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Targeting the PD1/PD-L1 axis in melanoma: Biological rationale, clinical challenges and opportunities

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CriticalReviewsinOncology/Hematology89(2014)140–165

Targeting

the

PD1/PD-L1

axis

in

melanoma:

Biological

rationale,

clinical

challenges

and

opportunities

Barbara

Merelli

a

,

Daniela

Massi

b

,

Laura

Cattaneo

c

,

Mario

Mandalà

a,

aUnitofMedicalOncology,DepartmentofOncologyandHematology,PapaGiovanniXXIIIHospital,Bergamo,Italy bDivisionofPathologicalAnatomy,DepartmentofSurgeryandTranslationalMedicine,UniversityofFlorence,Italy

cDivisionofPathologicalAnatomy,PapaGiovanniXXIIIHospital,Bergamo,Italy Accepted15August2013

Contents

1. Introduction... 141

1.1. Immuneevasionashallmarkincancer... 141

2. Tcellrecognitionandimmunecheckpoints... 141

3. PD-1/PD-L1expressionandbiologicalfunction... 143

3.1. FunctionalimplicationsofPD1/PD-L1interaction... 144

4. PD-1/PD-L1inmalignantdiseases... 144

5. PrognosticroleofPD-L1andPD-1inmalignancies... 146

6. Clinicaltrialsandfuturedevelopments... 156

7. Conclusion... 162

Reviewers... 162

Conflictofintereststatement... 162

References... 162

Biographies ... 164

Abstract

Adynamicinterplayexistsbetweenhostandtumor,andtheabilityofthetumortoevadeimmunerecognitionoftendeterminestheclinical courseofthedisease.Significantenthusiasmcurrentlyexistsforanewimmunotherapeuticstrategy:theuseofimmunomodulatorymonoclonal antibodiesthatdirectlyenhancethe functionofcomponentsoftheanti-tumorimmuneresponsesuchasTcells,orblockimmunologic checkpointsthatwouldotherwiserestraineffectiveanti-tumorimmunity.Thisstrategyisbasedontheevidencethatdevelopmentofcancer isfacilitatedbythedis-regulationandexploitationofotherwisephysiologicalpathwaysthat,undernormalcircumstances,down-regulate immuneactivationandmaintaintolerancetoself.AmongthesepathwaysanimportantroleiscoveredbytheProgrammeddeath-1 (PD-1)/PD-Ligand(L)1axis.AnemergingconceptincancerimmunologyisthatinhibitoryligandssuchasPD-L1areinducedinresponseto immuneattack,amechanismtermed“adaptiveresistance”.Thispotentialmechanismofimmuneresistancebytumorssuggeststhattherapy directedatblockingtheinteractionbetweenPD-1andPD-L1mightsynergizewithothertreatmentsthatenhanceendogenousantitumor immunity.Theanti-PD-1strategycanbeeffectiveinseveralsolidtumorssuchasrenalcellcarcinoma(RCC)ornon-smallcelllungcancer (NSCLC),howeverinthisreviewwesummarizethebiologicalroleofPD-1/PD-L1oncancerbyfocusingourattentioninthebiological rationale,clinicalchallengesandopportunitiestotargetthePD-1/PD-L1axisinmelanoma.

©2013ElsevierIrelandLtd.Allrightsreserved.

Keywords:PD-1;PD-L1;Melanoma

Correspondingauthorat:UnitofMedicalOncology,DepartmentofOncologyandHaematology,PapaGiovanniXXIIIHospital,PiazzaOMS1,24100 Bergamo,Italy.Tel.:+390352673687;fax:+390352674985.

E-mailaddresses:bmerelli@hpg23.it(B.Merelli),daniela.massi@unifi.it(D.Massi),lcattaneo@hpg23.it(L.Cattaneo),mariomandala@tin.it (M.Mandalà).

1040-8428/$–seefrontmatter©2013ElsevierIrelandLtd.Allrightsreserved. http://dx.doi.org/10.1016/j.critrevonc.2013.08.002

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B.Merellietal./CriticalReviewsinOncology/Hematology89(2014)140–165 141

1. Introduction

1.1. Immuneevasionashallmarkincancer

Cellular transformation and tumor development result fromanaccumulationofmutationalandepigeneticchanges thatalternormalcellgrowthandsurvivalpathways [1].In 2000,HanahanandWeinbergreportedsixbiological capabil-ities(hallmarks)acquiredduringthemultistepdevelopment ofhumantumors:sustainingproliferativesignaling,evading growthsuppressors,resistingcelldeath,enablingreplicative immortality,inducingangiogenesis,andactivatinginvasion andmetastasis[2].Inthelasttwodecades,basedonthe emer-genceofnewexperimentaldata,twofurtherhallmarkshave beenaddedtothislist:reprogrammingofenergymetabolism andevadingimmunedestruction[3].

This Copernican revolution had its foundation on the awarenessthatcompleteknowledgeofcancerdevelopment cannotbe achievedwithout recognizingtheimportance of thetumormicroenvironment,averyimportantpartofwhich isplayedbytheimmunesystem.Adynamicinterplayexists betweenhostandtumor,andtheabilityofthetumortoevade immunerecognition(immunesurveillance)oftendetermines theclinicalcourseofthedisease[4].

Theconceptof cancerimmunesurveillanceisbasedon the hypothesis that the immune system can suppress the developmentorprogressionofspontaneousmalignancies[5]. Severaldata,firstfromanimalmodelsandlaterfromstudies incancerpatients,confirmedthisoriginal conceptthat the immunesystemcanrecognizeandrejecttumors,supporting thehypothesisthatimmuneevasionbycancercellsplaysan importantroleinthedevelopmentandprogressionoftumors [6].

Basedonthisbiologicalbackground,cancer immunother-apyfocusesonthedevelopmentofagentsthatcanactivatethe immunesystemtorecognizeandkilltumorcells.However, untilrecently,alltheeffortstotherapeuticallymodulatethe immunesystemweretemperedbydisappointingresultsfrom clinicaltrialswithcancervaccinesandbiochemotherapy regi-mens,as wellas bythe lowresponse rates andhightoxic effectassociated withthese two strategies[7–14]. Athird approachknownasadoptivecelltherapyhasshownclinical benefitforsomepatientsalthoughtechnicalaspectsandthe complexityoftheprocedureshavelimitedmorewidespread use[15].Despitetheinitiallackofclinicalsuccess,extensive researchoverthepasttwentyyearsyieldedtheidentification of innovativewaysto manipulatethe immune response to cancer.

Theregulationoftheimmunesystemdependsonafine controlsysteminwhichakeyroleisplayedbycellular recep-torsthatensuretheactivationorinhibitionofcellsinvolved inthecontrolofinfectionsandtumorsanddevelopmentof autoimmunity.Someofthesemechanismsareactivatingand dictatewhethertheresponsearises,whileothersplaytherole ofpowerfulrepressors.Antagonistantibodiesactingonsuch repressorsresultinenhancedimmuneresponses,agoalthat

mayalsobeachievedwithagonistantibodiesactingonthe activatingreceptors.

More recently,significantenthusiasmarosefor afourth immunotherapeuticstrategy:theuseofimmunomodulatory monoclonalantibodiesthatdirectlyenhancethefunctionof components ofthe anti-tumorimmune responsesuch as T cells or block immunologic checkpoints that would other-wise restrain effective anti-tumor immunity. This strategy is based on the evidence that development of cancer is enabledbythedis-regulationandexploitationofotherwise physiological pathways that, under normal circumstances, down-modulate immuneactivation and maintain tolerance to self. A series of therapeutic agents are under clini-cal development, and one of them, which is directed at theCTL-associatedantigen4(CTLA-4)inhibitoryreceptor (Ipilimumab,Yervoy®),hasbeenapprovedforthetreatment of metastaticmelanoma. Thelist of antagonist agents act-ingonrepressorsunderdevelopmentincludesanti-CTLA-4, anti-Programmeddeath-1(PD-1),anti-PD-Ligand1(L1)or (B7-H1), anti-KIR (killer cell Ig-like receptor), and anti-TGF-␤. Agonist antibodies currentlybeing investigated in clinicaltrialstargetCD40,CD137(4-1BB),CD134(OX40), andglucocorticoid-inducedTNFreceptor (GITR)(Fig.1). Amongthesepathwaysanimportantroleiscoveredbytwo immunologiccheckpoints:theCTLA-4andthePD-1/PD-L1 axis.

An emerging concept in cancer immunology is that inhibitory ligands such as PD-L1 are induced in response toimmuneattack,amechanismtermedadaptiveresistance. Thispotentialmechanismof immuneresistancebytumors suggeststhattherapydirectedatblockinginteractionbetween PD-1andPD-L1mightsynergizewithothertreatmentsthat enhanceendogenousantitumorimmunity.

In thisreviewwe summarizethebiologicalroleof PD-1/PD-L1incancerbyfocusingourattentiononthebiological rational, clinicalchallenges andopportunitiestotarget the PD-1/PD-L1axisinmelanoma.

