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Association of genotypes and haplotypes of multi-drug transporter genes ABCB1 and ABCG2 with clinical response to...

Article in Biomedicine & pharmacotherapy = Biomedecine & pharmacotherapie · April 2014

DOI: 10.1016/j.biopha.2014.01.009

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Original article

Association of genotypes and haplotypes of multi-drug transporter genes ABCB1 and ABCG2 with clinical response to imatinib mesylate in chronic myeloid leukemia patients

Anthony Au

^a,

*, Abdul Aziz Baba

^b,c

, Ai Sim Goh

^d

, S. Abdul Wahid Fadilah

^e

, Alan Teh

^f

, Hassan Rosline

^g

, Ravindran Ankathil

^a,

*

aHumanGenomeCentre,SchoolofMedicalSciences,UniversitiSainsMalaysia,16150,KubangKerian,Kelantan,Malaysia

bDepartmentofInternalMedicineandClinicalHaematology,UniversitiSainsMalaysia,16150,KubangKerian,Kelantan,Malaysia

cSchoolofMedicine,InternationalMedicalUniversity,57000,BukitJalil,KualaLumpur,Malaysia

dDepartmentofMedicine,HospitalPulauPinang,10990,Georgetown,Penang,Malaysia

eCellTherapyCentre,UKMMedicalCentre,UniversitiKebangsaanMalaysia,43600,Bangi,Selangor,Malaysia

fDepartmentofHaematology,SimeDarbyMedicalCenter,47500,SubangJaya,Selangor,Malaysia

gDepartmentofHaematology,SchoolofMedicalSciences,UniversitiSainsMalaysia,16150,KubangKerian,Kelantan,Malaysia

1. Introduction

Imatinibmesylate(IM),acompetitiveinhibitoroftheBCR-ABL tyrosine kinase, has proven to be an extremely effective and generally well tolerated drug, producing durable responses in patients with chronic myeloid leukemia (CML). Despite the outstandingresultsobtained withIMforthetreatmentofCML, asignificantproportionofpatientsshowsuboptimalresponseor developresistancetoIM.MechanismofresistancetoIMinCML

patients involve BCR-ABLdependent and BCR-ABLindependent pathways.BCR-ABLdependentmechanismwhichmainlyinvolve point mutations in the tyrosine kinase domain (TKD) and amplification of BCR-ABLgene, account for approximately 50%

ofpatientswhodevelopresistance[1].FortheCMLpatientswho donotfitintotheBCR-ABLdependentmechanismsofresistance, several other BCR-ABL independent mechanisms have been postulated. Pharmacogenetic variability, which influences the pharmacokineticsofIM,couldbeapossibleBCR-ABLindependent mechanismmediatingresistance.

IM is a substrate for the adenosine triphosphate binding cassette (ABC) transporters, ABCB1 and ABCG2. ATP Binding CassetteB1(ABCB1)genelocatedatchromosome7q21.1,consists ARTICLE INFO

Articlehistory:

Received24December2013 Accepted20January2014

Keywords:

ABCB1 ABCG2

Chronicmyeloidleukemia Imatinibmesylate

Singlenucleotidepolymorphisms

ABSTRACT

Theintroductionandsuccessofimatinibmesylate(IM)hasbecomeaparadigmshiftinchronicmyeloid leukemia(CML)treatment.However,thehighefficacyofIMhasbeenhamperedbytheissueofclinical resistancethatmightduetopharmacogeneticvariability.Inthecurrentstudy,thecontributionofthree commonsinglenucleotidepolymorphisms(SNPs)ofABCB1(T1236C,G2677T/AandC3435T)andtwo SNPsofABCG2(G34AandC421A)genesinmediatingresistanceand/orgoodresponseamong215CML patientsonIMtherapywereinvestigated.Amongthesepatients,thefrequencydistributionofABCG2 421CC,CAandAAgenotypesweresignificantlydifferentbetweenIMgoodresponseandresistant groups(P=0.01).ResistancewassignificantlyassociatedwithpatientswhohadhomozygousABCB1 1236CCgenotypewithOR2.79(95%CI:1.217–6.374,P=0.01).ForABCB1G2677T/Apolymorphism,a bettercomplete cytogeneticremissionwas observedforpatientswithvariantTT/AT/AAgenotype, compared to other genotype groups (OR=0.48, 95%CI: 0.239–0.957,P=0.03). Haplotype analysis revealedthatABCB1haplotypes(C1236G2677C3435)wasstatisticallylinkedtohigherrisktoIMresistance (25.8%vs.17.4%,P=0.04),whileABCG2diplotypeA34A421wassignificantlycorrelatedwithIMgood response(9.1%vs.3.9%,P=0.03).Inaddition,genotypicvariantinABCG2421C>Awasassociatedwitha majormolecularresponse(MMR)(OR=2.20,95%CI:1.273–3.811,P=0.004),whereasABCB12677G>T/

