Retinal metabolic and structural alterations in response to aflibercepttreatment in neovascular age-related macular degeneration

Jakobsen, D. B., Torp, T. L., Stefansson, E., Peto, T., & Graussland, J. (2018). Retinal metabolic and structuralalterations in response to aflibercept treatment in neovascular age-related macular degeneration. ActaOphthalmologica.

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1

Title

Retinalmetabolicandstructuralalterationsinresponsetoaflibercepttreatmentinneovascularage‐

relatedmaculardegeneration.

Authors

DitteBorupJakobsen1,2

ThomasLeeTorp1,2

EinarStefansson3,4

TundePeto2,5

JakobGrauslund1,2

Affiliations

1. DepartmentofOphthalmology,OdenseUniversityHospital,Odense,Denmark.

2. DepartmentofClinicalResearch,UniversityofSouthernDenmark,Odense,Denmark.

3. UniversityofIceland,Reykjavik,Iceland.

4. LandspitaliUniversityHospital,Reykjavik,Iceland.

5. CentreforPublicHealth,Queen'sUniversityBelfast,Belfast,UK.

Correspondingauthor

ProfessorJakobGrauslund,MD,PhD,DMSci

DepartmentofOphthalmology

OdenseUniversityHospital

Sdr.Boulevard29

DK‐5000OdenseC,Denmark

Phone:+4565412782

Fax:+4566126387

E‐mail:jakob.grauslund@rsyd.dk

2

Abstract

Purpose

Non‐invasive retinalmarkers of disease activity couldpave theway for individualized treatment in

neovascularage‐relatedmaculardegeneration(nAMD).Weaimedtoevaluateifretinalvascularoxygen

saturationandcalibrescouldpredicttheinitialtreatmentresponseafteraloadingphaseofintravitreal

afliberceptinnAMD.

Method

A total of 149 eyeswere included (nAMD, n=76; dry AMD, n=30; normal eyes n=43). Of these, 57

treatment‐naïveeyeswithnAMDreceivedthreemonthlyinjectionswith2.0mgafliberceptandwere

subsequentlystratifiedaccordingto functionalandstructuralresponseaccordingtodevelopment in

best‐correctedvisualacuityandmacularretinalthickness.Theretinalvascularoxygensaturationand

calibresweremeasuredpriortotreatmentandonemonthafterthethirdinjection.

Results

PatientswithnAMDanddryAMDhadhigherretinalarteriolaroxygensaturationascomparedtonormal

eyes(94.3%vs.95.2%vs.92.6%,p=0.04).Thirty‐nine(68.4%)and12(21.1%)eyeswithnAMDwere

functionalandstructuralresponders.Aftertheloadingphase,structuralnon‐respondersdevelopeda

higher retinal arteriolar (95.3% vs. 93.3%, p=0.03) and venular (64.7 vs. 59.4%, p=0.02) oxygen

saturation,andrespondersdevelopedalowerretinalarteriolarcalibre(118.0µmvs.114.3µm,p<0.01).

Inamultiplelogisticregressionmodel,increasingretinalvenularoxygensaturationassociatedwitha

negativetreatmentoutcome(oddsratio1.17foreach1%increment,95%confidenceinterval1.01‐1.36,

p=0.03).

Conclusion

Changes in the retinal venular oxygen saturation associate independently with initial treatment

response innAMD,but functionalandstructural retinalmeasurementsprior to treatmentcouldnot

predictthetreatmentresponse.

Keywords

Neovascular age‐related macular degeneration, choroidal neovascularization, aflibercept, retinal

oximetry,retinalvascularcalibre,diseaseactivity.

3

Introduction

Neovascularage‐relatedmaculardegeneration(nAMD)isthemostcommoncauseofblindnessinthe

elderly population (Ferris et al. 1984;Hoeg et al. 2016) affecting annually 6100 eyes per 1million

inhabitantsintheDanishpopulation(Buchetal.2005).

Repetitive intravitreal injections of vascular endothelial growth factor (VEGF) inhibitors has

demonstratedefficacy(Rosenfeldetal.2006;Brownetal.2009;Heieretal.2012),buttreatmentwith

frequentandcontinuouslyinjectionsisahugeburdenforthepatientsandthehealthcaresystem.

MultiplemechanismsareinvolvedinthedevelopmentofnAMD,andhypoxiahasbeenproposedasa

centralmediator.ItstimulatestheproductionofVEGF,whichpromotesneovascularizationandvascular

leakage(Folkman& Klagsbrun1987;Adamis& Shima2005;Ng& Adamis2005;Stefanssonetal.

2011).