2. Tcellrecognitionandimmunecheckpoints

InductionofimmuneresponserequiresTcellstoreceive twosetsofsignalsfromantigen-presentingcells:theTcell receptormustrecognizecomplexesofMHCwiththeantigen onthesurfaceofanantigen-presentingcell(APC).Tcells andtheTcellreceptorcomplexdonotrespondtoantigenin solution,butevenforthespecificantigentheyonlyrespondto antigen-MHCcomplexesonthecellsurface.Thisinteraction isnecessary,butnotsufficient,forTcellactivation.

T cell activation also requires a co-stimulatory signal involving interaction of CD28 on the T cell with CD80 or CD86 (B7 family genes) on the APC, which pro-motesTcellclonalexpansionandcytokinesecretion[16]. CD28activatesasignaltransductionpathwayactingthrough Phosphatidylinositide3-kinases(PI3K),lymphocyte-specific protein tyrosine kinase (Lck) and adaptor proteins GRB2

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142 B.Merellietal./CriticalReviewsinOncology/Hematology89(2014)140–165

Fig.1.Agonistantibodies(ontheleft)andantagonistantibodies(ontheright).Antagonistagentsunderdevelopmentactingonrepressorsincludeanti-CTLA-4 andanti-Programmeddeath-1(PD-1).

andGRB2-related adaptorprotein (Grb-2/ITK) toprovide itsco-stimulatorysignalforTcellactivation.

Tcell-mediatedimmunityisregulatedbybalancing stimu-latoryandinhibitorysignalsthatregulatetheresponse[17]. Intheabsenceofco-stimulatorymolecules,theTcellsenter anunresponsivestateknownasclonalanergy inwhichthe Tcellsareincapableofprovidingantigen-specificimmune responses[18].

Inphysiologicalconditions,immunecheckpointsare cru-cialformaintainingself-toleranceandforprotectingtissues fromthedamageoftheimmuneresponsetopathogenic infec-tion.

There is evidencethat tumorsresist immune attack by inducing tolerance toward tumor-specific T cells and by expressing ligands that bind inhibitory receptors such as immunecheckpoints.Tumorscanresult inderegulation of theimmune-checkpointproteinsanddevelopamechanism for immune resistance, especially against T cells that are specificfortumorantigens[19].

Preliminaryclinicaldatashowthatantibodyblockageof immunecheckpoints cansubstantiallyenhancetherapeutic antitumor immunity[19,20].Unlike conventional antibod-iesusedfor the treatment oftumors, antibodies thatblock immunecheckpointsdonotbinddirectlytothetumorcells, buttargetlymphocytereceptorsortheirligands,inorderto modulatetheirantitumoractivity.

Several immunologic treatment options, such as the induction of an immuneresponse or the administration of antibodies, have been investigated in melanoma and have showninterestingresults[21].CTLA-4andPD-1arethetwo mostinvestigatedimmunecheckpointreceptorsinmelanoma and cancer immunotherapy (Table 1) [22]. CTLA-4 is an inhibitory membrane receptor expressed exclusively on T cells,whereitprimarilyregulatestheamplitudeoftheearly

stagesofTcellactivation,counteractingtheactivityoftheT cellco-stimulatoryreceptor,CD28.

Asreportedabove,theengagementoftheTcellantigen receptorbyitselfisnotsufficientforfullTcellsactivation;a secondco-stimulatorysignalisrequired.Thisco-stimulation ismediatedbyengagementofCD28ontheTcellsurfaceby membersoftheB7familyonAPC[23].

These co-stimulatory molecules are integral membrane proteinsexpressedonseveralcellswithAPCfunction.These molecules,includingB7.1(CD80)andB7.2(CD86),bindto otherligands onT cellsandprovide the second signalfor T-cells activation.Limited expressionof B7 on APCsisa mechanismformaintenanceofperipheralTcellstolerance, ensuring that T cells activation canonlybe stimulated by appropriatecells[24].

Interestingly,tumorcellsdonotexpressB7,andthis con-tributestotheir poorcapacitytoinduceimmuneresponses [25,26].Afteractivation, Tcells expressCTLA-4, aclose homologuetoCD28.CTLA-4bindsmembersoftheB7 fam-ilywithamuchhigheraffinitythanCD28[27].Accordingly, CTLA-4 expressiononthesurfaceofTcellsdecreasesthe activationofTcellsbycompetingwithCD28forbindingwith CD80andCD86.CTLA-4exertsdistincteffectsonthetwo majorsubsetsofCD4+Tcells:down-modulationofhelper TcellactivityandenhancementofregulatoryT(TReg)cell

immunosuppressiveactivity.

ThespecificsignalingpathwaysbywhichCTLA-4blocks T cell activation are still under investigation, although a number of studies suggest that activation of the protein phosphatases,SHP2(alsoknownasPTPN11)andPP2A,is importantincounteractingkinasesignalsthatareinducedby TCRandCD28.

The centralrole of CTLA-4 for keeping T cell activa-tion in check is dramatically demonstrated by the lethal

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B.Merellietal./CriticalReviewsinOncology/Hematology89(2014)140–165 143 Table1

SimilaritiesanddifferencesbetweenCTLA-4andPD-1.

PD-1 CTLA-4

Biologicalfunction Inhibitoryreceptor Inhibitoryreceptor Expressionon ActivatedTcells,activatedBcells,activatedNK

cells,TILsindifferenttumortypes

Tcellsatthetimeoftheirinitialresponsetoantigen (activatedCD8+effectorTcells)

Majorrole •LimitationofTcellsactivityinperipheraltissues followinginflammatoryresponses

•Limitationofautoimmunity

RegulationoftheearlystageofTcellsactivation

Ligands •PD-L1(B7-H1/CD274) •PD-L2(B7-CD/CD273)

•CD80(B7.1) •CD86(B7.2)

Mechanismofaction •PD-1bindstotheligand •CTLA-4interactswiththeligand

↓ ↓

•RecruitmentofphosphataseSHP-2 •BindingwithPI3K,phosphatasesSHP-2and PP2A

•Decreasedexpressionofthecellsurvivalprotein Bcl-xL

•Blockadeoflipid-raftexpression ↓

•PD-1inhibitskinases(PI3K/AKT)thatare involvedinTcellsactivation

•Blockadeofmicroclusterformation

NKcells,naturalkillcells;CTLA-4,cytotoxicT-lymphocyteantigen4;PD-1,Programmeddeath-1;PD-L1,Programmedcelldeath1ligand1;PI3K, Phosphatidylinositide3-kinase.

systemic immune hyperactivation phenotype of CTLA-4-knockoutmice.

In experimental models, mice rapidly develop lym-phoproliferative disease with multiorgan lymphocytic infiltration and tissue destruction, including particularly severemyocarditisand pancreatitis,anddie at 3–4 weeks ofage[28].TheseverephenotypeofmicelackingCTLA-4 impliesacriticalroleforCTLA-4indown-regulatingTcell activationandmaintainingimmunologichomeostasis.Inthe absenceofCTLA-4,Tcellsareactivated,canspontaneously proliferate,andmaymediatelethaltissueinjury.

3. PD-1/PD-L1expressionandbiologicalfunction

ThePD-1receptorisa50–55kDatypeItransmembrane glycoprotein of the Ig superfamily, with an extracellular domainshowing21–33%sequenceidentitywithCTLA-4, CD28 and the inducible co-stimulatory (ICOS) molecule, but distinct function and ligand specificity [29,30]. PD-1 showsanextracellularIgVregion,atransmembranedomain, andanintracellular tail. Thecytoplasmic domainpresents two tyrosine residues: one represents an immunoreceptor tyrosine-based inhibitory motif (ITIM), and the other an immunoreceptor tyrosine-based switch motif (ITSM) that mayrecruitphosphatases,similartoothernegativeregulators [31,32].

PD-1 expression is mostly detected at the cell surface (membrane)level,whereitexertsitsfunctionofinhibitory receptor,however,PD-1expressionhasalsobeenobserved inthecellcytoplasm[33].Itisasyetunclearwhether cyto-plasmicPD-1exertsbiologicalfunctionsormerelyservesas

arepositoryoftheprotein,allowinganimmediateresponse assoonasitsexpressionatthemembraneisrequested.

PD-1isexpressedonCD4−/CD8−thymocytesin transi-tiontoCD4+/CD8+stageandonmatureT-andBcellsupon activation,whileitisnotdetectableonrestingTcells.Thus, in comparisonwith CTLA-4, PD-1has a slightlybroader expressionprofile,alsopresentonactivatedmyeloidlineage cellssuchasmonocytes,dendriticcellsandNKcells[34,35]. PD-1isquicklyup-regulatedonTlymphocytesafter expo-suretocognate antigenanditsexpressioniscontrolledby several cytokines including IFN-␥, IL-2, IL-7, IL-15, and IL-21[36].Uponantigenclearance,PD-1expressionwanes accordingly.Innormaltissues,PD-1signalinginTcells regu-latesimmuneresponsestodiminishdamage,andcounteracts thedevelopmentofautoimmunitybypromotingtoleranceto self-antigens.

