Breast cancer prognostication and prediction in the postgenomic era

14 Pages·2007·0.82 MB·English

review AnnalsofOncology18:1293–1306,2007
doi:10.1093/annonc/mdm013
Publishedonline21February2007
Breast cancer prognostication and prediction
in the postgenomic era
P. E. Lønning1,2*, S. Knappskog1,3, V. Staalesen1,3, R. Chrisanthar1,3 & J. R. Lillehaug3
1SectionofOncology,InstituteofMedicine,UniversityofBergenand2DepartmentofOncology,HaukelandUniversityHospital;
3DepartmentofMolecularBiology,UniversityofBergen,Bergen,Norway
Received3January2007;accepted11January2007
D
o
Expanding knowledge,together with implementation of newtechniques, hasfuelled the area oftranslational w
n
medicalresearchaimingatimprovingprognosticationaswellaspredictionincancertherapy.Atthesametime, loa
d
newdiscoveries haverevealed abiologicalcomplexity wewere unaware ofonly adecade ago.Thus,weare e
d
facedwith novel challengeswith respect tohow wemayexplore issues suchas prognostication andpredict fro
m
drugresistance invivo.Whilemicroarray analysisexploringexpression ofthousandsof genesinconcert h
represents amajor methodological advancement, discoveries suchas the findingof different mechanisms of wttps
eaplteigrnenateivtiecsspilelicnicnignga,nindtrtohnaitchmypuetarstipolnicsin,gthsaetemmossttogebneeatrtaunmsocruipr-trseliantethdephhuemnoamnegneonno,meexeamreplsifiuebsjeactctoomplex vie://aca
ed
pathologythat may notbe explored with useofsingle analytical methodsonly.Thispaper discusses clinical rem
settingsfor studying drugresistance invivo together with adiscussion ofcontemporary biology inthis field. ic
.o
Notably,eachindividualparameterwhichhasbeenfoundcorrelatedtodrugresistanceinvivosofarrepresents up
.c
either adirect drug targetor afactorinvolved inDNArepair orapoptosis. Onthe basis ofthese findings, o
m
wesuggest drugresistance maybe explored onthe basis ofupfront biologicalhypotheses. /a
n
Keywords:breastcancer,microarray,mutation,prediction,splicing,therapyresistance n
o
n
c
/a
rtic
introduction wewereunawareofjustadecadeago.Sequencingofthe le
-a
humangenomehasrevealedittocontainonly(cid:1)30000genes; b
Mostoftheclassicalstudiesintranslationalresearchrelatesto ontheotherhand,mergingevidencehasrevealedthe stra
theissueof‘prognostication’.Thus,asimpleliteraturesearchon c
complexityofthisgenomewithindividualgenescodingfor t/1
‘prognosis’and‘breastcancer’inconcertrevealed>10000 8
multipletranscriptsthroughmechanismssuchasalternative /8
publishedtitlesfromtheWebofScience(6December2006) splicing[1,2]andseveralpromoterswithdifferentligand- /1
2
duringthelasttwodecades. 9
bindingpreferencesforeachgene[3].Also,forsomegene 3
Contrastingthislargenumberofpublications,andrarely /1
products,severalthousandDNA-bindingsites[4,5]ordirect 7
mentioned,isthelimitationsof,or,rather,thespecific andindirecttranscriptionalregulationofseveralhundred 813
requirementsforaprognosticfactortobeofvaluetoclinical 5
downstreamgenes[6,7]havebeendiscovered. b
therapy.Whiledevelopmentofmoreeffectivebutalsomore y
Thisreviewisdividedintothreemainparts;first,wewill g
toxictherapyformsunderlinestheneedofidentifyingpatients brieflydiscusstheissuesofprognosticationandpredictionin ues
withagoodprognosisthatmaynotneedadjuvanttreatment, ageneralperspective;secondly,wewilldiscusscontemporary t o
thisdoesnotimplythatallpatientswithapoorprognosismay findingsinmolecularbiologywithparticularrelevanceto n 1
0
benefitfromtherapy.Thus,aparameterpredictingpoor cancerand,thirdly,wefocusonhowourpresentknowledge A
pborothgntoosiisndmivaiydupareldpircotgdnrousgtircefsaiscttaonrcseanasdwperlol.gTnohsisticre‘lgaetense canhelpimprovingtherapy. pril 2
0
1
profiles’generatedbytechniquessuchasmicroarrays. 9
Thankstotheextendedbiologicalknowledgeand,in
particular,novelmethodstomeasurebiologicalvariablessuch prognostication in breast cancer
astheuseofcomplementaryDNA(cDNA)microarrays,we
Theimportanceof—anddifferencesbetween—aprognosticand
haveforthefirsttimetheopportunityofidentifyingthe
apredictivefactorisdiscussedelsewhere[8]andmaybe
mechanismsofdrugresistance.Thisexpandingbiological
illustratedbyexaminingrelapse-freeoroverallsurvivalcurves
knowledge,however,hasalsorevealedabiologicaldiversity
fromtheOxfordmeta-analysis[9].Fortheexampleshere,we
havechosencurvesillustratingimprovementinrelapse-free
*Correspondenceto: ProfP.E.Lønning,DepartmentofOncology,SectionofOncology,
survivalwithrespecttoadjuvantversusnoadjuvant
InstituteofMedicine,HaukelandUniversityHospital,N-5021,Bergen,Norway.
