Nras
in
melanoma:
Targeting
the
undruggable
target
Mario
Mandalà
a,∗,
Barbara
Merelli
a,
Daniela
Massi
baUnitofMedicalOncology,DepartmentofOncologyandHematology,PapaGiovanniXXIIIHospital,Bergamo,Italy bDivisionofPathologicalAnatomy,DepartmentofSurgeryandTranslationalMedicine,UniversityofFlorence,Italy
Accepted9May2014
Contents
1. HistoricalperspectiveonNRASincancerandafocusonmelanoma... 108
2. BiologicalfunctionsofNRAS... 109
2.1. Cellproliferation... 109
2.2. Suppressionofapoptosis ... 111
2.3. Metabolism... 111
2.4. Remodelingthemicroenvironment ... 111
2.5. Evasionoftheimmuneresponse... 111
2.6. Metastasis... 111
3. NRASinmelanocyticcellneoplasms ... 112
4. NRAS:prognosticorpredictivebiomarkerinmelanoma?acriticalanalysisofcurrentliterature... 113
4.1. IsNRASaprognosticbiomarkerinmelanoma?... 113
4.2. IsNRASapredictivebiomarkerinmelanoma?... 115
5. NRASasamechanismofresistancetoBRAFinhibitorsinmelanoma... 116
6. Futuredirections... 118
Reviewers... 119
References... 119
Biographies ... 122
Abstract
RASbelongstotheguanosine5-triphosphate(GTP)-bindingproteins’family,andoncogenicmutationsincodons12,13,or61ofRAS familyoccurinapproximatelyonethirdofallhumancancerswithN-RASmutationsfoundinabout15–20%ofmelanomas.Theimportance ofRASsignalingasapotentialtargetincancerisemphasizednotonlybytheprevalenceofRASmutations,butalsobythehighnumber ofRASactivatorsandeffectorsidentifiedinmammaliancellsthatplacestheRASproteinsatthecrossroadsofseveral,importantsignaling networks.Rasproteinsarecrucialcrossroadsofsignalingpathwaysthatlinktheactivationofcellsurfacereceptorswithawidevarietyof cellularprocessesleadingtothecontrolofproliferation,apoptosisanddifferentiation.Furthermore,oncogenicrasproteinsinterferewith metabolismoftumorcells,microenvironment’sremodeling,evasionoftheimmuneresponse,andfinallycontributestothemetastaticprocess. After40yearsofbasic,translationalandclinicalresearch,muchisnowknownaboutthemolecularmechanismsbywhichthesemonomeric guanosinetriphosphatase-bindingproteinspromotecellularmalignancy,anditisclearthattheyregulatesignalingpathwaysinvolvedinthe controlofcellproliferation,survival,andinvasiveness.InthisreviewwesummarizethebiologicalroleofRASincancerbyfocusingour attentiononthebiologicalrationalandstrategiestotargetRASinmelanoma.
©2014ElsevierIrelandLtd.Allrightsreserved.
Keywords:NRAS;Melanoma;Prognostic;Predictive;Resistance
∗Correspondingauthorat:UnitofMedicalOncology,DepartmentofOncologyandHaematology,PapaGiovanniXXIIIHospital,PiazzaOMS1,Bergamo
24100,Italy.Tel.:+390352673687;fax:+390352674985.
E-mailaddress:mariomandala@tin.it(M.Mandalà).
http://dx.doi.org/10.1016/j.critrevonc.2014.05.005
Ras proteins are crucial crossroads of signaling
path-ways that link the activation of cell surface receptors
with a wide variety of cellular processes leading to the
control of proliferation, apoptosis anddifferentiation.
Fur-thermore,oncogenicrasproteinsinterferewithmetabolism
oftumorcells,microenvironment’sremodeling,evasionof
theimmuneresponse,andfinallycontributestothemetastatic process.
TheimportanceofRASsignalingasapotentialtargetin
cancer is emphasized not onlyby the prevalence of RAS
mutations,butalso bythe highnumberof RASactivators
andeffectorsidentifiedinmammaliancellsthat placesthe
RASproteinsatthecrossroadsofseveral,importantsignaling networks.
After40yearsofbasic,translationalandclinicalresearch,
much is now known about the molecularmechanisms by
which these monomeric guanosine triphosphatase-binding
proteinspromotecellularmalignancy,anditisclearthatthey
regulatesignalingpathways involvedinthe controlof cell
proliferation, survival,andinvasiveness. In thisreviewwe
summarizethebiologicalroleofRASincancerbyfocusing
ourattentiononthebiologicalrationalandstrategiestotarget
RASinmelanoma.
1. HistoricalperspectiveonNRASincanceranda focusonmelanoma
Thirty yearsago a pioneering study demonstrated that
small fragmentsofDNA fromhumancancer-derivedcells
couldinducemalignantcharacteristicsinmousefibroblasts
[1].ThecellularhomologofanoncogenefoundintheHarvey
ratsarcomaretrovirus(H-RAS)wasidentifiedas theDNA
sequenceresponsibleforsuch malignanttransformation.A
newstepintumorbiologyhadbeenputinplace:thiswasthe
first demonstrationthat humantumors containedactivated
oncogenes,relatedtothosepickedupbyretrovirusesfrom
theirhostgenomes[2,3].Genesequencingrevealedthatthe
differencebetweenthewild-type(wt)humanH-RASgene
andtheoncogenicformfoundintumorswasasinglepoint
mutation.Subsequently, threeRAS genesand
correspond-ingproteinsweredescribed:N-RAS(neuroblastoma-RAS),
H-RASandK-RAS(Kirsten-RAS)[4–6].
RAS belongs to the guanosine 5-triphosphate
(GTP)-binding proteins’ family. When acted upon by specific
factors,suchasextracellularligandsthatbindspecific
mem-brane receptors,theseproteins cycle between anactivated
and inactivated form, RAS-GTP and RAS-GDP,
respec-tively[7].Activationrequiresdissociationofproteinbound
GDP, aprocess that is accelerated byguanine
nucleotide-exchangefactors(GEFs).Thisswitch-onprocessinvolvesthe
reversibleexchangeofGDPforGTP.Theswitch-offprocess
isentirelydifferentandinvolveshydrolysisofGTPtoGDP,
the guanosine triphosphatase (GTPase) reaction, which is
basicallyirreversible.ThisprocessisacceleratedbyGTPase activatingproteins(GAPs)(Fig.1a).
Inphysiologicalconditions,RASproteinsaretetheredto
the innercellmembrane,coupling growthfactorreceptors
to downstream signalingpathways andregulate important
cellularfunctionssuchascellgrowth,proliferation,and
sur-vival.Muchisnowknownaboutthemolecularmechanisms
bywhichthesemonomericguanosinetriphosphatase-binding
proteinspromotecellularmalignancy,anditisclearthatthey
regulate signalingpathwaysinvolved inthecontrol ofcell
proliferation,survival,andinvasiveness.