MostbiologicalfunctionsofPD-1havebeenelucidatedby generatingPD-1-deficientmicewithgeneknockout technol-ogy.PD-1-knockoutmicespontaneouslydevelopphenotypes of lymphoproliferativeautoimmunediseases,accompanied byamarkedaccumulationofinflammatorycellsinaffected organs, including CD4+ and CD8+ T cell subsets [37]. AutoimmunemanifestationsinPD-1micearedifferentfrom those observed in CTLA-4 −/− mice, which die at 3–4 weeksofagefrommassivelymphocyticinfiltrationand tis-sue destruction in multiple organs[38]. The PD-1related autoimmunephenomenaareoverallmilderandlessfrequent thanthoseobservedfollowinganti-CTLA-4therapy.Table1 summarizes the main differences between CTLA-4 and PD-1.

TwoligandsforPD-1,designatedPD-1ligand1(PD-L1; B7-H1/CD274)andPD-1ligand2(PD-L2;B7-DC/CD273), have been identified based on the similarity to other B7

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144 B.Merellietal./CriticalReviewsinOncology/Hematology89(2014)140–165

superfamilies.PD-L1andPD-L2aretypeItransmembrane glycoproteins composed of IgC- and IgV-type extracellu-lardomainsthatpresent40%aminoacididentitysequence [34,39–41]. In contrast to the limited expression of PD-L2onactivatedmacrophages[42],PD-L1ismorebroadly expressedonimmuneandnon-hematopoieticcells. Specif-ically,PD-L1isconstitutivelyexpressedonT-andBcells, macrophages,anddendritic cells,andis upregulated upon stimulationbyproinflammatorycytokinesincludingIFN.

DespitethefactthatPD-L1mRNAhasbeenreportedin awiderangeofhumanhealthytissuesandcelltypes(with highexpressioninplacenta,heart,lungandliver,low expres-sioninspleen,lymphnodesandthymus,andnoexpression inthe centralnervous system),constitutive PD-L1protein is much less ubiquitous in normal tissues.PD-L1 protein hasalsobeenreportedtobeexpressedonparenchymalcells suchaspancreaticisletcells,endothelialcells,musclecells andtrophoblasts.DiscrepanciesbetweenmRNAandprotein expressionare most likely attributabletoa seriesof post-transcriptional controls that have not yet been completely clarified[43].

PD-L1-deficientmicedonotdevelopspontaneous autoim-mune diseases, mild-to-moderate levels of lymphocyte accumulationareevidentinthekidneys,liver,andlung,with apredominant CD3+/CD8+ component exhibiting signifi-cantlydecreased apoptosis [44]. Although the mechanism underlyingselectiveaccumulationofCD8+Tcells in PD-L1−/−miceremainstobeclarified,thesefindingsimplicate aroleforPD-L1inthemaintenanceofTandcellhomeostasis inperipheralorgans.Overall,thesedatasupportthe hypoth-esisthattheexpressionofthisligandinnon-lymphoidtissue cellscanpreventimmune-mediatedtissuedamage[45].

3.1. FunctionalimplicationsofPD1/PD-L1interaction

PD-1/PD-L1 interactioninhibitsTlymphocyte prolifer-ation,survival,andeffectorfunctions,inducesapoptosisof antigen-specificTcells,andpromotesthedifferentiationof CD4+TcellsintoFoxp3+regulatoryTcells.Themechanism bywhichPD-1exertstheinhibitoryeffecthasbeenpartially explained.Assoon as PD-L1interactswithPD-1,there is arecruitmentofSrchomologyregion2domain-containing phosphatase-1 (SHP-1) and SHP-2, which dephosphory-latemultiplemembersof theTCRsignalingpathway.This abrogatesdownstreameffectsofT-cellactivation,including cytokineproduction,cell-cycleprogression,andthe expres-sionofsurvivalproteins.Furthermore,theinhibitionofRAS andPI3K/AK3pathwayscooperatestoinhibitlymphocyte’ functionandsurvival(Fig.2).

Consideringthat(1)PD-L1isupregulatedin hematopoi-eticandreticuloendothelialcells,inresponseto proinflam-matorycytokinessuchasIFN-␥,and(2)PD-1isexpressedto variousdegreesonactivatedTcells,itisreasonableto specu-latethattheco-expressionofligandandreceptorininflamed tissues mitigatesagainst collateraltissue destruction byT cellsatthesesites.ThePD-1/PD-L1axiswouldthereforebe

importantinmitigatingtheinflammatoryresponseandinturn potentialautoimmunefociresultingfromthedysregulationof theeffectorphaseoftheimmuneresponse.Thismechanism isimportantinvariousphysiological andpathological pro-cesses including feto-maternaltolerance, graft-versus-host disease,andvariousautoimmunediseases.

Anotherpieceofthepuzzleforunderstandingthecentral roleofthePD-1/PD-L1axisintheimmuneresponsederives fromstudiesevaluatingthedevelopingofTcellexhaustion. WhilePD-1isexpressedonactivatedlymphocytesduringan acuteinflammatoryprocess tolimittissue damage and, as soonastheexogenousstimulusiscleared,thePD-1wanes accordingly, the persistent antigen exposure may prevent the down-regulation of PD-1. Since the immune response playsan important rolein stavingoff cancer,mechanisms ofimmunosuppressionhinderproductiveanti-tumor immu-nity.Tcellexhaustionisonesuchmechanism.PD-1hasbeen identifiedasamarkerofexhaustedTcellsinchronicdisease states,includingcancer,andblockadeofPD-1–PD-L1 inter-actions hasbeenshowntopartiallyrestoreTcellfunction. ThedevelopmentofTcellexhaustiontranslatesintoamajor immuneresistanceandpromotestheimmuneevasion.

4. PD-1/PD-L1inmalignantdiseases

Via immunohistochemistry, PD-L1 is constitutively expressedinmanyhumancancers[46].Thefindingthat PD-L1iscommonlyupregulatedonmanydifferenttumortypes including melanoma[47],ovariancancer[48],lungcancer [49], clearcellrenal carcinoma [50],urothelial carcinoma [51],squamouscellcarcinomasoftheheadandneck[52,53], esophageal cancer[54], cervical cancer[55], breast carci-noma[56],pancreaticcancer[57],gastriccancer[58],Wilms tumor[59]andglioblastoma[60],andthatPD-1isexpressed inTILs,hascreatedanimportantrationaleformAbblockade ofthispathwayforcancerimmunotherapy.

In additiontoexpressionontumorcells,PD-L1canbe detectedoncellslocatedinthetumormicroenvironment,and highlevelsofPD-L1expressionhavebeenreportedinTILs andtumor-associatedmacrophages[49,61,62].However,the ultimate meaning of the PD-L1expression ontumor cells andothercellsinthemicroenvironmentremainstobefully determined.

PD-L1 expression on cancer cells may be an adaptive response to immune attack and induced by cytokines, or constitutiveasaresultofoncogenicprocesses,asoccursin

BRAFV600 mutated melanoma. The role of oncogenes to

driveimmunosuppressionhasbeensuggestedbutnotasyet clarifiedsofar.

Interestingly, in melanoma models there is evidence

that BRAFV600 mutation, along with the STAT3 signal,

is essential for immune evasion by human melanomas. Recently,Khalilietal.demonstratedthattheactivationofthe MAPKsignalingpathwayinmelanomacellsbyoncogenic BRAFV600Eleadstotheproductionofinterleukin1(IL-1)␣/␤

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B.Merellietal./CriticalReviewsinOncology/Hematology89(2014)140–165 145

Fig.2.UponinteractionofPD-L1withPD-1,thereisarecruitmentofSrchomologyregion2domain-containingphosphatase-1(SHP-1)andSHP-2,which dephosphorylatemultiplemembersoftheTCRsignalingpathway.ThisabrogatesdownstreameffectsofT-cellactivation,includingcytokineproduction, cell-cycleprogression,andtheexpressionofsurvivalproteins.TheinhibitionofRASandPI3K/AK3pathwayscooperatestoinhibitlymphocytes’function andsurvival.

[63].Tumor-associated fibroblasts(TAFs) respond toIL-1 byupregulatinganimmunomodulatorytranscriptional pro-gramresultingintheproductionofCOX-2,PD-1ligands,and chemokines,aswellasintheamplificationofIL-1signaling (Fig.3).Collectively,thesefactorsandsignalingcircuitries suppress cytotoxic T lymphocyte functionsand ultimately promotetumorgrowth.Thesedatahighlightthehypothesis thatbothmechanisms(constitutiveandinduced)may con-comitantlycontributetothePD-L1expressionontumorcells andinothercellsinthemicroenvironment,potentiallyfueling animmunetolerancestate.

