ContentslistsavailableatScienceDirect
Leukemia Research
jo u r n al ho me p ag e :w w w . e l s e v i e r . c o m / l o c a t e / l e u k r e s
BCR-ABL kinase domain mutations, including 2 novel mutations in imatinib resistant Malaysian chronic myeloid leukemia
patients—Frequency and clinical outcome
Marjanu Hikmah Elias
a, Abdul Aziz Baba
b, Husin Azlan
b, Hassan Rosline
c, Goh Ai Sim
d, Menon Padmini
e, S. Abdul Wahid Fadilah
f, Ravindran Ankathil
a,∗aHumanGenomeCentre,SchoolofMedicalSciences,HealthCampus,UniversitiSainsMalaysia,Malaysia
bHaemato-OncologyUnit,DepartmentofInternalMedicine,SchoolofMedicalSciences,HealthCampus,UniversitiSainsMalaysia,Malaysia
cHematologyDepartment,SchoolofMedicalSciences,HealthCampus,UniversitiSainsMalaysia,Malaysia
dHospitalPulauPinang,Malaysia
eHospitalRajaPermaisuriBainun,Malaysia
fMedicineDepartment&CellTherapyCentre,UKMMedicalCentre,Malaysia
a r t i c l e i n f o
Articlehistory:
Received13October2013 Receivedinrevisedform 26December2013 Accepted29December2013 Availableonline6January2014
Keywords:
Chronicmyeloidleukemia Imatinibmesylate
BCR-ABLdependentmechanisms Tyrosinekinasedomain Mutation
a b s t r a c t
Discoveryofimatinibmesylate(IM)asthetargetedBCR-ABLproteintyrosinekinaseinhibitor(TKI)has resultedinitsuseasthefrontlinetherapyforchronicmyeloidleukemia(CML)acrosstheworld.Although highresponseratesareobservedinCMLpatientswhoreceiveIMtreatment,asignificantnumberof patientsdevelopresistancetoIM.ResistancetoIMinpatientshasbeenassociatedwithaheterogeneous arrayofmechanismsofwhichpointmutationswithintheABLtyrosinekinasedomain(TKD)arethe frequentlydocumented.Thetypesandfrequenciesofmutationsreportedindifferentpopulationstudies haveshownwidevariability.Wescreened125MalaysianCMLpatientsonIMtherapywhoshowed eitherTKIrefractoryorresistancetoIMtoinvestigatethefrequencyandpatternofBCR-ABLkinase domainmutationsamongMalaysianCMLpatientsundergoingIMtherapyandtodeterminetheclinical significance.Mutationalscreeningusingdenaturinghighperformanceliquidchromatography(dHPLC) followedbyDNAsequencingwasperformedon125IMresistantMalaysianCMLpatients.Mutations weredetectedin28patients(22.4%).Fifteendifferenttypesofmutations(T315I,E255K,G250E,M351T, F359C,G251E,Y253H,V289F,E355G,N368S,L387M,H369R,A397P,E355A,D276G),including2novel mutationswereidentified,withT315Iasthepredominanttypeofmutation.Thedatageneratedfrom clinicalandmolecularparametersstudiedwerecorrelatedwiththesurvivalofCMLpatients.Patients withY253H,M351TandE355GTKDmutationsshowedpoorerprognosiscomparedtothosewithout mutation.Interestingly,whentheprognosticimpactoftheobservedmutationswascomparedinter- individually,E355GandY253Hmutationswereassociatedwithmoreadverseprognosisandshorter survival(P=0.025and0.005respectively)thanT315Imutation.Resultssuggestthatapartfromthose mutationsoccurringinthethreecrucialregions(catalyticdomain,P-loopandactivation-loop),otherrare mutationsalsomayhavehighimpactinthedevelopmentofresistanceandadverseprognosis.Presenceof mutationsindifferentregionsofBCR-ABLTKDleadstodifferentlevelsofresistanceandearlydetectionof emergingmutantclonesmayhelpindecisionmakingforalternativetreatment.SerialmonitoringofBCR- ABL1transcriptsinCMLpatientsallowsappropriateselectionofCMLpatientsforBCR-ABL1KDmutation analysisassociatedwithacquiredTKIresistance.IdentificationoftheseKDmutationsisessentialinorder todirectalternativetreatmentsinsuchCMLpatients.
©2014ElsevierLtd.Allrightsreserved.
∗ Correspondingauthorat:HumanGenomeCentre,SchoolofMedicalSciences, HealthCampus,UniversitiSainsMalaysia,16150KubangKerian,Kelantan,Malaysia.
Tel.:+6097676968;fax:+6097658914.
E-mailaddress:[email protected](R.Ankathil).
