Thymidylate synthase expression and genotype have no major impact on the clinical outcome of colorectal cancer patients treated with 5-fluorouracil

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ContentslistsavailableatScienceDirect

Pharmacological

Research

j o u r n al hom ep ag e :w w w . e l s e v i e r . c o m / l o c a t e / y p h rs

Thymidylate

synthase

expression

and

genotype

have

no

major

impact

on

the

clinical

outcome

of

colorectal

cancer

patients

treated

with

5-ﬂuorouracil

Marina

Vignoli

a,b,1

_,

_Stefania

_Nobili

c,1

_,

_Cristina

_Napoli

c

_,

_Anna

_Laura

_Putignano

a,b

_,

_Maria

_Morganti

c

_,

Laura

Papi

b

_,

_Rosa

_Valanzano

d

_,

_Fabio

_Cianchi

e

_,

_Francesco

_Tonelli

d

_,

_Teresita

_Mazzei

c

_,

_Enrico

_Mini

c

_,

Maurizio

Genuardi

a,b,∗

a_Fondazione_{Farmacogenomica}_Fiorgen,_Sesto_Fiorentino,_Italy

b_Dipartimento_di_{Fisiopatologia}_Clinica,_Sezione_di_Genetica_Medica,_Università_di_Firenze,_Firenze,_Italy

c_Dipartimento_di_{Farmacologia,}_Unità_di_{Chemioterapia,}_Università_di_Firenze,_Firenze,_Italy

d_Dipartimento_di_{Fisiopatologia}_Clinica,_Sezione_di_Chirurgia,_Università_di_Firenze,_Firenze,_Italy

e_Dipartimento_di_Area_Critica_Medico_Chirurgica,_Università_di_Firenze,_Firenze,_Italy

a

r

t

i

c

l

e

i

n

f

o

Articlehistory:

Received16February2011

Receivedinrevisedform12April2011

Accepted14April2011 Keywords: Colorectalcancer Thymidylatesynthase mRNAexpression Genotype 5-Fluorouracil

a

b

s

t

r

a

c

t

Backgroundandobjectives:Thymidylatesynthase(TS)expressionlevelsappeartoberelatedtoresponse to5-ﬂuorouracil-(5-FU)-basedchemotherapyincolorectalcancer(CRC)patients.Threepolymorphisms havebeenproposedasmodulatorsofTSexpression:atandemlyrepeatedsequence(2R/3R)inthe5UTR, aSNP(G>C)withinthe3Ralleleanda6bpdeletioninthe3_UTR.

ToevaluatetheinﬂuenceofTSexpressionandpolymorphismsonclinicaloutcomeof5-FU-treated patientsweperformedacomprehensivegeneticanalysison63CRCpatients.

Methods:TSexpressionlevelswereanalyzedinnormalandtumortissues.TScodingsequenceandUTR polymorphismswereinvestigatedonDNAfromnormaltissue.LOHanalysiswasperformedtodetermine tumorgenotype.

Results:Adifferenceindisease-freesurvival(DFS),althoughnotstatisticallysigniﬁcant,wasobserved betweenhighandlowmRNAexpressionlevels:patientswithlowlevelsshowedlongerDFS.The2R2R genotypeshowedsigniﬁcantlylowerexpressionthanthe3R3Rand2R3Rgenotypesinnormaltissue.No otherTSpolymorphismwasassociatedwithmRNAexpressionorclinicaloutcome.

Conclusions:Theresultsobtainedinthispilotstudyindicatethatthenumberof5UTRrepeatsisthe majorgeneticdeterminantofTSexpression.Thelackofassociationwithotherpolymorphismsmight bepartiallyexplainedbytheexistenceoflinkagedisequilibriumintheTSgene.Ourdatasupportthe growingevidencethatTScontrolmayrequiremultiplemechanismsactinginclosecoordinationwith oneanotherandsuggestthatTSgenotypingaloneintumorsamplesisnotsufﬁcienttoaccuratelypredict responseto5-FU.

1. Introduction

Morethan50yearsafteritsintroductionintoclinicalpractice, 5-ﬂuorouracil(5-FU)isstillafundamentaldruginthetreatment ofcolorectal cancer(CRC)and manyothertumors, eitheralone orincombinationwithotherdrugs[1,2].Severalvariables associ-atedwithgenesinvolvedinthe5-FUmetabolicpathwayhavebeen studiedfortheirpotentialrelationshipwithclinicaloutcomeand

∗ Correspondingauthorat:SectionofMedicalGenetics,DepartmentofClinical

Pathophysiology,UniversityofFlorenceMedicalSchool,VialeG.Pieraccini6,50139

Florence,Italy.Tel.:+390554271421;fax:+390554271413.