Mutationsat positions12, 13,or 61of the H-RAS,
N-RAS,andK-RASimpairtheGTPaseactivityofthecarrier
RAS proteinsandlockthemintoaconstitutivelyactivated
stateinwhichtheyelicitdownstreameffectors,eveninthe absenceofligandsthatbindspecificmembranereceptors[8]. Thispeculiaroncogenicactivation–disablingtheenzymatic
activity –differentiatesRASfromotheroncogenickinases
such as EGFR or B-RAF, whichare typically mutated to
produceahyperactiveenzyme.
TheimportanceofRASsignalingasapotentialtargetin
cancer is emphasizednot only by the prevalence of RAS
mutations, butalsoby thehighnumberof RAS activators
andeffectorsidentified inmammaliancells thatplaces the
RASproteinsatthecrossroadsofseveral,importantsignaling networks(Fig.1b).
The first RAS effector identified is the RAF
ser-ine/threonine kinase [9–12].Activation of RAFinitiates a
phosphorylationcascadethat progressesthrough MEKand
ERK(p42/p44MAPK),andultimatelyleadstofine
adjust-mentsindownstreamtargetsthatregulatecellproliferation,
survival, and differentiation [13]. A second RAS effector
isthe p110catalyticsubunitof thephosphatidylinositol 3-kinase(PI3K)[14].Phosphorylationofphosphatidylinositol byPI3KbringstheAKTserine/threoninekinasetotheplasma
membrane,whereitbecomesactivatedandtransmits
down-streamsignalstoregulatecellsurvival,proteinsynthesis,and
metabolism[15].RAFandPI3Karealsocommonlymutated
in melanoma, suggesting that theseproteins might be the
primaryoncogeniceffectorsofRASsignaling[16].
Interestingly,whilemutationsinRAFandRASare
gen-erally mutually exclusive, this is not the case for PI3K
mutation.Thesebiologicaldifferencessuggestthat
endoge-nouslevelsofactivatedRASdonotefficientlyactivatePI3K
signaling,whileRASandRAFmutationsappearfunctionally
equivalent.AnotherexplanationisthattheRAS/RAFdouble
mutationislethalforthecellwhereasRAS/PI3Karenot.
Since oncogenic mutations incodons 12, 13, or 61 of
RASfamilyoccurinapproximatelyonethirdofallhuman
cancerswithN-RASmutationsfoundinabout 15–20%of
melanomas,RASandthesignalingpathwaysunderits
con-trol have been kept firmly in focus as therapeutic targets
(Fig.2).However,after40yearsofresearch,manyproblems
remain open.First,whathaspreventedthedevelopmentof
drugsagainstRAS?
Several factors have hampered the development of
therapies that are able to inhibit RAS in a specific and
Fig.1.(a)MechanismofRASactivation.Receptortyrosinekinase(RTK)-mediatedactivationrequiresdissociationofproteinboundGDP,aprocessthatis
acceleratedbyguaninenucleotide-exchangefactors(GEFs).Thisswitch-onprocessinvolvesthereversibleexchangeofGDPforGTP.Theswitch-offprocess
isentirelydifferentandinvolveshydrolysisofGTPtoGDP,theguanosinetriphosphatase(GTPase)reaction,whichisbasicallyirreversible.Thisprocess
isacceleratedbyGTPaseactivatingproteins(GAPs).(b)EffectorsidentifiedinmammaliancellsthatplacetheRASproteinsatthecrossroadsofseveral,
importantsignalingnetworks.(TIAM1:T-celllymphomainvasionandmetastasis1;PI3K:phosphoinositide3-kinase;PDK1:phosphoinositide-dependent
kinase-1;ERK:extracellularregulatedkinase;RALGDS:RALguaninenucleotidedissociationstimulator;PLD:phospholipaseD;PLC:phospholipaseC;
PKC:proteinkinaseC).
highintracellularconcentrationsofGTP;(3)theattemptto
inhibit farnesylation, a key posttranslational modification
step of RAS that is essential for RAS function, through
thefarnesyltransferaseinhibitors(FTIs),was ineffectivein
clinical trials;(4) targetingmutant N-RASwith siRNAis
still limited to preclinical models because of the
signifi-cant challenge in delivering antisense oligonucleotides in
vivo.
In this review we summarize the biological role of
RASincancerbyfocusing ourattention onthe biological
rationalandstrategiestotargetRASinmelanoma.Forthis
purpose,weperformedanextensive“Medline”andCancerlit
literature review (1995–2012). Various combinations of
searchterms were used depending onthe requirements of
thedatabasebeingsearched.Thesetermsincluded“RAS”,
“MAPK”, “target therapy”, “MEK” in combination with
“cancer patients”, “melanoma”, “incidence”,
“pathogen-esis”, “management”, “cancer”, “tumors”, “resistance”,
“trials”,“prospective”,“phase”,“retrospective”.Inaddition,
we manually researched all relevant review articles and
the references of the retrieved papers. Finally, trials were excludedifrelevantdatacouldnotbeextracted.
2. BiologicalfunctionsofNRAS
Ras proteins are crucial crossroads of signaling
path-waysthat linkthe activationof cellsurfacereceptorswith awidevarietyofcellularprocessesleadingtothecontrolof proliferation,apoptosisanddifferentiation(Fig.3).
Further-more, oncogenic ras proteinsinterferewithmetabolismof
tumorcells,microenvironment’sremodeling,evasionofthe
immune response,andfinallycontributes tothemetastatic
process.
2.1. Cellproliferation
Three decades ago Feramisco et al. demonstrated that
Fig.2.Potentialtherapeuticstargetsinmelanoma.
Fig.3.Rasproteinsarecrucialcrossroadsofsignalingpathwaysthatlinktheactivationofcellsurfacereceptorswithawidevarietyofcellularprocesses
bymicroinjectionintoavarietyof somaticcells determine
dramaticmorphologicalchangesfollowedbytransient cell
proliferation [17]. Proliferation is a check and balances
process,being the result of different stimuli, that elicit or inhibitcellcycle[18].OncogenicRASfuelscell
prolifera-tionthroughfourdistinctbiologicalmechanismsthat carry
thebalanceofdifferentstimulitohangonthesideofthecell
cycle:upregulationofgrowthfactors,expressionofgrowth
factorreceptors,upregulationofintegrinsthatpromote pro-liferation anddownregulation of anti-proliferative signals. Thesecomplexandstillunclarifiedmechanismsleadto
acti-vationofseveraltranscriptionfactorssuch serumresponse
factor(SRF),JUN,activatingtranscriptionfactor2(ATF2)
andnuclearfactor-B(NF-B)[19,20].Inturn,thesefactors triggertheexpressionofcyclinD1[21].Theexpressionofthe
G1cyclinseemsacrucialdeterminantofRAS-induced
trans-formation.IthasbeenreportedthatcyclinD1-deficientmice areresistanttodevelopingepithelialtumorsthatareinduced
bytheHRASoncogene.Pharmacologicalinterferencewith
cyclinD1orcyclin-dependentkinaseinhibitors(CKIs),such
asp27andp21,whichwouldotherwiseassociatewithand
inhibitcyclin-dependentkinases(CDKs),couldbean
excit-ingavenueofcancerresearchinthecomingyears.