Measurement of the retinal oxygen saturation can be performed non‐invasively by a

spectrophotometric retinal oximeter. In a cross‐sectional study, Geirsdottir et al. reported that in

patientswithtreatment‐naïvenAMD,retinaloxygensaturationandagewerepositivelycorrelatedas

compared to normal controls (Geirsdottir et al. 2014). This could indicate that the retinal oxygen

metabolismisalteredinnAMDandthatitmightserveasapotentialbiomarkeroftreatmentresponse.

Previous studies have also investigated structural changes in retinal vessel calibres after VEGF

inhibitionwithranibizumaborbevacizumabinnAMD(Papadopoulouetal.2009;Fontaineetal.2011;

Micielietal.2012;Tatlipinaretal.2012;Wickremasingheetal.2012;Mendrinosetal.2013;Pekeletal.

2015;Kurtetal.2017).Resultshavebeenconflicting,andasfarasweknow,thishasnotbeentestedin

responsetoaflibercepttreatment,whichhasreplacedranibizumabandbevacizumabinmanycentres.

Thisstudyaimedtoinvestigateifretinalmetabolismandstructureasgivenbyretinalvascularoxygen

saturation and calibresmay be used as non‐invasivemarkers for early treatment response after a

loadingphaseofthreemonthlyintravitrealafliberceptinjectionsineyeswithtreatment‐naïvenAMD.

Materialsandmethods

Recruitmentofpatients

AllpatientswererecruitedattheDepartmentofOphthalmologyatOdenseUniversityHospitalbetween

October2016andJanuary2017.Patientswithtreatment‐naïveeyeswithnAMDwereincludedinthe

prospectivestudy,andinacross‐sectionalcomparison,patientswithdryAMDaswellaseyeswithout

maculardiseasewererecruitedforbaselinecomparison.

Eyeswithpreviousretinallasertreatmentofanykindwereexcludedfromthestudy.Allpatientswere

only allowed to participate in the studywith one eye. If patients in the non‐nAMDgroupshad two

eligible eyes,we included the eyewith best imagequality (and chose the right eye if therewas no

differenceinimagequality).IntheprospectivestudyofnAMD,iftwoeyeswereeligible,weincluded

4

theeyethathadfollow‐upimaging,orifthiswasobtainedinbotheyes,weusedthesamealgorithmas

forthenon‐nAMDgroups.

Datawerecollectedonage,gender,smoking,diabetesandformercataractsurgery.Questionsabout

smokingweredivided into four categories: current smokers, smokingcessation<10years, smoking

cessation>10years,andnon‐smokers.Inthedataanalysis,thefirsttwogroupswerecombinedand

definedasthe“smoking”groupandthelasttwogroupsasthe“non‐smoking”group.

AllparticipantswereCaucasian.

Baseline

Allexaminationsweredoneineyesdilatedwithtropicamide10mg/mlandphenylephrinhydroclorid

10%. All diagnoses were initially given by trained physicians at the department, based upon

ophthalmoscopyandsweptsourceopticalcoherencetomography(SSOCT)(DRIOCTTriton,Topcon,

Tokyo, Japan). If nAMDwas suspected, fluorescein and indocyanine green angiography (Heidelberg

Spectralis, Heidelberg Engineering, Heidelberg, Germany) were performed. For all patients, the

diagnosiswassubsequentlyconfirmedbyaretinalspecialist(JG).DryAMDweredefinedasanynumber

ofmaculardrusenorgeographicatrophyseenonadisc‐centredfundusimage.

Best corrected visual acuity (BCVA) was measured according to the Early Treatment Diabetic

Retinopathy Study (ETDRS) scale. Intraocular pressure (IOP)was recorded (Icare tonometer, Icare

FinlandOy,Helsinki,Finland),andcentralretinalthicknesswasmeasuredbySSOCT.

Non‐invasiveretinalmeasurements

Measurement of retinal oxygen saturation and calibre was performed on disc‐centred 50° fundus

imagescapturedbyOxymapT1andanalysedwiththeOxymapAnalysersoftware(OxymapmodelT1,

Oxymap,softwareversion2.5.0,Reykjavik,Iceland).

Thecamera isbasedona funduscamera, takingtwo fundus imagessimultaneouslyat twodifferent

wavelengths. The 570 nm wavelength image is insensitive to oxygen as opposed to the 600 nm

wavelength. By calculating the difference in absorbance of light in the two images, the software

measuresandvisualises theoxygensaturation inone single imageusinga colourgridonall traced

vessels(Figure1)(Geirsdottiretal.2012).

