bathroomevent arm
Odor—Non- PGxsubjectsindarapladibtreated >0.5% Darapladib:340/6173 Darapladib:244/4769 Darapladib:584/10942
bathroomevent arm
*MAF=MinorAlleleFrequency;Analysesincludedimputedvariants(imputationr2(cid:21)0.5)inbothSTABILITYandSOLID-TIMI52studies.AnalysisofLp-
PLA enzymeactivitywasperformedusing13,572,687variants,whilst9,334,585variantswereusedfortheefficacyandtolerabilityanalyses
2
#Meta-analysesweredonewithinplaceboordarapladibtreatedarmseparately.
§Inordertoreduceasignaltonoiseratio,thediarrheaendpointwasdefinedasasubsetthatwasmostlikelyassociatedtotreatmentwithdarapladib.For
moredetails,seeMaterial&Methods(Phenotypedefinitionandmeasurements).
MI=Myocardialinfarction;MCE=MajorCoronaryEvent
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andraregenome-widevariantswascarriedouttocombinetheassociationresultsfromboth
studies.Wedetected578variantswithconsistentdirectionofeffectinbothSTABILITYand
SOLID-TIMI52studiesthatsurpassedthegenome-widesignificantP-valuethresholdof5E-
08inregionsofchromosomes1,6(2clusters),9(2clusters),12,15and19.Resultsforthe
mostsignificantvariantfromeachchromosomalclusteraresummarizedinTable3;withdata
forothervariantsinS1Table.Allgenomiccontrol(λ )estimateswerecloseto1,suggesting
gc
noinflationduetouncontrolledpopulationstructure(seehistograms,QQandManhattan
plotsinS2Fig).
Ofthe578genome-widesignificantvariants,25werecontainedwithintheexomecompo-
nentofthegenotypingarray,while171wereconsideredrarevariants(minorallelefrequency
[MAF]<0.5%).Themoststrikingassociationdetectedisduetothewell-knownPLA2G7
V279Fnullvariant(Table3)[15;18;25–36].Asexpectedthisvariantwasmostlyobservedin
Asiancountries(Table4).Wealsofoundgenome-wideassociations(P(cid:20)5.0E-08)forthreeaddi-
tionalrarePLA2G7nullvariants(S2Table),whichtendedtobespecifictocertainethnicgroups
(Table4).TwocommonPLA2G7codingvariantsalsoshowedgenome-wideassociationsstatis-
ticallyindependentoftheassociationbetweenPLA2G7V279FvariantandbaselineLp-PLA
2
activity.TheV379AcommonvariantwasassociatedwithaslightlyhigherLp-PLA activityand
2
theR92HcommonvariantwasassociatedwithaslightlylowerLp-PLA activity(S11Fig).
2
Table3outlinesthemostsignificantvariantsforeachlocusassociatedwithbaselineand/or
changefrombaselineinLp-PLA activityandhowthiscorrelateswithvariousbaselinelipid
2
andC-reactive-proteinlevels.Thestrongestassociationatthe1p13locuswasforrs12740374,
locatedinthe3’UTRoftheCELSR2gene.Thisvariantwasalsosignificantlyassociatedwith
baselineLDLandHDLlevels.FoursignificantvariantswerelocatedintheLPAgene.Variant
rs10455872wasthemostsignificantandwasalsoassociatedwithbaselineLDLlevels.Lowfre-
quencyvariantrs57578064isthemostsignificantinthelocusthatincludestheTOMM5and
FBXO10genesandhasnotbeenpreviouslydescribed(S3Fig).Anothernovellocusonchro-
mosome9containedtheintergenicrarevariantrs189889864withaverylargeeffect.Acluster
ofsignificantvariantswerelocatedwithintheSCARB1gene,withthemostsignificanteffect
observedforrs11057830.Anotherlargeclusterofsignificantvariantswasobservedonchro-
mosome15,ofwhichintronicvariantrs2733201locatedintheFRMD5genewasthemostsig-
nificant(S3Fig).ThevariantsinthislargeclusterareinhighLDwitheachotherandfall
withinseverallargerearrangements/copynumbervariationsintheregion.FortheAPOE/
APOC1locusvariantrs7412(APOEε2allele)intheAPOEgenewasthemostsignificantvari-
ant,whichwasalsoassociatedwithbaselineLDLandtriglyceridelevels.