However,mostofthedatasofarreportedinliteratureare heterogeneousinterms ofPD-L1expression, variabilityin theassaysandcellimmunolocalization,aswellascut-off val-uesforpositiveversusnegativePD-L1immunohistochemical expression.Furthermore,PD-L1expressionhasbeen inves-tigated in primary tumors and metastatic tissue samples, but little conflicting data is available with regard to the concordance/discordance of PD-L1and PD-1 immunohis-tochemicalexpressioninpairedhumantissuesfromprimary melanomasandrespectivemetastases.

Anotherroleinthiscomplicatedpuzzleisplayedby PD-1.Similartoexhaustedvirusantigen-specificTcells,which

havebeenreportedinchronicviralinfections,themajority ofTILsinmelanomaexpresshighlevelsofPD-1compared withTcellsfromnormaltissuesandperipheralblood.

Previous studies on several human cancer types ele-gantlyshowedthatoverexpressionofPD-L1intumorcells mayallowimmune evasion[46].WhenPD-L1bindswith PD-1 receptor on T-cells, T-cell function is compromised through induction of apoptosis, suppression of prolifera-tion, and inhibition of T -cell cytokines release, such as IFN-␥,IL-4andIL-2.Furthermore,the PD-1/PD-L1 inter-action inhibits T lymphocyte proliferation, survival and cytokines secretion, promotes the differentiation of CD4-positive/CD25-negative/Foxp3-negativeTcellsintoFoxp3+ regulatory Tcells (Treg), andinducesapoptosis of tumor-specificTcells.Tregcellsallowtumorcellstogrowlocally, andPD1/PD-L1pathwayontumorcellscauses immunosup-pressionbyPD-L1-inducedTregcells.

One study showed that the majority of CD8+ TILs specific for the melanoma antigen MART-1 expresses significant levelsofPD-1comparedwithlowerexpression onMART-specificTcellsfromtheperipheralbloodof the samepatients[64].PD-1expressionbyTILsinmelanoma correlatedwithanexhaustedTcellphenotypeandimpaired

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146 B.Merellietal./CriticalReviewsinOncology/Hematology89(2014)140–165

Fig.3.TheactivationoftheMAPKsignalingpathwayinmelanomacellsbyoncogenicBRAFV600Eleadstotheproductionofinterleukin1(IL-1)␣/␤.

Tumor-associatedfibroblasts(TAFs)respondtoIL-1byupregulatinganimmunomodulatorytranscriptionalprogramresultingintheproductionofCOX-2,PD-1 ligandsandchemokinesaswellasintheamplificationofIL-1signaling.Collectively,thesefactorsandsignalingcircuitriessuppresscytotoxicTlymphocyte (CTL)functionsandultimatelypromotetumorgrowth.

effectorfunction.Thesedataunderscoreonceagainthatthe assessmentofcirculatinglymphocytesdoesnotnecessarily reflectwhathappensinthetumormicroenvironment,atthe biological interface between immune system and cancer. Therefore, the evaluation of the immunological profile in thecirculationmaybemisleadinginthe assessmentofthe interaction between lymphocytes, APC, other cells in the microenvironment,andtumorcells.

In conclusion, all the above reported data suggest that the constitutively or the cytokine-induced PD-L1 expres-sionwouldprovideaselectionadvantageofcancercellsby inhibitingtumor-specific recognitionandelimination byT cells.PD-L1/PD-1axisisapotentialpointofcontactbetween targetmolecules(BRAFV600),immuneevasionandtumor growth.Therefore,therapeuticantibodiesthatblockPD-1in orderprevent the inhibitoryinteraction betweenPD-1and PD-L1maypartiallycircuitthecancerimmuneevasionand elicittheimmunesystemtorecognizeandkilltumorcells.

5. PrognosticroleofPD-L1andPD-1inmalignancies

Tables 2 and 3 summarize studies on the prognostic roleof PD-L1andPD-1indifferent tumortypes.Overall, the results published so far are heterogeneous in terms

of patient’ selection criteria and methodology used for immunohistochemical analysis. Specifically, difficultiesin comparing resultsarise fromthefollowing considerations: (i)availabledataareretrospective;(ii)patientswithdifferent tumorstageshavebeenincluded;(iii)primaryormetastatic sites have been included; (iv) different tumor histotypes havebeen comparedwithin thesamestudies;(v) different typesoftissueshavebeenevaluated(frozenversus paraffin-embedded samples); (vi) monoclonal and/or polyclonal antibodies have been used; (vii) both cytoplasmic and/or membranousimmunostaininghavebeenevaluatedtodefine positivity;andfinally(viii)differentscoresofPD-L1and/or PD-1 positivity have been reported. Thus, the prognostic role of PD-L1 andPD-1inthe context of differenttumor typesrequires furtherinvestigation andonlyfuturestudies will establish whether PD-L1is an immune correlate and predictivemarkerforresponsetoantiPD-1antibody.

Accordingtotheaimofthepresentreviewwewillfocus onmelanoma.Atthetimeofthepresentmanuscript,three studiesreportedtheroleofPD-L1inmelanomapatients.

Hino et al. evaluated the intensity of PD-L1 expres-sioninmelanomaspecimens(59primarytumors,16lymph nodes,and4 in-transit metastases).By multivariate analy-sis,immunohistochemicalPD-L1expressioncorrelatedwith Breslowthickness,diseasefree,andoverallsurvival[47].

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B. Mer elli et al. / Critical Re vie ws in Oncolo gy/Hematolo gy 89 (2014) 140–165 147 Table2

PD-L1expressionandprognosisinmalignancies.

Author Na Tumor Tissues/DetectionMethod Immunostaining Antibody Prognosticrole

Thompson[79] 306 Renalcellcarcinoma FFPEtissues/IHC Cell-surfacemembrane (≥5%tumorstaining=PD-L1 positivity) Anti-PD-L1 MoAb (5H1) Yes: Association PD-L1positivitytumor/ -Death (RR,3.92;P<0.001) -Overallmortality (RR,2.37;P<0.001)

PD-L1independentprognosticfactor ofcancer-specificdeath

Thompson[50] 429 Renalcellcarcinoma Fresh-Frozentissues/IHC Cell-surfacemembrane (≥10%tumorstaining=PD-L1 positivity) Anti-PD-L1 MoAb (5H1) Yes: Association PD-L1positivitytumor/ death (RR,4.53;P<0.001)

PD-L1independentprognosticfactor ofcancer-specificdeath

Taube[66] 150 Melanoma FFPEtissues/IHC Cell-surfacemembranePD-L1 expression (≥5%tumorstaining=PD-L1 positivity) Anti-PD-L1 MoAb (5H1) Anti-PD-L1polyclonalAb (4059) Yes: Association PD-L1positivitytumor/ OS (P=0.032)

Gadiot[65] 63 Melanoma FFPEtissues/IHC N/A

(≥1%tumorstaining=PD-L1 positivity) SeveralAnti-PD-L1 MoAbs Anti-PD-L1polyclonalAb (4059) No

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148 B. Mer elli et al. / Critical Re vie ws in Oncolo gy/Hematolo gy 89 (2014) 140–165 Table2(Continued)

Author Na Tumor Tissues/DetectionMethod Immunostaining Antibody Prognosticrole

Hino[47] 59 Melanoma FFPEtissues/IHC Cytoplasmarea Anti-PD-L1

MoAb (27A2) Yes: Association PD-L1positivitytumor/ -OS (P=0.0402) -PFS (P=0.0522)

PD-L1independentprognosticfactor ofOSandPFS

Ohigashi[54] 41 Esophagealcancer Frozentissues/IHC; mRNAanalysis(Real-time quantitativePCR) Cell-surfacemembrane or Cytoplasmarea (≥10%tumorstaining=PD-L1 positivity) Anti-PD-L1 MoAb (MIH1) Anti-PD-L2 MoAb (MIH18) Yes: Association -PD-L1mRNAexpression positivity/ OS (P=0.025) -PD-L2mRNAexpression positivity/ OS (P=0.003) -PD-L1positivityandPD-L2 positivitystatus/ OS (P=0.0008) PD-L1andPD-L2=independent PrognosticfactorsofOS Wu[58] 102 Gastriccancer FFPEtissues/IHC Cytoplasmarea

(somenuclearmembrane localization) AntiPD-L1 MoAb (2H11) Yes: Association PD-L1positivitytumor/ OS(P<0.01) PD-L1=

independentprognosticfactorofOS (RR=2.803;P=0.040)

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B. Mer elli et al. / Critical Re vie ws in Oncolo gy/Hematolo gy 89 (2014) 140–165 149

Konishi[49] 52 Non-smallcelllung carcinoma

Frozentissues/IHC Cell-surfacemembrane and/or

Cytoplasmarea

(focalorscatteredpattern)