E-mailaddress:m.genuardi@dfc.uniﬁ.it(M.Genuardi).

1 _These_authors_contributed_equally_to_this_work.

responsetochemotherapy.Todatethemostwidelystudied molec-ularmarkeristhymidylatesynthase(TS),thebiologicaltargetof 5-FUandrelateddrugs.TSisthekeyenzymeofthedenovo syn-thesisofdeoxythymidinemonophosphate(dTMP),thatcatalyzes themethylationofdeoxyuridinemonophosphatetodTMP[3,4],an essentialstepinDNAsynthesis.

TheexpressionleveloftheTSgeneappearstoberelatedto clin-icaloutcomeandresponseto5-FUchemotherapyandhasbeen suggestedasapotentialprognosticand/orpredictivemarker. John-stonet al. [5] ﬁrstdemonstrated a correlation betweenlow TS levelsandimproved5-yeardisease-freesurvival(DFS)and over-allsurvival(OS)inrectalcancerpatientsreceiving5-FUadjuvant chemotherapy.Ameta-analysisbyPopatetal.[6]showedthatCRC patientswithadvanceddiseasetreatedwithTSinhibitorshada sig-niﬁcantlybetterOSiftheyhadlowTSexpressioninprimarytumors

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ormetastases,whereasapredictiveroleofTSexpressionwasnot establishedfortheadjuvantsetting.

ThreedifferentpolymorphismsintheTSuntranslatedregions (UTRs)havebeenproposedasmodulatorsofTSmRNA transcrip-tionalandtranslationalefficiency.The5UTRcontainsavariable numberof28bptandemrepeats(VNTR)[7,8].Althoughuptonine repeatshavebeendescribed,thevastmajorityofTSalleles har-boreither 2 or3 repeats,creating genotypesdefinedas 2R/2R, 2R/3Rand3R/3R,respectively.The3RallelespresentaG>Csingle nucleotidepolymorphism(SNP)atthe12thpositionofthesecond repeat[9].ThetwoallelesofthisSNParedefinedas3RGand3RC, respectively.Thethirdpolymorphismisa6bpinsertion/deletionat nucleotide1494withinthe3UTR[10].Inaddition,afurtherSNPin theVNTRregion,consistingofaC>Gsubstitutioninthefirstrepeat ofthe2Rallele,hasrecentlybeendescribed[11].

ManystudieshavebeenconductedtoinvestigatewhetherTS genotypesmightexplaindifferencesinmRNAexpressionlevels, buttheresultsareheterogeneousandevencontroversial.

Invitroexperimentshaveshown thatthe3R/3Rgenotype is associatedwithhigherlevelsofTSgeneexpressionthanthe2R/2R genotype[8].SubsequentexperimentsonDNAsamplesfromCRC patientsprovidedsupporttotheseresults,indicatingthatthe3R sequencehasgreatertranscriptionalefﬁciencythanthe2Rallele

[12,13]. The influence of the VNTR on TSexpression has been ascribedtothepresenceofaUSFfamilyE-boxconsensuselement inrepeatunitscontainingtheGnucleotideatposition12.TheG>C substitutioneliminatestheUSF-1bindingsite,thusabolishingthe translationenhancereffectofthe3Rallele[9].Eachofthefirsttwo repeatsofthe3RGallelehasaGatthisposition;therefore,3RG allelescontaintwopotentialUSF-1bindingsites,whereastheGis presentonlyinthefirstrepeatof2Rand3RCalleles,that conse-quentlyhaveasingleUSF-1site.

Theins/delpolymorphismwithinthe3UTRseemstomodulate TSexpressionbyaffectingmRNAstability;the6bpdelallelehas beenassociatedwithdecreasedmRNAstabilityinvitroandlower intratumoralTSexpressioninvivo[14,15].

Bycontrast,otherstudiesdidnotdetectanycorrelationbetween TSpolymorphismsandmRNAorproteinexpressionlevels[16–18], orevenasigniﬁcantlydecreasedTSmRNAexpressioninsamples frompatientswiththe3R/3Rgenotype[19].

Discrepanciesinresultsamongdifferentstudiesmaybedue tomethodologicaldifferencesor incompleteanalysisleadingto partial results. Some groups analyzed TS expression levels by immunohistochemistry (IHC) [14,15,20] while others used real timequantitativePCR[12,21,22].Inaddition,lossofheterozygosity (LOH),thatmodiﬁesTSgenotypeintumorcells,hasbeenreported toaffecttumorresponseandsurvivalandTSexpression[21,23].