Tel:[phone];Fax:[phone];E-mail:[email protected] chemotherapyamonglymphnodenegativeandnode-positive
ª2007EuropeanSocietyforMedicalOncology
review
AnnalsofOncology
breastcancerpatients(Figure1A)obtainedfromanearlier Whileprobably>100individualprognosticfactorshavebeen
meta-analysis[10,11]. reportedinbreastcancer,ingeneralcombiningseveralfactors
Theoriginalmessagefromthisfigurewastoillustratethe addsmarginalbenefits.Thus,bycombiningthreetofourof
benefitsofadjuvantchemotherapy(Figure1B)inalowaswell themostimportantfactors,littlemaybegainedbyadding
asahigh-riskpopulationdefinedbylymphnodestatus.The additionalinformation.Therearetwomainreasonsforthis.
figure,however,illustratesinadditiontheprognosticvalueof First,manysingleprognosticfactorsarenot‘independent
studyinglymphnodestatusinearlybreastcancerpatients. variables’;lookingattraditionalfactorssuchasmitoticindex,
Lookingat10-yearoutcome(Figure1C)forpatientswith flowcytometryindicators,vascularinvasionandtumourgrade,
metastasestoaxillarylymphnodes(onenodeormore)atthe thereisastrongcorrelationbetweenthesefactors[13–22].
timeofsurgeryversusthosewithout,predictedriskofhaving Thesecondissueisapotentialconfounding‘predictive’effect;
arelapsewithin10yearsfromoperationwithaspecificityinthe patientsstratifiedforrelapseriskonthebasisofthe
rangeof70%andasensitivityof(cid:1)60%[10,11]inthe prognosticfactormay,inaddition,expressadifferential
populationnothavingadjuvantchemotherapy(calculations sensitivitytosystemictreatment(seebelow).
onthebasisoffigurespresentedintheoriginalpublications). Figure1Billustratesthepredictivevalue,orratherlackof
Whilethegroupofnode-positivepatientsmaybesubsequently such,oflymphnodestatuswithrespecttoeffectof D
o
w
stratifiedonthebasisofthenumberofmetastaticnodes[12], chemotherapy.Thedataclearlyrevealedthatthebenefit, n
wearestillleftwiththeproblem;howtoidentifyagroupof determinedbyreductioninhazardratioofhavingarelapse,was loa
d
patientswithsuchalowriskofrelapsethatitmakesadjuvant similarwithinbothgroups[9].Thefactthatthiswasthecase e
d
therapyredundant?Thefigurespresentedhereillustratethe wasnotsomethingpredictedupfront;actually,itwasrealised fro
m
needtodevelopbetterprognosticfactorsbeyondlymphnode longafterimplementationofadjuvantchemotherapyonthe
h
negativity. basisofriskprofile.Consideringotherprognosticfactors,we ttp
s
://a
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a
d
A B e
Relapse-free survival (%) Relapse-free survival (%) m
19000 CTroenattreodl 10900 TCroenattreodl ic.ou
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80 80 .c
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70 70 /a
n
60 61.5 Node 60 61.5 Node no
54.5 negative 54.5 negative nc
50 50 /artic
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40 38.5 40 38.5 -ab
Node Node s
positive positive tra
29.5 29.5 ct/1
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50 0
A
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40 38.5 ril 2
Node 0
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positive Relapse-free survival 9
29.5
0 Time
0 5 10 years
Prognostic factor Effect of adjuvant therapy
Figure1. Relapse-freesurvivalamongpatientstreatedforearlybreastcancerinphaseIIItrialsstratifiedfornodalstatusand±chemotherapyaccordingtothe
OxfordOverviewAnalysis[10,11];reproducedwithpermission.(A)Originalfigure.(B)Illustratingtheeffectofchemotherapyamongnode-positiveand
node-negativepatients.(C)Illustratingprognosisamongnode-negativeandnode-positivepatientsnotexposedtoadjuvanttherapy(prognosis).(D)
Theoreticalexampleillustratingeffectofchemotherapyamongagroupofpatientswithagoodprognosis(blueline)versusapoorprognosis(blackline)
definedbyaspecificprognosticfactor.Inthiscase,theprognosticfactorinadditionpredictedforpoorresponsetotherapy,illustratingthedangerofpotential
selectionofpatientsforadjuvanttherapyonthebasisofprognosiswithoutinformationwhethertheprognosticfactorcouldalsopredictdrugresistance.
1294|Lønningetal. Volume18|No.8|August2007
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maybelesslucky.Thus,itmaynotbeautomaticallyinferred therapyapplied,(ii)asgeneproductsmodifyingtherapy
thatanovelprognosticfactormaynotinvolveapredictive responsethroughDNAmetabolismor(iii)asgeneproducts
componentor,saidinotherwords,wemaynottakefor participatingintheexecutionofapoptosisand/orgrowth
grantedupfrontthattheimprovementwithrespectto arrest/senescence.
percentagereductioninrelapserateduetotherapyshouldbe Belowisasummaryofindividualpredictivefactorsfoundto
similarinthe‘highrisk’and‘lowrisk’patientpopulations. beassociatedwithresponsetodifferenttypesofchemotherapy
Thus,whileTP53mutationstatusaswellasHER-2 invivo.Notably,withtheexceptionofamplificationofthe
amplificationarebothknowntobeassociatedwithapoor topoisomeraseIIgene,forwhichmergingevidenceindicate
prognosisinbreastcancer[23–25],TP53mutationspredict diagnostictestingfortherapyselectionmaybecloseto
reducedsensitivitytoanthracycline-containingtherapy[26,27], implementation[31–34],fornoneoftheindividualfactors
whileHER-2amplificationsconferlowsensitivitytoregimens identifiedsofardowehavedataindicatingclinicaluseinbreast
suchasthecombinationchemotherapywithcyclophosphamide, cancerinthenearfuture.However,thefindingsdescribedin
methotrexateandfluorouracil(CMF)[28]. detailsbelowarechallenging,indicatingweareapproaching
Thepotentialnegativeconsequencesofselectingpatientsfor aconceptualunderstandingofbiologicalmechanismsand
adjuvanttreatmentusingTP53mutationstatusoranyother pathwaysplayingakeyroleintherapyresistance[35]. D
