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Comprehensive evaluation of risk factors for lymph node metastasis with papillary thyroid carcinoma in Southwest China patients

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Abstract

Background: With the increasing incidences of papillary thyroid cancer(PTC), it is important to risk-stratify patients who may have more aggressive tumor biology. This study aimed to evaluate the risk factors for lymph node metastasis with PTC in Southwest China Patients which may provide a substantial reference for clinical diagnosis and treatment. Methods: 1045 PTCs (313 PTMC and 732 non-PTMC) between August 2016 and August 2019 were examined totally (including one Tibetan). BRAF V600E mutation was tested in all samples. The clinical data (gender, age, tumor location, sample source and pathological features) were retrospectively analyzed. Logistic regression analysis was performed to evaluate independent risk factors for LNM. Results: 181 out of 313 PTMC cases (57.8%), 145 out of 732 non-PTMC cases (19.8%) had BRAF V600E mutation, the Tibetan had a double mutation of BRAF L597Q and V600E in two separate lesions. In PTMC, significant difference in gender and sample source was found (BRAF V600E mutation vs. wild-type). In non-PTMC, significant difference in gender was found (BRAF V600E mutation vs. wild-type). The female (OR=1.952; 95% CI= 1.373-2.774; P= 0.00), age (31-59 years) and diameter of tumor ≤1cm (OR=3.273; 95% CI= 2.417-4.432; P=0.000) were significant independent predictors of LNM in all PTCs. In PTMC, the female (OR= 3.002; 95% CI= 1.654-5.446; P= 0.00) was a significant independent predictor of LNM. The tumor in left and right lobes simultaneously was an independent protective factor of LNM in each group (PTCs: OR=0.287; PTMC: OR=0.170; non-PTMC: OR=0.441, respectively). The BRAF V600E mutation rate of US-FNAC was much higher than FFPE in PTMC (P=0.018). Conclusions: Unlike previous research, our findings suggested that the female patients and diameter of tumor ≤1cm were risk factors for LNM and the BRAF V600E wild-type of PTMC might be more aggressive than others. Interestingly, the position of tumor in bilateral thyroid simultaneously was an independent protective factor for LNM. The US-FNA should be recommended for gene analysis (BRAF V600E) in PTMC. The BRAF L597Q mutation may be an independent aggressive factor in the Chinese Tibetan population. Hence, clinicians should consider an individualized treatment according to gene mutation, gender, age, tumor size and location of tumor in order to achieve a better therapeutic efficacy.
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Preprint:Pleasenotethatthisarticlehasnotcompletedpeerreview.
Comprehensiveevaluationofriskfactorsforlymph
nodemetastasiswithpapillarythyroidcarcinomain
SouthwestChinapatients
CURRENTSTATUS:POSTED
YanDong
ChongqingMedicalUniversityFirstAffiliatedHospital
DanWang
ChongqingMedicalUniversityFirstAffiliatedHospital
YishengLuo
ChongqingMedicalUniversityFirstAffiliatedHospital
LingChen
ChongqingMedicalUniversity
HuiliBai
ChongqingMedicalUniversityFirstAffiliatedHospital
YifanShen
ChongqingMedicalUniversityFirstAffiliatedHospital
YangliZhang
ChongqingMedicalUniversityFirstAffiliatedHospital
XuepingChen
ChongqingMedicalUniversityFirstAffiliatedHospital
XinliangSu
ChongqingMedicalUniversityFirstAffiliatedHospital
JinqiuZhao
ChongqingMedicalUniversityFirstAffiliatedHospital
HuandongLiu
TibetUniversityMedicalCollage
JungaoLu
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GuizhouMedicalUniversity
ZuoyiYao
ChengduFirstPeople'sHospital
YajingZhao
ChongqingMedicalUniversityFirstAffiliatedHospital
ChanglongHe
ChongqingMedicalUniversityFirstAffiliatedHospital
XiaosongLi
ChongqingMedicalUniversityFirstAffiliatedHospital
250894831@qq.comCorrespondingAuthor
10.21203/rs.3.rs-26100/v1
SUBJECTAREAS
CancerBiology Oncology
KEYWORDS
Papillarythyroidcarcinoma(PTC);Papillarythyroidmicrocarcinoma(PTMC);non-
papillarythyroidmicrocarcinoma(non-PTMC);BRAFmutation;genetictesting
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Abstract
Background:Withtheincreasingincidencesofpapillarythyroidcancer(PTC),itisimportanttorisk-
stratifypatientswhomayhavemoreaggressivetumorbiology.Thisstudyaimedtoevaluatetherisk
factorsforlymphnodemetastasiswithPTCinSouthwestChinaPatientswhichmayprovidea
substantialreferenceforclinicaldiagnosisandtreatment.