2.2. Suppressionofapoptosis
OncogenicRASmayhavebothpro-apoptoticand
anti-apoptoticfunctions.Theanti-apoptoticfunctionofoncogenic
RAS is mediated by several effector pathways, including
theRAS–PI3KandtheRAS–RAFpathway.Bothpathways
have been implicated in phosphorylating and inactivating
thepro-apoptoticprotein BCL-2-associatedagonist of cell
death(BAD).There isevidencethat RASisimplicated in
both the development and maintenance of melanoma. In
experimental models, melanoma genesis and maintenance
arestrictlydependentuponexpressionofHRasV12Gandon
theoppositeHRasV12Gdown-regulationresultsinclinical
andhistologicalregressionofprimaryandexplantedtumors
[22].Theinitialstagesofregressioninvolvedmarked apopto-sisinthetumorcellsandhost-derivedendothelialcells.These
dataclearlysupport thehypothesis of anoncogenic
RAS-drivenerosionoftheapoptoticpathwaysanditscontribution
tomelanomadevelopment.
2.3. Metabolism
RAS-drivenactivationofMAPKandPI3Keffector
path-waysstimulatemTORactivitywhich,inturn,up-regulates
thehypoxia-induciblefactor1␣(HIF1␣),whichiswell
rec-ognizedforitsabilitytostimulateaglycolyticshift[23].RAS
dependentupregulationofHIF1␣enhancesthetranscription
oftheglucosetransporterGLUT1,thusconferringcellswith anincreasedcapacitytotakeupglucose.Inaddition, onco-genicRASleadstoanincreaseinthelevelsofkeyglycolytic
enzymes[24].Thus,oncogenicRASdirectlycontributesto
metabolicreactionsthatstimulatetheuseof glucoseasan
anabolicsubstrateinproducingbuildingmaterialforcellular
growth.OncogenicRASinterfaceswithcellularmetabolism
andthisinteractionincreasesultimatelythe glycolyticrate andcellularviability,supportingtumorgrowthinvivo[25].
2.4. Remodelingthemicroenvironment
RASactivationsustainspro-angiogenicprocessesthrough
modulation of endothelial growth factors levels,
enhance-ment of local inflammation and stromal remodeling [26].
RAS upregulates VEGFA via multiple effectors,
includ-ing,HIF1␣,cyclooxygenase2(COX2)andprostaglandins’
production[27].Furthermore,RAS-mediatedproductionof
pro-inflammatory cytokines, such as IL-6 and IL-8, has
emergedasanothercontributortotheinductionof angiogen-esis[28].Finally,upregulationofmatrixmetalloproteinase2
(MMP2),MMP9andurokinase-typeplasminogenactivator
(uPA)hasbeendescribed[29].
2.5. Evasionoftheimmuneresponse
Oncogenic RAS can disrupt antitumor immunity by
essentially two mechanisms: first,by reducing the surface
expression of antigen-presenting major histocompatibility
complexes (MHC) on tumor cells, resulting in decreased
immunogenicityoftheRAS-transformedcells[30].Second,
byovercominghost-protecting adaptiveimmuneresponses
[31].Upon oncogenic RAS expression, the recruitmentof
immunosuppressiveregulatoryTcellsandmyeloid-derived
suppressor cells attumorsitemay leadtoa compromised
antitumorimmuneresponse[32].
2.6. Metastasis
Metastasisisamulti-stageprocessinvolvingamultitude of cellular activities such as cancer cell motility, intrava-sation, transit intheblood orlymph vessels,extravasation
andgrowthatanewsite.RASpromotestheseprocessesby
engaging adiverseandbroad platform of effector
mecha-nisms.OncogenicRASinducesalterationsincell–celland
cell–matrix interactionsandtheacquisitionof amigratory
phenotypeultimatelycontributingtothemetastaticprocess.
OncogenicRASreducesE-cadherinlevelsandinducesthe
destabilization of E-cadherin – -catenin complexes and
the  catenin relocalization [33]. In addition, oncogenic
RAScontributestotheenhancedmotilityoftumorcellsby
affecting changes in the polymerization, organization and
contraction of actin;the polymerization and/or stabilityof microtubules;andthetranscriptionalregulationofmitogenic geneproducts[34].OncogenicRASprotectstumorcellsfrom matrixdeprivation-inducedapoptosis,oranoikisthereby con-tributingtotheircapacityofmigrationthroughthecirculatory system[33–35].
3. NRASinmelanocyticcellneoplasms
Oneof the unresolvedissues concerningthe oncogenic
activationofRASpertainstowhetherspecificoncogenic
out-putsare drivenbymutations inaparticular RASisoform.
This hypothesisis supported by the well-recognized
non-random distribution pattern of activated isoforms of RAS
amongdifferentcancertypes.
NRAS mutations have been found in approximately
15–20% of human melanomas while HRAS and KRAS
mutationsarerare(1%)[36].Arationalexplanationforthe greateroccurrenceofNRASmutationsreliesondistinct
dif-ferences between the signaling capabilities of NRAS and
KRASinmelanocytes [37].Whenthe transformation
effi-ciencies of mutant NRAS and KRAS were compared in
immortal,non-transformedInk4a/Arf-deficientmelanocytes,
it was shown that in contrast to KRAS mutation, NRAS
mutationleadstoincreasedcellularproliferationandismore
potently tumorigenic [37]. Furthermore, NRAS mediates
activation of bothMAPK andPI3K/AKT/MYCsignaling.
Specifically, although both NRAS and KRAS efficiently
activate the classical MAPK pathway, only NRAS
effec-tivelypreventsglycogensynthasekinase3(GSK3)-mediated
phosphorylation of Myc via PI3K/AKT, which results in
enhancedactivityofendogenousMycprotein[37].In
con-trasttoKRAS,NRASandHRASalsoshowamorepotent
activation of PI3K/AKT likely due to the fact that both
NRASandHRAScolocalizetolipidrafts,whereasKRAS
isexcludedfromlipidraftsandlocalizes tothedisordered plasmamembrane[38],resultinginalessefficientactivation oralimitedaccesstoadefinedsubsetofdownstreameffector proteins.
ThereisagreatdebatewhetherspecificRASisoforms dic-tatespecificclinico-pathologicalmelanocyticcellneoplasms.
AnextraordinarilyhighNRASmutationfrequencyseemsto
becharacteristicofmedium-sized(≥1.5cm)andlarge-giant
congenital nevi whereas common acquired nevi and Spitz
nevihaverareNRASmutations(4.6%and4%,respectively)
[39].