Avariantwasassociatedwithasignificantlylowermolecularresponse(OR=0.49,95%CI:0.248–0.974, P=0.04).However, therewas nosignificantcorrelationof theseSNPs withIMintoleranceand IM inducedhepatotoxicity.Our resultssuggesttheusefulnessofgenotypingofthesesinglenucleotide polymorphismsinpredictingIMresponseamongCMLpatients.

ß2014ElsevierMassonSAS.Allrightsreserved.

* Correspondingauthors.Tel.:+6097676968.

E-mailaddresses:auzlanthony@gmail.com(A.Au),rankathil@hotmail.com (R.Ankathil).

Availableonlineat

ScienceDirect

www.sciencedirect.com

0753-3322/$–seefrontmatterß2014ElsevierMassonSAS.Allrightsreserved.

http://dx.doi.org/10.1016/j.biopha.2014.01.009

of28intronsand28exonsandencodesforP-glycoprotein(P-gp), with170kDaor1280aminoacids.ABCG2,alsoknownasBreast CancerResistanceProtein(BCRP)isthesecondmemberoftheG familyofABCtransporters.Locatedonchromosomalregion4q22, ABCG2geneconsistsof16exonsthatspanover66kbandencodes a72-kDamembraneproteinthatiscomposedof655aminoacids.

Accordingly, ABCB1 and ABCG2 might be influencing the pharmacokineticsandintracellularorsystemiclevelofIM.Both ABCB1andABCG2displaya highaffinityforIMandhavebeen demonstratedtoconferresistanceinvitrobyextrudingIMfrom haematopoieticcells[2].Singlenucleotidepolymorphisms(SNPs) of these drug transporters could be potential determinants of variabilityindrugdispositionandefficacy.

ABCB1ishighlypolymorphicwithexisting50andmoresingle nucleotidepolymorphisms(SNPs)yettobeidentified.SNPswithin ABCB1genehavebeenassociatedwithPGpover-expression.Three polymorphisms(T1236C,G2677T/AandC3435T)ofABCB1have significanteffectonP-gpexpression,functionalityandsubstrate distribution[3].The3435C>Tlocatedon exon26, isin linkage disequilibriumwiththeothertwoSNPs,2677G>T/A(exon21)and 1236C>T (exon 12), of which 2677G>T/A is responsible for a substitutionintheaminoacidsequence(Ala893Ser/Thr).Genetic polymorphismsin ABC transporters have beenassociated with alteredtransporterfunctionsofvariousdrugs[4].Moreover,the ABCB1 SNPs are highly polymorphic within different ethnic groups.BCRPexpressionandfunctioncanbealteredbySNPsin ABCG2gene.Twomostcommon ABCG2polymorphismsare 34 G>A,which codes for Val12Metand 421C>A which codes for Glu141Lys(Zamberetal.,2003).

Pharmacogeneticstudieshavebeenhelpfulintheevaluationof sensitivityprofileofdrugs.SNPsofABCB1andABCG2genescan causeinter-individualvariations inthepharmacokineticsforIM.

Therefore,weaimedtodeterminewhetherdifferentgenotypeand haplotype pattern of SNPs ABCB1 (1236T>C, 2677G>T/A, and 3435C>T)andABCG2(34G>Aand421C>A)haveanyinfluencein mediating clinical response in CML patients undergoing IM treatment.IMpharmacogeneticsmayhaveanobviouscorrelation withthecytogeneticandmolecularresponseofIM.ApartfromIM response,theeffectsofpharmacogeneticcovariatesonhematolo- gical,non-hematological adverseside effectsand hepatotoxicity amongCMLpatientsundergoingIMtreatmentwerealsoexamined.