1. Introduction
Chronicmyeloid leukemia(CML) iscaused bythereciprocal translocation,t(9;22)(q34;q11),involvingchromosomes9and22 whichresultsinfusionofBCRandABL1genes.Asaconsequence ofthistranslocation,anovel,BCR-ABLfusiongeneiscreatedwhich resultsinexpressionoftheconstitutivelyactivetyrosinekinase BCR-ABLprotein.Theconstitutiveactivityofthistyrosinekinase 0145-2126/$–seefrontmatter©2014ElsevierLtd.Allrightsreserved.
http://dx.doi.org/10.1016/j.leukres.2013.12.025
playsacentralroleinthepathogenesisofthedisease.Theexpres- sionofchimericBCR-ABLproteinwithderegulatedtyrosinekinase activityhasbeenshowntobenecessaryandsufficientforthetrans- formedphenotypeofCMLcells[1].
Imatinibmesylate (IM),themoleculartargeted drugfor the treatment of all phases of CML is a 2-phenylaminopyrimidine derivativethatworksbybindingtothetyrosinekinasedomainof BCR-ABLandblockingitsfunction[2].TargetedinhibitionofBCR- ABLkinasewithIM hasbecomethefrontlinetherapy fornewly diagnosedpatientswithCMLandotherleukemiasthatexpressBCR- ABLkinase[3].Despitethesuccessfultreatmentresultsobtained withIM,achievementofprolongedresponsetoIMisstilladaunt- ingproblem,thechiefobstaclebeingdevelopmentofresistanceto IMinasignificantproportionofpatients.Someofthechronicphase CMLpatientsandmostoftheCMLpatientswithlatestage(accel- eratedorblastphase) developresistancetoIMand thedisease progressescreatingconcerninthetreatmentofCML.
Amongthemultiplemechanismsofresistanceidentified,the dominantmechanismappearstobeacquisitionofpointmutations inthekinasedomainofBCR-ABLwhichresultsinalteredaffinityof IMfortheBCR-ABL1protein.BCR-ABLmutationshadbeenreported tooccurinapproximately50%ofpatientswhodevelopresistance toIM[4].
Defining the mechanisms of resistance in large number of patientsisthebestoptiontobeginwiththeclinicaleffortstoover- comeIMresistance.Asafirststep,weconductedcomprehensive BCR-ABLkinasedomainmutationanalysisof40Philadelphia(Ph) positiveCMLpatientswhodemonstratedIMresistance.Mutations weredetectedin32.5%(13/40)ofpatients[5].Additional85IM resistantCMLpatientsweresubsequentlyscreenedformutations.
Thisreportencompassesmutation screeningresultscarriedout in125PhpositiveCMLpatients(inclusiveof40patientsalready reported)whodevelopedresistancetoIM.
2. Methods
2.1. Studysubjects
ThestudywasundertakenatHospitalUniversitiSainsMalaysia, during the period from June 2008 to September 2013, after getting approval from the Research and Ethics Committee of UniversitySainsMalaysiaandMinistryofHealth,Malaysia(NMRR- 10-1207-7183 and NMRR-10-1206-7127). For this comparative cross sectional study, 125Philadelphia (Ph)chromosome posi- tiveChronicMyeloidLeukemiapatientswererecruitedfromfour hospitalsin Malaysia(Hospital University Sains Malaysia,Hos- pitalPulauPinang,HospitalIpohandPusatPerubatanUniversiti KebangsaanMalaysia)thataltogethercanbeconsideredtorepre- sentCMLpatientsofpeninsularMalaysiacoveringthewest,east andcentralregionsofthecountry.
The Philadelphia chromosome positive Chronic Myeloid Leukemiapatientsincludedwerethoseinchronic,acceleratedor blastphase,treatedforatleast12months,withIMdoseof400mg asfrontline treatment.Patients wereenrolledby random sam- plingaccordingtothephase2extendedaccessprotocolsandwho showedonlysuboptimalresponseorsignsofclinicalresponseto IM. ThoseCML patientswho were Phnegative and those who didnotoptforIMtreatmentwereexcludedfromthisstudy.The medical recordsof all patientswere reviewed until September 2013.Thebasicdemographic,diseasecharacteristicsandtreatment management detailswerecollected. For each patient,diagnosis wasconfirmedby hematological,cytogeneticas wellasmolec- ularanalysis.The response toIM therapy was evaluatedbased onthemeasurementofhematologic,cytogeneticand molecular responses.Hematologicalresponsewasevaluatedevery3rdmonth
oftreatmentandcytogeneticresponse wasevaluatedevery6th monthoftreatment.