Inordertotakeintoaccountallmajorintrinsicfactors poten-tiallyinvolvedintherelationshipbetweenTSgenotype,expression andclinicalresponseto5-FU,weperformedacomprehensivepilot study;thisinvolvedinvestigationofthewholeTScodingsequence, 5 and 3 UTRpolymorphisms andmRNAexpression in normal and matchingtumor tissuesof a series of CRCpatients receiv-ingtreatmentwith5-FUonly.Weanalyzedpossibleassociations betweenTSgenotypeand expression aswellas thosebetween theseexperimentaldataandsurvival parameters (DFSand OS). AdditionalassociationsbetweenTSgenotype,expressionand clin-ical/pathologicalcharacteristicswereexplored.

2. Materialsandmethods

2.1. Tissuesamples

Primary tumor and corresponding colonic mucosa explants obtainedfrom63 CRCpatientsatsurgery werefrozenin liquid

nitrogen until molecularanalysis. Normal colonic mucosa was takenatadistanceof∼10cmfromthetumors.Immediatelyafter resection,thetumorsamplewasdividedintoequalportionsafter washingandremoval ofnecrotictissues.Somespecimenswere freshfrozeninliquid nitrogen,and oneportionwasembedded inparaffintoconfirmhistologically thatitwasnotsignificantly contaminatedbynormalornecrotictissue,orlymphocytes.

Allsampleswerecollectedbeforecombinationchemotherapy became standard practice both in the adjuvant setting and for advanceddiseaseinCRCpatients.Patientstreatedintheadjuvant setting receivedfolinic acidand 5-FU according to the sched-uledescribedinthepooledanalysisbytheIMPACTinvestigators

[24].Patientstreatedforadvanceddiseasereceivedchemotherapy accordingtotheMachoverregimen[25].Inbothcaseshigh-dose folinicacidwasadministered.

Informedconsentwasobtainedfromallpatientsfortheuse ofspecimensandclinical/pathologicaldataforresearchpurposes accordingtotheguidelinesestablishedbythelocalethical com-mittee.

2.2. Geneexpressionanalysis

TotalRNAwasisolatedfromtumorandnormaltissuesusing a Trizol RNA isolation kit with glass-fiber filter purification methodology(RiboPure kit, Ambion Inc.,Austin, TX,USA). RNA concentrationandpuritywereverifiedbyaGeneQuantII spec-trophometer(PharmaciaBiotech,Cambridge,UK).

cDNAwasgenerated from10_␮gof totalRNA usingrandom primers and the M-MLV reverse transcriptase RNase H minus (PromegaCorporation,Madison,WI,USA)accordingtothe man-ufacturer’sprotocol.

Real-time polymerase chain reaction (RT-PCR) analysis was performedwiththeABIPRISM7900HTFastSequenceDetection System(AppliedBiosystems,FosterCity,CA,USA).

Apredesignedandvalidatedgene-speciﬁcprobe-basedTaqMan GeneExpressionAssayfromAppliedBiosystems(FosterCity,CA, USA)wasusedfortheTSgene.Reactionswereperformedusing TaqmanFastUniversalPCRMasterMixNoAmpEraseUNG;twoto threereplicatesforeachreactionwereplatedonto96-wellplates. ThePCRprogramwas95◦Cfor20sand40cyclesof95◦Cfor1s and60◦Cfor20s.

The housekeeping gene glyceraldehyde 3-phosphate dehy-drogenase (GAPDH) was used as endogenous reference for standardization (TaqMan Endogenous Control concentration-limitedprimer,AppliedBiosystems,FosterCity,CA,USA),andthe HumanReferenceTotalRNA(Stratagene,LaJolla,CA,USA)wasused asacalibratorsample.TheexpressionlevelsofTSmRNAwere nor-malizedtotheendogenousreferenceandexpressedrelativetothe calibratoras2−CT,accordingtothecomparativeCTmethod[26]. Avalidationexperimentwasperformedtodemonstratethatthe efﬁcienciesofTSandreferencegeneampliﬁcationswere approxi-matelyequal,usingastandardcurvemethodwithseveraldilutions ofthecDNAcalibratorsample.

2.3. TSgenotyping

GenomicDNAextractionwasperformedusingBIOROBOTEZ1 (Qiagen,Italy)andEZ1DNATissueKit(Qiagen,Italy)accordingto themanufacturer’sprotocol.