Twocircleswereplacedontheopticdisccentredimage.Thefirstcirclewasmanuallyplacedaround

theedgeoftheopticdisc,20‐35pixelsawayfromtheedgeofthedisc.Thiswasdonetoavoidreflection

oflightfromtheretinalnervefibrelayeraroundthedisc,asthiscouldinfluencethemeasurementof

oxygensaturation.Thesecondcirclewasplacedconcentricaroundthefirstcircle,withadiameterat

threetimesthediameterofthefirstcircle.Allvesselmeasurementsweredonebetweentheinnerand

theoutercircle.Ineachimage,vesselmeasurementsweredoneon8vessels(oneretinalarterioleand

venuleineachquadrant).Vesselslargerthan6μmwereautomaticallydetectedandtracedforalength

of50‐200pixels.Measurementswereperformedineachvesselfromtheinnercircleuntilalengthof

5

maximumof200pixelswasreachedortothefirstbranchingofthevessel.Ifthebranchingoccurred

beforereachinga lengthof50pixels, thebranchwasmeasured instead.Thesoftwareautomatically

measured the mean retinal vascular oxygen saturation and vascular calibre in each chosen vessel

segment.Retinalarteriolarandvenularsaturationandcalibreweredefinedas themeanof the four

arteriolesandvenulesineachimage.

Measurementsandgradingwerecarriedoutbythesametrainedgrader(DBJ),accordingtoapredefined

protocol.Eyeswithanimagequalityoflessthan6.0(duetoblurrymedia)wereexcludedfromthestudy.

Atfollow‐up,measurementswererepeatedatthesamevessel‐segmentsthatwereusedatbaseline.Ifa

vesselgradedatbaselinewasungradableatfollow‐up,thequadrantwasleftungradedatfollow‐up.

Follow‐upandoutcomemeasurements

EacheyediagnosedwithnAMDreceived threemonthly injectionsof intravitreal2.0mgaflibercept.

Follow‐upwas conducted approximately onemonth after the last injection.At follow‐up, eyeswith

nAMDwereclassifiedasfunctionalandstructuralrespondersandnon‐respondersbasedonchangein

visualacuityandmacularthicknessfrombaselinetofollow‐up.Eyeswereclassifiedasfunctionalnon‐

responders if any decrease in BCVA were seen (≥1 ETDRS‐letters). In addition, structural non‐

respondersweredefinedasnochangeorincreaseinmacularthickness.

Statisticalanalyses

Demographicdataarepresentedasmedianwithinterquartilerange(IQR)forcontinuousdataandas

percentageforcategoricaldata.ComparisonofthethreegroupsatbaselinewasdoneusingKruskal‐

Wallisequalityofpopulationsranktestforcontinuousdata,andchi‐squaretestforcategoricaldata.For

statistical significant differences between groups,Wilcoxon rank‐sum test was used to identify the

groupthatdifferedfromthetwoothergroups.Forcontinuousdata,differencesbetweenresponders

andnon‐responderswerecalculatedbyWilcoxonrank‐sumtest,andforcategoricaldata,weusedchi‐

squaretest.Within‐groupdifferencesfrombaselinetofollow‐upwerecalculatedbyWilcoxonsigned‐

rank test for paired data. We tested the association between retinal oxygen saturation and best

correctedvisualacuity(continuousvariables)byusingthePearsoncorrelationcoefficient.

Multiplelogisticregressionanalysiswasperformedwithretinalvascularoxygensaturationandcalibre

as predictors for treatment outcome in a model adjusted for age, sex, smoking, diabetes and time

betweenlastinjectionandfollow‐up.P‐valuesunder0.05weredefinedasstatisticallysignificant,and

STATA14.2(StataCorp,CollegeStation,TX)wasusedforallstatisticaltests.

Approvals

TheprojectwasapprovedbytheRegionalScientificEthicsCommittee(IDS‐20160102),andtheDanish

Data Protection Agency (ID 16/35476). The project was conducted according to the tenets of the

DeclarationofHelsinkiandstandardgoodclinicalpractice.Allparticipantsgavewrittenconsentbased

onbothwrittenandoralinformationpriortoinclusion.

6

Results

Atotalof149eyesfrom149patientswereincludedinthestudy.Ofthese,76weretreatment‐naïveeyes

withnAMD,30wereeyeswithdryAMD,and43wereeyeswithoutretinalpathology(Table1,Figure

2).InthenAMDgroup,10eyesdidnotreceivetreatment,fiveeyeswerelostduringfollow‐up,retinal

imaging at follow‐upwas not possible in two eyes, and two eyes received two injections andwere

therefore excluded. Hence, 57 eyes with nAMDwere included in the prospective part of the study

(Figure2).Alleyesreceivedthreeinjections,andthetimebetweenthelastinjectionandfollow‐upwas

37.0±13.0days(median±IQR).