TheEuropeanLeukemiaNet2013[6]wasusedasaguidelinefor theassessmentofclinicalresponseoftheCMLpatients.Complete hematologicremissionwasclassifiedaswhenapatientshowed transition ofperipheralblood cell countsas wellasbone mar- rowmorphologybacktonormalinwhichtotalwhitebloodcell countwouldbelessthan10×109L–1and plateletcountwould be less than 450×109L–1. Absence of peripheral blast, imma- turegranulocyteslikepromyelocytesormyelocytes,lessthan5%
peripheralbasophilsandnonpalpablespleenwereconsideredfor co-definingthecompletehematologicalremission[7].Cytogenetic remissionwascategorizedintocomplete,major,partial,minorand non-responsegroups.AtotaldisappearanceofPhchromosomein cytogeneticanalysisconfirmedthecompletecytogeneticresponse (CCyR)whilepresenceoflessthan35%Ph+cellsinbonemarrow confirmedthepartialcytogeneticresponse(PCyR).Patientswith minorcytogeneticresponseshowed36–65%ofPh+cellsinbone marrowwhilethosewhoshowed66–95%Ph+chromosomeposi- tivitywerecategorizedunderminimalcytogeneticresponsegroup.
Patientswhosebonemarrowshowedmorethan95%Ph+chromo- somepositivitywereclassifiedasnocytogeneticresponsetoIM followingBaccaranietal.[6,7].
AccordingtoLeukemiaNet2013,primaryresistance,alsoknown asintrinsicresistancetoIMisdenotedbyfailuretoachievecom- pletehematologicresponse(CHR)and/orhavingmorethan95%Ph+ chromosomewithin3months;havinglessthanpartialcytogenetic response within6months; andhaving incompletehematologic responseornocytogeneticresponse(CyR)within12months.Sec- ondaryresistanceoracquiredresistanceisdefinedaswhenpatients initiallyrespondtotherapy,buteventuallyloseCHR,completeCyR aswellasmajormolecularresponse(MMR)[8].
2.2. BCR-ABLmutationsanalysis
BCR-ABLmutationanalyseswerecarriedoutinallCMLpatients employingdenaturing highperformance liquidchromatography (dHPLC)anddirectDNAsequencingmethod.Forthis,totalRNAwas extractedfromtheperipheralbloodbyusingQIAampRNAblood miniextractionkit(QIAGEN,Germany)accordingtothemanual withaslightmodification,followedbycDNAsynthesisusingcDNA SynthesisKit(Bioline,UnitedKingdom).
Subsequently,amplificationof3overlappingfragmentscover- ingtheentiretyrosine kinasedomainwasgenerated bynested PCRusing theprimers describedbySoveriniet al.[9].Thefirst PCR(BCR-A)with1475bpampliconlengthwasamplifiedfromthe synthesizedcDNA,followedbythesecondandthirdPCR(ABL-B andABL-C)with393bpand482bpampliconlengthrespectively, amplifiedfromBCR-Aamplicon.AllthePCRwereperformedusing AccuSureMix(Bioline,UnitedKingdom)[9].
Then,thepresenceofsequencevariationwasscreenedbydHPLC (ProStarHelixSystem,Varian,USA).ThePCRproductsofsamples thatshowedaltereddHPLCprofile,indicative ofmutation,were directlysequencedwithbothforwardandreverseprimers,after purificationstepsusingPCRpurificationkit(QIAGEN,Germany)to characterizethemutation.
2.3. Bioinformaticsanalysis
An online program available at http://genetics.bwh.
harvard.edu/pph2/, called PolyPhen-2 was used to predict the potential consequence of each mutation on the BCR-ABL pro- tein structure. ClustalX program version 2.0.12 was used for multiple alignment of Homo sapiens ABL1 protein sequence (CAA34438) with ABL1 protein sequence of chimpanzee (Pan troglodytes; XP001166213.2), pig (Sus scrofa; XP003122293.3),
mouse(Mus musculus; NP001106174.1), rat(Rattus norvegicus;
NP001094320.1),cow(Bostaurus;NP001193789.1)andchicken (Gallusgallus;XP001233812.1).
2.4. Statisticalanalysis
Fisher’sexacttestwasusedtotestcategoricaldifferences,and studentt-testwasperformedtocheckdifferencesofmeasurable variationamongpatients.Unconditionallogisticregressionanal- ysiswasusedtoassesstherelationshipbetweentyrosinekinase domainmutationandtheCMLpatients’responsetoIMaswellas thecytogeneticresponsebycalculatingtheoddratios(ORs)and 95%confidence interval(CI). Thetests wereconductedbySPSS software(version20.0)andallP-valuesweretwo-sided.Survival analysiswasdonebycalculatingtheoverallsurvivalthatincluded theintervalbetweentheinitiationofimatinibtreatmentanddeath regardlessofdurationofdiscontinuationofimatinibtreatment.For overallsurvivalcomparison,Kaplan–Meiercurvesforeachcate- gorywereplottedandcomparedusingthelog-ranktest.