o
w
factorassociatedwithdrugresistanceasaprognosticfactorto n
definea‘highriskgroup’isdepictedinFigure1D.Perhaps amplification of topoisomerase II loa
d
theextremeconsequenceofselectingpatientsonthebasisof e
relapseriskonlywithoutattentiontoapotentialpredictiverole Aafnfetchtriancgycselivneersalseinemtrascteolluaclahriepvreotchesesiresa,nctaiutusmingouDrNefAfectsby d from
oftheprognosticfactormaybeillustratedtakingendocrine
intercalationaswellasgenerationoffreeradicals[36–38]. h
treatmentwithtamoxifenasanexample.Whilelong-termrisk Akeymechanism,however,seemstobetheinhibitionofthe ttps
ofrelapseremainssimilaramongpatientswithoestrogen DNAdamagerepairenzymetopoisomeraseII(Topo-II)[39]. ://a
receptor(ER)-positiveandER-negativebreastcancernot c
Notably,itwasrevealedmorethanadecadeagothat ad
undergoingadjuvanttherapy,short-termprognosis,alsofor e
anthracyclinesadministeredindifferentdosesintheadjuvant m
patientsnotundergoingadjuvantendocrinetherapy,isworse ic
fortheER-negativeones[29,30].Thus,selectingpatientsfor settingwereassociatedwitha‘dose–response’effectamong .ou
tumoursamplifiedforHER-2.Thiseffectwasnotseenamong p
adjuvanttamoxifentreatmentonthebasisofriskprofilealone .c
HER-2-negativetumours[40].Thisfindingleadtosuggestions o
payingnoattentiontoknowledgeaboutthebiologyoftheER m
thatHER-2maybeassociatedwithsensitivitytoanthracycline /a
couldmeantotreatER-negativebutnottheER-positive n
therapy;yet,findingsfrominvitrostudiesdidnotsupportthe n
tumourswiththeantioestrogen;theoutcomewouldbe on
concept[41].However,theTopo-IIislocatedonchromosome c
predictable. /a
Whilethereisaneedforbetterprognosticfactors,no 17q12incloseproximitytoHER-2,andthetwogenesare rtic
frequently,butnotunivocally,coamplified[32].Thus,thereis le
prognosticfactorshouldbeimplementedforclinicaluse -a
nowmergingevidencefromstudiesintheneo-adjuvantaswell b
withoutaproperevaluationofitspredictivepowerwith s
respecttothekindoftherapyunderconsideration.Therefore, astheadjuvantsettingindicatingthatTopo-IIamplificationmay trac
toimprovenotonlypredictionbutalsoprognostication,we sensitizetumourstotreatmentwithanthracyclinesinearly t/1
needtounderstandpotentialassociationsbetweenavailable bitreseaesmtcsanthcaetrT[3o1p–o3-I4I,d4e2l–e4ti5o]n.sFcoarurseeastohnessa(msoefaser)nsuintiezxaptiloaninteod, 8/8/1
parametersanddrugresistance.Thisrelatestomultiple-gene 29
anthracyclinesthatisobservedforTopo-IIamplifications[33]. 3
signaturesaswellastoindividualbiologicalfactors. /1
7
8
1
TP53 mutations 3
predicting sensitivity to therapy in 5
b
breast cancer Inadditiontoitsroleinbreastcancer,TP53mutationsor, y g
rather,p53proteinimmunostaininghasbeenshowntobe u
e
Contrastingthepanelofprognosticfactorsavailable,only aprognosticfactorindifferentmalignancies,includingsoft st o
alimitednumberofindividualfactorsassociatedwith tissuesarcomas,colorectalcancers,non-small-celllungcancer, n
1
chemotherapysensitivityorresistancehavebeenidentified. ovariancancerandhaematologicalmalignanciesamongothers 0
A
Forreasonsdiscussedindetailbelow,wefinditusefulto [46–54].Normally,p53,theproteincodedbytheTP53gene, p
discriminatebetweenfactorspredicting‘sensitivity’and existsatverylowconcentrationsinthecellandisnotdetected ril 2
0
‘resistance’totherapy.Thefindingthat,forexample, byimmunostaining.p53degradationoccursthroughMDM2 19
expressionofafactorpredictstherapyresistancemaynot binding,followedbyubiquitination[55,56].Asmanymutated
implicatethatalltumoursdevoidofthesameparameterare p53variantsmaynotbindtoMDM2,currentopinionis
sensitive.Further,consideringsensitivity,thereisevidence thatanextendedhalf-lifewithintracellularaccumulationof
thatsomefactors,suchasamplificationoftopoisomeraseII, mutatedp53explainsincreasedlevelsoftheproteinallowing
donotpredictsensitivityinabsolutetermsbut,rather, detectionbyimmunostaining[57].Thus,p53immunostaining
agradedsensitivityorresponsivenesstoanthracycline- hasbeenusedasasurrogatemarkerfortheexistenceofTP53
containingchemotherapy(seebelow). mutations.
Consideringfactorsthathavebeenfoundcorrelatedto Thereare,however,severalpitfallsusingthisassumption.
resistanceordrugsensitivitysofar,theyallhaveapotential First,thepossibilityexistthatincellsexposedtophysiological
biologicalexplanation:(i)asspecifictargetmoleculesforthe stresstheremaybeanaccumulationofnormalp53protein.
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Secondly,itiswellrecognisedthatinbreastcancermutatedp53 asimilarassociationhasbeenrecordedindifferent
maynotalwaysbedetectedbyimmunostaining[58–60];about haematologicalmalignancies[69–72].
30%oftumoursharbouringTP53mutationsdonotexpress Whileseveralstudieshavereportedanassociationbetween
immunostainingwhenexposedtothemostcommonlyused TP53mutationsandresistancetoanthracyclinesandsimilar
antibodies;similarfindingshavebeenmadeinothercancers, compounds,notably,evidencepresentedsofardoesnot
suchascancerofthelargebowel,endometriumandstomach providesupportforTP53mutationstobeusedasaselection
[60].Inparticular,weobservedthatmanyofthemutations criterionwithrespecttotherapeuticregimens.Theunderlying
associatedwiththerapyresistancedidnotstain[59].Some reasonisthelowsensitivityandspecificityofsuchtesting.
mutations,suchasbaseinsertionsordeletions,maycreate Apotentialexplanationtothisfindingispresentedlaterinthis
anonsensereadingframeoranearlystopcodon,resultingin paper,illustratingtheneedtoevaluatefunctionalpathways
lossofthefull-lengthproteinproduct(Figure2).Other andnotonlyindividualgenesinthissetting.