Methods:1045PTCs(313PTMCand732non-PTMC)betweenAugust2016andAugust2019were
examinedtotally(includingoneTibetan).BRAFV600Emutationwastestedinallsamples.Theclinical
data(gender,age,tumorlocation,samplesourceandpathologicalfeatures)wereretrospectively
analyzed.LogisticregressionanalysiswasperformedtoevaluateindependentriskfactorsforLNM.
Results:181outof313PTMCcases(57.8%),145outof732non-PTMCcases(19.8%)hadBRAF
V600Emutation,theTibetanhadadoublemutationofBRAFL597QandV600Eintwoseparate
lesions.InPTMC,significantdifferenceingenderandsamplesourcewasfound(BRAFV600Emutation
vs.wild-type).Innon-PTMC,significantdifferenceingenderwasfound(BRAFV600Emutationvs.wild-
type).Thefemale(OR=1.952;95%CI=1.373-2.774;P=0.00),age(31-59years)anddiameterof
tumor≤1cm(OR=3.273;95%CI=2.417-4.432;P=0.000)weresignificantindependentpredictorsof
LNMinallPTCs.InPTMC,thefemale(OR=3.002;95%CI=1.654-5.446;P=0.00)wasasignificant
independentpredictorofLNM.Thetumorinleftandrightlobessimultaneouslywasanindependent
protectivefactorofLNMineachgroup(PTCs:OR=0.287;PTMC:OR=0.170;non-PTMC:OR=0.441,
respectively).TheBRAFV600EmutationrateofUS-FNACwasmuchhigherthanFFPEinPTMC
(P=0.018).
Conclusions:Unlikepreviousresearch,ourfindingssuggestedthatthefemalepatientsanddiameter
oftumor≤1cmwereriskfactorsforLNMandtheBRAFV600Ewild-typeofPTMCmightbemore
aggressivethanothers.Interestingly,thepositionoftumorinbilateralthyroidsimultaneouslywasan
independentprotectivefactorforLNM.TheUS-FNAshouldberecommendedforgeneanalysis(BRAF
V600E)inPTMC.TheBRAFL597QmutationmaybeanindependentaggressivefactorintheChinese
Tibetanpopulation.Hence,cliniciansshouldconsideranindividualizedtreatmentaccordingtogene
mutation,gender,age,tumorsizeandlocationoftumorinordertoachieveabettertherapeutic
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efficacy.
Background
Papillarythyroidcarcinoma(PTC),isthemostcommonhistologicalsubtypeofthyroidcancer,which
occursinmorethan90%ofallthyroidmalignancies,hasincreasedincidenceatanalarmingpacein
recentyears[1-4].Thisrecentdramaticchangeisprimarilyattributabletotheincreasedusingofthe
fine-needleaspiration(FNA)orultrasonography-guidedbiopsyastheearlydiagnosismethodsin
patientswithoutpalpablethyroidnodes[5,6].AlthoughthemortalityrateofPTCisrelativelylow,20-
50%ofpatientsaccompanywiththeriskoftheworstclinicaloutcomes(e.g.distantmetastases[7],
thehighrateoflong-termpersistenceofthediseaseandpossibilityofrecurrence[8]).Papillarythyroid
microcarcinoma(PTMC,diameteroftumor≤1cm),accountsinmorethan50%ofallnew-onset
thyroidcancers,hasbeenincreasingrapidlyduringthelastseveraldecadesallaroundtheworld[9,10].