3. Results
Inthepresentstudy,mutationalanalysiswasperformedon125 IMresistant,MalaysianCMLpatients.Thedemographicprofile,dis- easecharacteristics,cytogeneticresponseandmolecularresponse detailsofthese125CMLpatientswererecordedandareshownin Tables1and2.
Interestingly,fromallthemutationsidentified(Table3),2muta- tionsappearedtobenovelmutationswhichhavenotbeenreported sofar,inanycancerdatabasenamelyCatalogueofSomaticMuta- tionsin Cancer(http://www.sanger.ac.uk/genetics/CGP/cosmic/), InternationalCancerGenomeConsortium(http://www.icgc.org/) andHumanGeneMutationDatabase(http://www.hgmd.cf.ac.uk/
ac/index.php).So,thesetwomutationscanbeconsideredasnovel mutations.Fornucleotidepositioned251,asubstitutionfromGly (G)toAsp(D)wasreported[10].However,inourstudy,insteadof G251D,weidentifiedanewmutationofG251E,involvingasubsti- tutionfromGly(G)toGlu(E).
Associationriskofthetyrosinekinasedomainmutationwith cytogeneticresponseafter12monthsofIMtreatmentamongthe IM resistant CML patients wasevaluated. Presence of tyrosine kinasedomainmutationwasfoundtobeassociatedwithahigher riskforhavingnocytogeneticresponseamongtheCMLpatients (OR,3.2;95%CI,1.267–8.083;P-value,0.014).
Table1
DemographicprofileoftheCMLpatientsshowingIMresistance.
Demographic No.ofpatients(%)
Gender
Male 48(38.4%)
Female 77(61.6%)
Age
Mean(range) 42.1(13–76)
Male 41.5(13–76)
Female 42.5(15–65)
Race
Malay 90(72.0%)
Chinese 22(17.6%)
Indian 13(10.4%)
IMtreatmentduration(months)
13–24 36(28.8%)
25–36 15(12.0%)
37–48 23(18.4%)
49–60 13(10.4%)
>61 38(30.4%)
Mean(range) 45.4(12–120)
Table2
DiseasecharacteristicsandtreatmentmanagementoftheIMresistantCMLpatients.
Diseasecharacteristicandtreatmentmanagement No.ofpatients(%) CMLphaseduringsamplecollection
Chronicphase 89(71.2%)
Acceleratedphase 24(19.2%)
Blastphase 12(9.6%)
Hematologicresponse(HR)
Complete 109(87.2%)
Suboptimal 3(2.4%)
LossofHR 13(10.4%)
Cytogeneticresponse(CyR)
CCyR 27(21.6%)
MCyR 22(17.6%)
mCyR 20(16.0%)
noCyR 32(25.6%)
CompletebutlossCyR 24(19.2%)
IMresponse
Suboptimalresponse 43(34.4%)
Primaryresistance 52(41.6%)
Secondaryresistance 30(24.0%)
3.1. Survivalanalysis
The data generated from clinical and molecular parameters studiedwerecorrelatedwiththeprognosisandsurvivalofCML patients.IntheKaplan–Meiersurvivalanalysis,patientsinchronic phase showedsignificantlyhigher overall survival comparedto patientsinacceleratedphase(P=0.001)andpatientsinblastcri- sis(P<0.001).Whenconsideringthecytogeneticresponseofthe patientsagainstIM treatment, patientswho achievedcomplete cytogeneticresponseshowedsignificantlyhigheroverallsurvival comparedtopatientswhodidnotachieveanycytogeneticresponse (P=0.005).
Basedonthepresenceofmutationattyrosinekinasedomainof BCR-ABL1gene,mutationfreepatientsshowedsignificantlyhigher overallsurvivalcomparedtopatientswhoshowedthepresence of M351T,Y253Hand E355G mutation(P=0.028,P<0.001and P=0.022,respectively).Thus,itisreasonabletosuggestthatthe classificationofIMresistantCMLpatientsbasedonthepresence andtypeofBCR-ABLmutationsmaybeassociatedwithdiseaseout- come.Interestingly,amongtheMalaysianCMLpatientsstudied, mutationssuchasY253HandE355Gwereassociatedwithlower survivalthanT315Imutation(P=0.005andP=0.025respectively) (Fig.2).
Table3
BCR-ABLTKDmutationanalysisresultsoftheCMLpatients.
Mutation No.ofpatients(%)
MutationatBCR-ABLgene
Absent 97(77.6)
Present 28(22.4)
Typeofmutation
T315I 9(7.2)
E255K 4(3.2)
G250E 2(1.6)
M351T 2(1.6)
F359C 2(1.6)
G251Ea 1(0.8)
Y253H 1(0.8)
V289F 1(0.8)
E355G 1(0.8)
N368Sa 1(0.8)
L387M 1(0.8)
H396R 1(0.8)
A397P 1(0.8)
E355A 1(0.8)
D276G 1(0.8)
aNovelmutations.