In order todetect rarecoding sequence variations,genomic DNA samples were screened by denaturing high-performance liquid chromatography (dHPLC) on a Transgenomic Wave Sys-tem(TransgenomicCo.,Omaha,NE,USA).Primersandconditions usedforPCRampliﬁcationanddHLPCanalysisareavailableupon request.

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Amplificationofthe5UTRtractcontainingtheVNTRandG>C SNPwasperformed aspreviously described [22]. PCR products wereelectrophoresedontoa2.5%agarosegel.Amplification prod-uctsof213and241bpwereobservedforthe2Rand3Ralleles, respectively.Tenmicrolitersoftheamplificationproductsfromall samplesweresubsequentlydigestedwiththerestrictionenzyme HaeIII,whichallowsrecognitionoftheG/CSNPinthe3Rallele. HaeIIIdigestionalsoallowstoinvestigatetheC/GSNPlocatedin theproximalrepeatofthe2Rallele.DigestedPCRproductswere electrophoresedontonon-denaturingpolyacrylamidegels.

For 1494del6bp analysis, genomic DNA was ampliﬁed by PCR using the following primers: forward 5 -CAAATCTGAGG-GAGCTGAGT,reverse5-TGAGCAGATAAGTGGCAGTACAina reac-tion containing100ng DNA, 1.5mM MgCl2, 0.25mM

deoxynu-cleotidetriphosphates,30pmolofeachprimerand1.25UofTaq polymerase.Cyclingconditionswere:denaturationat95◦Cfor30s, annealingat60◦Cfor30sandelongationat72◦Cfor30s,for35 cycles.TheampliﬁedfragmentsweredigestedwithDraIandthe productsseparatedona2.5%agarosegel.Theexpectedfragment sizeswere92bp+60bpforthecommonallele(6bpins)and146bp fortherareone(6bpdel).

LOH was investigated by comparing genotypes of matched tumorandnormaltissuefromthesamepatient.Patients homozy-gousfortheVNTR,G>Cand6bpdeletionpolymorphismswere analyzedforothermicrosatellitemarkerslocatedupstreamand downstreamoftheTSgeneregion(D18S170,D18S1140,D18S1372, D18S498, D18S59). Primerswere labelled with6-FAM to allow detectionoftheampliﬁedproductsbyanABIPrism310Genetic Analyser.LOHwasdeterminedbyassessmentofpeakheightratios betweentumorandconstitutionalallelesusingthefollowing for-mula:

constitutionalallele2/constitutionalallele1 tumorallele 2/tumorallele 1 .

Aratio>1.5indicatedlossofallele2,aratio<0.5indicatedloss ofallele1,andaratiobetween0.51and1.49indicatedretentionof bothalleles.

LOH analysis couldbe performed on60 out of 63 patients because of unavailability of two tumor DNA samples and due tothepresenceofmicrosatelliteinstability,thatprevented LOH

Table1

NumberofUSF-1bindingsitesinTS5_UTR_alleles_and_genotypes.

Genotype No.ofUSF-1bindingsitesa

2R/loss 1(1/0) 2R/2R 2(1/1) 2R/3RG 3(1/2) 2R/3RC 2(1/1) 3RC/loss 1(1/0) 3RG/loss 2(2/0) 3RG/3RC 3(2/1) 3RG/3RG 4(2/2)

a_In_brackets:_numbers_of_USF-1_binding_sites_for_each_allele_comprised_in_the

genotypecombination.

assessment,inathirdsample.SamplesshowingLOHweredeﬁned as2R/lossand3R/losstoindicatetheallelethatwasretainedin tumorDNA.

2.4. Statisticalanalysis

Thecorrelations betweenTSmRNAexpressionintumorand normaltissueandclinical/pathologicalcharacteristics(sex, histo-type,tumorsite,stageandgrading)andgenotypeswereanalyzed usingANOVAandt-test.Theassociationbetweengenotypesand clinical/pathologicalfeatureswasanalyzedbythe2_test.

Inordertoevaluatetherelationshipsbetweenresponseto ther-apyandgenotypesorpathologicalcharacteristics,patientswere categorizedasrespondersornonrespondersonthebasisoftheir disease-freeanddiseaserecurrencestatus,respectively,andthe2

testwasused.

ThepairedStudent’st-testwasusedtoanalyzethecorrelation ofTSgeneexpressionbetweennormalandtumortissues.