Anti-PD-L1 MoAb (MIH1) Anti-PD-L2 MoAb (MIH14) No

Mu[80] 109 Non-smallcelllung carcinoma

FFPEtissues/IHC Cell-surfacemembrane and/or

Cytoplasmarea

(focalorscatteredpattern)

Anti-PD-L1 (clonenotspecified)

Yes: Association PD-L1positivitytumor/ 3yearsOS(P=0.034) Gao[81] 240 Hepatocellular carcinoma

FFPEtissues/IHC Cell-surfacemembrane and/or

Cytoplasmarea

(focalorscatteredpattern)

Anti-PD-L1 MoAb Anti-PD-L2 MoAb Yes: Association PD-L1positivitytumor/ -DFS (P=0.047) -OS (P=0.029)

PD-L1independentprognosticfactor ofDFS

(P=0.015)

Cariani[82] 42 Hepatocellular carcinoma

Frozentissues/mRNA analysis(RealTime quantitativePCR) FFPEtissues/IHC(on15 HCCsamples)

Cytoplasmarea Anti-PD-L1polyclonalAb Yes: Association

PD-L1mRNAexpressionpositivity/ OS

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150 B. Mer elli et al. / Critical Re vie ws in Oncolo gy/Hematolo gy 89 (2014) 140–165 Table2(Continued)

Author Na Tumor Tissues/DetectionMethod Immunostaining Antibody Prognosticrole

Zeng[83] 109 HBV-related Hepatocellular carcinoma FFPEtissues/IHC NA (≥50%tumorstaining=PD-L1 positivity) Anti-PD-L1 MoAb Yes: Association PD-L1positivitytumor/ -TFS (P≤0.001) -OS (P≤0.001)

PD-L1independentprognosticfactor ofTFS(P≤0.001)andOS (P=0.007)

Karim[55] 115 Cervicalcancer FFPEtissues/IHC Cell-surfacemembrane (PD-L1/PD-L2) Anti-PD-L1 MoAb (5H1) Anti-PD-L2 MoAb Anti-PD1 MoAb No

Nomi[57] 51 Pancreaticcancer Frozentissues/IHC mRNAanalysis(Real-time quantitativePCR)

Cell-surfacemembraneand Cytoplasmarea (≥10%tumorstaining=PD-L1 positivity) Anti-PD-L1 MoAb (MIH1) Anti-PD-L2 MoAb (MIH18) Yes: Association PD-L1positivitytumor/ OS (P=0.016)

PD-L1independentprognosticfactor ofOS

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B. Mer elli et al. / Critical Re vie ws in Oncolo gy/Hematolo gy 89 (2014) 140–165 151

Nakanishi[51] 65 Urothelialcancer Frozentissues/IHC Cell-surfacemembraneand/or Cytoplasmarea (focalpattern) Anti-PD-L1 MoAb (MIH1) Yes: Association: PD-L1positivitytumor/ -OS (P=0.021) -Post-resectionrecurrence (P=0.026)

Cho[62] 45 Oralsquamouscell carcinoma

FFPEtissues/IHC Cell-surfacemembraneand/or Cytoplasmarea Anti-PD-L1polyclonalAb (ab82059) No Hsu[84] 74 Nasopharyngeal carcinoma FFPEtissues/IHC (46samples) Snap-frozentissue (28samples) NA Anti-humanPD-1polyclonalAb Anti-PD-L1 MoAb (MIH1) Anti-PD-L2 MoAb (MIH18) No

Droeser[85] 1420 Colorectalcancer FFPE tissues-TMA/IHC

NA

(Score2–3tumorstaining=PD-L1 positivity;

IntensityofPDL1 Staining=

Score0:negative

Score1:veryweakexpression Score2:moderateexpressionbut weakerthanplacenta)

Score3:equivalenttoorstronger expressionthanplacenta)

Anti-PD-L1 MoAb (27A2) Anti-PD-L1 PolyclonalAb (ab82059) Yes: Association

PD-L1positivitytumor(MoAb)/ OS

(P=0.0001)

PD-L1positivitytumor(polyclonal Ab)/

OS (P=0.008)

PD-L1trendforindependent prognosticfactor(P=0.052)

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152 B. Mer elli et al. / Critical Re vie ws in Oncolo gy/Hematolo gy 89 (2014) 140–165 Table2(Continued)

Author Na Tumor Tissues/DetectionMethod Immunostaining Antibody Prognosticrole

Hamanishi[48] 70 Ovariancancer FFPEtissues/IHC NA

(Score2–3tumorstaining=PD-L1/ PD-L2positivity;

IntensityofPD-L1andPDL2 staining=-Score0:negative -Score1:veryweakexpression -Score2:moderateexpressionbut weakerthanplacenta(PD-L1)and tonsil(PD-L2)

-Score3:equivalenttoorstronger expressionthanplacentaortonsil)

Anti-PD-L1 MoAb (27A2)

Yes:

PD-L1independentprognosticfactor of: -OS(RR,4.26;95%CI,1.39–12.94; P=0.011) -PFS(RR,2.57;95%CI,1.11–5.93; P=0.027) Topalian[67] 42 18 Melanoma 10

Non-smallcelllung carcinoma 7

Colorectalcancer 5

Renalcellcarcinoma 2 Prostatecancer FFPEtissues/IHC NA (≥5%tumor staining=PD-L1positivity) Anti-PD-L1 MoAb (5H1) Yes: Association

PretreatmentPD-L1positivitytumor/ ObjectiveResponseaftertreatment withanti-PD-1antibody

IHC,immunohistochemistry;FFPE,formalin-fixedandparaffin-embedded;OS,overallsurvival;DFS,diseasefreesurvival;PFS,progressionfreesurvival;RR,riskratio;Ab,antibody;MoAb,monoclonal antibody;TMA,tissuemicroarray;TFS,treatmentfreesurvival;NA,notapplicable;PCR,polymerasechainreaction.

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B. Mer elli et al. / Critical Re vie ws in Oncolo gy/Hematolo gy 89 (2014) 140–165 153 Table3

PD-1andprognosisinmalignancies.

Author N◦ Tumor Tissues/detection

method

Cell

immunolocalization/cut-offfor

positivity

Antibody Prognosticrole

Hsu[84] 74 Nasopharyngeal carcinoma FFPEtissues/IHC(46 samples) Snap-frozentissue(28 samples)

NA Goatpolyclonalantibodyanti-human PD-1(R&DSystems,Minneapolis)

YES→ Correlation:

PD-1+-CD8expression/OS(P=0.05). PD-1+-CD8expression/DFS(P=0.007). PD-1+CD8expression/locoregional recurrencefreeSurvival(P=0.004). PD-1+-CD8

expression/locoregionalrecurrence-free survival(fourgroups)(P=0.004). HighPD-1+-CD8grouphada6.5times higherriskoflocoregionalrecurrence (P=0.005),a6.5timeshigherriskof treatment

failure(P=0.013),anda9.5timeshigher riskofdeath(P=0.015);

PD-1expression

inintratumoralCD8cells:independent effectonthepost-treatmentoutcome ---NO→ NoCorrelation: PD1+-CD8expression/distant metastasis-freesurvival(P=0.31). PD-1+-CD4expression/ OS(P=0.56). PD-1+-CD4expression/ DFS(P=0.90)

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154 B. Mer elli et al. / Critical Re vie ws in Oncolo gy/Hematolo gy 89 (2014) 140–165 Table3(Continued)

Author N◦ Tumor Tissues/detection

method

Cell

immunolocalization/cut-offfor

positivity

Antibody Prognosticrole

Wahlin[86] 70 Follicularlymphoma FFPEtissues/IHC Tissuemicroarray (TMA) Non-nuclear Quantification:cell count=PD-1positive cells/sumoftotal cellulararea Compartment: total/follicular/interfollicular

Anti-PD-1Abnon-specified YES→ Correlation

FollicularPD1positivity/

favorableprognosis(RR=0.34,P=0.01) alongwithinterfollicularCD8+cells (RR=0.86,P=0.014)independentofFLIPI

Takahashi[87] 82(patientstreatedby standardR-CHOPas initialtherapy) Follicularlymphoma NA NA PD-1positivity staining:>14.4% Anti-PD1 MoAb (Abcam52587) NO→ Nocorrelation

CRrate/PD-1positivitypatients NocorrelationPD-1positivecells (≥7.5%)/PFS(P=0.20) NocorrelationPD-1positivecells (≥7.5%)/OS(P=0.60)

PD-1isnotaIndependentprognosticfactor Amongmalepatients(n=43):correlation PD-1positivecells/worsePFS(P=0.03) Richendollar[88] 91 Follicularlymphoma Tissuemicroarray

(TMA)/IHC NA PD-1positivity: >35.6PD-1positivity cells/HPF Anti-PD1 Polyclonal(Abcam) YES Association

PD1positivityTcells/decreasedOSl (HR=1.64,P=0.10)