Clinically,manycaseshavedemonstratedthatPTMChadagoodprognosisinmostinstances
followingsurgicalinterventions.What'smore,PTMCtumorgrowthwasusuallyveryslowandsome
patientsdevelopedclinicallyproblematictumorgrowthaftermanyyearsofobservation[11,12].In
addition,mostPTMCalsohaveaveryindolentnatureandexcellentoutcomes,expertconsensus
recommendedthatPTMCshouldbeidentifiedandmanagedseparately[13,14].
TheB-typeRafkinase(BRAF)mutationhasfrequentlybeenthesubjectofintensiveresearchto
investigatethetumorigenicroleandclinicalimplicationsinthyroidcancers,particularlyPTCs.
Approximately,90%ofBRAFmutationswasT1799Atransversepointmutation,resultinginavalineto
glutamicacidswitchatcodon600(V600E)[15,16].ThekinaseactivityofBRAFV600Ewas460-fold
higherthanthewild-typeBRAF,andthisactiveconformationcanconstitutivelyactivateits
downstreameffectstotransformnormalcellsorinducecancerproliferationwithouttheneedofRAS
foractivation[17],whichsuggeststhatthemutationisaearlyeventduringPTCsdevelopment,and
thereisacomplexprocessthatmighteffecttumorigenesisandaggressiveness.TherareBRAFL597Q
(c.T1790A)pointmutation,whichisregardedasanoncogene,hasbeenpreviouslyreportedand
describedinchildhoodacutelymphoblasticleukemia[18].
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AsinPTCingeneral,lymphnodemetastasishasbeenreportedtobeariskfactorforincreasedtumor
recurrenceratesandalsobecloselyinconnectionwithreducedsurvivalrate[19].Inaddition,in2018,
Lutz.etal[20]reportedthatanimbalanceinDNArepairgeneexpressionisassociatedwithaggressive
clinicopathologicalfeaturesinPTCs.Giventhecontroversiesabove,atotalof1045PTCspatients
wereenrolledinthisstudy,including313patientswithPTMCand732patientsinnon-PTMC(diameter
oftumor1cm).Theaimofthisretrospectiveobservationalstudywastoverifytheassociationsof
BRAFV600EmutationswithclinicopathologicfeaturesandnexttoidentifytheriskfactorsforLNMof
PTCpatients.Onecaseofsolitarybrainmetastasisfromoccultpapillarythyroidcarcinomainthe
ChineseTibetanpopulationwasreportedfirstlyindetail.Thisinformationmaybringbenefitsfor
clinicianstomakecorrectclinicaldecisionsforPTCpatientsinfuture.
Methods
Patientpopulation
Inthisstudy,theclinicaldataof1045SouthwestChinapatients(includingoneTibetan)withPTCs
werecollectedforanalysisfromAugust2016toAugust2019.Accordingtotumordiameter,patients
werediagnosedwithPTMCornon-PTMC.Amongthesepatients,313werediagnosedwithPTMC,732
werediagnosedwithnon-PTMC.AllparticipantsofthisstudywereChinesewithoutbloodrelationship
witheachotherandhavesignedinformedconsent.
Allpatientsmettheinclusioncriteria,whichwerethefollowing:(1)underwenteitheraresectionora
diagnosticprocedure(biopsyorcytologicalspecimen);(2)confirmedasPTCbyintraoperativerapid
pathologyorpostoperativepathologydetection;(3)analysisofgenemutation.Differentlocationsof
thyroidtumorweredividedintosevenregions:leftlobe,rightlobe,bothleftandrightlobes,isthmus,
leftlobeandisthmus,rightlobeandisthmus,bilaterallobesandisthmusaccordingtoUSimaging
results.Gender,ageanddiagnosticdate,samplesourcewereavailablefor1045patients.
Pathologicalexamination
PTCtissueswereembeddedinparaffinandweresectionedat4umaccordingtostandardprocedures.
ThesectionswereprocessedforHEstainingandwereusedforobservationbylightmicroscopy.
DifferenttypeofPTCsandpresenceoflymphnodemetastasiswerereviewedbytwopathologists
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independentlyinablindedmanner.Theinconsistentdiagnosticcaseshavebeendiscussedwitha
thirdpathologist.