5 UTRTSgenotypesweregroupedaccordingtothenumber of USF-1 binding sites (Table 1).Comparisons were performed betweenpatientswith1–2USF-1TSbindingsitesand with3–4 USF-1TSbindingsitesforbothnormalandtumortissue.

Genotypesofthe3UTRwerealsoconsidered,aloneandin com-binationwith5genotypes.Thepresenceoflinkagedisequilibrium (LD)between5 and3 UTRTSpolymorphismswasinvestigated

Fig.1. RelationshipsbetweenTSexpressionand5_UTR_{polymorphisms}_in_normal_tissue._(A)_Relationship_with_VNTR_number_(2R2R_vs_3R3R,_p₌_0.020;_2R2R_vs_2R3R,

p=0.049;2R3Rvs3R3R,p=0.322);(B)Relationshipwithcomplete5UTRhaplotype(VNTRnumberandG>CSNP;2USF-1bindingsitevs3–4USF-1bindingsites,p=0.258);

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Table2

Mainclinical/pathologicalfeaturesofcolorectalcancerpatients.

No.ofpatients 63 Age Medianvalue 61 Range 23–76 Sex M 32 F 31 Stage(AJCC) II 27 III 25 IV 11 Grading G2 52 G3 11 Histotype Adenocarcinoma 58 Colloid 5

Siteofprimarytumors

Leftcolon 23

Transversecolon 6

Rightcolon 9

Rectum 25

Typeof5-FUchemotherapy

Adjuvant 52

Palliative 11

usingMIDASsoftware[27];D values>0indicatethepresenceof

LD.

PatientsweredividedintohighandlowTSmRNAlevelgroups usingthemedianvalueasacut-off.Patientswhodiedduetocauses unrelatedtocolorectalcancerwereexcluded.OSwascalculated fromsurgerytothedateoflastfollow-upordeath,DFSfromsurgery totheﬁrstevidenceofdisease.Medianfollow-uptimewas com-putedforallpatientsaliveatthetimeofanalysis.Survivalcurves wereestimatedbytheKaplanMeiermethodandcomparedwith thelog-ranktest.

AnalyseswerecarriedoutusingtheSPSSversion15Software.P values<0.05wereconsideredsigniﬁcant.

3. Results

Clinical/pathologicalcharacteristicsofthepatientsinvestigated arereportedinTable2.Theserieswascomprisedof32malesand31 females,withamedianageatdiagnosisof61years(range23–76). Allpatientsreceived5-FUchemotherapy:52asadjuvantand11as palliation.

Interindividual variation in TS mRNA expression was 25.5 (TS/GAPDHratios:0.35–8.93;median value1.53)and231.5fold (TS/GAPDHratios:0.04–9.26;medianvalue1.93)intumorand nor-maltissue,respectively(datanotshown).However,overall,mean TSmRNAexpressionwasnotsigniﬁcantlydifferentbetweentumor andnormaltissue(p=0.076,mean±SD:1.96±1.82vs2.67±2.27, respectively).

GenotypedistributionsoftheVNTR,G>CSNP,ins/del6bp poly-morphismsandtheircombinationsintumorornormaltissueare shownin Table3.LOH wasfoundin 29/60(48%) samples.The recentlydescribedC>GSNPintheﬁrstrepeatofthe2Rallelewas absentinthisseries.Genotypefrequenciesofthe5VNTRand3 6bpdeletionpolymorphismswerewithinHardy–Weinberg equi-librium(datanotshown).Analysisofalleliccombinationsatthese sitesshowedthepresenceofLD:the3RGallelewasassociatedwith the6bpdelalleleinthe3UTR(D=0.68)and,ontheotherhand, the2Rallelewasassociatedwithabsenceof6bpdel(D=0.67).

NosequencevariationwasfoundwithintheTScodingsequence byextensivedHPLCscreening.

TheVNTRgenotypewasassociatedwithTSexpressionlevels innormaltissues,withthe2R2Rgenotypeshowingsigniﬁcantly

Table3

ConstitutionalandtumorTSgenotypesincolorectalcancerpatients.