PD1:independentprognosticfactorfor decreasedOS

Muenst[89] 189 Hodgkinlymphoma Tissuemicroarray (TMA)/IHC

NA

PD-1positivity: >23PD-1positivity cells/mm2

Anti-PD-1GoatantihumanMoAb (AF1086R&DSystems)

YES→

PD1positivityTcells/worseOS (P=0.005)

NO→

PD1isnotaIndependentprognosticfactor (P=0.022)

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B. Mer elli et al. / Critical Re vie ws in Oncolo gy/Hematolo gy 89 (2014) 140–165 155 Shi[90] 56 Hepatocellular carcinoma FFPEtissues/IHC, FACS Both cell-surface membraneand cytoplasmarea Anti-PD1

MouseantihumanMoAb(R&D Systems)

Anti-PD-L1

MouseantihumanMoAb (Biolegend),FACSwith PE-conjugatedPD-1andPD-L1 (eBiosciences)

YES→ Correlation

HighlevelsofintratumoralPD1+CD8+T cells/ shorterDFS (P<0.001) Zeng[83] 109(cryoablation) 23(IHC) HBV-related Hepatocellular carcinoma

FFPEtissues/IHC NA NA YES

Correlation

PD-1/TFS(P≤0.001) PD-1/OS(P=0.001)

CirculatingPD-1expression=independent poorprognosticfactorforTFS(P=0.046) N◦,numberofpatients;FLIP,FollicularLymphomaInternationalPrognosticIndex;R-CHOP,treatmentwithRituximab,Cyclophasfamide,Doxorubicin,VincristineandPrednisolone;FFPE,formalin-fixed andparaffin-embedded;CR-rate,completeresponserate;OS,overallsurvival;PFS,progressionfreesurvival;TFS,treatmentfreesurvival;FACS,flowcytometryanalysis;RR,riskratio;MoAb,monoclonal antibody.

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156 B.Merellietal./CriticalReviewsinOncology/Hematology89(2014)140–165

Gadiotandcolleagues evaluatedparaffin-embedded tis-suesof63patientswithstagesIII-IVmelanomadiagnosedby theNetherlandsCancerInstitutebetween2000and2004. PD-L1wasanalyzedinbenignnevi(n=10),primarymelanomas (n=43), satellite metastases (n=8), in transit-metastases (n=21),anddistantorganmetastases(n=22).Interestingly, theauthorsshowedthat20%ofthebenignnevisampleswere PD-L1-positive,whileonly5%ofprimarymelanomaswere PD-L1positive. Satellite metastases,in-transit metastases, lymphnodesanddistantmetastaseswerePD-L1positivein 25%,40%,14%,and18%ofcases,respectively.PD-L1was moreexpressed insatellite andin-transit metastases com-paredwithlymphnode,anddistantmetastases.Inthisstudy, PD-L1didnotcorrelatewithdiseasefreeoroverallsurvival [65].

Finally,Taubeetal.demonstratedacorrelationbetween PD-L1andthepresenceofTILsinbothneviandmalignant melanoma:98%ofPD-L1positivemelanomaswere associ-atedwithTILscomparedwithonly28%ofPD-L1negative melanomas.Furthermore,PD-L1positivemelanocyteswere almostalwayslocalizedadjacenttoTILs.Interestingly,a pos-itivecorrelationbetweensurfacePD-L1expressionbytumor cells(≥5%)andoverallsurvivalinmetastaticdiseasein56 patientswasreported.Ontheotherhand,therewasno signif-icantdifferenceinsurvivalinrelationtoPD-L1expression inprimarymelanoma[66].

There are important differences in the first two above referencedstudies[47,65].PD-L1wasevaluatedwith differ-entimmunostainingprotocols.InHino’sstudy,itisunclear whether theauthors testedtheir antibodies againstisotype controls and PD-L1Fc protein blockade to ensure lack of backgroundorunspecificsignals.

Anotherlikelyexplanationfortheconflictingresultscould be that small series of fewerthan 100 melanoma patients from diverse origin were analyzed. The most noticeable differencebetweentheAsianmelanomapatientpopulation [47]and the cohort populationevaluated byGadiot isthe tumor type. In the latter, superficial spreading and nodu-larmelanomas were predominantlydiagnosed, whereas in thecohortofHinoetal.,mainlypatientswithacral lentigi-nousmelanomaswereincluded.Thelatterhavebeenshown topreferentially expressc-kit amplifications or mutations, whereasthemelanomasfromtheEuropeancohortaremore likely to express BRAF mutations. Finally, more women wereincluded inHino’sstudy.Women areknown tohave abetterprognosisinmelanomaandmayhaveinfluencedthe prognosticanalysisofPD-L1.

Finally,Taubeetal.suggestthatpatients withboth PD-L1expressionandTILs inmelanomamay haveimproved prognosiscomparedwiththegroupwithPD-L1expression withoutTILs[66].Thisstudyseemstosuggestthatthe eval-uationofPD-L1expressionisimportantbutnotsufficientto understandtheintricateinterplaybetweenmelanomaandthe immunesystem. PD-L1 overexpressioninmelanoma with TILScouldbesuggestiveofanactiveandefficientimmune response,andthereforemaycorrelatewithabetterprognosis.

6. Clinicaltrialsandfuturedevelopments

We performed an extensive “Medline” and Cancerlit literature review (1995–2013). Various combinations of search terms were used depending onthe requirements of the database being searched. These terms included “PD-1” or “PD-L1” or “B7-H1”, in combination with“cancer patients”or“melanoma”or“tumor”or“tumour”or “activ-ity” or “safety” or “phase 1” or “immunotherapy” or “immunecheckpoints”or“lymphocytes”or“randomized”or “prospective”or“clinical”or“earlyphase”or“nivolumab” or “lambrolizumab”. In addition, relevant references in each article werescanned,andwe didmanual searchesof abstractsfromtheannualmeetingsoftheAmericanSociety ofHematology(1993–2012),AmericanSocietyforClinical Oncology(1993–2013),EuropeanHaematologyAssociation (1993–2012),andEuropean SocietyforMedicalOncology (1998–2013).

Table 4 summarizes the main prospective phase 1 studies evaluating anti PD-1/PD-L1 antibodies in cancer patients. Topalian and coll. enrolled 296 patients with advancedmelanoma,non-small-celllungcancer, castration-resistant prostate cancer, or renal-cell or colorectal cancer to receive BMS-936558 (anti-PD-1 antibody) at a dose of 0.1–10.0mg/kg of body weight every 2 weeks for 8 week-treatment cycles for 12 cycles until progression or completeresponse[67].Commontreatment-relatedadverse events included fatigue, rash,diarrhea,pruritus, decreased appetite, andnausea. Grade 3 or 4 drug-related toxicities occurred in 14% of patients; moreover, there were three deathsfrompulmonarytoxicity.Drug-relatedseriousadverse events occurred in 32 of 296 patients (11%). The spec-trum, frequency, and severityof treatment related adverse eventsweregenerallysimilaracrossthedoselevelstested. Drug-related adverse events of special interest (e.g., those withpotentialimmune-relatedcauses)included pneumoni-tis, vitiligo,colitis, hepatitis, hypophysitis,andthyroiditis. In this trial,objective responseswere observedin 28% of melanomapatients.Interestingly,partialsustained,longterm responseswereobservedateachcohortlevel(0.3–10mg/kg). At adoseof 3.0mg/kg, objectiveresponses werenotedin 7 of17patients (41%).AsmeasuredbystandardRECIST criteria,objectiveresponseswerelonglasting,withresponse durationsof1yearormorein13outof26patientswhohad aresponsewith1yearormoreoffollow-up.

Inthesamestudyimmunohistochemicalanalysiswas per-formedonpretreatmenttumorspecimensobtainedfrom42 patients. Objectiveresponsewasreportedinasubgroupof patients (9/25 patients:36%) withPD-L1-positive tumors. Thesepreliminarydatawouldsuggestarelationshipbetween PD-L1expressionontumorcellsandobjectiveresponse.

Hodietal.reportedtheactivityandsafetyofBMS-936558 inpatientswithpreviouslytreatedadvancedmelanoma[68]. BMS-936558wasadministeredeverytwoweeksatdosesof 0.1–10mg/kg during dose-escalation and/or cohort expan-sion. Patients received up to 12 cycles (4 doses/cycle) of

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B.Merellietal./CriticalReviewsinOncology/Hematology89(2014)140–165 157 Table4

Mainprospectivephase1studiesevaluatingantiPD-1/PD-L1antibodiesincancerpatients.