mutationsmayleadtoanunstableproteinproductasrevealed
forsomeproteinproductsgeneratedfrommutationsinthep53
BRCA1, BRCA2 and the Fanconi proteins
upstreamactivatorsATMandCHEK2[61,62].Finally,mutated
andwild-typep53proteinmayexistindifferentthree- WhilegeneproductsinvolvedinDNArepairaredescribedin D
o
w
dimensionalfolding,leavingtheepitopeexposedorunexposed thesectionbelow,duetotheirimportanceinbreastcancer n
[63–66]. theBRCA1and-2aredescribedseparately,includingthe loa
d
ReviewingtheliteraturewithrespecttoTP53as relatedFanconicomplex.Relevanttoourdiscussionisthe e
d
apredictivefactor,moststudiesusingimmunostaininghave observationthatsomaticmutationsaffectingBRCA1or-2 fro
m
notdetectedanycorrelationbetweenp53stainingand arerareinbreastcancer.
h
responsetochemotherapyinbreastcancer[67].Incontrast, Whileseveralstudieshavereportedapoorprognosisin ttp
s
studiesexaminingTP53mutationstatusatthegenomiclevel tumoursarisinginBRCA1mutationcarriers[73–75],the ://a
havereportedanassociationbetweenfailuretoanthracycline- prognosisforpatientsharbouringBRCA2mutationsseemsto c
a
d
ormitomycinC-containingchemotherapyandTP53 benotmuchdifferentfromspontaneousbreastcancers[76]. e
m
mutationsinbreastcancer[27,59,68].Interestingly, Interestingly,breastcancersappearinginBRCA1carriershave ic
.o
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n
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2
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7
8
1
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5
b
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u
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s
t o
n
1
0
A
p
ril 2
0
1
9
Figure2. Differenttypesofgenemutationsandmechanismsofepigeneticsilencing.
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beenreportedtocarrydistinctgeneprofiles[77]andfrequently summary
tobeassociatedwithbasaltumour-likecharacteristics[78], Thefindingsdiscussedhereareexcitingfromascientificpoint
ageneprofileknowntobeassociatedwithageneralpoor ofview,inasmuchastheypointtodistinctbiological
prognosis[79,80].Withrespecttochemoresistance,anecdotic mechanismsbeingthecauseofdrugresistance.However,
evidencehasindicatedimprovedsensitivitytoanthracyclines withtheexceptionofTopo-IIamplification,noneofthese
intumoursexpressinghighmRNAlevelsofBRCA1[81];in individualgenealterationsidentifiedsofarhasrevealed
contrast,lowlevelsofBRCA2messengerRNA(mRNA)hasbeen apredictivepowerwithrespecttosensitivityaswellas
foundassociatedwithenhancedsensitivitytotaxanes[82]. specificitywarrantingtestinginalarge-scaleclinicalsetting.
Thenumberofpatientsinthesestudiesislowandthese Nevertheless,theyindicatebiologicalexplanationstodrug
findingsneedfurtherconfirmation. resistance.Thus,thesegeneticchangesmaybeconsidered
TheFanconisyndromeisarecessivesyndromecharacterised lighthouses,orbeacons,identifyingfunctionalpathwaysof
bycongenitalanomaliesandanaemiasthat,inaddition, majorimportance[35].
includesanenhancedriskofdevelopinghaematologicalas
wellasnon-haematologicalmalignancies[83].Whilethe
Fanconiproteincomplexexertsseveralfunctions,amajoreffect pathologic changes leading to D
o
seemstobeinteractionswithBRCA1and-2withrespect disturbed gene functions wn
toDNArepair.Recently,hypermethylationofFANCF,an loa
Thefindingthatchangesinindividualgenesareassociated d
upstreamgeneintheFanconigenecomplexwasfound e
associatedwithsensitivitytoplatinumdrugsinovarian whaitshlebaudttnooitnfcurlelaysepdrefdoiccutisveonfotrhderpurgorbeasbisiltiatnycoefmteescthinagninsomts d from
cancercelllines[84].
individualgenesbut,rather,multiplegenesinconcertusing h
ttp
other enzymes involved in DNA repair methodssuchascDNAoroligonucleotidearrays.Before s
discussingsuchoptions,wewillbrieflyconsiderthe ://a
DNArepairinvolvesseveralmechanismssuchasbase c
mechanismsleadingtodisturbedgenefunction. ad
excisionrepair,nucleosideexcisionrepair,mismatchrepairas e
m
wellasO6-methylguanineDNAmethyltransferase;thereaders ic
arereferredtoreviewsonthesubjectsfordetails[85–87]. somatic mutations .ou
p
Sofar,therearelittledataonalterationsintheseenzyme Somaticmutationsmaybeconsideredthe‘classicalway’of .c
o
systemswithrespecttobreastcancer.However,there geneinactivation.Thismayoccurassinglebasedefects m
/a
areseveralinterestingfindingsinothercancerforms. (substitutions,leadingtoaminoacidchangeinthecoded n
n
Themismatchrepairsystemisresponsibleforremoving protein),singlebaseinsertionsordeletionsordeletionsor on
c
incorrectlypairednucleotidesinthegenome[86].Thesystem insertionsofsmallerorlargerfragments(Figure2).Obviously, /a
containsseveralenzymesmakingheterodimers[86],and anyinsertionordeletionofanumberofbasepairsnotdividable rtic
le
mutationsaffectingthissystemareresponsibleforinherited bythenumberofthreewillleadtoashiftofreadingframe -a
b
nonpolyposiscancerofthelargebowel,theLynchsyndrome and,thus,acompletechangeoftheaminoacidsequenceof s
[88,89].Whilereportedtobeassociatedwithagood theproteinC-terminaloftheaffectedcodon.Suchnonsense trac
prognosisincancerpatientsnotexposedtoadjuvant mutationsoftenleadtotruncatedformsoftheproteinin t/1
8
chemotherapy[90],mutationsinmismatchrepairgeneshave questionduetonovelstopcodons.Lossofwildtypeactivityis /8/1