Fig.1. Themulti-alignmentofhumanABL1proteinwithitsorthologsofvariousspeciesbyusingClustalXprogramversion2.0.12andthepositionofeachmutationinthe tyrosinekinasedomain.Allthemutationsdetectedarehighlyconservedamongdifferentspeciesandlocatedinconservedblockofaminoacids.
Fig.2. OverallsurvivalofIMresistantCMLpatientsbasedonthepresenceofmutationinTKDofBCR-ABLgene.
4. Discussion
The development of resistance to IM in CML patients has beenassociatedwithaheterogeneousarrayofmechanisms.These includeBCR-ABLdependentpathwaysandBCR-ABLindependent pathways. BCR-ABLdependentpathwayshave beenreportedas oneofthemostfrequentmechanismthatcontributetoIMresis- tanceamongCMLpatientstreatedwithImatinib[11].TheBCR-ABL dependentpathwaymostlyinvolvestheTKDmutationsoftheBCR- ABL1gene[12,13].Drukeretal.[15]demonstratedthatblockingof
theBCR-ABL1proteinintotheinactiveconformationbyImatinib preventsthetransferofphosphatefromAMP(ATP)tosubstrates andinhibitsthedownstreamsignalingpathways.However,accord- ingtoCorbinetal.[14],somemutationsseemtodisruptcritical contactpointbetweenImatinibandBCR-ABL1.Inaddition,other mutationsappeartoinduceatransitionfromtheinactivetothe activestate,aconformationwhich imatinibwouldbeunableto bind[14,15].
Inthisstudy,28(22.4%)MalaysianIMresistantCMLpatients showedthepresenceofmutationintheirtyrosinekinasedomain
Table4
FrequencyofBCR-ABLkinasedomainmutationindifferentpopulation.
Author Population Totalpatients
(N)
Mutation frequencyinCML patients(%)
CPCMLpatients(%) APCMLpatients(%) BPCMLpatients(%)
Branfordetal.(2003)[16] Australian 144 19(n=27) 72(n=104) 28(n=40) 0(n=0)
Jabbouretal.(2006)[4] Caucasian 171 36(n=62) 78(n=134) 20(n=34) 2(n=3)
AiLeenAngetal.(2008)[17] Singaporean 94 45(n=42) 56(n=53) 16(n=15) 28(n=26)
Kimetal.(2009)[18] Korean 137 63(n=79) 25(n=34) 26(n=36) 49(n=67)
Sherbenouetal.(2010)[19] Caucasian 98 30(n=29) Notmentioned Notmentioned Notmentioned
Qinetal.(2011)[20] Chinese 127 58(n=74) 55(n=70) 22(n=28) 23(n=29)
Presentstudy(Eliasetal.,2013) Malaysian 125 22(n=28) 75(n=94) 20(n=25) 5(n=6)
ofBCR-ABL1gene.Oncomparingthemutationfrequencyofthe presentstudy withthefrequency reportedinother studypop- ulations, a lower frequency was observed among IM resistant MalaysianCMLpatients(Table4).
AccordingtoKimetal.[18],thefrequencyofmutationsiscor- relatedwiththeproportionofpatientsindifferentphasesofthe disease.InthestudybyKim etal.[18],25%oftherecruitedIM resistantCMLpatientswereinchronicphase(CP),whereastherest 75%ofCMLpatientswereinacceleratedphase(AP)orblastphase (BP).Consequently,Kimetal.[18]observedahigh(63%)frequency ofmutationamongtheirIMresistantCMLpatients.Inthepresent study,75%ofthepatientsstudiedwereinCPandonly25%werein APandBP.Thus,therelativelylowoverallfrequencyofmutations inourstudycomparedtootherpublishedstudiesmaybeexplained bythepredominanceofchronicphaseCMLpatientsincludedfor mutationanalysis.AlowpercentageofTKDmutation(19%)was alsoobserved byBranford et al.[16] in IM resistantAustralian CMLpatients.ThelowpercentageofTKDmutationsobservedin Brandfordetal.’sstudy(2003)alsocouldbeduetothemajority ofpatientsscreenedbeingintheCP.Thepresentstudyresultsas wellasotherquotedreports(Table4)areinagreementwithBran- fordetal.[21]whoreportedthattheestimatedpercentagesofIM resistantpatientsinCML-CP,CML-APwhocarriedclinicallyrele- vantmutationswouldbeapproximately18%and31%respectively whereasitwouldbe29–52%inpatientswithCMLBP[21].