PreoperativeskullCT/MRIscanandmolecularpathologydiagnosis
TheCT,MRI,colorultrasounddiagnosis,ultrasound-guidedfine-needleaspiration(US-FNA)andHE
stainingwereusedformorphologicaldetection.Thespecificexpressionofthyroidcancer-related
proteins(cytokeratin,CK;thyroglobulin,Tgandthyroidtransformingfactor-1,TTF-1)weredetected
bytheimmunohistochemical(IHC)analysis.
Samplecollection,DNAextractionandMutationScreening
DNAwereextractedfromformalin-fixedparaffin-embedded(FFPE)orFNACbyTRIzolreagent
(InvitrogenU.S.Cat.No.15596-026)accordingtothemanufacturer’sprotocols.DNAconcentrationsof
allsamplesweredeterminedbytheNanoDropND-1000spectrophotometerat280nm(Thermo
Scientific,Waltham,andMass).Thegenemutationwasdetectedbythreedifferenttechnology
platforms(ARMS-PCR,NGSandSangersequencing).TheARMS-PCRreagentswereprovidedbyAmoy
DiagnosticsCo.,Ltd(P215101901X,Xiamen,China).TheNGSandSangersequencingwereperformed
bylaboratorydevelopedtests(LDTs),andsomehigh-frequencymutationandtargeteddrug-related
genesofthyroidcancerhavebeendetectedbyNGS(Table7),theforwardprimerofBRAFinSanger
sequencing:5'-GCTTGCTCTGATAGGAAAATGAG-3',thereverseprimerofBRAFinSangersequencing:
5'-GGGCCAAAAATTTAATCAGTGG-3'andtheprimersweresynthesizedbyInvitrogenBio-TechCo.,Ltd.
(Shanghai,China).
Statisticalanalysis
StatisticalanalysiswasperformedusingIBMSPSS22.0software(IBMCorp.,Version22.0,Armonk,
NY,USA).Quantitativedatawereexpressedasmean±SD.Qualitativedatawererepresentedasa
percentageorfrequency.TheChisquaretestorFisher’sexacttestwasusedtoevaluatethe
differenceinclinicalfeaturesbetweentwodifferentgroups.Theunivariateandmultivariatelogistic
regressionanalysiswereperformedtoassessindependentriskfactorsforpresenceofLNMinPTCs,
resultsarereportedasoddsratios(OR)with95%confdenceintervals(CI).Ap-valuelessthan0.05
wasconsideredstatisticallysignificant.
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Results
ClinicopathologicalCharacteristicsof1045PatientswithPTMCornon-PTMC
Aretrospectivestudyof732patientswithnon-PTMCand313patientswithPTMCduringtheperiodof
2016.08-2019.08wasperformedtoassesstheclinicopathologicalcharacteristicsatdiagnosis,
includinggender,ageatdiagnosis,samplesource(FFPEtissuesorFNAC),lymphnodemetastasis,
differentlocationsofthyroidtumor(leftlobe,rightlobe,bothleftandrightlobes,isthmus,leftlobe
andisthmus,rightlobeandisthmus,bilaterallobesandisthmus)andBRAFV600Emutationstatus,
etc(Table1).TheBRAFV600EmutationratesofPTCshavebeenincreasingduring2016.08-2019.08
(Figure5),inaddition,themutationrateofPTMCwassignificanthigherthannon-PTMC(P<0.05).
298maleand747femalepatientshavebeenanalyzedinthisstudy.Themeanagewas41.97±
12.94.Thepatientsweredividedintothreesubgroupsaccordingtoage:youngsubgroup(<30years,
n=206),middlesubgroup(31-59years,n=735),andoldsubgroup(≥60years,n=104).Sample
sourcewasconsistofFFPE(n=742)andFNAC(n=303).Lymphnodemetastasiswaspresentin181
cases(57.8%)ofPTMCand145cases(19.8%)ofnon-PTMC.Thelymphnodemetastasiswaspresentin
31.2%patients.Forlocationofthyroidtumor:382PTCpatientsinleftlobe,480inrightlobe,6in
isthmus,150inbothleftandrightlobes,7inleftlobeandIsthmus,15inrightlobeandIsthmus,5in
bilaterallobesandisthmus.TheBRAFV600Emutationoccurredin273cases(87.2%)ofPTMCand
566cases(77.3%)ofnon-PTMC.ThetotalmutationrateofBRAFV600Ewas80.3%.The
clinicopathologicalcharacteristicsandsamplesourcebetweenPTMCandnon-PTMCwascomparedin
thisstudy(Table1).TheBRAFV600EmutationrateinPTMCgroupwasmuchhigherthanthenon-
PTMCgroup(P=0.00).ThefrequencyoflymphnodemetastasisinPTMCgroupwasalsosignificantly
higherthanthenon-PTMCgroup(P=0.00).Otherclinicalparametersshowednosignificant
differencesbetweenthetwogroups.