beenassociatedwithpoorsensitivityto5-fluorouracil[91], themostcommonconsequence. 29
3
underliningthefactthatprognosticinformationcannotbe Manysinglebasesubstitutionsmaynotleadtoanaminoacid /1
7
usedtomakeassumptionsabouttherapysensitivity. substitutionand,thus,remainnonfunctionalpolymorphisms, 8
1
Nucleotideexcisionisanimportantmechanismforthe exceptfortherarecaseswheresinglebasesubstitutions 35
b
protectionofcellsfromexogenouscarcinogens.Thus, modulatemRNAstabilityorsubcellularmRNAtargeting.For y
g
mutationsaffectingthenucleotideexcisionenzymecomplex manysinglebasesubstitutionsleadingtoasingleaminoacid u
e
arethecauseofXerodermapigmentosum[86],asyndrome substitution,thisleadstoaproteinwithabiologicalfunction st o
associatedwithaveryhighriskofUV-inducedskincancers deviatingonlymoderatelyfromtheoriginalprotein.Lookingat n
1
andothermalignancies.Thefactthatthiscomplexremoves germlinepolymorphisms(definedbyconventionasasinglebase 0
A
DNA-platinumadducts[92]offersanexplanationwhyhigh substitutionoccurringin>1%ofthegeneralpopulation), p
mRNAlevelsofERCC1,oneoftheenzymesinthiscomplex, forexamplethep53arg72proleadstoamarginalchangein ril 2
0
hasbeenfoundassociatedwithresistancetoplatinum biologicaleffects[100].Incontrast,othersinglebase 19
compoundsinbothovarianandgastriccancer[93,94]. substitutionsinTP53affectingcriticalproteindomainsmay
TheO6-methylguanineDNAmethyltransferaseisresponsible leadtononfunctionalproteins.Wefoundsinglebase
forremovinglargeDNAadducts[95].Interestingly,silencing substitutionssuchasthecodon249AGG-GGG(Arg-Gly)to
ofthisgenethroughpromoterhypermethylationhasbeen causelackofp53proteinstainingaswellastobeassociatedwith
reportedtoenhancesensitivitytoalkylatingagentssuchas resistancetodoxorubicininprimarybreastcancer[59].Most
BCNUandtemozolomideinhumanglioblastomas[96–98]. ofthegermlinemutationsdetectedinLi–Fraumenifamilies
Whilepolymorphismsinthebaseexcisionpathwaygenes aresinglebasesubstitutions[101],soisthecasefor(cid:1)50%of
havebeenassociatedwithbreastcancerrisk[99],wearenot thegermlinemutationsoftheRBgene,identifiedin
awareofanystudyreportingmutationsorpolymorphismsin retinoblastomas[102].Basesubstitutionsalsorepresentthe
thesegenestobeassociatedwithdrugsensitivity. mostfrequentformofdefectidentifiedintheCDKN2Agenein
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familiesatriskofmalignantmelanomas[103].Currentwork truncatedRNAtranscripts[120].FortheTP53analoguesTP63
isconcentratingonmethodstodeterminethepathogenic andTP73,afamilyoftruncatedtranscriptsaregenerated[121],
effectofdifferentsingleaminoacidsubstitutions[104]. andfunctionalactivealternativetranscriptsforTP53havebeen
Forothergenes,suchasBRCA1and-2,themostcommon identifiedaswell[121,122].Formanyalternativetranscripts
germlinemutationsappeartobenonsensemutationsresulting itisnotknownwhethertheyaretranslatedintoaprotein
inatruncatedprotein[105]. productor,ifso,whethertheproteinmaybefunctional,while
Notably,largerdeletionsmaybedifficulttodetectby forsomegenes,suchastheTP53analogueTP73,alternative
standardDNAsequencing,andtraditionalmethods,suchas transcriptscodingforproteinswithaneffectantagonisticto
Southernblotting,arelessspecific,andrequiressubstantial thewild-typeproteinhasbeenidentified[123].
amountsofmaterial,makingitunfitfortestingontumour
materialwithalimitedtissueamountsupply.Recently, gene amplification
improvedmethods,suchasmultiplexligationamplification
Chromosomaldisturbanceisahallmarkofcancer[124,125].
(MLPA)havebecomeavailable[106–109].Yet,deletionsmay
Thus,formanygenessuchasTopo-II(seeabove),MDM2[126]
escapedetectioniftheyarenotproperlycoveredbythe
aswellascyclinsDandE[127,128]amplifications,andnot
probes[110].Insuchcases,combinedanalysisonDNAand D
mutations,arethepathologicaleventrecordedinneoplasias. o
w
cseDqNueAncisehailgsohlmybayenbeeficdiuaelatsolionstsroonfipcamrtsutoaftitohnescnoodtindgetected Notably,amplificationseemstoimplicatemRNAup-regulation nloa
inabouttwoofthreeofcasesinbreastcancer[129],underlining d
withanyofthesemethods. e
thatinoneofthreecasesamplificationofalargegenesegment d
maynotbeassociatedwithmRNAup-regulationofeachgene fro
m
mutations involving noncoding regions inthatdomain.Theimportanceofgeneamplificationmaybe h
Recently,therehasbeenfocusonmutationsaffecting illustratedusingTopo-IIasanexample(seeabove)aswell ttps
noncodingregions.Consideringintronicmutations,both asMDM2anditssistergeneMDMX.Themainfunctionof ://a
c
mutationsdirectlyaffectingthesplicingsitesanddeep theMDM2andMDMXgeneproductsistomediatep53 ad
e
intronicmutationsmayleadtoaberrantsplicing(seebelow), degradation,andamplificationofMDM2,andmorerecently m
severelyaffectingthefunctionofthetranslatedprotein MDMX[130],hasbeenrevealedasanalternativewayofp53 ic.o
u
[111,112].Inaddition,mutationsaffectingpromoterareas inactivation. p
.c
mayleadtoeitheranonfunctionalor,insomecases, o
m
‘hyperfunctional’promotersequences,asrecentlyillustrated epigenetic mechanisms /a
n
withrespecttotheMDM2gene[113]. n
Geneimprintingisacriticalphysiologicalprocess,andthe o
n
Inrarecases,mutationsmayleadtocreationofanew c
readersarereferredtocontemporaryreviewsonthesubject /a
functionalpromoter.Thelatterisillustratedbytheobservation [131,132].Geneticimprintingplaysacrucialrolein rtic