Author Tumors Drug Dose/schedule(i.v.) Pts(n) OR(%) PFS OS

AntiPD-1antibody Topalian[67] Melanoma NSCLC CRPC RCC CRC BMS936558 0.1mg/kg 0.3mg/kg 1.0mg/kg 3mg/kg 10mg/kg Every2weeks 296 – 6% 32% 18% At24weeks 47% 67% 56% NR Brahmer[91] Melanoma NSCLC CRPC RCC CRC MDX1106 0.3mg/kg 1.0mg/kg 3mg/kg 10mg/kg 39 0 0 17% 10% NR Berger[92] Advanced Hematologic malignancies CT-011 0.2mg/kg 0.6mg/kg 1.5mg/kg 3mg/kg 6mg/kg 17* 6% NR At21days 76% McDermott[93] RCC BMS936558 1.0mg/kg 10mg/kg Every2weeks 34 28% 31% At24weeks 50% 67% NR Patnaik[94] Melanoma NSCLC RC Sarcoma Carcinoid MK3475 1.0mg/kg 3mg/kg 10mg/kg Every2weeks 9 NR NR NR Kudchadkar[95] Melanoma BMS936558 Plusvaccines# 1.0mg/kg 3mg/kg 10mg/kg Every2weeks 30 NR NR NR Hodi[68] Melanoma BMS936558 0.1mg/kg 0.3mg/kg 1.0mg/kg 3mg/kg 10mg/kg Every2weeks 95 20% 20% 29% 41% 20% NR NR Brahmer[96] NSCLC BMS936558 1.0mg/kg 3mg/kg 10mg/kg Every2weeks 75 6% 28% 19% NR NR AntiPD-L1antibody Brahmer[70] Melanoma NSCLC CRPC RCC CRC OC PC BMS936559 0.3mg/kg 1.0mg/kg 3mg/kg 10mg/kg 160 0 6% 29% 19% At24weeks 42%31%22%NR NR Tykodi[97] Melanoma NSCLC OC PC RCC CRC BMS936559 0.3mg/kg 1.0mg/kg 3mg/kg 10mg/kg Every2weeks 162 10% NR NR

NSCLC,non-smallcelllungcancer;CPRC,castration-resistantprostatecancer;RCC,renalcellcarcinoma;CRC,colorectalcancer;OC,ovariancancer;PC, pancreaticcancer;RC:rectalcancer.

#MART-1/gp100/NY-ESO-1peptideswithadjuvantMontanideISA51. *8outof17patients:acuteleukemia.

:Melanomapatients. •:NSCLC.

∞:ovariancancer. NR:notreported. N:number.

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158 B.Merellietal./CriticalReviewsinOncology/Hematology89(2014)140–165 Table5

OngoingclinicaltrialsevaluatingantiPD-L1incancerpatients. Monotherapy

Non-randomized

Drug Phase Trial Disease Primaryoutcomemeasures

MPDL3280A 1 NCT01375842 Solidtumorsorhematologic malignancies

-DLTs

BMS-936559 1 NCT01452334 Non-Hodgkin’slymphoma

Hodgkinlymphoma Multiplemyelomachronic Myelogenousleukemia

-Safety -Tolerability

MPDL3280A 2 NCT01846416 PD-L1-positiveNSCLC -OR

BMS-936559 1 NCT00729664 Solidtumors -Safety

-MTD -DLT

BMS-936559 1 NCT01455103 Melanoma -Immunomodulatoryeffects

Combinationtherapy Non-randomized -MPDL3280A -Vemurafenib 1 NCT01656642 Melanoma -IncidenceofDLTs -NatureofDLTs

-Incidence,natureorseverity ofadverseeventsand laboratoryabnormalities -MPDL3280A

-Bevacizumab -Chemotherapy

1 NCT01633970 Advancedsolidtumors -Safety -DLTs/MTD DLT,doselimitingtoxicity;NSCLC,non-smallcelllungcancer;OR,objectiveresponse;MTD,maximumtolerateddose.

treatmentoruntilPDorCR.Ninety-fivemelanomapatients withEasternCooperativeOncologyGroup(ECOG) Perfor-mance Status (PS)≤2 were treated with BMS-936558 at 0.1(n=13),0.3(n=17),1(n=28),3(n=17),or10mg/kg (n=20). The majority of patients (60/95) had received interferon-alpha or IL-2 (prior anti-CTLA-4 excluded). Sevenoutof95patientspreviouslyreceivedB-rafinhibitor therapy.Sixtyoutof95patientshadpreviouslyreceivedmore than2linesoftreatment.Theincidenceofgrade3–4related adverse events (Aes) was 19% andincluded gastrointesti-nal(4%),endocrine(2%),andhepatobiliarydisorders(1%). There were no drug-related deaths. Clinical activity was observedatalldoselevels,including patientswithvisceral orbonemetastases.Of20patientswithobjectiveresponse, 12hadobjectresponseduration≥1year,and6patientswere onstudywithobjectiveresponsedurationbetween1.9and 11.3months.Severalpatientshadprolongedstabledisease.

RecentlyHamidetal.evaluatedtheanti-PD-1antibody lambrolizumabatadoseof10mg/kgofbodyweightevery 2or3weeksor2mg/kgevery3weeksin135patientswith advancedmelanoma[69].

The confirmed response rate across all dose cohorts was 38%, withthe highest confirmedresponse rate, 52%, observedinthecohortthatreceived10mg/kgevery2weeks. Theresponseratedidnotdiffersignificantlybetweenpatients pretreatedornotwithipilimumab.Atamedianfollow-upof 11 months, among patients achieving a response to treat-ment,longtermresponseswerereportedin81%ofpatients.

Theoverallmedianprogression-freesurvivalamongthe135 patientswaslongerthan7months.Commonadverseevents treatment-relatedwerefatigue,rash,pruritus,anddiarrhea; mostoftheadverseeventswerelowgrade.

Finally, Brahmer et al.evaluated207 patients (75with non-small cell lung cancer, 55 with melanoma, 18 with colorectal cancer, 7 withgastriccancer, and4 withbreast cancer),usinganti-PD-L1antibody(BMS-936559)ina mul-ticenter PhaseI trialatmultiple escalatingdose (from0.3 to 10mg/kg) [70].Anti-PD-L1antibody was administered every14daysin6-weekcycles,forupto16cyclesoruntil thepatienthadacompleteresponseorconfirmeddisease pro-gression.Grades3–4immune-relatedtoxiceffectsoccurred in9%ofpatients.Anobjectiveresponse,completeorpartial, wasobservedin9/52patientswithmelanoma(29%response ratesat3mg/kg),2/17withrenalcellcancer,5/49with non-smallcelllungcancer(mostlynon-squamoussubgroup)and 1/17withovariancancer.Prolongedstabilizationofdisease wasobservedfor12–41%lastingat24weeks[70].

Overall,withantiPD-1orantiPD-L1antibodies,the inci-denceofserious(grade3–4)adverseeffectswasatarange similartothat withCTLA-4-blockingantibodies,butmost wereless clinicallysignificantwiththe exceptionof pneu-monitis,whichledtodeathasaresultoftoxicity.Blocking theligandPD-L1withthefullyhumanIgG4antibody BMS-936559 resultedin aslightlylowerfrequencyof objective tumorresponsesandalsofeweradverseeffectsinphaseI test-ing.Theseinitialexperiences,togetherwiththecurrentinitial

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B.Merellietal./CriticalReviewsinOncology/Hematology89(2014)140–165 159 Table6

Ongoingclinicaltrialswithanti-pd-1antibodies. Monotherapy

Non-randomized

Drug Phase Trial Disease Primaryoutcomemeasures

BMS-936558 1 NCT01621490 Melanoma -Immunomodulatoryeffects

BMS-936558 1 NCT01592370 -Non-Hodgkin’slymphoma -Hodgkinlymphoma -Multiplemyeloma -CML -Safetyand -Tolerability BMS-936558 2 NCT01721759 NSCLC -OR BMS-936558 1 NCT01658878 HCC -Safety

BMS-936558 1 NCT00836888 Solidtumors -Safety

-Pharmacokinetic

AMP-224 1 NCT01352884 Solidtumors -Safety

-Pharmacokinetic -Tolerability Randomized BMS-9365580.3mg/kg versus BMS-9365582mg/kg versus BMS-93655810mg/kg (pretreatedpatients) versus BMS-93655810mg/kg (naïvepatients) 1 NCT01358721 RCC -Immunomodulatoryactivity BMS-936558 versus Dacarbazine 3 NCT01721772 Melanoma -OS MK-3475

atdifferentdoselevels: Low(0.1mg/kg) Intermediate(3mg/kg) High(10mg/kg)

PatientswithMelanomaand NSCLCwillberandomizedto: Lowdose

versus

Intermediatedose versus

Highdose

1 NCT01295827 Anytypeofcarcinomaor MelanomaorNSCLC MelanomaandNSCLC

-Safety -OR

-Changefrombaselinein candidatebiomarkerexpression levelsinmelanomaandNSCLC participants -MK-3475 versus -Carboplatinor -Carboplatin+ -Paclitaxel -Paclitaxelor -Dacarbazineor -Temozolomide 2 NCT01704287 Melanoma -OR -PFS -OS -BMS-936558 versus -Docetaxel 3 NCT01673867 NSCLC -OS

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160 B.Merellietal./CriticalReviewsinOncology/Hematology89(2014)140–165 Table6(Continued)