Patientswithtreatment‐naïvenAMD,dryAMDandnormaleyesarecomparedinTable1.Thegroup

withnormaleyeswereyounger thantheothergroups(p<0.01andp<0.01)andhada lowerretinal

arteriolar oxygen saturation thanpatientswith nAMD (p=0.02) anddryAMD (p=0.03). In addition,

patientswithnAMDhad lowerBCVAascompared toeyeswithdryAMD(p<0.01)andnormaleyes

(p=0.02).Thethreegroupsdidnotdifferaccordingtogender,smoking,diabetes,IOP,retinalvenular

oxygensaturationorretinalvascularcalibres.

Atbaseline,patientswhowere laterclassifiedas functional respondersandnon‐respondersdidnot

differaccordingtoage,sex,smoking,diabetes,formercataractsurgery,CNVtype,BCVA,IOP,andcentral

retinalthickness(Table2).However,atfollow‐up,BCVAwasbydefinitionhigheramongrespondersas

comparedtonon‐responders(70.0±24.0ETDRSlettersvs.56.5±23.0ETDRSletters,p=0.02).

Atbaselineandfollow‐upfunctionalrespondersandnon‐respondersdidnotdifferaccordingtoretinal

oxygensaturationorvascularcalibre,butbetweenbaselineandfollow‐uprespondersshowedatrend

towardsan increase inretinalarteriolaroxygensaturation(94.3±5.4%vs.95.0±4.4%,p=0.05)and

decreaseinretinalarteriolarcalibre(115.3±15.0μmvs.110.5±12.3μm,p<0.03)(Table3).

Atbaseline,patientswhowerestratifiedasstructuralrespondersandnon‐respondersdidnotdiffer

accordingtoage,sex,smoking,diabetes,formercataractsurgery,CNVtype,BCVA,andIOP.However,

macularthicknesswashigherinrespondingthannon‐respondingpatients(293.0±101.0μmvs.230.0

±44.5μm,p<0.01)(Table2).Nodifferenceswerefoundatfollow‐upbetweenthetwogroups.

Structuralnon‐respondershadanincreaseinarteriolarandvenularoxygensaturationfrombaselineto

follow‐up,(arteriolar93.3±3.3vs.95.3±2.8,p=0.03,venular59.4±11.1vs.64.7±6.5,p=0.02)andthis

incrementwashigherwhencompared tonon‐responders (arteriolar1.6±2.3.vs.0.5±1.7,p=0.03,

venular 3.9 ±7.9 vs. 0.1 ±5.3, p=0.03). In addition, the arteriolar calibre decreased frombaseline to

follow‐upinresponders(118.0±14.5.vs.114.3.±14.5,p=0.01)(Table3).

Inamultiplelogisticregressionmodel,adjustedforage,sex,smoking,diabetesandtimebetweenlast

injection and follow‐up, an increase in retinal venular oxygen saturation associatedwith anegative

7

initial structural treatment outcome (odds ratio for a full treatment response 1.17 for each 1.0%

increment,95%confidenceinterval1.01‐1.36,p=0.03,Table4).

Finally,anegativecorrelationwasseenbetweenbaselineretinalvascularoxygensaturationandchange

inretinalmacularthickness.Patientswithahigherretinaloxygensaturationatbaselinehadalarger

decreaseinmacularthicknessatfollow‐up(arteriolarp<0.01,venularp=0.03)(Datanotshown).

Discussion

Inthisstudy,weprospectivelydemonstratedretinalmetabolicandstructuralalterationsasashort‐

termresponsetointravitrealaflibercepttherapyinpatientswithtreatment‐naïvenAMD.

Theretinalarteriolarandvenularoxygensaturationwere foundto increaseat follow‐upinpatients

withanegativestructuralresponsetotheinitialtreatment.Inaddition,increasedretinalvenularoxygen

saturation independently associated with a 17% increased risk of an unchanged/higher macular

thickness after the loading phase. This could reflect underlying damage of the retinal vasculature

inducedbythepersistentdiseaseactivityinpatientswithoutapositivetreatmentresponseoftheinitial

treatment.

Inpreviousstudies,higherretinalvenularoxygensaturationshavebeendemonstratedinpatientswith

ischemicmaculardiseasessuchasnAMDanddiabeticretinopathy(DR)(Geirsdottiretal.2014;Rilven

etal.2017).Theremightalsobehigherretinalarteriolaroxygensaturationinsuchpatients,butthishas

notbeendemonstratedconsistently,potentiallyduetotheceilingeffectoftheretinalarteriolaroxygen

saturation,whichisoftencloseto100%(Rilvenetal.2017).Thefactthatwedidfindmetabolicchanges

in the retinal arteriolarandvenularoxygensaturationcouldbe seenas anexpressionof the subtle

vascularchangesseeninnAMD.Thediseasemechanismshouldbetakenintoaccountwhencomparing

studiesofnAMDandDR.IncontrasttothedisturbancesofthechoroidalbloodsupplyinAMD,DRaffects

theinnerretina,whichissuppliedfromtheretinalcirculation(Stefanssonetal.2011).