Interestingly,amongtheninedifferenttypesofmutationsiden- tified,two(G251EandN368S)werenovelmutationsandhadnot beenreportedpreviously.TheG251Emutationislocatedinthe P-loopregionwhere ATP binds.Severalstudies[18,22,23] have reportedthatmutationintheP-loopregionplayanimportantrole inthedevelopmentofresistancetoIMamongtheCMLpatients andhave70-foldto100-foldless sensitivitytoIM comparedto nativeBCR-ABL[22].Althoughoneofthenovelmutations,N368S, islocatedoutsidethethreecrucialregions(IMbindingsite,P-loop anda-loop)ontheBCR-ABLkinasedomain,stillitwaspredictedto beclinicallyrelevantviain-silicoanalysis.
To estimate the effect of the novel mutations on the out- come of the CML patients, in-silico analyses were performed onthemutations. TheG251EaswellasN368Smutationswere predicted to be possibly damaging by using PolyPhen2 with a score of 0.898 for both mutations, using HumDiv model (http://genetics.bwh.harvard.edu/pph2/).So,bothBCR-ABLG251E andN368S mutationsin thepresentstudy waspredictedtobe clinicallyrelevant.Hence,itisreasonabletosuggestthatBCR-ABL mutationsthathavenoclinicalsignificancemightbequiterare.
However,thesenovelmutationswereunabletobeclassifiedas acquiredmutationsbecausepatientsampleatthepointofresis- tanceonlywasavailableandnotatthepointofdiagnosis.Thus,the presenceofthesemutationsatthetimeofdiagnosiscouldnotbe proved.
Themulti-alignmentofhumanABL1proteinwithitsorthologs amongvariousspeciesbyusingClustalXprogramversion2.0.12 revealed that both G251E and N368S mutations are conserved
amongspeciesinahighlyconservedblock.Fig.1showsthemulti- alignment ofhumanABL1protein withitsorthologs ofvarious speciesbyusingClustalXprogramversion2.0.12andthepositionof eachmutationinthetyrosinekinasedomainthatwerediscovered amongthepatients.Fromthis,itisreasonabletopresumethatthese mutationsmayleadtoalterationoftheBCR-ABLproteinstructure orimportantstructureforitsbiologicalfunctionthatmayaffectthe actionofImatinibonthisprotein.However,therealimpactofthe twonovelmutationsidentified,inmediatingresistanceisyettobe determinedasfurtherfunctionalstudiesarewarrantedtoelucidate thisissue.
On survival analysis, IM treated CML patients withBCR-ABL mutations,especiallythosewithT315I,M351T,Y253HandE355G TKDmutationsshowedshortersurvivalperiodcomparedtothose withothertypesofmutationsandthosewithoutmutations.Loca- tionofT315ImutationattheIM bindingsite oftyrosinekinase stronglyexplainstheshortersurvivalandpoorprognosisofCML patientswithT315I mutation.Moreover,therearereports stat- ingthatpatientswithT315ImutationarenotonlyresistanttoIM butalsotoothersecondandthirdgenerationofTKIlikeDasatinib [24],Nilotinib[25]andBosutinib[26].Thus,T315Imutationconfers trueresistancetoTKIandtherebypromotesshorteningofpatients’
survival.
However,wheninter-individualcomparison ofpatientswith these four mutations that were associated with shorter sur- vivalwasmade,patientswithY253HandE355GTKDmutations showedworstoutcomecomparedtopatientswithT315Imuta- tion(P=0.005andP=0.025respectively).Inthispresentstudy,the overallsurvivalofpatientswithT315Imutationwasnottheworst asbeinggenerallyexpected.Thiscouldbeattributedtothefact thatsomeofthepatientsidentifiedwithT315Imutationhadbeen subjectedtobonemarrowtransplantation.Duringtheoverallsur- vivalanalysis,thepatients’statuswascensoredinconsequenceto thebonemarrowtransplant.Thismusthaveresultedintheover- allsurvivalbeinghigherthanexpected,forthepatientswithT315I mutation.
ThelocationofY253Hmutationatthenucleotidebindingloop (P-loop)highlightsitsimpactonIMresistancedevelopment,which alsoexplainsthereasonforpoordiseaseoutcome.Withdrawalof IMisthebestoptionforpatientswithY253Hmutationwhichalso confersatrueresistantphenotypetoIM.AlthoughE355Gisnot locatedwithinthethreecrucialregionsoftyrosinekinasedomain, itisprobablethatitstillcouldbehavingsomeeffectonthebinding towardIMasE355Gislocatedneartheactivationloop.Thus,the resultssuggestthatapartfromthosemutationsoccurringinthe threecrucialregions(catalyticdomain,P-loopandactivation-loop), mutationsinotherregionsalsomayhaveadverseimpactresulting inthedevelopmentofresistanceandpooroutcome.