2. Materialsandmethods 2.1. Samplecollection

Thismulti-centricstudyprotocolwas approvedby Universiti Sains Malaysia Human Ethics Committee and registered under National Medical ResearchRegister (NMRR), Ministry of Health Malaysia.Atotalof215Philadelphia(Ph)chromosomepositiveCML participants(agerangebetween11to78years)undergoing400mg IMdailyforatleast6monthsinseverallocalhospitalsandmedical centersinMalaysiawereenrolledafterobtainingwritteninformed consent.Outofthe215CMLpatients,106weremalesand109were femaleswithameanageof41.5years.WhentheseCMLpatients werecategorizedbasedonIMtreatmentresponse,107wereIMgood respondersand108IMwereresistantPh+CMLpatientsbelongingto chronicandacceleratedphase.Bloodsamplesofthesepatientswere collectedaftergettingwritteninformedconsentandstoredinEDTA vacutainertubesuntilanalysis.

2.2. Evaluatingimatinibresponseandtolerance

Hematologic,cytogeneticandmolecularcriteriawereaccessed inordertojustifyclinicalresponsetoIMbyreferring‘‘European Leukemia Net: guideline for managing CML patients’’ [5].

MolecularresponsewasclassifiedbasedonBCR-ABLcontrolgene transcriptratios,expressedontheInternationalScale,wheremajor molecular response (MMR) and complete molecular response (CMR)weredefinedasratios0.1%and0.0032%respectively.

Cytogeneticresponsewasclassifiedascomplete (0%Ph+meta- phases), partial(>0to35% Ph+cells), minor(>35 to65%Ph+

metaphases), minimal (>65–95% Ph+ metaphases), and none (>95–100%Ph+metaphases)basedonGTGbandedanalysisofa minimum 20 bone marrow metaphases. CML patients were categorizedas incompletehematological response(CHR)when they achieved<45010⁹/L platelet count, <1010⁹/L WBC countand<5%basophils.ThosepatientswithCHRin3months, MCgRin12monthsandMMRin18monthswereconsideredasIM goodresponders.Thosepatientswhodidnotachievetheabove responsecriteriawithinthespecifiedtimeframewerecategorized undernonresponders/resistantgroup.Inthisstudy,commonIM adversesideeffectswerenon-hematologicaltoxicity(nauseaand/

orvomit,skinhypo-pigmentation and/orrash, joinpain and/or muscle cramp, headache and/or fatigue) and hematological toxicity(anemia,thrombocytopeniaandneutropenia)inallgrades, afteradministrationwith400mgIM.Parametersofhepaticinjury wereassessedbybiochemicalparameterslikelevelsofbilirubin, alkalinephosphatase(ALP)andalaninetransaminase(ALT).

2.3. Genotyping

DNA from the peripheral blood of the study subjects were extractedusingQIAGENampDNAextractionkit(Qiagen,Hilden, Germany)followingthemanufacturer’sprotocol.DNAyieldwas standardizedinto50ng/

m

laftermeasurementwithInfinite¹200 PRONanoQuant.PolymeraseChainReaction-RestrictionFragment LengthPolymorphism(PCR-RFLP)wasdesignedforamplification oftheSNPsABCB11236T>C,2677G>T/A,3435C>T,andalsothe ABCG2G34AandC421ASNPs.Forwardandreverseprimerswere self-designedfor1236T>C(F:5⁰-CGAAGAGTGGGCACAAACCAG-3⁰ andR:5⁰-GCATGGGTCATCTCACCATC-3⁰)and2677G>T/A(F:CCT TCATCTATGGTTGGC-3⁰andR:5⁰-GCATAGTAAGCAGTAGGG AG-3⁰).Primersfor3435C>TwasobtainedfromAmeyawetal.[6]

while34G>Aand421C>AwerereferredtoKobayashietal.[7].

EachPCRmixtureconsistedof1MyTaqReactionBuffer(Bioline Ltd, London, UK), 1 unit MyTaq DNA Polymerase (Bioline Ltd, London,UK),50nggenomicDNAtemplatesandddH2Oinatotal volumeof20

m

l.PCRconditionsinvolveddenaturationat958Cfor 1min,andrepeated35cyclesconsistingof3steps:denaturationat 958Cfor15seconds;annealingat638C/568C/618C/618C/608Cfor 15secondsand;extensionat 728Cfor10seconds, followed by 3minsfinalextensionat728C.PCRproductsweresubsequently digested with Fermentas FastDigest (Fermentas, Lithuania) Eco0109Ifor30mins,378C(1236T>C),BanI378Cfor30mins (2677G>T),RsaIfor30mins,378C(2677G>A),MboIfor30mins, 378C(3435C>T),BseMIfor558C,20mins(34G>A)andTaaIfor10 mins at 658 C(421C>A)respectively. Digested DNA fragments wereelectrophoresedon3.5%BiolineAgaroseHiResgel(Bioline Ltd,London,UK)andstainedwithSyBrGreen.Aspartofquality controlchecking,genotypingresultsweredirectlysequenced(First BASELaboratoriesSdnBhd,Malaysia)in10%ofsamplesafterthe purificationsteps(GeneJETPCRPurificationKit,Fermentas).