Inaprospectiveclinicalstudy,Bek&Jorgenseninvestigatedretinalmarkersfortreatmentresponsein

eyeswithdiabeticmacular oedema treatedwith three injectionsof ranibizumab (Bek& Jorgensen

2016).Theauthors foundthathighermeanarterialbloodpressureandarteriolaroxygensaturation

before treatmentwas associatedwith a decrease in visual acuity and an increase in central retinal

thickness. In contrast, innAMDwe foundhigher retinal arteriolarandvenularoxygensaturationat

baselinetobecorrelatedwithalowermacularthicknessatfollow‐up.Furthermore,developmentofa

higher arteriolar oxygen saturation after aflibercept loading associated with a negative structural

treatmentresponse.

As far as we know, the concept of retinal oxygen metabolism in nAMD, has only been tested by

Geirsdottir et al. that compared retinal oxygen saturation in eyes with nAMD and healthy eyes

(Geirsdottiretal.2014).Theyfoundtheretinalvenularoxygensaturationtobepositivelycorrelated

8

withageineyeswithnAMD(andviceversainhealthyeyes),butincontrasttoourstudy,theyfoundno

differences in retinal arteriolar oxygen saturation between eyes with nAMD and healthy eyes.

Geirsdottiretal.pointstowardsacorrelationbetweenhighervenularoxygensaturationandamore

severestateofnAMD.Methodologicaldifferencesbetweenthestudiescouldaccountforthedifferent

results.

Severalpreviousstudieshaveinvestigatedtheeffectofranibizumaborbevacizumabonretinalvascular

calibreineyeswithnAMD,andonerecentstudyhasinvestigatedtheeffectofaflibercept(Tetikogluet

al.2017).Thelatterwasaretrospectivestudythatincluded15treatment‐naïveeyeswithnAMDthat

weretreatedwiththreeafliberceptinjectionswith30daysfollow‐upafterthelastinjection.Theyfound

astatisticallysignificantretinalarteriolarvasoconstrictionaftertreatment,inpatientswithapositive

treatmentresponse,thesameresultswerefoundinourstudy.Furthercomparisonsbetweenthestudies

werenotpossible,giventhatonlytheirabstracthasbeenpublished.

The same pattern was also seen in studies of other VEGF‐inhibiting agents. Papadopoulou et al.

demonstrated adecrease in retinal arteriolar calibre in nAMDeyes treatedwith three ranibizumab

injections(n=11)(Papadopoulouetal.2009).Fontaineetal.alsofoundadecreaseinretinalarteriolar

calibreafterthefirstbevacizumabinjection,andthedecreasewasstillpersistentattheendofthestudy,

fiveweeksafterthethirdinjection(n=23)(Fontaineetal.2011).InthestudybyMicielietal.,therewas

asignificantdecreaseinarteriolarcalibreineyeswithnAMD,whichpreviouslyhadthreeorlessanti‐

VEGFinjectionswithranibizumab,butnochangewasseeninthegroupwithmorethanthreeinjections

priortotreatment.Onlytwoeyesinthestudygroupweretreatment‐naïve(n=15)(Micielietal.2012).

InthestudybyMendrionosetal.,decreasedretinalarteriolarcaliberwereseenafterthefirstofthree

injections with ranibizumab, and the difference was still present at 12 months follow‐up (n=10)

(Mendrinosetal.2013).Incontrasttothesestudies,Pekeletal.reportedofunchangedretinalvessel

calibrethreemonthsafterthreeinjectionswithranibizumab(n=32)(Pekeletal.2015).

The two biggest studies investigating the effect of ranibizumab and bevacizumab treatment on the

retinalcalibreinnAMDeyeswereperformedbyWickremasingheetal.(n=88)andKurtetal.(n=68)

(Wickremasingheetal.2012;Kurtetal.2017).InthestudybyKurtetal.,theycomparedbevacizumab

withranibizumabtreatmentineyeswithnAMD(Kurtetal.2017).Patientsweregivenoneinjectionand

followedforonemonth.Theyfoundanarteriolarvasoconstrictionineyestreatedwithranibizumab,

butnotineyestreatedwithbevacizumab.Nochangeswerefoundintheretinalvenularcalibreineither

group.InthestudybyWickremasingheetal.,patientswithnAMDweretreatedwiththreeranibizumab

injectionsandfollowedfor12months(Wickremasingheetal.2012).Theirstudypopulationwasvery

similartooursaccordingtoage(79.1±7.9years)andsex(64.2%females).Theyfoundthatpatients

whohaddecreasedvisualacuityatfollow‐uphadhigherretinalvenularcalibreatbaselinecomparedto

9

thosewhohadstableorimprovedvisualacuity.Noassociationwasfoundbetweenthearteriolarcalibre

andtreatmentoutcome.