ThefrequencyofNRASmutationsinmedium-sized
con-genital nevi is 64–70% [39–41] and raises to 94.7% in
large-giant congenitalnevi whereithasbeen recently
rec-ognizedas thesolerecurrentsomatic mutation[42].It has
beensuggestedthatNRASmutationsexertstrongergrowth
signals,resultingintheformationoflargernevi thanthose linkedtoBRAFmutations[43].Incontrast,smallcongenital nevi(<1.5cm)aregeneticallysimilartocommonacquired
neviandtendtoshowalowerincidenceofNRASmutations
andhigherincidenceofBRAFmutations[40].Inaddition,
ithasbeen reportedthatnevi thatdisplayhistological
fea-tures frequently found in nevi present at birth (so-called
“congenital pattern nevi”) but lack a definitive history of
presenceatbirthshowedonly25%ofNRASmutationsand
71%of BRAFmutations[44].NRASmutationswerealso
foundin48%to70%ofproliferativenodulesthatdeveloped
withincongenitalneviearlyinlife,butthepresenceofsuch
mutationsdoesnotseemtoconferanincreasedriskof malig-nanttransformation[44,45].
Recently, differentstudieshavedemonstratedthat early
embryonic/post zygotic somatic mutations in the NRAS
gene areimplicated inthe developmentofneurocutaneous
melanocytosis,ararecongenitaldisorder,inwhichaffected patientshaveanincreasednumberofmelanocytesinthe lep-tomeningesandtheskin,withalargecongenitalmelanocytic
nevus usually associated with so-called “satellites” in the
vicinity, and childhood melanoma of the central nervous
system[46–48].Inlinewiththeseobservations,recentlyit
hasbeenshownthatprimarymelanomaoftheCNSin
chil-drencarriesoncogenicmutationsinNRAS,unlikeprimary
melanomaofthecentralnervoussysteminadults,inwhich
NRASisnotacommondriveroncogene[46].
So-called “dysplasticnevi”donotseemtocarryNRAS
mutations[49–51].However,inanotherstudy5/7
“dysplas-tic nevi”from individualswithahereditarypredisposition
to melanoma (whocarried germline CDKN2Amutations)
were reported to be NRAS mutated and it was suggested
thatNRASmutationsareimplicatedduringearlymelanoma
development[52].Overall,giventhelimitednumberofcases
analyzed and the lack of interobserver agreement for the
morphology-based diagnosis of “dysplastic nevi” it is too
early to draw significant conclusions. A recent study has
shownthatnevus-associatedmelanomasshowasimilar
fre-quencyofBRAFV600-andNRASQ61-mutationscompared
topublishedreportsofmelanomasoftheskiningeneral[53].
SuchresultsdonotsupporttheconceptthatoncogenicBRAF
orNRASmutationsplayamajorroleinthedevelopmentof
melanomafromnevianddonotsustainthemultisteptheory
ofmelanomaprogressionfromabenignmelanocyticnevus
through“dysplasticnevus”andeventuallytomelanoma[53].
RAShasbeenextensivelyinvestigatedinmelanomaand
severalstudieshaveassessedwhetherspecificRASisoforms
correlatewithrace,patternofsunexposure,clinical
presen-tation,andconventionalmorphologicalfeatures,whichare
commonlyreportedinhistopathologicalreports.
NRASismutatedinapproximately15–20%of primary
cutaneousmelanomasinCaucasianpatients[54–58].Inblack
Africans andAsianpopulationsthereisalowerfrequency
(12%and7.2%,respectively)[59,60].Patientswith
NRAS-mutatedmelanomaswerereportedtobeolderincomparison
withindividualswithBRAF-mutatedtumors[61]althoughin
arecentmeta-analysison31studiesinvolving1972patients,
noassociationbetweenageandNRASmutationswasfound
[55].Similarly,nocorrelationwasfoundbetweengenderand
NRASmutations[58].
In moststudies,NRASmutationwassignificantlymore
frequentinmelanomasarisinginchronicsun-damagedskin
[55,62].TheincidenceoftheNRASmutationaccordingto tumorsitewashighestintheextremities(25%),followedby
thefaceorscalp(18%)andtrunk(18%)[55,61,63].NRAS
mutationshavealsobeenfoundinconjunctivalmelanomas
(18% frequency) [63], sinonasal melanomas (22%) [65],
1, which is a rare mutation site for cutaneous melanoma
[66]. Interestingly, melanoma of unknown primary sites
showedNRAS mutationsin32% of casesassociated with
highsomaticmutationrates,highratiosofC>T/G>A
transi-tions,anda45%ofBRAFmutations,collectivelyindicating
a mutation profile consistent with cutaneous sun-exposed
melanomas[67].
NRASmutationsareoverallmorefrequentlyevidentin
patients with nodular melanoma [55]. From 25% to 31%
of NRAS mutations occurred in this melanoma subtype
[55,59,68].AhigherincidenceofNRASmutationswasfound
in non-acral fast growing melanomas in comparison with
non-fastgrowingmelanomas(26.5versus12.1%)[69].
WhileinsomestudiesNRASmutatedmelanomaswere
reportedtobesignificantlythickerandhigherClark’slevel
thanwttumors [61,62,64,68]otherreports could not
con-firm any association between NRAS mutation and tumor
thickness[70,71]. Ulceration was reported to be lower in
NRAS-mutated tumors in comparison to BRAF mutated
tumors(9.7versus22.4%,respectively)[63]butnoobvious
effectofmutationalstatusonthepresenceofulcerationwas
reportedbyothers[68].MelanomasharboringNRAS
muta-tionshave showngreater mitotic rates thanBRAF mutant
melanomas[63,68].
Inconclusion,theretrospectivenatureofthestudiesand
theheterogeneity of patients’ populationsmayexplainthe
differentresultsobtainedso far,anditshouldbe
acknowl-edgedthatphenotypic-genotypic correlationsinmelanoma
isstillaworkinprogress.
4. NRAS:prognosticorpredictivebiomarkerin melanoma?acriticalanalysisofcurrentliterature
Theprognostic andpredictive significanceof NRAS in
melanomaisstillamatterofintensedebate.
Abiomarker is,by definition, an objectively measured
andevaluated parameter that provides information on the
naturalhistoryofaspecificdisease,itspathogenicprocess
oronpharmacologicalresponsestoaspecifiedtherapeutic
intervention.Aprognosticbiomarkerprovides information
onoverallcanceroutcome,regardlessoftherapy.Inthe med-icalliteraturetwotypesofprognosticbiomarkershavebeen
reported:biomarkersthatgiveinformationonrecurrencein
patientswhoreceivecurativetreatment andthosethat
cor-relatewiththemedianoverallsurvival(OS)inpatientswith
metastaticdisease.AccordingtoaNIHConsensus
Confer-ence,aclinicalusefulprognosticmarkermustbeaproven
independent,significantfactor,thatiseasytodetermineand interpretandhastherapeuticconsequences[72].
Prognosticbiomarkersthatprovideinformationontherisk ofrelapseareimportantnotonlytobetterstratifypatientsin clinicaltrialsbutalsotosparemanypatientsthe
treatment-relatedtoxicitywithoutcompromisingsurvival.Abiomarker
withpredictive value gives information on the effectof a
therapeuticinterventioninapatient.Twotypesofpredictive
biomarkershavebeenreported:(1)upfrontand(2)early pre-dictive markers.The firstcanbe usedfor patientselection
andthe second provides informationearly duringtherapy.