thatanintronicsinglebasesubstitutiongeneratedanovel le
embryoniclifebutduringthewholelifetimeoftheindividual -a
promoterelementallowinggeneactivationleadingto aswell[133].Basically,twomainmechanisms;promoter bs
avariantofthalassaemia[114]. hypermethylations[134]andhistonedeacetylations[135], tra
c
havebeendiscovered(Figure2). t/1
8
alternative splicing Importantly,deregulatedpromoterhypermethylations /8
/1
Thelastdecadehasfocusedinparticularontheissueof isafrequentformofgeneinactivationincancer,and 29
alternativesplicing,i.e.thegenerationofseveralprocessed hypermethylationhasbeendetectedincriticalgenessuch 3/1
7
RNAtranscriptsfromasingleprimaryRNAtranscript(Figure3 asCDKN2A,thegenecodingforthecdkinhibitorp16, 8
1
and4).Formostgenes,thisismanifestedbysynthesisofamain inmelanomas[136],andtheDNArepairenzymeO6- 35
full-lengthtranscriptwiththeadditionofseveraltruncated methylguanineDNAmethyltransferaseinglioblastomas. by
variants.Alternativesplicingisaphysiologicalphenomenon;the Interestingly,O6-methylguanineDNAmethyltransferase gu
e
readersarereferredtorecentcomprehensivereviewsonthe promoterhypermethylationwasfoundtocorrelatetoresponse st o
subject[115].Whilethereisevidencethatperhapsasmuchas totemozolomideinthissetting[97,98]. n
1
75%oftranscribedgenesinthehumangenomemaybesubject Whilepromoterhypermethylationisrelativelyeasytodetect, 0
A
toalternativesplicing[1],thephenomenonseemsmore wecurrentlylackconvenientmethodsfordetectingprotein p
extensiveincancercomparedwithnormaltissue[116]. deacetylations.However,substantialexperimentalevidence ril 2
0
ConsideringMDM2andChk2,knowntobeofcritical linkshistonedeacetylationtogenetranscriptionalcontrol[135, 19
importancetop53degradationandactivationinresponseto 137],anddeacetylaseinhibitorsarecurrentlyindevelopment
DNAdamage,respectively[56,117],>40and60alternative forcancertherapy[138].Thus,appropriatediagnosisnotonly
transcriptshavebeenidentified[118,119];forChk2,the ofgenesilencingbutalsothespecificmechanismresponsible
numberoftranscriptsidentifiedwerehigherinbreastcancer maybeakeyissueforfuturetargetedtherapies.
comparedwithbenigntissue.Thefindingofhypersplicingin
cancerislikelyduetomechanismsotherthanmutationsinthe exploring gene dysfunction using
splicingsites,asthesplicevariantsoftenaredetectedtogether
microarrays
withthemaintranscriptandnomutationmaybeidentified,
Interestingly,forMDM2,itseemsthatactivationofthedifferent Theinventionofmicroarrays,allowinginvestigationsof
promotersmayleadtoadifferentratiobetweenmainand multiplegenesinconcert,representsanimportantstep
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Figure3. Alternativesplicing.(I)Ageneistranscribedintoapre-messengerRNA(mRNA)copycontainingthecompleteDNAsequencefromthepromoter 1
3
5
regiontothepolyadenyltail.(II)Eitherduringpre-mRNAtranscriptionorimmediatelyafter,intronsequencesareexcisedleavingonlytheexons(red), b
y
formingthemRNAgeneproduct.(III)Commonalternativesplicingeventsareskippingofoneorseveralexonsorinsertionofoneorseveralalternateexons; g
u
sometimestermed‘‘crypticexons’’or‘‘pseudo-exons’’(x,yandzonfigure).Thelatterhavethesequenceofanormalexon,butarecalled‘alternate’asthey e
s
arenotpartofthepredominanttranscript.Partsofexonsmaybelostduetosplicingatuncommonsplicesites.Alternativesplicingalsoincludesexon t o
n
substitution,oftenduetotwoexonsbeingsituatedsocloseonthegenethattheysharesplicingsignalsequences,andmaythereforenotbothbeincludedin 1
0
themRNAproduct. A
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forwardinourattemptstoexplorebiologicalmechanismsof onlytobedevoidofthesefactorsbut,actually,torepresent 1
9
chemoresistanceincancer.Thus,microarraystudieshavebeen adistinctclassoftumours,identifiedbyaspecificgeneprofile.
usedtoexplorethebiologyofmosttumourtypes.Sofar,two Secondwasthefindingthathierarchical[79]aswellas
majorgoalswithrespecttobreastcancerhavebeenachieved: supervised[142–144]clusteringwasabletorevealstrong
First,usinghierarchicalclustering,Perouetal.[139]and prognosticsignatures.WhilethespecificityoftheAmsterdam
Sørlieetal.[79]wereabletoshowthatbreastcancersmay 70geneprofilehasbeenchallenged[145],thisgeneprofileas
beclassifiedonthebasisofglobalgene‘signatures’.This wellasthe‘Rotterdam’profile[146]providesprognostic
classificationhasbeenfoundrobust,confirmedinseveral informationexceedingwhatisgeneratedbylymphnode
studies[80,140,141].Themainfindingprobablywasthe statusonly.
identificationofthe‘basal’,or‘triple-negative’class,revealing Themajordangerofusingsuchprofilesselectingpatients
tumourslackingexpressionoftheERaswellasHER-2not fortherapyliesinthefactthatwehere,asforindividualgenes,
Volume18|No.8|August2007 doi:10.1093/annonc/mdm013|1299
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AnnalsofOncology
lackinformationwhetheritalsopredictssensitivitytotherapy. Whilethe‘21-gene’signaturedevelopedbytheNSABPwas
Thus,whiletheAmsterdam70genesignature[143]revealed foundassociatedwithbenefitfromCMFintheadjuvantsetting
prognosticinformationinbothnode-negativepatients [155],itspredictivevaluetotaxaneandanthracycline-
(ingeneralnotexposedtoadjuvanttreatment)aswellasin containingchemotherapyintheneo-adjuvantsettingdidnot
node-positivepatients(ingeneralexposedtoadjuvant exceedthepowerofothergeneprofiles[152].