Asinourfunctionalfindings,Kurtetalfoundarteriolarvasoconstrictiononeweekandonemonthafter

treatmentwithranibizumab,butnotbevacizumab,ineyeswithnAMD(Kurtetal.2017).

Asasecondaryendpoint,wefoundtheretinalarteriolaroxygensaturationtobehigherinAMD(dryand

neovascularalike)ascomparedtohealthyeyes,eventhoughthegroupswerenotperfectlybalanced

accordingtoage.Thus,wecannotconcludeifthehigheroxygensaturationwascausedbythedisease

orreflectedbythehigherageintheAMDgroups.

Strengthsofourstudyincludetheprospectivedesignwithastrictclassificationoftreatmentresponse

basedonclinicallyrelevantwell‐definedcriteria.Inaddition,allpatientsweretreatedwiththesame

drugatthesamehospital.Ontheotherhand,limitationsshouldbeacknowledged.Intheprospective

partofthestudy,weonlyfollowedpatientsaftertheloadingphasewithnointermediarymeasurements,

thenumberofparticipantswaslimited,andinfluencebymasssignificancecannotberuledoutinthe

statisticalanalysis.Inaddition,systemicmeasurementslikearterialbloodpressurewerenotavailable.

In conclusion, we found higher retinal arteriolar and venular oxygen saturation in patients with a

structuralnegativetreatmentresponse,andadecreaseinretinalarteriolarcalibreinpatientswitha

positive treatmentresponse toaflibercept treatment.Even thoughretinalmetabolismandstructure

prior to therapy could not be used to predict treatment response after three monthly aflibercept

injections, our findings points to a metabolic alteration in eyes with nAMD who do not respond

sufficiently.Long‐termstudieswouldbeneededtotellifthiscouldbeusedasapotentialmarkerthat

canbeusedforclinicalguidanceintherapy.

Acknowledgements

ThestudywasfinanciallysupportedbytheVeluxfoundation.ThestudywaspresentedattheEuropean

AssociationforVisionandEyeResearch(EVER)congressinNicethe29thofSeptember2017.

10

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bevacizumabinjectiononretinalvesselcalibre.Clin.Exp.Optom.95:94‐98.

TetikogluM,MMKurt,HMSagdik,SAktas,MAYildirim&FOzcura(2017):RetrospectiveAnalysisof

theEffectofAfliberceptLoadingDoseontheRetinalVesselDiametersinPatientswith

Treatment‐NaiveNeovascularAMD.Cutan.Ocul.Toxicol.:1‐18.

WickremasingheSS,LBusija,RHGuymer,TYWong&SQureshi(2012):Retinalvenularcaliber

predictsvisualoutcomeafterintravitrealranibizumabinjectiontreatmentsforneovascular

AMD.Invest.Ophthalmol.Vis.Sci.53:37‐41.

12

Figurelegends

Figure1:

A:OpticdisccentredimagetakenbyOxymapT1ofthelefteye(withoutoverlayingcolour‐grid). B:

Sameopticdisccentredpictureas(A)butwithoverlayingcolour‐grid.Thehighlightedpartoftheretinal

vesselsbetweenthetwocirclesisusedformeasurementofretinaloxygensaturationandcalibre.The

oxygenation‐colour‐scaleisshowntotheright.

Figure2:

Flowchart showing the included eyes divided in the three groups (neovascular age‐relatedmacular

degeneration(AMD),dryAMDandnormaleyes).

13

Table1

Comparisonofthethreegroupsatbaseline.

nAMD

Median±IQR

DryAMD

Median±IQR

Normaleyes

Median±IQR

Pvalue

Eyes,n 76 30 43

Age,years 79.0±12.5 77.0±9.0 71.0±9.0** <0.01*

Sex(men),% 38.2 43.3 34.9 0.77

Smokers,% 32.0 14.3 35.7 0.17

Diabetes,% 12.0 10.7 14.6 0.94

BCVA,ETDRSletters 61.0±32.5** 75.0±22.0 75.0±15.0 <0.01*

IOP,mmHg 15.0±6.0 16.0±6.0 15.0±4.0 0.50

Retinalvasculararteriolar

saturation,%94.3±5.2 95.2±5.5 92.6±5.8** 0.04*

Retinalvascularvenular

saturation%60.4±13.6 62.0±15.1 58.3±10.5 0.28

Retinalvasculararteriolar

calibre,μm114.6±17.0 111.1±18.0 113.5±16.0 0.20

Retinalvascularvenular

calibre,μm151.9±22.8 151.9±25.0 155.0±29.5 0.21

ContinuousdataarecalculatedusingKruskal‐Wallisequalityofpopulationsranktestandpresentedas

median and interquartile range (IQR). Categorical data are calculated using chi‐squared test and

presentedaspercentage.Forstatisticallysignificantpvalues,**marksthegroupthat isstatistically

differentfromthetwoothergroups(calculatedusingWilcoxonrank‐sumtest).*p<0.05

nAMD:neovascularage‐relatedmaculardegeneration.BCVA:Best‐correctedvisualacuity.ETDRS:

EarlyTreatmentDiabeticRetinopathyStudy.IOP:intraocularpressure.

