! " # $ " %' ! ( $ ) * "!"#$%&'(#)%"Te molecular basis of lung cancer is complex and heterogenous. Improvementsinourunderstandingofmolecularalterationsat multiple levels (genetic, epigenetic, protein expression) and their functionalsignifcancehavethepotentialtoimpactlungcancer diagnosis,prognosticationandtreatment.Lungcancersdevelop throughamultistepprocessinvolvingdevelopmentofmultiple geneticandepigeneticalterations,particularlyactivation ofgrowthpromotingpathwaysandinhibitionoftumour suppressorpathways.Greaterunderstandingofthemultiple biochemical pathways involved in the molecular pathogenesis of lung cancer is crucial to the development of treatment strategies thatcantargetmolecularaberrationsandtheirdownstream activatedpathways(1).Specificmolecularalterationsthat drivetumourgrowthandprovidetargetsfortherapyhave beenbestdefinedinadenocarcinomas(ADC)butthereis increasinginterestinthemolecularlandscapeofsquamous cellcarcinoma(SCC)highlightingnewpotentialtherapeutic targets.Inlungcancerasinothermalignancies,tumourigenesis relatestoactivationofgrowthpromotingproteins[e!g.,v-Ki-ras2Kirstenratsarcomaviraloncogenehomolog(KRAS), epidermalgrowthfactorreceptor(EGFR),BRAF,MEK-1, HER2,MET,ALKandrearrangedduringtransfection(RET)] aswellasinactivationoftumoursuppressorgenes[e!g!"P53, *%+,('+-$ /)%+%01 %2 +'"0 (-"(,$Wendy A. Cooper1,2, Davld C. L. Lam3, Sandra A. O'Too|e1,4,5, [obn D. Mlnna61TlssuePatbo|ogyandDlagnostlcOnco|ogy,Poya|PrlnceA|lredHosplta|,Camperdown,Sydney,NSW,Austra|la, 2Scboo|olMedlclne, Unlverslty ol Western Sydney, NSW, Austra|la, 3Department ol Medlclne, Unlverslty ol Hong Kong, Hong Kong SAP, Cblna, 4Klngborn Cancer Centre and Garvan |nstltute ol Medlca| Pesearcb, Dar|lngburst, Sydney, NSW, Austra|la, 5Sydney Medlca| Scboo|, Unlverslty ol Sydney, NSW, Austra|la, 6Hamon Center lor Tberapeutlc Onco|ogy Pesearcb, Unlverslty ol Texas Soutbwestern Medlca| Center at Da||as, Texas, USA34567386Lungcancersarecharacterisedbyabundantgeneticdiversitywithrelativelyfewrecurrentmutationsoccurringathigh frequency.However,thegeneticalterationsoftenaffectacommongroupofoncogenicsignallingpathways.Therehave beenvastimprovementsinourunderstandingofthemolecularbiologythatunderpinslungcancerinrecentyearsand thishasledtoarevolutioninthediagnosisandtreatmentoflungadenocarcinomas(ADC)basedonthegenotypeof anindividualstumour.Newtechnologiesareidentifyingkeyandpotentiallytargetablegeneticaberrationsnotonlyin adenocarcinomabutalsoinsquamouscellcarcinoma(SCC)ofthelung.Lungcancermutationshavebeenidentifedin v-Ki-ras2Kirstenratsarcomaviraloncogenehomolog(KRS),epidermalgrowthfactorreceptor(EGFR),BRFandthe parallelphosphatidylinositol3-kinase(PI3K)pathwayoncogenesandmorerecentlyinMEKandHER2whilestructural rearrangementsinALK,ROS1andpossiblyrearrangedduringtransfection(RET)providenewtherapeutictargets. AmplifcationisanothermechanismofactivationofoncogenessuchasMETinadenocarcinoma,fbroblastgrowthfactor receptor1(FGFR1)anddiscoidindomainreceptor2(DDR2)inSCC.Intriguingly,manyofthesegeneticalternations areassociatedwithsmokingstatusandwithparticularracialandgenderdiferences,whichmayprovideinsightintothe mechanismsofcarcinogenesisandroleofhostfactorsinlungcancerdevelopmentandprogression.Teroleoftumour suppressorgenesisincreasinglyrecognisedwithaberrationsreportedinTP53,PTEN,RB1,LKB11andp16/CDKN2A. Identificationofbiologicallysignificantgeneticalterationsinlungcancerthatleadtoactivationofoncogenesand inactivation of tumour suppressor genes has the potential to provide further therapeutic opportunities. It is hoped that these discoveries may make a major contribution to improving outcome for patients with this poor prognosis disease. 