treatment),itisnotpossibletoinferfromthedatathatthe Microarraydataprovideenormousamountsofinformation
signatureisnotassociatedwithsensitivitytotherapy. revealinggeneexpressionofpotentiallyallhumangenesin
Incontrasts,studiesaimingatdevelopingsignatures asingleanalysis.However,itisclearthatsucharraysmaynot
predictingdrugresistancehavenotbeensuccessful.While identifyallmechanismsofgeneinactivation.Thismaybe
severalstudieshavereportedgenesignaturesassociatedwith illustratedbyafewexamples(Figure5).Assumingtheprobe
sensitivitytotreatmentwithbothtaxanes[147,148]and onthearrayhybridisestothe59endofanmRNAtranscript,
anthracycline-containingregimens[149,150]aswellas pathologicalchangessuchasasinglebasemutationoreven
combinationscontainingboth[149,151–154],noneofthese adeletionlocateddownstreamoftheprobingareawouldbe
signatureshavecomeintogeneralclinicaluseforacoupleof overlooked.Forsinglebasesubstitutionswithinthearea
reasons.First,thesesignatureshavebeendevelopedinstudies coveredbytheprobe,theresultislesspredictable;the D
o
w
enrollingalimitednumberofpatientsonly.Secondly, transcriptmaynotbindtheprobeatall,butinothercasesit n
theyconsistentlyreportedsensitivityaswellasspecificity maystillbindpartlyorcompletelyand,thus,thesubstitution loa
d
predictingtherapyoutcomeof(cid:1)70%–80%,whichisbelow maybeoverlooked.Whilearraystoexploresplicevariants e
d
whatshouldberequiredifaparameteristobeusedtoselect havebeenconstructed[1],asalreadymentioned,RNAsplicing fro
m
patientsfortherapy. seemstobeparticularlyextensiveincancertissue[116,119].
h
Thirdly,whiletheyreportlikelihoodforapatienttorespond Thus,whileintheoryexpressionofeachgeneinthehuman ttp
s
toacertaintreatmentregimen,noneofthestudiesatthesame genomecouldbequantifiedwithanarraycontaining30000 ://a
timeexploredthepowerofthesignaturepredictingsensitivity probes,toassessthefullRNAsplicingprofileforeachgene, c
a
d
toalternativetreatmentoptions.Bydefiningapatientas weprobablymayneedmillionsofprobesonthearrays. e
m
chemoresistantwemayavoidthetoxicside-effectsofauseless ic
therapy;however,wemaynotallocatethepatienttoany .ou
p
alternativetherapywithoutknowledgeaboutitsefficacy. chemoresistance—a conceptual .c
o
approach m
/a
n
n
Cytostaticsingeneralworkbyinterferingwithcritical o
n
processesrelatedtocellcycleandmitosis.Formost c/a
compoundstheydisturbDNAsynthesisor,fortaxanes,the rtic
mitoticspindle.Thecellularresponseanticipatedshouldbe le-a
growtharrest,apoptosisoralternativewaysofgrowtharrest, bs
probablyincludingsenescence[156].Thecellularmechanisms tra
c
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Figure5. Microarraysignalsaregeneratedbyhybridisingaprobeto
Figure4. Splicingmechanism:splicingofexonsissignalledbythe thegeneproduct[complementaryDNA(cDNA)].Alternativesplicing,as
consensussequences:MAG|GTRAGTinthe39endofoneexonand wellasdifferenttypesofmutationsmayinfluencetheresultbutmaygo
CAG|Ginthe59endofthenextexon.Thesesequencesarebrought undetectedaswellpendingontheexacttypeoflesionaswellasitslocation
togetherbytheSpliceosome,loopingtheintronbetweenout.Irregular withrespecttotheareahybridisingwiththeprobe.Themarks‘‘X’’and‘‘-’’
splicingduetomutationsintheseconsensussequencesarenormally indicatepotentialerrors:‘‘X’’indicateswronglydetectedtranscriptlevels,
labelledmutations,andnotalternativesplicing.Alternativesplicingis while‘‘-’’signifiescorrectlevelsreported,butwithotherundetectable
variationinwhichsplicesitescombinewhenallsplicesitesareintact. lesionspresentinthegene.
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ofrescueandsurvivalisDNArepair,alternatively,lackof lessthana25%increaseinthesumoftheproductsoftwo
growtharrest/apoptosiscausedbydefectsinthegenes perperdiculardiametersofallmeasuredlesionsandthe
regulatingthesecellularfunctions. appearanceofnonewlesions).Forboththerapeuticcompounds
Thelasttwodecadeshaveprovidedenormousamountsof studied,wedetectedanassociationwithTP53mutations
informationregardingthemechanismsregulatinggrowth affectingtheDNA-bindingL2andL3domainsandresistanceto
controlaswellastheprocessesofapoptosis/senescence.Our therapy(Table1).Toexplaintheseresultsweneedtomake
challengeishowtoimplementthisinformationtoour certainassumptions.Consideringthegroupoftumours
understandingoftherapyfailure.Theideaofexploring harbouringTP53mutationsintheL2orL3DNA-binding
disturbancesingenesinvolvedinapoptosis,aswellasinDNA domainsthataresensitivetotherapy,wehypothesisethat
repair,suchastheTP53gene,asacauseofdrugresistanceinvivo, apoptosiscouldbeachievedbyoneoftwopathways,oneof
wasonthebasisofelegantpreclinicalexperiments[157,158]. whichcontainsTP53.Thus,tumoursbeingsensitivetotherapy
Whileitismandatorytotakeintoaccounttheresultsgenerated despitetheirTP53mutationmayhavethesecondpathway
fromexperimentalsystems,theneedforproperverificationin intact,whiletumoursharbouringTP53mutationsandbeing
vivocannotbeemphasisedstronglyenough.Thestoryofthe resistantmayhavethissecondpathwayinactivated.Secondly,
MDR1gene,encodingtheP-glycoprotein,aswellasgenes tumoursthatareresistanttotherapydespiteexpressingwild- D
o
w
codingforothermembranepumps,maydepictthis;whilethe typeTP53mayhaveagenelocatedup-ordownstreamof n
theoreticalconceptsarewellfoundedanddatafrom TP53inactivatedinadditiontoinactivationofthealternative loa
d
experimentalsystemsconvincing[159,160],invivostudies pathway.ThedifferentscenariosaredepictedinFigure6. e