14

Table2

Characteristicsofpatientswithtreatment‐naïveneovascularage‐relatedmaculardegenerationasstratifiedbyinitialfunctionalandstructuralresponseto

loadingwiththreemonthlyinjectionswithintravitrealaflibercept.

Functionalresponse Structuralresponse

Yes

Median±IQR

No

Median±IQRPvalue

Yes

Median±IQR

No

Median±IQR Pvalue

Baseline

Eyes,n 39 18 12 45

Age,years 79.0±11.0 81.5±14.0 0.40 78.0±7.5 8.1±13.0 0.79

Sex(men),% 46.2 27.8 0.18 50.0 37.8 0.44

Smoking(smokers),% 30.8 22.2 0.60 25.0 28.9 0.83

Diabetes(diabetes),% 16.7 2.6 0.48 25.0 6.6 0.16

Cataractsurgery(former),% 64.1 50.0 0.31 41.7 64.4 0.15

TypeofCNV(classic/occult/other),% 38.5/61.5/0.0 44.4/55.6/0.0 0.66 25.0/75.0/0.0 44.4/55.6/0.0 0.22

BCVA,ETDRSletters 60.0±31.0 67.5±23.0 0.91 66.5±25.0 64.0±29.0 0.75

IOP,mmHg 15.0±6.0 15.5±7.0 0.65 15.0±4.0 15.0±7.0 0.43

Centralretinalthickness,μm 284.0±97.0 277.0±145.0 0.91 293.0±101.0 230.0±44.5 <0.01*

Follow‐up

Timefromlastinjectiontofollow‐up,

days37.0±12.0 35.5±13.0 0.16 40.0±14.0 36.0±13.0 0.20

BCVA,ETDRSletters 70.0±24.0 56.5±23.0 0.02* 67.5±20.5 65.0±33.0 0.71

Centralretinalthickness,μm 237.0±55.0 226.0±61.0 0.20 232.0±57.0 249.0±62.0 0.19

StructuralandfunctionalresponsedefinedaccordingtochangeinBCVAandretinalmacularthicknessbetweenbaselineandfollow‐up.Atfollow‐uppatients

withapositivestructuralandfunctionalresponsehadnolossinBCVAandthinnerretinalmacularthickness,respectively.Continuousdataarecalculatedusing

15

Wilcoxonrank‐sumandarepresentedasmedianandinterquartilerange(IQE).Categoricaldataarecalculatedusingchi‐squaredtestandarepresentedas

percentage. CNV: Choroidal neovascularization. BCVA:Best‐corrected visual acuity. ETDRS: Early TreatmentDiabeticRetinopathy Study. IOP: intraocular

pressure.*p<0.05

16

Table3

Retinalmetabolicandstructuralmeasurementsasstratifiedbyinitialfunctionalandstructuralresponseto

intravitrealafliberceptloadinginpatientswithtreatment‐naïveneovascularage‐relatedmaculardegeneration.