Constitutionalgenotype(%)a _Tumor_genotype_(%)b

5_UTR 2R/LOH – 9(15) 2R2R 11(17) 9(15) 2R3R 30(48) 12(20) 2R3RC 14 7 2R3RG 16 5 3R/LOH – 20(33) 3RC – 8 3RG – 12 3R3R 22(35) 10(17) 3RG3RG 13 5 3RG3RC 8 5 3RC3RC 1 3_UTR 6bpdel/LOH – 12(20) 6bpins/LOH – 17(28) 6bpdel/6bpdel 15(24) 7(12) 6bpins/6bpins 22(35) 12(20) 6bpdel/6bpins 26(41) 12(20) a_n₌_63.

b_n₌₆₀_(tumor_genotype_was_not_analyzed_in₃_patients_due_to_{unavailability}_of

DNAfromtumortissue).

lower mRNA expression than the 3R3R and 2R3R genotypes (p=0.020and 0.049, respectively;Fig.1a).However, there was nosigniﬁcantassociationinnormalcolonicmucosabetweenTS expressionandthecomplete 5UTRgenotypes(VNTRcombined withtheG>CSNP),whentheseweredividedinto2groups cor-responding to the presence of 2 and 3–4 USF-1 binding sites, respectively(Fig.1b).

NootherrelationshipwasobservedbetweengenotypeandTS expressionlevelseitherinnormalortumortissue.Inparticular,TS expressionlevelsweresimilarbetweentumorsampleswith1–2 and3–4USF-1bindingsites(Fig.2).

Although not signiﬁcant, a difference in DFS was observed betweenhighandlow tumorTSmRNAexpressionlevelsinthe groupofpatientswithcompletelyresectedtumors(Fig.3):patients withlowTSmRNAlevelshadalongerDFS(p=0.122).Ontheother

Fig.2.RelationshipbetweenTSexpressionand5_UTR_genotype_(VNTR_and_G_>_C

SNP)intumortissues(1–2USF-1bindingsitevs3–4USF-1bindingsites,p=0.626);

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Fig.3.Survivalparametersofpatientswhoreceived5-FUchemotherapyaccording

toTSgeneexpressionlevels.LowTSmRNAexpression<1.53(medianvalue);high

TSmRNAexpression>1.53(medianvalue);n=52,p=0.122.

hand,nodifferencewasobservedbetweensurvivalparameters(OS andDFS)andTSexpressioninnormaltissue(datanotshown).

CombinedanalysisofTSVNTRandG>Cgenotypesbothin nor-malandtumortissueshowedthatcompletelyresectedpatients (n=52) with 1–2USF-1 bindingsites had a prolonged survival comparedtopatientswith3–4USF-1bindingsites.Howeverthis differencewasnotstatisticallysigniﬁcant(Fig.4).Similarly,no sig-niﬁcantdifferencewasobservedwhenOS wasevaluatedinthe entirecaseseries(n=63)(datanotshown).

EvaluationofTS3UTRpolymorphismaloneorincombination withthe5UTRgenotypedidnotshowanysigniﬁcantdifference inrelationtosurvivalparameters(datanotshown).

Norelationshipwasobservedbetweenclinical/pathological fea-turesandTSexpressionlevelseitherinnormalortumortissues. Likewise,nocorrelationbetweenalleleandgenotypefrequencies inthe5 or3 UTR,includingrepeatnumberintheVNTR,G>C SNPand 6bpdeletionpolymorphisms, and clinical/pathological featureswereobserved(datanotshown).

Finally, no association between clinical features/TS geno-types/TSexpressionandresponsetotherapywasobserved,with theexceptionofastatisticallysigniﬁcantvalue(p=0.007)between Duke’s stageand response:patientswithDuke’s stage Bhad a

lowerincidenceofdiseaserecurrence(47.8%)comparedtostage Cpatients(69.3%).

4. Discussion

In thepresent study we have extended ourprevious analy-sisoftherelationshipbetweenTSgenotype,TSmRNAlevels,and responseto5-FUtreatmentinCRCpatients[22].Tothispurpose, weinvestigatedexpressionandgenotypesinbothnormalcolonic mucosaandtumortissueandweanalyzedfurthergeneticvariables, includingthewholeTScodingsequenceinconstitutionalDNA,LOH intheTSregion,3UTRpolymorphisms,andestimateofLD.

Overall,nosigniﬁcantcorrelationsbetweenTSalleles,genotypes orexpression,andclinicalparameters,includingresponseto5-FU, wereobserved.Althoughthedifferencewasnotstatistically signiﬁ-cant,resultsontherelationbetweensurvivalandTSexpressionare inkeepingwiththoseobtainedinourpreviousstudy[22]aswell asbyotherauthors[5,12,28,29]:patientswithlowTSexpression levelstendtohaveaprolongedDFScomparedtothosewithhigh TSexpressionlevelsintumortissue.