9:; 5Lung cancer; mutation; molecular pathology; oncogene; tumour suppressor gene! #$%&'( )*+ ,-./012314536789368-: ;%*5 .-:/87:*++?:,-7,9.6/8:,-./:-HR8>9QR74 J-#OS-1Thep16INK4A/RBpathwayregulatescellcycleprogression fromG1toSphase.RB1isatumoursuppressorgenethat encodesRBproteinwhichregulatescellcycleG1/Stransition bybindingthetranscriptionfactorE2F1.RB1wasthefirst TSGdescribedinlungcancer(121)andisinactivatedin about90%ofsmallcelllungcarcinomasbutonlyabout 10-15%ofNSCLC(1).InNSCLC,thepathwayismostly switchedoffthroughalterationsofcyclinD1,CDK4andthe cyclindependentkinaseinhibitorp16(CDKN2A)(105). p16INK4AinhibitscyclinD1dependentphosphorylationofRB protein,therebypreventingcellcycletransitionthroughthe G1/Scheckpoint(122).p16INK4Aisinactivatedinabout80% ofNSCLC(123,124)andwasalteredin72%oflungSCCs examinedbyTCGA,mostlythroughhomozygousdeletion, methylationorinactivatingmutations(12).Inaddition,there isoverexpressionofcyclinD1throughgeneamplificationor other mechanisms in about 40% of NSCLC (123).*%+,('+-$ #-$0,#)"0 )" L58G8Thepresenceofthesemoleculartargetsasdescribedabovenow defnes the characteristics of NSCLC, with EGFR mutation and ALK rearrangements being the most clinically relevant at present (125). Teprevalenceofthesemutationsvariesinlungcancerarising frompatientindifferentregions(126).ActivatingEGFR mutationswerefoundinupto20%ofCaucasianswhileinthe AsianpopulationstheseEGFRmutationscanbepresentinup to40%ofpatientswithNSCLC(127).Teseethnicdiference inNSCLCpropertiesappearstobenotlimitedtothepresence ofactivatingEGFRmutationsbutisalsoevidentinotherdriver oncogenicmutationprofles(includingALK,KRS,METetc.), histologyandhencetumourresponsetotargetedtherapy treatment(63,126,128).Tepresenceofthesedrivermutations is generally found to be mutually exclusive to others in the same tumour (126). In lung ADC among Asians, ALK rearrangement isseeninupto7%ofpatientswithlungADC(79).Lung tumoursbearingEML4-ALKrearrangementarenon-responsive toconventionalchemotherapyorEGFR-tyrosinekinase inhibitors but are sensitive to a specifc tyrosine kinase inhibitor namedcrizotinib(129).Basedonourcurrentunderstandingof therapeuticmoleculartargetsofEGFRmutationandALKgene rearrangementinNSCLCandtheavailabilityofcorresponding targetedagents,analgorithmoftestingformoleculartargetsin NSCLCisproposedasinFigure1,whichrepresentsastepwise approachtotestingforindividualtargets,beginningwithEGFR then,ifnegative,ALKfusiongeneorotherpotentialtargetsif appropriate.AmongNSCLC,adenocarcinomaaccountsforupto80% ofhistologicalsubtypes(130).Therearepreviousreportsof correlations between histological subtypes of ADC demonstrating micropapillaryfeatureswithpresenceofactivatingEGFR mutations,leadingtothesuggestionsthatthepresenceof specificmutationsinNSCLCactuallyrepresentheterogeneity incancerbiologyandalsoresponsetotherapy(131).Giventhe heterogeneityoflungcancerhistology,however,histological subtypesaredifficulttobeusedasthesolereliablemarkerfor guidancetomolecularphenotypingandselectionoftargeted therapy (132,133).TargetingtherapeuticoncogenicmutationslikeEGFRand ALKcangivedramaticinitialtreatmentresponseoratleastan initial stable clinical disease. Te response rate is up to 70% in lung ADCbearingfavourableactivatingEGFRmutations(134).The median progression free survival is usually quoted as 9-11 months withdifferenttyrosinekinaseinhibitors(135,136),after whichmostpatientswithEGFRmutationswillexperience diseaseprogressionanddrugresistance.Aproportionofsuch drugresistanceisattributedtothedevelopmentofasecond mutation, usually T790M at exon 20 (137). It is hard to explain theeventuallossofdrugsensitivityintumoursbearingthose !""