nBaseline

Median±

IQR

Follow‐up

Median±

IQR

Difference

Median±

IQR

Baselineto

follow‐up,

paired

Pvalue

Functionalresponse

Retinal

arteriolar

saturation,%

Responders 39 94.3±5.4 95.0±4.4 0.8±1.8 0.05

Non‐responders 18 94.7±4.1 95.2±3.6 0.3±2.8 0.39

Pvalue 0.93 0.98 0.70

Retinalvenular

saturation,%

Responders 39 61.5±12.8 63.5±12.1 1.4±6.7 0.37

Non‐responders 18 59.5±17.0 60.0±17.5 0.3±6.0 0.14

Pvalue 0.71 0.55 0.82

Retinal

arteriolar

calibre,μm

Responders 39 115.3±15.0 110.5±12.3 ‐1.3±7.3 0.05

Non‐responders 18 118.6±20.5 118.5±24.5 ‐1.9±4.3 0.19

Pvalue 0.63 0.28 0.69

Retinalvenular

calibre,μm

Responders 39 152.0±22.8 149.8±21.9 1.0±9.3 0.34

Non‐responders 18 155.3±16.8 155.5±22.0 ‐1.0±9.3 0.98

Pvalue 0.63 0.51 0.59

Structuralresponse

Retinal

arteriolar

saturation,%

Responders 12 95.2±4.9 95.0±4.9 0.5±1.7 0.23

Non‐responders 45 93.3±3.3 95.3±2.8 1.6±2.3 0.03*

Pvalue 0.14 0.86 0.03*

Retinalvenular

saturation,%

Responders 12 61.7±14.9 62.0±14.3 0.1±5.3 0.50

Non‐responders 45 59.4±11.1 64.7±6.5 3.9±7.9 0.02*

Pvalue 0.55 0.53 0.03*

Retinal

arteriolar

calibre,μm

Responders 12 118.0±14.5 114.3±14.5 ‐1.3±5.0 0.01*

Non‐responders 45 111.8±12.6 111.0±12.6 ‐1.0±6.9 0.45

Pvalue 0.39 0.52 0.68

17

Retinalvenular

calibre,μm

Responders 12 152.0±18.8 152.0±19.0 1.0±7.5 0.27

Non‐responders 45 156.2±25.1 147.3±23.7 ‐1.5±12.6 0.78

Pvalue 0.72 0.68 0.40

Functionalandstructuralresponsedefinedaccordingtochangeinbest‐correctedvisualacuity(BCVA)andretinal

macularthicknessbetweenbaselineandfollow‐up.Atfollow‐uppatientswithapositivestructuralandfunctional

response had no loss in BCVA and thinner retinal macular thickness, respectively. Data is calculated using

Wilcoxonrank‐sumtest.Thepvalueforthechangesfrombaselinetofollow‐upforbothgroupswascalculated

usingWilcoxonsigned‐ranktest (paireddata).Dataarepresentedasmedianwith interquartilerange(IQR). *

p<0.05

18

Table4

Multiplelogisticregressionmodelindicatingoddsratios(ORs)and95%confidenceintervals(CIs)of

structuralandfunctionaltreatmentresponseafterintravitrealafliberceptloadingintreatment‐naïve

neovascularage‐relatedmaculardegenerationaccordingtoretinalvascularoxygensaturationand

calibre.

OR(95%CI) Pvalue

Functionalresponse

Retinalarteriolaroxygen

saturation,%

Baseline 1.03(0.87‐1.22) 0.69

Follow‐up 1.06(0.91‐1.23) 0.43

Difference 1.08(0.86‐1.34) 0.50

Retinalvenularoxygen

saturation,%

Baseline 1.04(0.95‐1.13) 0.34

Follow‐up 1.05(0.97‐1.13) 0.19

Difference 1.05(0.93‐1.18) 0.44

Retinalarteriolarcalibre,

μm

Baseline 0.98(0.92‐1.02) 0.36

Follow‐up 0.97(0.92‐1.02) 0.24

Difference 0.97(0.86‐1.09) 0.67

Retinalvenularcalibre,μm

Baseline 0.97(0.93‐1.01) 0.16

Follow‐up 0.98(0.94‐1.01) 0.22

Difference 1.01(0.97‐1.84) 0.07

Structuralresponse

Retinalarteriolaroxygen

saturation,%

Baseline 0.91(0.75‐1.08) 0.28

Follow‐up 1.02(0.86‐1.20) 0.85

Difference 1.34(0.97‐1.84) 0.07

Retinalvenularoxygen

saturation,%

Baseline 0.98(0.90‐1.07) 0.65

Follow‐up 1.05(0.95‐1.16) 0.28

Difference 1.17(1.01‐1.36) 0.03*

Retinalarteriolarcalibre,

μm

Baseline 0.98(0.92‐1.03) 0.39

Follow‐up 0.98(0.91‐1.04) 0.44

19

Difference 1.02(0.90‐1.16) 0.74

Retinalvenularcalibre,μm

Baseline 0.99(0.95‐1.03) 0.70

Follow‐up 0.99(0.95‐1.03) 0.49

Difference 0.95(0.86‐1.06) 0.38

StructuralandfunctionalresponsedefinedaccordingtochangeinBCVA(bestcorrectedvisualacuity)

andretinalmacular thicknessbetweenbaselineand follow‐up.At follow‐uppatientswithapositive

structuraland functionalresponsehadno loss inBCVAand thinnermacular thickness,respectively.

Dataiscalculatedusingmultiplelogisticregressionanalysis,adjustedforage,sex,smoking,diabetes

andtimebetweenlastinjectionandfollow‐up.*p<0.05

20

Figure1

(A)

(B)

21

Figure2

nAMD

76eyes

nAMDfollow‐up

57eyes

Functionalresponse

57eyes

Responders

39eyes

slet

Non‐responders

18eyes

Structuralresponse

57eyes

Responders

12eyes

Non‐responders

45eyes

Notreatment

10eyesslet

Nofollow‐up

5eyesslet

Retinalimagingnotpossible

2eyes

Slet

Lessthan3injections

2eyes

DryAMD

30eyes

Normal

43eyes