Besidesthegenome-wideanalysis,reviewoftheresultsforthepre-specifiedcandidategene
variantsforLp-PLA activityrevealedtwoadditionallocithatsurpassedthecandidategene
2
significancethresholdof5E-04,butwerenotgenome-widesignificant(Table3):LPLcoding
variantrs328,whichchangestheaminoacidatposition474fromaserinetoastopcodon,was
associatedwithbaselineLp-PLA activity,HDLandtriglyceridelevels(S3Fig);intronicvari-
2
antrs6511720intheLDLRgenewithbaselineLp-PLA activityandLDLlevels.
2
Meta-analysisofchangefrombaselinelipoprotein-associated
phospholipaseA2activity
AnalysisofchangefrombaselineLp-PLA activityafteronemonthoftreatmentrevealedthree
2
significantlociaroundtheCELSR2,APOEandPLA2G7genes,noneofwhichwereassociated
withchangefrombaselineLp-PLA activityafteradjustingforbaselineLp-PLA activity
2 2
(Table3;unadjustedP-valuesinS1Table),indicatingthatthebaselineLp-PLA geneticassoci-
2
ationwasconfoundingthechangefrombaselineanalysis.
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Pharmacogeneticanalysisofdarapladibclinicaltrials
P 1 8 9 9 3 9 7 3 4 8
eline eCRP 0.44 0.5 0.62 0.27 0.49 0.62 0.98 2.4E-0 0.15 0.28
s n
omba Baseli Beta (SE) -0.02 (0.02) 0.08 (0.14) 0.02 (0.03) 0.25 (0.23) -0.3 (0.43) 0.01 (0.02) 0 (0.04) 0.1 (0.03) -0.04 (0.03) 0.03 (0.03)
fr P 3 9 3 7 7 5 2 0 5 5
hange eTRIG 0.1 0.8 6.8E-0 0.7 0.2 0.5 0.6 1.5E-3 2.3E-3 0.9
c n
eand Baseli Beta (SE) -0.02 (0.01) 0.01 (0.07) -0.06 (0.02) 0.04 (0.12) 0.37 (0.34) 0.01 (0.01) -0.01 (0.02) 0.23 (0.02) -0.21 (0.02) 0 (0.02)
n
utingtobaseli BaselineHDL BetaP (SE) 0.081.5E-10 (0.01) -0.060.45 (0.07) 0.040.09 (0.02) -0.060.63 (0.12) -0.420.22 (0.34) -0.010.32 (0.01) -0.040.08 (0.02) 0.081.4E-04 (0.02) 0.27.6E-32 (0.02) 0.030.07 (0.02) otein(mg/L)
variantscontrib BaselineLDL BetaP (SE) -0.166.1E-40 (0.01) 0.020.81 (0.07) 0.123.6E-09 (0.02) 0.270.03 (0.12) 1.281.6E-04 (0.34) 0.030.048 (0.01) -0.060.004 (0.02) -0.411.3E-96 (0.02) 0.020.33 (0.02) -0.137.4E-15 (0.02) P=C-reactivepr
ncyandrare ChangefrombaselineLp-§◆PLA2 BetaP (SE) -0.460.38 (0.52) -0.240.92 (2.33) 2.160.02 (0.91) -1.420.77 (4.85) -2.210.89 (16.21) 0.150.79 (0.57) 0.490.60 (0.93) -0.040.96 (0.82) -1.040.14 (0.71) 0.620.39 (0.71) D-TIMI52. s(mg/dL);CR
atedcommon,lowfreque §BaselineLp-PLAactivity2adjustingforLDL *BetaPPerc (SE) -5.772.8E-280.22 (0.52) -85.648.2E-3015.57 (2.31) 5.727.7E-100.09 (0.93) 29.913.2E-080.11 (5.41) 52.62.4E-040.07 (14.33) 3.521.4E-090.11 (0.58) -6.725.2E-130.15 (0.93) -12.181.8E-470.36 (0.84) -3.32.9E-060.1 (0.7) -2.090.0040.004 (0.72) edinSTABILITYandSOLI ues. mol/L);TRIG=Triglyceride
specificfrequencyoftop-associ §BaselineLp-PLAactivity2 *ImpBetaPPerc Rsq#(SE) 1/-7.443.9E-390.77 1(0.57) 0.99/-86.711.6E-2234.08 0.99(2.72) 0.69/7.224.3E-130.24 1(1) 0.94/32.491.4E-080.16 0.93(5.72) 0.72/91.818.8E-090.16 0.65(15.96) 1/3.677.4E-090.15 0.94(0.63) 0.68/-7.35.9E-130.23 0.65(1.01) 1/-17.118.3E-781.55 0.94(0.92) 1/-3.71.5E-060.1 1(0.77) 1/-3.751.9E-060.11 1(0.79) theaverageof%varianceexplain seeS1TableforunadjustedP-val HDL=High-densitylipoprotein(m 003