Thelatterbiomarkerislessusefulthantheformerbecause
doesnotprovidereliableandusefulinformationtoselectthe beststrategytobeadoptedbeforestartingtherapy.
4.1. IsNRASaprognosticbiomarkerinmelanoma?
Severalstudieshavebeencarriedouttoexaminewhether
mutationsinNRASconferdifferentpathologicalfeaturesand clinicalbehavior. Theeffectof thesemutationsonclinical
outcome remainsuncertain[59,61,73,74].Table1
summa-rizesmostimportantstudiesontheprognosticroleofNRAS
inmelanoma[63,68,74–79,61,80–83].Themajorityofthese
studieshavebeenretrospectiveinnature,andmostofthem
includedpatientswithrecurrentormetastaticdisease.
WhenOSwasmeasuredfromthetimeofprimarytumor,
NRAS mutations were found to have no impact on OS
[59,63,61]. Akslen et al. evaluated 51 primary nodular
melanomas.InthisretrospectivestudyNRASmutationwas
foundin27%ofpatients[82].RASmutationwasnot associ-atedwithtumorcellproliferationbyKi-67expression,tumor thickness,microvesseldensity,orvascularinvasion,andthere werenodifferencesinpatientsurvival[82].
In anattempttocorrelateBRAFandNRASmutational
status with features known to influence tumor behavior,
including age, gender,Breslow depth, Clark level, mitotic
rate, the presence of ulceration, and AJCCstaging,
Eller-horstetal.performedastudyon223microdissectedprimary
melanomas[63].Patientswhosetumorscarriedeither
muta-tion presented with more advanced stages compared to
patientswithwttumors,andspecifically,weremorelikelyto
haveStageIIIdiseaseatdiagnosis.BRAFandNRAS
muta-tionsdid notinfluencesurvival.Furthermore, inthisstudy survivaldidnotdifferbetweenStageIIIpatientswhose
pri-marytumorsdoordonotcarrymutations,eventhoughthe
mutatedtumorstendedtoproducelargervolumenodal
dis-ease[63].
Recently, Devitt et al. reported data obtained from a
prospectivecohortof249patients[67].Whencomparedtowt
NRASpatients,multivariateanalysisofmelanoma-specific
survival identified NRASmutationsas anadverse
progno-sticfactor.Howeverinthemultivariate analysis,therewas
noevidencethatNRASmutationwasneitheranindependent
predictorofrelapsefreesurvival(RFS)norofOS[68].
However,intwostudieswhereOSwasmeasuredfromthe
timeofbiopsyofadvanceddisease,NRASmutationswere
associatedwithimprovedOSwhencomparedtotumorswith
BRAFmutationsorbothBRAF/NRASwttumors[73,74].
Mann et al. performed a comprehensive
clinico-pathologicalassessmentoffresh-frozenmacroscopicnodal
metastases and the preceding primary melanoma, somatic
mutation profiling, and gene expression profiling to
iden-tifydeterminantsofoutcomein79melanomapatients[81].
Table1
SummaryofmostsignificantstudiesaddressingtheprognosticsignificanceofNRASmutationsinmelanoma.
RASmutationandmelanomaprognosis
Author Patientsno. Stage Siteofprimary
melanoma
Genes Exons PFS OS
Demunter(2001)[75] 81 Allstages Skin NRAS 1 p=0.0130 –
Omholt(2002)[78] 72 Allstages Skin NRAS 2
3
– NS
Houben(2004)[76] 174 Allstages Skin BRAF 15
11
NS NS
p=0.02a
NRAS 1
2
Akslen(2005)[81] 57 Allstages Skin BRAF 15
11 – NS NRAS 2 1 Edlundh-Rose(2006) [79] 219 NA Skin BRAF 15 11 – NS NRAS 2 Ugurel(2007)[73] 109 III IV Skin Mucosa Occult NA BRAF 15 11 – p=0.006 NRAS 2 1
Ellerhorst(2010)[62] 223 I–III Skin BRAF 15 – NS
NRAS 2
Devitt(2011)[67] 244 I–III Skin BRAF 15 – p=0.04
(MSS)
NRAS 3
Jakob(2012)[77] 667 Allstages Skin
Mucosa Uvea Occult BRAF 15 – p=0.004 NRAS 1 2
Mann(2012)[80] 79 III Skin BRAF 15 – NS
NRAS 2 Bucheit(2013)[61] 438 IV Skin Mucosa Softparts Occult BRAF 15 – NS NRAS 1 2 Birkeland(2013)[74] 85 III IV Skin Mucosa Uvea Occult NRAS 3 p<0.01 p<0.001
Ekedahl(2013)[82] 203 IV Skin BRAF 15 – p=0.25
NRAS 2
OS:overallsurvival;PFS:progressionfreesurvival;MSS:melanoma-specificsurvival;NS:notsignificant.
aOSfrommetastasectomy.
NRAS mutation was independently associated with better
survival.Furthermore,a46-geneexpressionsignaturewith
strongoverrepresentationofimmuneresponsegeneswas
pre-dictiveofbettersurvival;inthefullcohort,mediansurvival
was>100monthsinthosewiththesignature,but10months inthosewithout.
Recently,inaretrospectivestudy,Jacobetal.testedfor
Table2
StudiesreportingonRASmutationsaspredictivebiomarkersinmelanoma.
RASaspredictivebiomarkerinmelanoma
Author Patients
no.
Stage Siteof
primary melanoma
Genes Mutations Drug(s) OS PFS TTP CCR/CB
Banerji(2008)
[83]
6 NR NR BRAF V600E 17-AAG NR – NR NR
NRAS G13D 17-AAG Joseph(2012) [84] 208 IIIc IV NR BRAF V600 HDIL2 NS NS NS NS – – p=0.05 NRAS G12 G13 Q61 HDIL2 Birkeland(2013) [74] 85 III IV Skin Mucosa Uvea Occult NRAS G12 G13 DTIC p<0.001 NS – NS Patelet(2013)[85] 18 III IV
Skin BRAF V600E
R603 S.+/−DTIC, TXT,E.orT. NR – NS NS NRAS Q61R Q61K G12S S.+/−DTIC, TXT,E.orT.
17-AAG:HSP90inhibitor17-allylamino-17-demethoxygeldanamycin;DTIC:dacarbazine;CB:clinicalbenefit:objectiveresponseorstablediseaserecorded
3monthsafterDTICtreatment;HDIL2:high-doseinterleukin2;CRR:clinicalresponserate;TTP:timetoprogression;NR:notreported;S.:selumetinib;
TXT.:docetaxel;E.:erlotinib;T.:temsirolimus.
significantassociationsofmutationwithtumorandpatient
characteristicsandwithsurvivalfromthediagnosisofstage
IVdisease[78].Tumormutationstatuswasassociatedwith
theriskofcentralnervoussysteminvolvementatthe
diag-nosis.PatientswithNRASmutationshadamediansurvival
of8.2monthsfrom stageIVdiagnosis, whichwasshorter
thanthe median survival of wtpatients (15.1 months).At
multivariateanalysis,afteradjustingforage,sex,metastatic
site,serumlactatedehydrogenaselevel,NRASmutationwas
independentlyassociatedwithdecreasedOS.