2.4. Statisticalanalysis

Hardy–Weinberg equilibrium was verified for all examined SNPs.Differenceingenotypefrequenciesamongthetwogroupsof CMLpatientsandtheassociationsofthevariousgenotypeswith good response and resistance to IM were determined using Pearson X² test. Odds ratios (OR) along with 95% confidence intervals(CI)andtwo-sidedP-valueswerecalculatedbyEpiInfo A.Auetal./Biomedicine&Pharmacotherapy68(2014)343–349

344

7 software. Chi-square or Fisher’s exact tests (if n<5) was performed to associate the clinical response (cytogenetic and molecular response) and tolerance (hematological and non- hematologicaltoxicity)withSNPs.Kruskal waillistestwasused tocomparethedistributionofmeanALT,ALPandbilirubinvalues inthedifferentgenotypepatterns.Allthesestatisticaltestswere carriedoutbySPSSsoftwareversion20.0.P-valueslessthan0.05 were considered statistically significant. Haplotype frequencies were determined between IM response groups and haplotype with>0.03%waspresented.CorrectedP-valuewasobtainedafter applying1000timesper-mutationtest,byusingtheHaploview4.2 software.

3. Results

3.1. ABCB1&ABCG2SNPsinIMresponse

ThegenotypicdistributionofABCB11236T>C,2677G>T/A,and 3435C>TandABCG234G>Aand421C>Apolymorphismsamong IMgoodresponseandresistantCMLpatientsareshowninTable1.

Withregard toABCB1 C3435T andABCG2 G34A, there wasno significantdifference inthegenotypefrequenciesbetween CML patientsshowinggoodresponseandresistanttoIM.However,the homozygous variants of ABCB1 G2677T/A and ABCG2 C421A genotypesweresignificantlyhigherinCMLpatientsshowingIM good response compared to IM resistant CML patients. The frequency of IM resistance was found to correlate with the numberofCalleleatlocusABCB11236,GalleleatABCB12677 locusandCallelesatABCG2allele.Resistancewashigheramong patientshomozygousfortheC1236Cgenotypewhencomparedto

patients withIM good response(20.4% vs. 8.4%)with OR2.79 (95%CI:1.217–6.374,P=0.01).FortheG2677T/Apolymorphism, thefrequencyofgenotypeTT/AT/AA(29.9%)washigheramongIM goodresponsegroupcomparedtoIMresistantgroup(13.9%)but statistically insignificant (P=0.14). However, this genotype showeda significantlower riskfor IMresistance withOR0.38 (95%CI:0.191–0.750,P=0.004).TheC3435Tgenotypewasfound nottoinfluenceIMresponsesincethegenotypefrequenciesofthis SNPinboth IMresponseandIMresistantgroupswerecloseto similarand withoutanysignificantdifference (P>0.05).Mean- whileforABCG2C421A,thehomozygousmajorCCgenotypewith afrequencyof53.7%,wasfoundtobesignificantlyhigher(P=0.01) among IMresistantgroupcompared toIMgood response CML patients(38.3%).RiskcalculationshowedthatCMLpatientswith ABCG2 C421C genotype had a higher risk for development of resistancewithOR1.87(95%CI:1.085–3.214,P=0.02).Whenthe frequencyofABCG2A421AwascomparedbetweenCMLpatients showinggoodresponseandresistancetoIM(16.8%vs.5.6%),the frequency was significantly higher in CML patients with good response (P=0.01)withOR0.29(95%CI:0.111–0.765,P=0.01).

Thisindicatesasignificantlowerriskofresistancedevelopmentfor CMLpatientswithABCG2A421Avariantgenotype.

Haplotypic frequencies of SNPs of ABCB1 and ABCG2 were determinedamongbothIMresponsiveandresistancegroupCML patients. ThemostfrequentlyobservedABCB1haplotypeswere 1236T/2677T/3435T (25%) and 1236C/2677G/3435C (21.7%).