Table3.Effectestimates,population-Lp-PLAenzymeactivity.2 VariantGeneAlleleFreq (Annotation)(%)# GWAS rs12740374CELSR2T17.7/ (3’UTR)19.8 rs76863441PLA2G7A1.0/ (V279F)0.5 rs10455872LPAG5.9/ (intron)6.9 rs57578064TOMM5A0.1/ (intron)0.2 rs189889864intergenicA0.02/ 0.02 rs11057830SCARB1A15.0/ (intron)15.2 rs2733201FRMD5T7.3/ (intron)8.2 rs7412APOET6.1/ (2)(R176C)6.8ε Candidategenes rs328LPLG9.2/ (S474X)9.4 rs6511720LDLRT8.3/ (intron)9.6 #STABILITY/SOLID-TIMI52 %varianceexplainedwasreportedas*§Lp-PLAactivity(nmol/min/ml)2◆AdjustedforbaselineLp-PLAactivity;2 LDL=Low-densitylipoprotein(mmol/L); https://doi.org/10.1371/journal.pone.0182115.t
PLOSONE|https://doi.org/10.1371/journal.pone.0182115 July28,2017 7/23
Pharmacogeneticanalysisofdarapladibclinicaltrials
Meta-analysisofefficacyendpoints
Genome-wideanalysisofefficacyfocusedontheMCEandMIendpoints,lookingatpotential
effectsinthedarapladibandplacebotreatedsubjectsseparately.Atotalof13,577STABILITY
and10,195SOLID-TIMI52subjectswereanalyzed(Table2)foratotalof~9.3millioncommon
andlowfrequencyvariants(MAF>0.5%).DuetothelowtreatmenteffectinboththeSTABIL-
ITYandSOLID-TIMI52clinicaltrialstherewaslimitedpowertoidentifygeneticvariantsthat
mayimpactefficacy(SeeS1Figforpowercurves).Onecommonlocusandsixlowfrequency
locimetthegenome-widesignificantassociationthresholdof5E-08withconsistentdirection
ofeffectinbothSTABILITYandSOLID-TIMI52studies,fiveofthesewereassociatedwith
MCEandtwowithMI(Table5;S5andS6Figs).Theassociatedcommonlocus(MAF24%),
rs181937009whichwasreasonablywellimputed(r2STABILITY0.84andSOLID0.75),hada
significantallelebytreatmentinteractioneffect(P=6.03E-9)butmarginalmaingeneticeffectin
individualtreatmentarms.CarriersoftheminoralleleintheplacebogroupshadanelevatedHR
1.22(95%CI1.11–1.33),meta-P=4.30E-5andinthedarapladibgroupsareducedHR0.79
(95%CI0.71–0.88),meta-P=2.07E-5.AllsixassociatedlowfrequencyvariantshadMAF<2.5%
andmanyhadimputationr2valuesbetween0.5and0.8,whichreducesthelevelofconfidencein
theseresults.Allgenomiccontrol(λ )estimateswerecloseto1,suggestingnoinflationdueto
gc
uncontrolledpopulationstructure(seeQQandManhattanplotsinS4Fig).
Noneofthepre-specifiedcandidategenevariantsforefficacy,includingthePLA2G7
V279Fnullvariant,wereassociatedunderthecandidategenesignificancethreshold(P=5E-
04)withMCEorMIineitherthedarapladiborplacebotreatmentarm.Alsononeofthevari-
antsthatwereassociatedwithLp-PLA activitywereassociatedwithefficacyendpoints(MCE
2
orMIevents)(S8Fig).
Meta-analysisoftolerabilityendpoints
Toinvestigategeneticvariantsthatcouldimpactdarapladibtolerability,weperformed
genome-wideanalysisof~9.3millioncommonvariantsforthediarrheaandodor-related
Table4. SummaryofPLA2G7variantsobserved(allelecountandeffectsize)inSTABILITYandSOLID-TIMI52studiesshowingsomeethnic
groupspecificity(inbold).