Since both in vitro and in vivo dataindicate that intragenic polymorphismsmayinfluenceTSexpressionlevels[8,9,12,30],we investigatedpotentialgenotype/mRNAcorrelationsinthisseries. AmongthedifferentintragenicTSvariantsanalyzed,asignificant associationwasonlyfoundbetween5UTR28bprepeatnumber andTSmRNAexpressioninnormal,butnotintumor,tissue.The intrarepeatG>CSNPapparentlydidnothavea majorinfluence ontheeffectsofrepeatnumbers,sincenodifferencewasobserved whencomplete5UTRgenotypeswereassessedagainstexpression, clinicalcharacteristics,survivalandresponseto5-FUtreatment.

Since the influence of theVNTR on TSexpression has been attributedtothepresenceofoneUSFfamilyE-boxconsensus ele-mentinrepeatunitscontainingtheGnucleotide,weclassifiedTS5 UTRgenotypesaccordingtothenumberofUSF-1bindingsites.The absenceofasignificantcorrelationbetweencomplete5UTR geno-typeandclinicalparametersindicatesthatthenumberofUSF-1 sitesdoesnothaveamajorinfluenceonsurvival.

Theseﬁndingssuggestthatthenumberofrepeatscouldbemore importantthantheirsequencedifferencesfortheregulationofTS mRNAexpressioninvivo.Otherauthorsreportedsimilarresults, conﬁrmedbothattheRNAandproteinlevel,onCRCtissuesamples

[31].Ontheotherhand,apositivecorrelationbetweenTSprotein expression/activityandthe3RGalleleinnormalmucosahasalso beenobserved[32].Thislatterobservationisconsistentwiththe experimentalresultsthatprovidedoriginalevidenceforaroleof theG>CSNPonTStranscriptionalefﬁciency[9].Ithasalsorecently beensuggestedthatthepositionoftheGnucleotideintherepeat

Fig.4.OverallsurvivalaccordingtothenumberofUSFbindingsitesintheTS5_UTR_observed_in_{constitutional}_(n₌_52,_p₌_0.090)_(A)_and_tumor_genotypes_(including_LOH

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clustermaybeimportantfortranscriptionalefﬁciency:its loca-tioninthemost5 repeathasbeenfoundtobeassociatedwith highexpressionlevels,regardlessofthepresenceofadditional G-containingrepeats[33].However,whileinvitrostudiesarevery importanttounderstandthepathophysiologicalmechanismsofTS regulation,theymaynotreﬂectmorecomplexinvivoconditions, sincetheregulationofTSexpressionislikelydependentonmultiple cisandtransfactors.

TheapparentlycontrastingresultsontheeffectsofVNTRrepeat numberandTSmRNAexpressionobtainedinmucosaandtumor samplescouldberelatedtotheoccurrenceofsomaticmutations andepigeneticalterationsinvolvingTSaswellasadditionalloci implicatedinthecontrolofTSexpressionintumorcells.Deletions ofchromosome18areafrequenteventincolorectalcarcinogenesis and,whentheyinvolvetheTSlocus,theycancauseareductionin itsexpressionlevels.Sofar,moststudiesanalyzingTSprognostic andpredictiverolehavenotconsideredthepossibleoccurrenceof LOHintumorcells.Inaddition,whilemostpreviousstudieslimited LOHanalysistocasesthatwereheterozygousforoneofthethree TSintragenicpolymorphisms[17,21,23,34],weinvestigated ﬂank-ingextragenicpolymorphismsinordertoincreasethenumberof informativesamples.Overall,theresultsobtainedindicatethatLOH alonecannotaccountforthedifferentcorrelationsbetween geno-typeandRNAexpressionobservedinnormalandtumortissue,and thatotherfactorsareimplicatedinthecontrolofTSexpressionin CRCcells.

Thelackofanyassociationbetweenindividualandcombined TSpolymorphismsandclinicaloutcomefollowing5-FUtreatment in this seriesof CRC patientsis in accordance withtheresults ofrecent studies[35,36].Otherstudieshave reported contrast-ingresultsontheclinicalsigniﬁcanceofthethreecommonUTR TSpolymorphisms[16,17,37,38].Itshouldbeconsideredthatsome authorsassignhighandlowexpressionlevelstothe3R3Rand2R2R genotype,respectively,onthebasisofpreviouslypublisheddata

[35–38].However,theseassignmentsmaynotbecorrect,sincewe, aswellasotherauthors[16,17],haveobservedthatTSexpression levelsdonotcorrelatewithTSgenotype.