Amonghaplotypes,thehaplotypeT1236T2677C3435withafrequency of 16.1% was significantly higher among IM responders with Pvalue0.04(Table1).WhereasthehaplotypeC1236G2677C3435with a frequency of 25.8% was significantly higher among the IM

Table1

AssociationofABCB1andABCG2genotypesandhaplotypeswithIMresponse.

SNPs Genotype n,frequency X² P OR 95%CI X² P

Responsive Resistant

ABCB11236 TT 43(40.2) 31(28.7) 7.39 0.25 0.60 0.339–1.058 3.14 0.08

CT 55(51.4) 55(50.9) 0.98 0.575–1.675 0.05 0.94

CC 9(8.4) 22(20.4) 2.79 1.217–6.374 7.38 0.01^*

ABCB12677 GG 19(17.8) 28(25.9) 8.54 0.14 1.62 0.841–3.126 2.10 0.15

GT+GA 56(52.3) 65(60.2) 1.38 0.802–2.364 1.35 0.25

TT+AA+TA 32(29.9) 15(13.9) 0.38 0.191–0.750 8.07 0.004^*

ABCB13435 CC 26(24.3) 31(28.7) 0.84 0.65 1.25 0.683–2.303 0.54 0.46

CT 64(59.8) 58(53.7) 0.78 0.454–1.338 0.82 0.37

TT 17(15.9) 19(17.6) 1.13 0.552–2.315 0.11 0.74

ABCG234 GG 52(48.6) 45(41.7) 1.73 0.42 0.76 0.441–1.295 1.04 0.31

GA 42(39.3) 52(48.1) 1.44 0.836–2.469 1.73 0.19

AA 13(12.1) 11(10.2) 0.82 0.350–1.922 0.21 0.65

ABCG2421 CC 41(38.3) 58(53.7) 9.09 0.01^* 1.87 1.085–3.214 5.12 0.02^*

CA 48(44.9) 44(40.7) 0.85 0.492–1.451 0.37 0.54

AA 18(16.8) 6(5.6) 0.29 0.111–0.765 6.88 0.01^*

Gene Haplotype Frequency X² P

Responsive Resistant

ABCB1 TTT 28.9 21.2 3.41 0.07

CGC 17.4 25.8 4.44 0.04^*

TTC 16.1 9.5 4.11 0.04^*

TGT 10.2 14.6 1.94 0.16

CTC 9.9 11.3 0.23 0.64

TGC 10.8 8.9 0.44 0.51

CGT 5.5 6.7 0.27 0.60

CTT 1.2 2.0 0.37 0.54

ABCG2 GC 38.1 43.7 1.4 0.24

AC 22.6 30.3 3.3 0.07

GA 30.1 22.0 3.7 0.06

AA 9.1 3.9 4.8 0.03^*

*P<0.05(Statisticallysignificant).

resistance group (P=0.04). For ABCG2 gene, only a single haplotypemarkerA34A421wasfoundtobesignificantlyassociated withIMgoodresponse(9.1%vs.3.9%,P=0.03).

ThedistributionofcytogeneticresponseamongCMLpatients withvariousABCB1andABCG2genotypesissummarizedinTable 2. There was no statistically significant association between cytogeneticresponseandgenotypefrequency,exceptforABCB1 2677.Thevariantgenotypes(TT, AAand TA)werefoundtobe associatedwithasignificantlylowerriskforCCyRwithOR0.48 (95%CI:0.239–0.957,P=0.03).For1236CCgenotypealso,anearly significantlowerriskassociationforcytogeneticresponsetoIM (OR:2.01,P=0.07),wasobserved.

The distributionof molecular response among CML patients withvariousABCB1andABCG2genotypesissummarizedinTable 3.Therewerestatisticallysignificantdifferencesinthemolecular responseofCMLpatientswith2677G>T/Aand421C>Agenotypes.

The presence of the variant genotype in ABCB1 2677 had a favorable impact on MMR achievement with OR 0.49 (95%CI:

0.248–0.974,P=0.04). Nearly significantlower risk association was observed for ABCB1 1236 CC genotype with molecular response (P=0.05). In ABCG2 421C>A, the frequency of MMR was44/99patients(44.44%)withtheCCgenotype,57/92(61.96%) withtheCAgenotypeand17/24(70.83%)withtheAAgenotype.

TheoddsratioofCCoverCA+AAwas2.20(95%CI:1.273–3.811, P=0.004).