Overall, the results published so far are heterogeneous
in terms of patients’ selection criteria and methodology.
Specifically,difficultiesincomparingresultsarisefromthe followingconsiderations: (i)mostof theavailabledataare retrospective;(ii)patientswithdifferenttumorstageshave
beenevaluated; (iii) primary or metastaticsiteshavebeen
tested;(iv)differenttumorhistotypeshavebeenincluded.
Hence,thereisnodefinitiveevidencethatNRAS
muta-tionisprognosticinpatientswithlimitedradicallyresected disease(stagesI–III) orinmetastaticsetting.Furthermore,
mostoftheobservationshavebeenconductedinCaucasian
populationswithscarcityofdatafromothergeographicareas (e.g.Asian).
4.2. IsNRASapredictivebiomarkerinmelanoma?
TheRASmutationalstatusdoesnotgiveinformationon
theeffectofatherapeuticinterventioninapatient,henceitis notapredictivemarkereitherupfrontorasearlypredictive
marker. Table 2 includes studiesaddressing the predictive
significanceofNRASmutationsinmelanoma[75,84–86].
So far, several different strategies of directly targeting RAShavenotresultedineffectivetherapeutics.Thereis
evi-dence that someNRAS-mutated celllines are sensitiveto
MEK inhibition invitro[87].However, inthismodel, the
sensitivitytotheMEKinhibitorofN-RASmutatedcellswas
significantlylowerthanthoseharboringBRAFmutation.
TheloweractivityofMEKinhibitorsinN-RAS-mutated
in comparison with BRAFV600-mutated melanoma cells
maybeexplainedbythecomplexityofpathwayswithwhich
RASinteractswithinthecell.
Itis wellknown thatRAS familymembershave
multi-ple other targets, such as PI(3)K andRalGDS; thesemay
exert more prominent oncogenic effects in certain tumor
subtypes,therebyreducingtherequirementforMAPK
acti-vation.Hence,single-agenttherapeuticstrategiesmayprove
insufficient in RAS mutant tumors. Instead, direct RAS
inhibitorsorcombinatorialstrategiesmayberequired.
Recently, anoral MEK inhibitor (MEK162)was tested
in patients with metastaticmelanoma harboring BRAF or
NRASmutationswithencouragingresultsinNRASmutated
patients [88]. In preclinical models MEK162 inhibited
growth of NRAS-mutated and Val600Glu BRAF-mutated
melanoma instudiesthat usedinvitroandinvivomodels
[89].
However,theresponseratewasreportedinonly20%of
patients and only in 10% of this population the response
was confirmed. Furthermore, the median progression-free
responsewas7.6weeks[88].Thesedataclearlyindicatethat
mostofthepatients rapidlydevelopresistancetotheMEK
inhibitor.
Atwo-arm,randomized, prospective,open-label,
multi-center,phaseIIIstudytocomparetheefficacyandsafetyof
MEK162(45mgbisindie)versusdacarbazine(1000mg/m2
IVevery 3 weeks) inpatients withadvanced (Stage IIIC)
unresectableormetastatic(StageIV)NRASQ61
mutation-positive cutaneous melanoma is currently underway. The
primaryendpointofthestudyisprogression-freesurvival,
whilesecondaryendpointisoverallsurvival(“NEMOtrial”
NCT01763164).
Anothersecond generationMEKinhibitor,selumetinib,
demonstratedmarkedinhibitionofpERK,eitherincelllines
harboring BRAF mutations as well as in thoseharboring
NRASmutations[90].
A randomized phase II study comparing the MEK
inhibitor Pimasertib (AS703026) with dacarbazine in
pre-viously untreated subjects with N-Ras mutated locally
advanced or metastatic malignant cutaneous melanoma is
currentlyunderway(NCT01693068).
Atthetimeofthepublicationofthismanuscriptthereare
norandomizedclinicaltrialscomparingMEK162withother
MEKinhibitorsinNRASmutatedmelanomapatients.
Recently thedevelopment of small moleculesthat
irre-versiblybindtoacommononcogenicmutant,K-Ras(G12C)
has been reported [91]. These compounds rely on the
mutantcysteineforbindingandthereforedonotaffectthe
wt protein. These inhibitors to K-Ras(G12C) subvert the
native nucleotide preference tofavor GDP over GTP and
impairingbindingtoRaf.Thesefindingsarerelevantsince
they reveal, for the first time, a new allosteric regulatory
site on Ras that is targetable in a mutant-specific
man-ner.
AsubgroupofmelanomaswithRASdependenceisthose
withlow-activity.
BRAFmutations,such asthosefoundatpositions466,
464and597.Celllineswithlow-activity BRAFmutations
showanimpairedactivationofMAPKsignalinginisolated
kinaseassaysandoftenharborconcurrentNRASmutations
at positions 12and 13. It cannot be excludedthat NRAS
melanomacellswithlowactivitymutantBRAFmaypartially
explainthesensitivityofasubgroupofNRASmelanomacells
toMEKinhibitors.
Inaccordance withthishypothesis,Dahlmanetal.
per-formedananalysisofBRAFexon15in49tumorswithlack
ofBRAFV600 mutationandshowedthat 2(4%)harbored
L597mutationsandother2 BRAFD594 andK601
muta-tions[92].InvitrosignalinginducedbyL597mutantswas
suppressedbyMEKinhibition.ApatientwithBRAFL597S
mutantmetastaticmelanomarespondedsignificantlyto
treat-mentwiththeMEKinhibitor,TAK-733.Collectively,these
data show clinical significance response to BRAF(L597)
mutationsinmelanoma.
ThefocusofindirectRASinhibitionhasthenshiftedto
interferewiththecomplexnetworkofactivateddownstream
cascades such as the MAPK, phosphoinositol 3-kinase
(PI3K),phospholipidC(PLC),RalGEF.
Posch et al. evaluated the sensitivity of RAS mutated
melanomacellsandxenograftstoMEKandPI3Kinhibitors
[93]. NRAS mutated cells were more sensitive to MEK
inhibition compared withthe PI3K/mTORcascade
inhibi-tion.CombinedtargetingofMEKandPI3Kwassuperiorto
MEKandmTORinhibitioninallNRASmutantmelanoma
celllines,suggestingthatPI3Ksignalingismoreimportant
for cell survival in NRAS mutant melanoma when MEK
is inhibited. However, targeting of PI3K/mTOR in
com-bination with MEK inhibitors is necessary to effectively
abolishgrowthofNRASmutantmelanomacellsinvitroand
regressxenograftedNRASmutantmelanoma.Inthismodel
MEK and PI3K/mTOR inhibition was synergistic. These
results indicate that combined targeting of the MEK/ERK
andPI3K/mTORpathwayshasantitumoractivityandcould
beavalidoptioninthetreatmentofNRASmutantmelanoma,
forwhichtherearecurrentlynoeffectivetherapies.