Monotherapy Non-randomized

Drug Phase Trial Disease Primaryoutcomemeasures

-BMS-936558 versus -Docetaxel 3 NCT01642004 SquamouscellNSCLC -OR -OS -BMS-936558 versus -Dacarbazineor -Carboplatinor -Paclitaxel 3 NCT01721746 Melanoma -OR -OS -BMS-936558 versus -Dacarbazine 3 NCT01721772 Melanoma -OS -BMS-936558 versus -Everolimus 3 NCT01668784 RCC -OS Combinationtherapy Non-randomized -BMS-936558 -NY-ESO-1 (peptidevaccine) -gp100:280–288 (peptidevaccine)

1 NCT01176474 Melanoma -Timetorelapseinpatientswith resectedStageIIIC/IVmelanoma

-BMS-936558 -MART-1 (peptidevaccine) -NY-ESO-1 (peptidevaccine) -gp100:209–217(210M) (peptidevaccine) -gp100:280–288(288V) (peptidevaccine) 1 NCT01176461 Melanoma -OR -BMS-936558 -BMS-986015 (ANTI-KIR): 1 NCT01714739 -NSCLC -Melanoma,-RCC -Colorectalcancer -Ovariancancer -Safetyand -Tolerability -BMS-936558 -Gemcitabine

2 NCT01313416 Pancreaticcancer -Feasibilityand -Safety -BMS-982470

(RecombinantInterleukin-21, rIL-21)

-BMS-936558

1 NCT01629758 Anytypeofcarcinoma -Safety

-BMS-936558 ± -DCRCCvaccine 2 NCT01441765 RCC -Safety -OR -BMS-936558 ± -DCAMLvaccine 2 NCT01096602 AML -Toxicity -BMS-936558 -DCfusionvaccine

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B.Merellietal./CriticalReviewsinOncology/Hematology89(2014)140–165 161 Table6(Continued)

Monotherapy Non-randomized

Drug Phase Trial Disease Primaryoutcomemeasures

-BMS-936558 -BMS-986015 (ANTI-KIR)

1 NCT01714739 SolidTumors -Safety

-Tolerability Randomized -Sipuleucel-T (vaccine) versus -Sipuleucel-T+ -BMS-936558 versus -Cyclophosphamide+ -Sipuleucel-T+ BMS-936558

2 NCT01420965 Prostatecancer -Feasibility -Immuneefficacy -BMS-936558or -BMS-936558Plus–Ipilimumab versus Ipilimumabalone 3 NCT01844505 Melanoma -OS

NSCLC,non-smallcelllungcancer;HCC,hepatocellularcarcinoma;RCC,renalcellcarcinoma;CML,chronicmyelogenousleukemia;AML,acute myel-ogenousleukemia;DC,dendriticcell;KIR,killer-cellimmunoglobulin-likereceptor;RR,responserate;PFS,progressionfreesurvival;OS,overallsurvival; OR,overallresponse.

clinicaltestingofaseriesofotherantibodies andblocking constructstothePD-1/PD-L1axis,provideanimpetusforthe continuedclinicaltestingofthesehighlyactive immunother-apiesformelanoma.

Tables5and6summarizeongoingclinicaltrialswithanti PD-1orantiPD-L1antibodies.

Basedonexperimentaldata,blockadeofB7-H1orPD-1 isnotexpectedtostimulatedenovoimmuneresponsesbut rathertoenhanceongoingimmuneresponsesagainsttumor antigens,sincethisaxisisimplicatedintheeffectivephase andnot in the priming phase of the immune response. A combinationalstrategytoelicittheimmuneresponsecould improvetheobjectiveresponserateaswellasthedurationof response.Inpatientswithadvanceddiseaseitislikelythat the majorityof TILshave an“exhausted” phenotype.The presence of inhibitory moleculeswould render these cells insensitivetotheactionofanti-PD-1.Onthebasisofthese considerations,preliminaryresultsinmicemodels suggest that combinationtherapies may be helpful inrestoring an activatingphenotypeinthetumormicroenvironment[71].In experimentalmodelscombiningB7-H1/PD-1blockadewith cancervaccines[72–75],adoptivetransferofpreactivatedT cells,orTcellstimulationwithanti-CD137[76]often pro-videsdramaticsynergistic antitumoreffects,insomecases eradicatingwell-establishedtumors.

Furthermorethere is preclinical evidencethat CTLA-4 and PD-1 could play complementary roles in regulating

adaptiveimmunity.Curranetal.reported,inmicexenograft, thatthecombinationofCTLA-4andPD-1blockade deter-mines the accumulation of CTLA-4/PD-1 double-positive TeffectorcellswithinB16melanomacelllines[77].These datasuggestthatTcellsthatwouldotherwisebefunctionally and proliferatively repressed are instead able to continue expandingandcarryingouteffectorfunctions.

InthesamemodelthecombinationofCTLA-4andPD-1 blockaderesultedmorethantwiceaseffectiveaseitheralone inpromotingtherejectionofB16melanomacelllines. Inter-estinglythefurtheradditionofPD-L1antibodyelevatesthe rateoftumor-freesurvivalto65%versus10%withCTLA-4 blockadealone[77].

Recently, onthe basis of these observations,a phase1 study to investigate the safety and efficacy of combined CTLA-4 and PD-1blockade [with the use of ipilimumab (anti-CTLA4antibody)andnivolumab(anti-PD-1antibody), respectively]inpatientswithadvancedmelanomahasbeen conducted[78].Inthisstudytheobjective-responseratefor allpatientsintheconcurrent-regimengroupwas40%.Atthe maximumtolerateddoses(nivolumabatadoseof 1mg/kg ofbodyweightandipilimumabatadoseof3mg/kg),53% ofpatientshadanobjectiveresponse,allwithtumor reduc-tionof80%ormore.Grade3or4toxicitiesoccurredin53% of patients and weregenerally reversible andqualitatively similartothosereportedwithmonotherapy(ipilimumabor nivolumabalone).

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162 B.Merellietal./CriticalReviewsinOncology/Hematology89(2014)140–165

7. Conclusion

Thepastdecadehasseenimportantstridesinthefieldof melanomaresearch.Extensiveresearchoverthepasttwenty yearsyieldedtheidentificationofnewandinnovativewaysto manipulatetheimmuneresponsetocancer.Negative regula-torsoftheimmunesystem,calledimmunologiccheckpoints, havebeenfoundtoplayimportantrolesinrestraining other-wiseeffectiveanti-tumorimmunologicresponses.Therapies thattargetthePD-1/PD-L1axishavedemonstrated promis-ing clinical results. Treatment is generally well tolerated, butanovelspectrumofsideeffects,termedimmune-related adverse events, has been experienced. Unfortunately, not all patients respond to these therapies, and evaluation of biomarkerspredictiveofresponseisongoing.

Someopenissuesandscarceinformationmayunfavorably impactthemanagementofpatientswithadvancedmelanoma, therefore, priority actions are needed in this direction. In particular:

1. A standardizedmethodology to define the PD1/PD-L1 positivemelanomaisstronglyneeded.Tumor heterogene-ityintermsofPD-L1expression,variabilityintheassays and cell immunolocalization as well as cut-off values forpositiveversusnegativePDL-1immunohistochemical expressionarenotuniformlydefinedanddonotcurrently allowappropriatepatient’sclassification.

2. The prognostic and predictive role of TILS (overall estimationand/orimmunophenotyping)inadditionto PD-1/PD-L1 remains to be determined in order to better stratify patients andto identify those whomay benefit fromtherapiesthattargetPD-1/PD-L1axis.

3. TheantiPD-1andPD-L1antibodieshavedemonstrated clinical activity inearly clinical trials, but randomized studieswithimmunecorrelatesarelackingsofar. 4. CombinationsofPD-L1/PD-1blockadewithcancer

vac-cines, adoptive transfer of pre-activated T cells, T cell stimulationwithanti-CD137orMAPKinhibitorsshould beinvestigatedastheycouldprovidesynergisticantitumor effects.

Reviewers

PiotrRutkowski,MD,PhD,Professor;Headof Depart-ment, Maria Sklodowska-Curie Memorial Cancer Center – Institute, Department of Soft Tissue/Bone Sarcoma and Melanoma,RoentgenaStr.5,PL-02-781Warsaw,Poland.

NitinChakravarti,PhD,Instructor,Departmentof Pathol-ogy– Research,Division ofPathology/LabMedicine,The UniversityofTexasMDAndersonCancerCenter,Houston, TX,TheUniversityofTexasMDAndersonCancerCenter, 1515HolcombeBlvd.,Houston,TX77030,USA.

OlivierMichielin, MD, PhD, Professor,UNIL, Depart-mentofOncology,ExperimentalOncologyUnit,CHUV,Rue duBugnon46,CH-1011Lausanne,Switzerland.

Conflictofintereststatement

Theauthorsdeclarethatthereisnoconflictofinterest.

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