3.2. ABCB1&ABCG2SNPsinIMtolerance

Table4showsassociationofABCB1andABCG2genotypeswith IM induced hemato- and non hemato-toxicity. However, the incidenceofsevereneutropeniawashigherinpatientswith1236 CCgenotype(13.3%,2/15)thaninpatientswithTC(11.9%,8/67)or TT(2.1%,1/47)genotypesbutstatisticallyinsignificant(P=0.14).

Nosignificantassociationofskinhypo-pigmentationorrashwas foundinpatientswithGG(30%,9/30),GA+GT(28.9%,22/76),and TT+TA+AA(8.7%,2/23)genotypes(P=0.12).

3.3. ABCB1&ABCG2SNPsinhepatotoxicitylevels

A comparison of ALT, ALP and bilirubinvalues among CML patientswithdifferentgenotypesofeach SNPissummarizedin Fig.1.NosignificantassociationwereobservedfortheseSNPs,as values of bilirubin, ALP and ALT did not differ significantly (P>0.05)amongpatientswithvariousgenotypes.Eventhough, differencesinthemeanvaluesofbilirubinwereobservedinABCG2 421 C>A, with 11.7

m

mol/L for CC, 9.7

m

mol/L for CA and 14.2

m

mol/L for AA genotype, it was statistically insignificant (P=0.10).Also,theALPvaluesshowedsomedifferencesinABCB1 2677G>T/A(ameanvalueof102.2U/lforGG,78.6U/IforGT+GA and89.8U/lforTT+TA+AAgenotypesrespectively)butstill,the differencewasnotstatisticallysignificant(P=0.28).

Table3

AssociationofABCB1andABCG2genotypeswithmolecularresponse.

SNPs Genotype MMR Non-MMR X² P OR 95%CI X² P

ABCB11236 TT 40 34 3.67 0.16 1.05 0.598–1.852 0.03 0.86

CT 66 44 0.65 0.381–1.123 2.38 0.12

CC 12 19 2.15 0.987–4.692 3.83 0.05

ABCB12677 GG 24 23 4.54 0.10 1.22 0.637–2.327 0.35 0.55

GT+GA 62 59 1.40 0.813–2.418 1.48 0.22

TT+AA+TA 32 15 0.49 0.248–0.974 4.23 0.04^*

ABCB13435 CC 30 27 0.05 0.98 1.13 0.616–2.077 0.16 0.69

CT 69 53 0.86 0.497–1.471 0.32 0.57

TT 19 17 1.11 0.540–2.269 0.08 0.78

ABCG234 GG 56 41 2.34 0.31 0.81 0.472–1.393 0.58 0.45

GA 47 41 1.11 0.641–1.909 0.13 0.72

AA 15 15 1.26 0.580–2.719 0.34 0.56

ABCG2421 CC 44 55 1.08 0.58 2.20 1.273–3.811 8.08 0.004^*

CA 57 35 0.60 0.349–1.047 3.25 0.07

AA 17 7 0.46 0.183–1.165 2.78 0.10

*P<0.05(Statisticallysignificant).

Table2

AssociationofABCB1andABCG2genotypeswithcytogeneticresponse.

SNPs Genotype CCyR Non-CyR X² P OR 95%CI X² P

ABCB11236 TT 46 28 3.67 0.16 0.71 0.401–1.265 1.35 0.25

CT 63 47 0.96 0.558–1.642 0.03 0.87

CC 13 18 2.01 0.930–4.353 3.24 0.07

ABCB12677 GG 24 23 4.54 0.10 1.34 0.701–2.567 0.79 0.37

GT+GA 65 56 1.33 0.768–2.293 1.03 0.31

TT+AA+TA 33 14 0.48 0.239–0.957 4.45 0.03^*

ABCB13435 CC 32 25 0.05 0.98 1.03 0.562–1.904 0.01 0.91

CT 70 52 0.94 0.547–1.624 0.05 0.83

TT 20 16 1.06 0.515–2.179 0.02 0.87

ABCG234 GG 52 45 2.34 0.31 1.26 0.734–2.171 0.71 0.40

GA 53 41 1.03 0.596–1.768 0.01 0.92

AA 17 7 0.51 0.199–1.268 2.18 0.14

ABCG2421 CC 55 44 1.08 0.58 1.09 0.637–1.879 0.11 0.75

CA 51 41 1.07 0.625–1.855 0.07 0.79

AA 16 8 0.62 0.255–1.527 1.08 0.30

*P<0.05(Statisticallysignificant).