EvaluatingCMLpatientswithclinicalsignsofresistanceforBCR- ABLmutationsisanimportantcomponentofdiseasemonitoring,in determininghowpatientsrespondtotreatmentandtounderstand theBCR-ABLmutationasthecauseofresistance.Notonlythepres- enceofmutations,butalsotheactualaminoacidchangeshouldbe
investigatedinCMLpatientsdisplayingresistancetoIMinorder tooptimize therapeutic response. Certain specific mutationsin BCR-ABLhavebeenlinkedwithpooroutcomeand hencemuta- tionscreeningisclinicallyrelevanttoidentifyCMLpatientswho arelikelytohavepooroutcomeandtofacilitateselectionofsub- sequenttherapy.Furthermore,presenceofmutationsindifferent regionsofBCR-ABLTKDleadstodifferentlevelsofresistance.The typeofmutationpotentiallycanindicatewhethersecondorthird generationBCR-ABLinhibitororalternativetherapeuticstrategies shouldbegiventosuchIMresistantpatientsandalsocanhelpto identifythosewhoneedIMdoseescalation.
Conflictofintereststatement
Allauthorshavenoconflictofinteresttoreport.
Acknowledgements
ThisstudywaspartlysupportedbyUniversitiSainsMalaysia– ResearchUniversityGrant(USM-RU)1001/PPSP/812070andFun- damentalResearchGrantScheme(FRGS)203/PPSP/6171104.The firstauthorisUniversitiSainsMalaysiaFellowshipholder.Theco- operationandsupportofstaffs,HumanGenomeCenter,Universiti SainsMalaysiaandallthepatientswhohaveparticipatedinthis studyaregratefullyacknowledged.
Contributions.M.H.E.carriedoutthelabworks,acquisitionof data,analysisandinterpretationofdata,draftingthearticle,revised itcriticallyforimportantintellectualcontentandfinal approval oftheversiontobesubmitted;A.A.B.,A.H.,R.H.,G.A.S.,P.M.,and F.A.W.suppliedtheacquisitionofdataandrevisedthearticle;R.A.
providedtheconceptionanddesignofthestudy,revisedthearticle criticallyforimportantintellectualcontentandfinalapprovalofthe versiontobesubmitted.
References
[1]UsuiN.Moleculartargetedtreatment–newtreatmentstrategyforpatients withchronicmyeloidleukemia.RinshoByori2004;52:136–44.
[2]DrukerBJ,GuilhotF,O’BrienSG,GathmannI,KantarjianH,GattermannN, etal.Five-yearfollow-upofpatientsreceivingimatinibforchronicmyeloid leukemia.NEnglJMed2006;355:2408–17.
[3]WuJ,MengF,KongLY,PengZ,YingY,BornmannWG,etal.Associationbetween imatinib-resistantBCR-ABLmutation-negativeleukemiaandpersistentactiva- tionofLYNkinase.JNatlCancerInst2008;100:926–39.
[4]JabbourE,KantarjianH,JonesD,TalpazM,BekeleN,O’BrienS,etal.Frequency andclinicalsignificanceofBCR-ABLmutationsinpatientswithchronicmyeloid leukemiatreatedwithimatinibmesylate.Leukemia2006;20:1767–73.
[5]EliasMH,BabaAA,HusinA,AbdullahAD,HassanR,SimGA,etal.Contri- butionofBCR-ABLkinasedomainmutationstoimatinibmesylateresistance inPhiladelphiachromosomepositiveMalaysianchronicmyeloidleukemia patients.HematolRep2012;4:e23.
[6]BaccaraniM,DeiningerMW,RostiG,HochhausA,SoveriniS,ApperleyJF, etal.EuropeanLeukemiaNetrecommendationsforthemanagementofchronic myeloidleukemia:2013.Blood2013;122:872–84.
[7]BaccaraniM,CortesJ,PaneF,NiederwieserD,SaglioG,ApperleyJ,etal.Chronic myeloidleukemia:anupdateofconceptsandmanagementrecommendations ofEuropeanLeukemiaNet.JClinOncol2009;27:6041–51.
[8]GorczycaW.Cytogenetics,FISHandmoleculartestinginhematologicmalig- nancies.UnitedKingdom:InformaHealthcare;2008.p.319.
[9]SoveriniS,MartinelliG,AmabileM,PoerioA,BianchiniM,RostiG,etal.
Denaturing-HPLC-based assayfor detection of ABL mutationsin chronic myeloidleukemiapatientsresistanttoImatinib.ClinChem2004;50:1205–13.
[10]PressRD,GalderisiC,YangR,RempferC,WillisSG,MauroMJ,etal.Ahalf- logincreaseinBCR-ABLRNApredictsahigherriskofrelapseinpatientswith chronicmyeloidleukemiawithanimatinib-inducedcompletecytogenetic response.ClinCancerRes2007;13:6136–43.