Race(N) rs76863441 rs201842579 rs1805018 rs1051931 rs140020965 rs1805017 rs147252565 rs34159425
V279F I317N* I198T V379A Q287X R92H T278M* L389S
All(22147) 339 10 2621 8483 12 12297 3 14
Effectsize(All)(Beta,SE)
Asian—East/Japanese/ 298 9 467 423 713
SouthEastAsianHeritage(1703)
White-White/Caucasian/European 37 1 1796 7453 12 10318 2 1
Heritage(18823)
AfricanAmerican/AfricanHeritage 2 159 221 300 12
(490)
AmericanIndianorAlaskanNative 23 77 185 1
(244)
Asian—Central/SouthAsian 1 112 153 557
Heritage(517)
White—Arabic/NorthAfrican 30 78 121 1
Heritage(195)
Mixedrace(143) 1 30 67 92
NativeHawaiianorOtherPacific 1 8 6
Islander(14)
*VariantcarrierobservedinSTABILITYonly
https://doi.org/10.1371/journal.pone.0182115.t004
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Pharmacogeneticanalysisofdarapladibclinicaltrials
Table5. Listofsignificantresultsforefficacyendpoints(MCEandMI).
Placebo Darapladib
Combined
Variant Gene Location Major Minor Study N MAF r2 HR# P HR P P P*
Allele Allele
(95%CI) (95%CI) Interaction (2df
test)
MajorCoronaryEvent
rs138741635 FHIT intron T A STABILITY 13577 0.019 0.7 1.89 3.36E- 0.79 3.85E-01 6.69E-03 2.41E-
(1.33, 04 (0.46, 03
2.7) 1.35)
SOLID-TIMI 10404 0.02 0.66 2.16 1.98E- 0.73 2.14E-01 3.89E-04 1.69E-
52 (1.52, 05 (0.44, 04
3.13) 1.2)
META 23981 2.02 2.87E- 0.75 1.33E-01 9.01E-06 1.90E-
(1.59, 08 (0.52, 06
2.56) 1.09)
rs192427471 intergenic C T STABILITY 13577 0.024 0.67 1.19 4.10E- 2.46 4.38E-08 4.03E-03 3.33E-
(0.79, 01 (1.79, 06
1.79) 3.45)
SOLID-TIMI 10404 0.021 0.68 0.97 8.81E- 1.61 9.57E-03 7.85E-02 5.35E-
52 (0.63, 01 (1.12, 02
1.49) 2.33)
META 23981 1.08(0.8, 6.23E- 2.04 6.31E-09 9.49E-04 1.80E-
1.45) 01 (1.61, 06
2.56)
rs181937009 intergenic A G STABILITY 13577 0.244 0.84 1.27 5.66E- 0.78 2.14E-03 1.46E-05 6.43E-
(1.11, 04 (0.67, 05
1.45) 0.91)
SOLID-TIMI 10404 0.236 0.75 1.18 1.95E- 0.8(0.69, 2.78E-03 9.74E-05 4.42E-
52 (1.03, 02 0.93) 04
1.33)
META 23981 1.22 4.28E- 0.79 2.07E-05 6.03E-09 1.00E-
(1.11, 05 (0.71, 07
1.33) 0.88)
rs12290663 intergenic G A STABILITY 13577 0.013 0.9 1.03 9.18E- 2.06 5.17E-04 1.16E-02 3.08E-
(0.64, 01 (1.37, 03
1.67) 3.13)
SOLID-TIMI 10404 0.018 0.65 1.07 7.72E- 2.16 1.62E-05 1.30E-02 3.32E-
52 (0.68, 01 (1.52, 04
1.67) 3.03)
META 23981 1.05 7.78E- 2.11 3.15E-08 4.07E-04 4.40E-
(0.76, 01 (1.61, 06
1.45) 2.78)
rs147204125 ACOT6 upstream A G STABILITY 13577 0.008 0.57 2.31 3.52E- 1.79 1.09E-01 5.49E-01 7.83E-
(1.32,4) 03 (0.88, 03
3.57)
SOLID-TIMI 10404 0.011 0.54 3.34 1.24E- 1.71(1, 5.11E-02 9.19E-02 2.40E-
52 (2.04, 06 2.94) 05
5.56)
META 23981 2.86 2.45E- 1.74 1.16E-02 9.14E-02 1.70E-
(1.96, 08 (1.14, 06
4.17) 2.7)
Myocardialinfarction
rs192476688, RP1- intron AT A STABILITY 13577 0.012 0.76 1.23 5.62E- 1.74 0.114879 5.54E-01 3.20E-
rs201052613 15D23.2 (0.63, 01 (0.88, 01
2.44) 3.45)
SOLID-TIMI 10404 0.011 0.66 0.83 6.48E- 4.29(2.7, 1.58E-09 6.95E-04 3.90E-
52 (0.38, 01 6.67) 06
1.82)
META 23981 1.04 8.94E- 3.21 4.47E-09 4.06E-03 1.50E-
(0.63, 01 (2.17, 04
1.75) 4.76)
(Continued)