Ingeneral,expressionandsurvivalanalysesinrelationto geno-typearecomplicatedbythepresenceofmultipleTSpolymorphisms thatoccurindifferenthaplotypecombinationscontainingalleles witheithersynergicoroppositeconsequencesonTSmRNAlevels. TheexistenceofLD,documentedbyusandotherauthors[14,35], maypartiallyexplainthediscrepanciesbetweenresultsobtained invitroandonclinicalsamples.Sincethe3RGalleleisassociated withthe36bpdeletion,thatisthoughttoreducemRNAstability, theoveralleffectshoulddependontheinteractionbetweenthese variants.ThesameappliestootherTShaplotypecombinations.

RareDNAvariantsintheTScodingsequencemayalso inﬂu-encetheeffectsoftheUTRpolymorphisms[39].However,noneof thepatientsincludedinthisstudy,thatistheﬁrstonetoscreen thewholeTScoding sequencefor prognosticpurposes,showed alterations.

Furthermore,TStranscriptionandtranslationarelikely inﬂu-enced by other genes, whose sequence (e.g. p53) [40] and expression(e.g.AEG-1)[41]canbealteredintumortissues.Ithas beensuggestedthatthep53statuscouldplayaroleinTS expres-sionin tumorcells, by altering transcription and/or translation levels[40].Ithasalsobeenshownthatastrocyteelevatedgene-1 (AEG-1),knowntoaugmentinvasion,metastasisandangiogenesis, directlycontributesto5-FUresistance,sinceitinducesthe expres-sionofLSF(lateSV40factor),atranscriptionfactorthatregulates theexpressionofTS[41].

TheTSproteinhasalsobeenshowntoinhibitthetranslation ofTSmRNAinanautoregulatorymanner[42],suggestingthatTS genotypingaloneisnotsufﬁcienttoaccuratelypredictresponse to5-FU. Itisalsolikelythatothergenescodingforenzymesor

proteinsinvolvedinthemechanismofactionandintheinactivating oractivatingmetabolismof5-FUareinvolvedintheoutcomeof colorectalcancerpatientstreatedwith5-FU[43].

Ithasalsobeenobservedthatanincreaseintheintracellular poolofreducedfolatesfollowingexposuretofolinicacidenhances 5-FUantitumoractivity[44,45].Thisisduetoanincreased inhi-bition of TS enzyme activity via the formation of a covalent ternary complex among the active 5-FU metabolite 5-ﬂuoro-2’-deoxyuridine-5’-monophosphate, TS and the 5,10-methylene tetrahydrofolatecofactor[3,4,46].Thisultimatelyleadstogreater sensitivityto5-FU[47]andimprovesresponse rateand overall survivalincolorectalcancerpatientscomparedwith5-FUalone

[48].Differentsizeandcompositionofcellularfolatecofactorpools maythusbeanotherimportantfactorofvariabilityto5-FU treat-ment.Theuseofhigh-dosefolinicacidinourcaseseries[24,25]

potentiallyallowedmaximal enhancementofclinical5-FU anti-tumoractivity.However,interindividualvariabilityinthesizeand compositionofcellularfolatepools,possiblymediatedby polymor-phismsoffolatetransportersormetabolicenzymes(e.g.MTHFR)

[49]mayhaveoccurredinourseries.Thisaspectwasnotstudied andwarrantsfurtherinvestigationonlargerseries.

Inconclusion,wedidnotﬁndsigniﬁcantcorrelationsformost parametersevaluatedinthisstudy,despitecomprehensive analy-sisofTSgenevariantsandRNAexpressioninbothnormalcolonic andtumortissue.However,duetotherelativelylimitedsample size,weakeffectsofadditionalgenotypicvariationscannotbe com-pletelyexcluded.Furtherinvestigationsonlargersampleseriesare neededtoclarifywhetherTShasarelevantroleindetermining responsetoFUtreatmentandcanbeusedintheclinicalsetting. MethodologicalissuesrelatedtoquantitationofTSRNA,protein expressionandenzymeactivity,aswellasthepotential involve-mentofadditionalconstitutionalandacquiredgeneticfactorswill alsoneedtobeaddressed.

Acknowledgements

SupportedbyagrantfromtheUniversityofFlorence(ex60%)to MG,agrantfromMinisterodell’Istruzione,dell’Universita’edella Ricerca,Rome(PRIN 2005)toTM, and bycontributionsofEnte CassadiRisparmiodiFirenzetoFiorgen,MGandEM,of Associ-azioneItalianaperlaRicercasulCancro,MilantoEMandofGruppo OncologicoChirurgicoCooperativoItaliano,FlorencetoEM.

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