#$% $' ()* +")$,-)(% ,.(/0$1 2/ )-/0 ,(/,$% S486favourable EGFR mutations (exon 19 deletions and L858R) even withouttheacquisitionofsecondarymutationslikeT790M orthepresenceofotheruncommonorlessfavourableEGFR mutations.Thiscouldreflectsuboptimaltherapeutictargeting andbetterunderstandingonthebiologyofEGFR-related tumoursignallingandotheroncogenicmutationswillimprove drugtargetingandgivepatientsbeterpredictionoftherapeutic response and prognostication.8%"(+'I)%"ITheidentificationofdrivermutationsinEGFRandALK heraldedaneweraoftargetedtherapyinlungadenocarcinoma andadvancedsequencingtechnologiesareprovidingeven moresophisticatedinsightsintothemolecularaberrations inoncogenesandtumoursuppressorgenesunderlyinglung cancer(12,138-142).Thesestudieshaveidentifiedarangeof potentiallytargetablegeneticaberrationsinlungcancerbut havealsohighlightedatroublingcomplexityandheterogeneity whichposessignificantchallengesformoleculardiagnosisand targetedtreatment.Greaterknowledgeofthemolecularbiology andgenomiclandscapeoflungcancerofferspromiseforthe future.Improvementsinoutcomefromlungcancerwillalmost certainlyrequiretheidentificationofincreasingnumbersof everrarerdrivermutations,anddiagnosticapproachesthatcan identify multiple therapeutic targets ofer signifcant advantages. However,theidentificationofdrivergenomicaberrationsalso requirestheparalleldevelopmentofefectivetargetedtherapies andformanyofthesechanges(suchasKRAS)suchtherapies arenotyetavailable.Resistancetotargetedtherapeuticsisan increasinglyrecognisedissueintowhichgenomicanalyses mayprovideimportantmechanisticinsightsunderlyingfuture rational therapeutic approaches.3(T"%S+,&0,F,"#IFundingsources:SydneyFoundationforMedicalResearch; Hong Kong SK Yee Medical Foundation; Cancer Institute NSW ClinicalResearchFellowship10/1/07;SydneyBreastCancer Foundation;LifehouseatRoyalPrinceAlfredHospitalGrant; Lung Cancer SPORE P50CA70907. Disclosure:WChasreceivedhonorariafromPzerOncology. Figure1.Asuggestedalgorithmformoleculartargettestingbasedonunderstandingofrelevantmolecularbiologyinnon-smallcelllungcancer (NSCLC).Test for alterations in other molecular targets or pathways e.g. ALK, ROS1, RE1, cME1TtaTKI resistant mutatione.g. T790MECFR wildtypeTest for ECFR mutationsNSCLC, in particular adenocarcinomaBiomarker-based personalized therapy for lung cancerECFR mutantTKI responsive mutatione.g. exon 19 deletion, L858R, L861QEGFR TKI New generation EGFR- TKI Targeted therapy e.g. crizotinibe.g. AfatinibFurther research into presence of other biomarkers:1. To understand the molecular biology of lung cancer2. Feedback for clinical management +/- prognostication!"#$%&' ") *+"$&,-, .-/0&/01 2"' 31 4#55' 3 6,7"80$ 9:;< S487SOTisamemberoftheRocheAustraliaMolecularPathology AdvisoryBoard.Theauthorsdeclarenootherconflictsof interest7,2,$,"(,I1.LarsenJE, MinnaJD. Molecularbiologyoflungcancer:clinical implications. 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