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Pharmacogeneticanalysisofdarapladibclinicaltrials
Table5. (Continued)
Placebo Darapladib
Combined
Variant Gene Location Major Minor Study N MAF r2 HR# P HR P P P*
Allele Allele
(95%CI) (95%CI) Interaction (2df
test)
rs117714106 intergenic C T STABILITY 13577 0.007 0.59 0.36 1.96E- 3.75 0.000162 5.79E-03 1.88E-
(0.08, 01 (1.89, 03
1.69) 7.69)
SOLID-TIMI 10404 0.007 0.61 0.72 5.80E- 3.82 8.00E-07 1.02E-02 1.75E-
52 (0.23, 01 (2.22, 04
2.27) 6.67)
META 23981 0.57 2.26E- 3.79(2.5, 5.23E-10 2.09E-04 1.50E-
(0.23, 01 5.88) 06
1.43)
#Hazardratioforminorallele
*A2-degree-freedomtestforbothgenotypemainandgenotypebytreatmentinteractioneffect
MAF=Minorallelefrequency
https://doi.org/10.1371/journal.pone.0182115.t005
endpoints,evaluatingdarapladibtreatedsubjectsonly(Table2).Inordertoreduceasignalto
noiseratio,thediarrheaendpointwasdefinedasasubsetthatwasmostlikelyassociatedto
treatmentwithdarapladib(limitedtosubjectswithdiarrheaexperiencedinthefirst90daysof
darapladibtreatment,excludingsubjectswhowerealsotreatedwithmetformin[diarrheaisa
well-knownside-effectofmetformintreatment]andsubjectsofWhiteraceonly.Threelocion
chromosome6,16andX(malesonly)respectivelyweresignificantlyassociatedwiththediar-
rheaendpointatthegenome-widelevel(S5Table).Afurthereightlocimetthegenome-wide
significantassociationthresholdforthemorestringentmoderate/severe(MS)diarrheaend-
point(S5Table).Mostofthesignificantvariantsatall12associatedlocihadlowallelefrequen-
cies,rangingfrom0.6to1.5%,withfewallelesobservedinthepopulationanalyzed.Onlythe
locionchromosomes5,9andX(females)includedsignificantvariantswithfrequencies
around3.5–4.5%.TherewasnocorrelationbetweenthediarrheaendpointsandLp-PLA
2
activitylevelsinthedarapladibtreatmentarmattheseloci(S8Fig).
Analysisofthebathroomandnon-bathroomrelatedodorendpointsdidnotidentifyany
significantgenome-wideassociations,althoughwedidobservesomeregionaldifferencesin
reportingoftheodorevents(seeS1Textfordetails).
Discussion
WehaveperformedacomprehensivePGxanalysisofpharmacodynamic,efficacyandtolera-
bilityphenotypesintwolargedarapladibcardiovascularoutcomestrialswhichfailedtomeet
theirprimaryendpoints.Althoughthetrialsdidnotshowefficacyofdarapladib,understand-
ingwhethergeneticscoulddelineateresponsivesubgroupswasconsideredakeyquestion.
Additionally,itwasimportanttounderstandthetolerabilityphenotypesasanalternativeindi-
cationfordarapladibwasunderconsideration.
InadditiontotheV279FnullalleleweidentifiedthreeotherrarenullPLA2G7alleleswithin
variousethnicgroups,butdidnotobserveanyassociationwithclinicalendpoints.Werepli-
catedtheassociationsofsixpublishedlocionbaselineLp-PLA activitylevelsandidentified
2
threenovelloci.Theanalysisofdarapladibtolerabilityendpointsdidnotrevealanyrobust,
highconfidencegeneticassociations,sincemostofthesignificantvariantsattheassociated
locihadlowallelefrequencies,withfewallelesobservedandanalyzed.Efficacyanalysesre-
vealedonlyonecommonvariantwhichhadadifferentialeffectbytreatmentarm,conferring
PLOSONE|https://doi.org/10.1371/journal.pone.0182115 July28,2017 10/23