Finally, Johnson et al. reported that patients with
NRASmutatedmetastaticmelanomaachieveincreased
clin-ical benefit from immunotherapy compared to thosewith
BRAF/NRASwt[94].
These datasuggest that NRAS mutation statusmay be
a biomarker of response to immunotherapy in metastatic
melanomaandthatmolecularlytargetedimmunotherapymay
befeasible.Howeveralarger,prospectiveanalysisis neces-sarytovalidateandexpandontheseresults,includingthose
withBRAFmutandKITmutmetastaticmelanomatodraw
firmconclusions.
Overall, the above data suggest that:(i) a subgroup of
NRASmutatedmelanomamaybesensitivetoMEK
inhibi-tionbutinmostcasesresistancerapidlyoccur;(ii)asubgroup
of NRAS mutated melanoma harbor low activity BRAF
mutation,andthemeaningofthesemutationsshouldbe
fur-therinvestigated;(iii)single-agenttherapeuticstrategiesmay
proveinsufficientinRASmutanttumors.Instead,
combina-torialstrategiesmayberequiredtoovercomeresistance.
5. NRASasamechanismofresistancetoBRAF inhibitorsinmelanoma
AhighpercentageofpatientswithBRAFV600Emutant
melanomasrespondtoselectiveRAFinhibitorsbutresistance
eventuallyemerges.
Unlike what happens in othertumors whereadditional
mutationseventuallyoccurinthetarget(EGFRinnon-small
cell lung cancer, c-KIT in GISTs, BCR-ABL in chronic
myeloidleukemia)theearlyevidencefromdirect
sequenc-ingofBRAFexonssuggeststhatnewpointmutationsarenot
evidentandthatBRAFV600Epersists.
RAShasbeenconsistentlydescribedasamechanismof
resistancetoBRAFinhibitors.Itiswellknownthatthereisa
switchinRAFisoformusagedependingonwhetherBRAF
Table3
ResistancetoBRAFinhibitors.
ResistancetoBRAFinhibitors
Author Patientswithacquired
resistancetotherapyno.
Drug Mechanismofresistance NRASacquired
mutationspatientsno.(%)
Nazarian(2010)[95] 12 Vemurafenib NRAScodon61
mutations
PDGFRBoverexpression
1(8.3%)
Trunzer(2013)[96] 13 Vemurafenib IncreasedpERKlevels
MEK1mutations
NRAScodon61
mutations
3(23%)
McArthur(2011)[97] 11 Vemurafenib NRAScodon61
mutations
1(9.09%)
Poulikakos(2011)[98] 19 Vemurafenib IncreasedRAS-GTP
levels Increased RAS-independentRAF dimerization 6(31.6%) Wagle(2014)[99] 5 Dabrafenib Trametinib MutationinMEK2
BRAFspliceisoform
BRAFamplification
5(100%)
VanAllen(2014)[100] 30 Vemurafenib
Dabrafenib MAPKpathway Alterations MEK1Mutations MEK2Mutations MIFTAmplification 23/45(51%)
Rizos(2014)[101] 38 MutationinMEK2
MutationinMEK1
MutationinNRAS
MutationinAKT
BRAFspliceisoform
BRAFamplification
3(8%)
whichBRAFismutated,BRAFisprimarilyresponsiblefor
signalingtoMEKandERK.In presenceofRASmutation
anexcessiveERKsignalingthrough BRAFandingeneral
MAPKactivation would inducecellcycle arrest or
senes-cencethroughtranscriptionalup-regulationofproteinssuch
as p21, p27, and p16INK4A [102].To avoidthis, the cells
switch to CRAF,which provides weaker signaling andis
compatiblewithtumorprogression.
Nazarianetal.demonstratedthathighlevelsofactivated
N-RASresultingfrommutationsleadtosignificantMAPK
pathwayreactivationuponBRAFinhibitortreatment[95].In
aseriesofelegantexperiments,knockdownofNRASreduced
growth of the respective BRAF inhibitors resistant cells.
Ontheopposite,overexpressionofN-RASconferredBRAF
inhibitorresistancetoBRAFinhibitorsensitiveparentalcell lines.
Recently,Suetal.usedcelllinestoestablishBRAFV600E
melanoma clones with acquired resistance to a BRAF
inhibitor [103].The authors confirmedthat nosecond-site
mutationscouldbeidentifiedintheBRAFcodingsequence.
Inthismodel,resistancecorrelatedwithincreasedlevelsof
RAS-GTP,andsequencingofRASgenesrevealedarare
acti-vatingmutationinKRAS,resultinginaK117Nchange in
theKRASprotein.Elevatedlevels ofCRAFand
phospho-rylatedAKTwerealsoobserved.Interestingly,combination
treatmentwithBRAFinhibitorandeitheraMEKinhibitor
oranAKTinhibitorsynergisticallyinhibitedproliferationof resistantcells.Thesedatasupportclinicalstudiesinwhich
combination therapy with other targeted agents are being
strategizedtoovercomeresistance.
Trunzer et al. [96] evaluated serial biopsies to study
changes in mitogen-activated protein kinase (MAPK)
signaling,cell-cycleprogression,andfactorscausing
intrin-sicor acquired resistancebyimmunohistochemistry,DNA
sequencing, or somatic mutation profiling to a BRAF
inhibitorwithintheBRIM2study[104].Inthisstudy3/13
patients hadNRASQ61Kco-occurring mutationsintumor
samplestakenatprogression.Combiningthesefindingswith
thosepreviously reportedby Nazarian[95] andMcArthur
[97],among36patientsanalyzed,fivepatients(14%)hadan
NRASmutationinaprogressivelesion.Thisfurthersupports thehypothesisbyNazarianetal.[95]thattheNRASmutation
isonemechanismofescapefromvemurafenibtherapy.
Overall,theabovereporteddatasuggestthat:(1)A
con-comitantbaselinemutationintheupstreamNRASoncogene
israrebutmayresultinearlylackofclinicalbenefittoBRAFi;
(2) RAS mutation is a common mechanism of acquired
resistance; (3) whether a combination therapy with other
targetedagentscouldovercomeresistanceremainstobe elu-cidated.
Table4
RAStargetinlocallyadvancedormetastaticmelanoma:ongoingclinicaltrials(www.clinicaltrials.govaccessedJanuary26,2014).