[11]KantarjianHM,TalpazM,GilesF,O’BrienS,CortesJ.Newinsightsintothe pathophysiologyofchronicmyeloidleukemiaandimatinibresistance.Ann InternMed2006;145:913–23.
[12]LaRoseeP,DeiningerMW.Resistancetoimatinib:mutationsandbeyond.
SeminHematol2010;47:335–43.
[13]O’HareT,EideCA,DeiningerMW.Bcr-Ablkinasedomainmutations,drug resistance, and theroad to a cure for chronic myeloid leukemia.Blood 2007;110:2242–9.
[14]Corbin AS,Buchdunger E, PascalF, Druker BJ. Analysis ofthe structural basisofspecificityofinhibitionoftheAblkinasebySTI571. JBiolChem 2002;277:32214–9.
[15]DrukerBJ,TamuraS,BuchdungerE,OhnoS,SegalGM,FanningS,etal.Effectsof aselectiveinhibitoroftheAbltyrosinekinaseonthegrowthofBcr-Ablpositive cells.NatMed1996;2:561–6.
[16]BranfordS,RudzkiZ,WalshS,ParkinsonI,GriggA,SzerJ,etal.Detection ofBCR-ABLmutationsinpatientswithCMLtreatedwithimatinibisvirtu- allyalwaysaccompaniedbyclinicalresistance, andmutationsintheATP phosphate-bindingloop(P-loop)areassociatedwithapoorprognosis.Blood 2003;102:276–83.
[17]AngAL,WangZP,YangLY,NatesanV,PoonM,KohLP,etal.TheM351TBCR- ABLkinasemutationisuncommoninasianpatientswithimatinib-resistant chronicmyeloidleukemia:possiblerelationshipwithimatinibplasmalevels.
In:50thASHAnnualMeetingandExposition.2008.
[18]KimSH,KimD,KimDW,GohHG,JangSE,LeeJ,etal.AnalysisofBcr-Ablkinase domainmutationsinKoreanchronicmyeloidleukaemiapatients:poorclinical outcomeofP-loopandT315Imutationisdiseasephasedependent.Hematol Oncol2009;27:190–7.
[19]SherbenouDW,HantschelO,KaupeI,WillisS,BummT,TuragaLP,etal.BCR-ABL SH3-SH2domainmutationsinchronicmyeloidleukemiapatientsonimatinib.
Blood2010;116:3278–85.
[20]QinY,ChenS,JiangB,JiangQ,JiangH,LiJ,etal.CharacteristicsofBCR-ABL kinasedomainpointmutationsinChineseimatinib-resistantchronicmyeloid leukemiapatients.AnnHematol2011;90:47–52.
[21]BranfordS,MeloJV,HughesTP.Selectingoptimalsecond-linetyrosinekinase inhibitortherapyforchronicmyeloidleukemiapatientsafterimatinibfailure:
doestheBCR-ABLmutationstatusreallymatter?Blood2009;114:5426–35.
[22]CangS,LiuD.P-loopmutationsandnoveltherapeuticapproachesforimatinib failuresinchronicmyeloidleukemia.JHematolOncol2008;1:15.
[23]LeeTS,PottsSJ,KantarjianH,CortesJ,GilesF,AlbitarM.Molecularbasisexpla- nationforimatinibresistanceofBCR-ABLduetoT315IandP-loopmutations frommoleculardynamicssimulations.Cancer2008;112:1744–53.
[24]SoveriniS,ColarossiS,GnaniA,CastagnettiF,RostiG,BosiC,etal.Resistance todasatinibinPhiladelphia-positiveleukemiapatientsandthepresenceorthe selectionofmutationsatresidues315and317intheBCR-ABLkinasedomain.
Haematologica2007;92:401–4.
[25]JabbourE,HochhausA,CortesJ,LaRoseeP,KantarjianHM.Choosingthebest treatmentstrategyforchronicmyeloidleukemiapatientsresistanttoimatinib:
weighingtheefficacyandsafetyofindividualdrugswithBCR-ABLmutations andpatienthistory.Leukemia2010;24:6–12.
[26]Khoury HJ, Cortes JE, Kantarjian HM, Gambacorti-Passerini C, Baccarani M,KimDW,etal. Bosutinibis active inchronic phasechronic myeloid leukemiaafterimatinibanddasatiniband/ornilotinibtherapyfailure.Blood 2012;119:3403–12.
The author has requested enhancement of the downloaded file. All in-text references underlined in blue are linked to publications on ResearchGate.
The author has requested enhancement of the downloaded file. All in-text references underlined in blue are linked to publications on ResearchGate.