A.Auetal./Biomedicine&Pharmacotherapy68(2014)343–349 346

4. Discussion

Although a number of factors may contribute to inter- individualvariabilityindrugresponse,thegenotypeofapatient is increasingly implicated in influencing drug disposition and activity.SNPsinABCB1and ABCG2have beendemonstrated to displayhighaffinityforIMandconferIMresistanceinvitroby extrudingIMfromhematopoieticcells [8]. IMis a substrateof ABCG2 and ABCB1in a narrow concentration range and actas inhibitorathigherconcentrationsduetoitshighaffinity[9].By contrast,thecontributionofABCB1polymorphismsinmodulating IMresistancehasyettobefullyclarified,astheeffluxofIMhave notbeenabletocorrelateABCB1over-expressioninIMresistant celllines.RNAisilencingofABCB1genewasshowntorestoreIM sensitivity and increase IM intracellular levels in multi-drug- resistantCML cellline [10,11].StudieswithCML patients have shown no association of 3435C>T polymorphism with the response to standard dose of IM (400mg/OD). In conjunction withourearlieranalysis,3435C>TSNPshowednoriskassociation with IM response in smaller patients group [12]. When the polymorphismwasevaluatedseparatelyeitherwiththecytoge- neticresponseor themolecularresponses, interestingly,ABCB1 2677 variant was associated with MMR in CML patients.

Furthermore, the wildtype ABCB1 haplotype (C₁₂₃₆G₂₆₇₇C₃₄₃₅) wasassociatedwithIMresistance,whichisinagreementwitha reportby Dulucqet al.inwhitepopulation [13].Althoughthey found a higher frequency of MMR in patients with non-G genotypesatposition2677,theycouldnotconfirmtheseresults in a larger patient cohort [14]. In contrast, our result is in accordancewiththefindingsofDeeniketal.[15],inwhichpatients withhomozygousABCB11236Tshoweda higherprobability to obtainMMR.Similarly,ahigherriskforIMresistancewasreported forCMLpatientswithhomozygousABCB13435Tallele,whereas better CCyR was observed for patients with the AG/AT/AA genotype at position 2677 by Ni et al. [16]. This has been attributed as possibly be due to thefact that carriers of 2677 variantgenotypehavelowerP-gpmessengerRNAexpressionthan

thosewhohad2677wildtypegenotype[17].Fewotherstudiesdid not findan associationbetween ABCB1 polymorphismsand IM response[14,18–20].

TheABCG2SNPsG34AandC421Aarethemostfrequentnon- synonymous polymorphisms in BCRP, which, like P-gp, causes removal ofIM.ABCG2 gene is over-expressed inCD34+ CD38–

humanhematopoieticstemcells.InCMLK-562celllines,higher levelsofABCG2mRNAandBCRPproteinwerereportedafterlong- termIMexposure and thelevelsdecreased graduallyat higher concentrations [21]. BCRP expression is lower in the livers of carriersof421AAgenotype[22].ExperimentsinvitrousingIM substratesshowedthattheA421AvariantofBCRPaffectstheIM accumulation[23].Consistentwiththis,alowerIMclearanceanda higherdose-adjustedIMtroughconcentrationwerefoundinCML patientswiththeABCG2A421Avariantgenotype[20,24],whereas theC421CgenotypewasassociatedwithincreasedIMresistance [19].OurresultsshowedthattheABCG2421variantAalleleis associatedsignificantlywithahigherrateofMMRinCMLpatients, andcouldbeutilizedasapredictivemarkerforIMresponse.Our resultsareinaccordancewiththestudybyKimetal.[19]which indicatedthattheA421AgenotypeexhibitedahigherMMRthan theCAorCCgenotype.Recently,Seongetal.[25]alsoreportedthat ABCG2 A421A genotype was significantly higher among CML patientswithMMRbutinsignificantlyassociatedwithcytogenetic response,whichis similarwithtothepresentstudy.Fewother previousreportsfailedtodeterminetheeffectofABCG2421C>A on the IM response and clearance [19,23,26,27]. Conversely, Takashietal.[20]demonstratedthatthedose-adjustedIMtrough concentration was significantly higher in patients with ABCG2 A421AthaninthosewithC421Cy.Therefore,theinter-individual genetic variation of the ABCG2 gene may contribute for the variabilityinthepharmacokineticsandclinicalresponseofIM.