d
correlatingP-glycoproteinexpressiontodrugsensitivity[161] Akeyproblemapplyingsuchamodelisthatwedonot fro
m
havenotsupportedtheconcept,andtheresultsfromclinical knowwhichgenesareinvolveddownstreamofp53inthe
h
trialsexploringpumpinhibitorsingeneralhavebeen apoptoticpathway.Consideringinvitroexperiments,p53has ttp
s
disappointing[162].Thus,whilethefindingofgenesignatures beenshowntoactivateseveralhundredgenes,dependingon ://a
predictingresistancetodifferentchemotherapycompounds theexperimentalsituation[6,7].However,thisrepresentsahuge c
a
d
acrossapanelofcelllinesisinteresting[163],thevalidityofthese reductioninthenumberofcandidategenestoexplorecompared e
m
profilestodrugresistanceinvivoremainstobeconfirmed. withuseofageneral‘hierarchical’clusteringor‘supervised’ ic
Assessingtumourgeneprofilesusingarraysdetectingmostof analysis,inwhichcasesmostgenesofthegenomeareinvolved. .ou
p
thegenesinthehumangenome,biostatisticalevaluationhas Exactlysimilarassumptionsmaybeappliedtoother .c
o
indicatedthousandsofindividualbiologicalsamplesmaybe systemsexploringcausesofresistancetoothercompounds. m
/a
neededtogeneraterobustgenesignatures[164].Supposethe MostDNArepairmechanismsinvolveproteincomplexes; n
n
averagenumberofRNAsplicevariantsforeachgenetobefive. thus,itislikelythatmutationsindifferentgenesinvolvedin on
c
Duetothesignificantsequenceoverlapbetweenindividual thesamecomplexmayachievesimilarbiologicaloutcomes. /a
splicevariantsfromasingletumour(Figure5),thismeansthat Inaddition,eachgeneissubjecttotranscriptional rtic
le
toevaluateexpressionofeachoutoffivesplicevariantswith stimulationbyupstreamfactors. -a
b
confidence,wemayprobablyneed10probes,perhapseven Thetechnicalproblemsassociatedwithidentifyinggene s
more,foreachgeneonaverage.Thus,thenumberofspotson defectswerediscussedabove.Ontheotherhand,itmaybe trac
thearraymayincreaseabove300000.Accordingly,thenumber arguedthatthetypeofgenedefectmaynotbeimportant t/1
8
ofbiologicalsamplesneededtogeneraterobustgeneexpression whenapplyingmicroarrayanalysis.Providedthegenedefect /8/1
signatureswouldinpracticaltermsbecomeunrealistic. leadstoanonfunctionalgene,theeffectshouldbedetectedin 29
3
Forthisreason,webelievethereisaneedtolimitthenumber /1
7
ofgenestobeexaminedonthebasisofbiologicalhypotheses 8
1
generatedupfrontthroughexperimentalwork.Asmentioned, 35
Table1. CorrelationsbetweenTP53mutationsaffectingtheDNA-binding b
eachindividualfactorfoundassociatedwithchemoresistance y
L2/L3domainandlackofresponse(progressivedisease;PD)in g
sofarhasbeeninvolvedinspecificbiologicalprocesses;either u
patientswithlocallyadvancedbreastcancertreatedwithdoxorubicin e
theyrepresenttargetsforthedrug,alternativelytheyare weeklymonotherapy[59]ormitomycinCand5-fluorouracil[68] st o
associatedwithprocessessuchasgrowtharrest/apoptosis,or n
(thedataprovidethebackgroundforhypothesisingthemodel 1
DNArepair.Mostoftheseexecutetheirfunctionaspartof 0
depictedinFigure5) A
aproteincomplexwithotherfactors.Likeothergenes,theyare p
activatedbycertainupstreamgenes(atthetranscriptionalor ril 2
Therapy TP53status Response Reference 0
posttranslationallevel)andactivatedownstreamgenesin 19
PR/SD PD
functionalcascades.Thus,itislikelytopostulatethat
inactivationofotherfactorsinthesamecomplex,orgenes Doxorubicina TP53wildtypeor 67 4 [59]
mutated‘non’L2/L3
involvedup-ordownstreaminthefunctionalcascade,may
TP53mutatedL2/L3 14 5
exertsimilareffects.
5-FUand TP53wildtypeor 22 3 [68]
TheissueofTP53mutationsinanthracyclineand
MitomycinCb mutated‘non’L2/L3
mitomycinCresistancemaybetakenasanexample.For
TP53mutatedL2/L3 3 6
reasonsdiscussedelsewhere[8],tostudyresistancefactorswe
decidedtocomparetumoursexpressingprimaryresistanceto PR,partialresponse;SD,stabledisease;PD,progressivedisease.
thecombinedgroupoftumoursobtaininganobjective aP=0.008.
responseorastabledisease(lessthana50%reductionand bP=0.006.
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Figure6. PotentialexplanationtothefindingspresentedinTable1relatingTP53mutationstatustodrugresistancetodoxorubicinormitomycinCin t on
breastcancer.Figureobtainedfrom[35]withpermission.Notethatallthefourtumoursexpressedadifferentgeneprofilebymicroarray;thesimilaritywas 10
greaterbetweentumourBandC(sensitiveandresistanttotherapy,respectively)thanin-betweenAandB(bothsensitive)orCandD(bothresistant). A
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asmuchasgeneslocateddownstreaminthepathwaywill sequencing,MLPAandexaminationofepigeneticevents,
remainunderexpressed.Thus,webelievegenearraysincluding suchashypermethylationstatus,toexploretheexactnature
genesinvolvedincertainbiologicalcascadesmaybeaway ofanypossiblegenedefect.
forward.However,lookingattheexamplepresentedinFigure6,
eachtumourexpresseddifferent‘geneprofiles’withrespectto
depicting the future
thegenesinvolvedintheapoptoticpathway.Curiously,the
twotumours(BandC)expressingthelargestsimilarityin Understandingchemoresistanceshouldrepresentagreatleap
geneprofilehadadifferentclinicaloutcome.Thus,webelieve forwardinourattemptstocurenotonlybreastcancerbutalso
thatarraysmustbeusedincombinationnotonlywith cancersingeneral.Sofar,theresultsofmicroarraystudieshave
RT-PCRtoverifygeneexpressionlevelsbutalsogene beendisappointingandtheresultsfromstudiesonindividual
1302|Lønningetal. Volume18|No.8|August2007