Drug Phase Trial Disease(s) Primaryoutcomemeasures
Monotherapynon-randomized
MEK162 II NCT01320085 BRAForNRAS
Mutatedmelanoma ORR RAF265 II NCT00304525 Melanoma MTD DLT Associationmutationsin NRAS/clinicalresponse(◦)
Selumetinib(AZD6244) II NCT00866177 BRAForNRAS
Mutatedmelanoma
Anti-tumorresponse
Monotherapyrandomized
Pimasertibversusdacarbazina II NCT01693068 NRASmutatedmelanoma PFS
MEK162versusdacarbazine III NCT01763164 NRASmutatedmelanoma PFS
AZD6244versustemozolamide II NCT00338130 Melanoma PFS
ORR* TTD
Durationofresponse
Assessmentoftheefficacyof
AZD6244versus
temozolomideBRAFor
NRASMMpatients(◦)
Combinationtherapynon-randomized
BKM120+MEK162 I NCT01363232 EGFRmutantNSCLCinPD
onEGFRinhibitors
Triplenegativebreastcancer
Pancreaticcancer
CRC Melanoma
NSCLC,withKRAS,NRAS,
and/orBRAFmutations
DLT
Trametinib
(GSK1120212)+GSK2141795
II NCT01941927 BRAFwtmelanoma ORR*inpatientswitheither
mutatedNRASorwt NRAS/wtBRAFM RAFinhibitor (BMS-908662)+immunotherapy (ipilimumab) I NCT01245556 Melanoma Toxicity PDwillbeassessedby evaluatingmarkersof
RAS/RAFpathwayactivity
(◦)
PI3K/mTORinhibitor
BEZ235+MEK1/2inhibitor
MEK162
Ib NCT01337765 EGFRmutantNSCLCinPD
onEGFRinhibitors
Triplenegativebreastcancer
Pancreaticcancer
Colorectalcancer
Melanoma NSCLC
Otheradvancedsolidtumors
withKRAS,NRAS,and/or
BRAFmutations
IncidenceofDLT
LEE011+MEK162 Ib NCT01781572 NRASmutatedmelanoma IncidenceofDLT
ORR*
MTD:maximumtolerateddose;EAS:ectopicACTHsecreting;wt:wildtype;NR:notreported;ORR:overallresponserate;PFS:progressionfreesurvival;
TTD:timetodeath;DLT:doselimitingtoxicity;ORR*:objectiveresponserate;CRC:colorectalcancer;(◦):secondaryoutcomemeasures.
The importance of RASin melanoma deserves clinical
andbiologicalinvestigationtooptimizetreatmentoflocally
advancedandmetastaticmelanoma.Although,inthelasttwo
decades,progresshasbeenslow,therearenowavarietyof
therapeuticstrategiesthatareprimedfor clinical
investiga-tion.Table4summarizesongoingtrialsinwhichRAS,and
preferentiallyNRAS,hasbeenselectedasatarget.
6. Futuredirections
Thirty yearsof basic, clinicalandtranslationalresearch
have producedalargeamount of knowledgepertainingto
theRASoncogenefamily(Fig.4).TheprevalenceofRAS
mutations, but also the high number of RAS activators
Fig.4. TimelineofkeyadvancesinNRASclinicalandtranslationalresearch.(a)Harveyetal.(1964)[97];(b)Kirstenetal.(1970)[98];(c)Shihetal.(1980)
[99];(d)Changetal.(1982)[2,3],(e)Deretal.(1982)[4],(f)Paradaetal.(1982)[5],(g)Santosetal.(1982)[100],(h)Milburnetal.(1990)[103];(i)Pai
etal.(1990)[102];(j)Boguskietal.(1993)[101];(k)Moodieetal.(1993)[9];(l)Vojteketal.(1993)[10];(m)Warneetal.(1993)[11],(n)Zhangetal.(1993)
[12];(o)Chinetal.(1999)[22];(p)Solitetal.(2006)[86];(q)Nazarianetal.(2010)[90];(r)Whitwametal.(2007)[37];(s)Asciertoetal.(2013)[87],(t)
Poschetal.(2013)[89].
proteinsatthecrossroadsofseveralsignalingnetworks.
Nev-ertheless, this extensive knowledge has not yet translated
intoclinicallyeffectivetherapiesformelanomasexpressing
mutantformsofRAS.
AsRAS is mutated in15–20% of melanomas, priority
actionsareneeded:
1. Future studies should focus on co-extinction strategies
otherthanreinforcinginhibitionofMAPKsignaling.
2. Inhibitionoftheactivateddownstreamcascadesincluding
MAPK,PI3K,PLC,RALshouldbepursuedinpreclinical
andearlyphaseclinicalstudies.
3. Mostof the downstreamtargets are not tumor specific
therapiesandbeartheriskofseveresideeffects.Hence,
well-designed clinical studies with appropriate
phar-macokineticsandpharmacodynamic endpoint between
combinationtherapiesareneeded.
4. MEKinhibitors as monotherapyshouldbe validated in
prospective,randomizedphaseIIIstudies.
Reviewers
OlivierMichielin,MD,PhD,CHUV–Multidisciplinary
OncologyCenter(CePO),UNIL–UniversityofLausanne,
Lausanne,Switzerland.
Matteo Carlino, PhD, Westmead Institute of Cancer
Research,TheUniversityofSydney,Australia.
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Biographies
MarioMandalàiscurrentlyamedical oncologistinthe
UnitofMedicalOncology,PapaGiovanniXXIIIHospitalin
Bergamo,Italy.HeisinchargeoftheClinicaland
Transla-tionalResearchUnit.HereceivedhisMDfromtheCatholic
University inRome,Italy in1995.He hascompleted
resi-denciesinMedicalOncologyattheCatholicUniversityin
RomeaswellasatMilanUniversityandtheEuropean
Insti-tuteofOncologyinMilan,Italy.Healsoobtaineddiplomas
inMedicalOncologyandHaematologyfromMilan
Univer-sity in2000 and2005,respectively. His researchinterests
include clinical and translational research on cancer and
thrombosis.Hismainoncologicalresearchfocuseson
gas-trointestinal cancerandmelanoma.He isafullmemberof
the European Society of Medical Oncology. He has
lead-or co-authored many scientific papers and is a reviewer
for several journals, including Lancet Oncology, Cancer,
British Journal of Cancer, Annals of Oncology, Cancer
TreatmentReview,JournalofThrombosisandHaemostasis,
Thrombosis and Hemostasis, Arteriosclerosis-Thrombosis
and Vascular Biology, Critical Reviews in Oncology and
Haematology.
DanielaMassi,MD,PhD,isassociateprofessorof pathol-ogyattheUniversityofFlorenceMedicalSchool,Italy.Her
pathologytrainingincludesadermatopathologyfellowship
attheInstituteforDermatopathology,ThomasJefferson Uni-versity,Philadelphia,PA,underthedirectionofA.Bernard Ackerman.Shethenreceivedapost-doctoralresearch
fellow-shipfromtheAmerican-ItalianCancerFoundation(A.I.C.F.)
with aresearchprogram incutaneousmelanoma. She has
beenmemberoftheExecutiveCommitteeoftheInternational
SocietyofDermatopathology(1997–2003),Chairmanofthe
DermatopathologyWorkingGroupoftheEuropeanSociety
of Pathology(2007–2011),andsheiscurrentlymemberof
the EORTC Melanoma Pathology Group. She is an
asso-ciate editor of VirchowsArchiv andscientificreviewer for
severalinternationalscientificjournals.Her research
inter-ests are focused onskintumorpathology,andparticularly
receptorsignalingandmoleculargeneticsofmelanoma.She
has authored more than 200 publications and contributed
to the volume ‘Pathology & Genetics of Skin Tumours’
of theWorldHealthOrganization(WHO)Classification of