TheroleofABCB1andABCG2polymorphismsinthedevelop- ment of IM intolerance and hepatotoxicity had not been documentedinearlierstudies.Thisstudyinvestigatedtheroles ofthesepolymorphismsinpatients’hematological/non-hemato- logical toxicityandliverfunctionparameters.However, present Table4

AssociationbetweenABCB1andABCG2genotypeswithhematologicalandnon-hematologicaltoxicitiesamongCMLpatientsundergoingIMtreatment.

SNPs Genotype NonHematologicalToxicities HematologicalToxicities

Nausea+ Vomit

Hypo- pigmentation+ SkinRash

JointPain+ Muscle Cramp

Fatigue+ Headache+ Fever

Anemia Thrombocytopenia Neutropenia

Yes No Yes No Yes No Yes No Yes No Yes No Yes No

ABCB11236 TT 14 33 11 36 8 39 4 43 4 43 7 40 1 46

CT 24 44 16 51 3 64 9 57 14 53 17 50 8 59

CC 5 11 6 9 1 14 3 12 2 13 0 15 2 13

P 0.82 0.39 0.71 0.47 0.19 0.53 0.14

ABCB12677 GG 7 24 9 21 4 26 2 28 7 23 7 23 4 26

GT+GA 31 46 22 54 6 70 10 65 10 66 13 63 6 70

TT+AA+TA 5 18 2 21 2 21 4 19 3 20 4 19 1 22

P 0.96 0.12 0.68 0.48 0.40 0.75 0.49

ABCB13435 CC 15 23 11 26 4 33 7 30 9 28 5 32 4 37

CT 21 47 14 53 5 62 5 61 8 59 16 51 6 67

TT 7 18 8 17 3 22 4 21 3 22 3 22 1 25

P 0.57 0.44 0.75 0.21 0.21 0.28 0.63

ABCG234 GG 16 37 18 35 4 49 6 46 6 47 7 46 3 50

GA 20 41 11 48 7 52 8 51 11 48 13 46 6 53

AA 7 10 4 13 1 16 2 15 3 14 4 13 2 17

P 0.70 0.18 0.64 0.95 0.55 0.42 0.61

ABCG2421 CC 23 45 15 52 7 60 7 60 10 57 12 55 8 59

CA 17 33 15 34 4 45 9 39 9 40 11 38 3 46

AA 3 10 3 10 1 12 0 13 1 12 1 12 0 13

P 0.73 0.59 0.90 0.15 0.63 0.47 0.28

*P<0.05(Statisticallysignificant).

Fig.1.Meanvaluesofbilirubin,ALPandALTindifferentgenotypegroupsofABCB1andABCG2.ThevaluesareshownasU/l.ALP:alkalinephosphatase;ALT:alanine aminotransferase.

A.Auetal./Biomedicine&Pharmacotherapy68(2014)343–349 348

study results showed no significant association between the studied polymorphisms and the incidence of severe toxicity inducedbyIM.Theseresultssuggesttheexclusionofthesedrug transportergenepolymorphismsasriskfactorsforseveretoxicity.

Thecurrentstudyexaminedalsotherelationshipsbetweengenetic variantsand hepatotoxicityduringIMtherapy. Butnoneof the SNPsshowedanyassociationwithbilirubin,ALPandALTlevels.

Manyfactors,otherthantheABCB1andABCG2genotypes(suchas IM dosage, the pharmacokinetics of IM in plasma and inter- individualvariationoftheseparameters),couldbeinfluencingthe livertoxicity.Furtherprospectivestudiesareneededtoelucidate theseunresolvedquestions.

5. Conclusion

OurresultsdemonstratedasignificantassociationoftheSNPs ABCB1 T1236C, G2677T/A and ABCG2 C421A withIM efficacy.

HaplotypepatternofC1236G2677C3435seemstobeassociatedwith higherrisk of IMresistance. Singlegeneticvariation marker of ABCB1G2677T/AandABCG2421C>Amayinfluencethemolecular response in CML with IMtherapy. Pretreatment genotypingof theseSNPsinCMLpatientsappearstobeusefulforpredictingIM resistancewhichmayallowthebestchoiceofdrugtreatmentfor CMLpatients.However,thesefindingsneedstobevalidatedwith otherpharmacogeneticvariantsandtheplasmaconcentrationof thedrugadministeredthroughprospectivestudieswithalarger patientpopulation.

Disclosureofinterest

The authors declare that they have no conflicts of interest concerningthisarticle.

Acknowledgements

Theworkwasfully supported byResearch University Grant (1001/PPSP/812067),Universiti SainsMalaysia.First authoris a recipientofUSMFellowship01/11.

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