BRAFV600EmutationalstatusandclinicalcharacteristicsinpatientswithPTMCornon-
PTMC
TherelationshipofBRAFmutationstatusandclinicalcharacteristicsof313PTMCpatientswere
analyzedinthisstudy.TheBRAFV600Emutationshowedsignificantassociationwithmalegender
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(P=0.026)andsamplesourcefromFFPEtissues(P=0.018)comparedwiththeBRAFV600Ewild-type
inPTMCpatients.However,therewasnodifferenceinlymphnodemetastases,ageandlocationof
thyroidtumorbetweentheBRAFV600EmutationandtheBRAFV600Ewild-type(Table2).
TheBRAFV600Emutationshowedsignificantassociationwithmalegender(P=0.003)comparedwith
theBRAFV600Ewild-typeinnon-PTMCpatients.However,therewasnodifferenceinotherclinical
featuresbetweentheBRAFV600EmutationandtheBRAFV600Ewild-type(Table2).
TheclinicopathologicalcharacteristicsandsamplesourceinallBRAFV600Emutationpatientswere
comparedadditionally(Table3).Alowerrateoflymphnodemetastasis(P=0.00,χ2=42.369)was
presentedinPTMCthaninnon-PTMC.Thenumberofmiddlesubgroup(31-59,P=0.004,χ2=11.306)
hadstatisticalsignificancebetweenPTMCandnon-PTMC.However,therewasnodifferenceinother
clinicalfeaturesbetweenPTMCandnon-PTMC.
UnivariateandmultivariateanalysisofriskfactorsforLNMinPTCs,PTMCandnon-PTMC
InPTCs(Table4),thefemale(OR=1.952;95%CI=1.373-2.774;P=0.00),middlesubgroup(31-59)
(OR=1.560;95%CI=1.050-2.318;P=0.028),PTMC(OR=3.273;95%CI=2.417-4.432;P=0.000)
werecharacterizedasindependentriskyfactorsforLNM.Moreover,thetumorinleftandrightlobes
simultaneously(OR=0.287;95%CI=0.166-0.497;P=0.000)wascharacterizedasaprotectivefactor
forLNM.However,therewasnodifferencebetweenBRAFV600EmutationandLNM(P>0.05).
InPTMC(Table5),thefemale(OR=3.002;95%CI=1.654-5.446;P=0.00)wascharacterizedas
independentriskyfactor,thetumorinleftandrightlobessimultaneously(OR=0.170;95%CI=
0.071-0.405;P=0.00)wascharacterizedasaprotectivefactor.Theage,BRAFV600Emutationdid
notshowstatisticaldifferenceswithLNM(P>0.05).
Innon-PTMC(Table6),thetumorinleftandrightlobessimultaneously(OR=0.441;95%CI=0.220-
0.882;P=0.00)wascharacterizedasaprotectivefactorforLNM.Thegender,ageandBRAFV600E
mutationdidnotshowstatisticaldifferenceswithLNM(P>0.05).
TheBRAFL597QmutationofPTCinoneTibetan
A57-year-oldTibetanmalepatient(withaprotrudingmassontheleftforehead)cametohospital
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withnumbnessintherightlimbforthreeweeks.ThepreoperativeskullCTandMRIscanshoweda
9.3cm×8.1cmmassintheleftfrontal(Fig.1AandB).Theskinonthesurfaceofthemasswas
normalandhardintexture.Thepreoperativeconventionalcolorultrasounddiagnosisshowedthatthe
sizeofthebilateralthyroidwasnormal,andtheechointherightlobewasuneven.Amass(1.2cm×
0.7cm)wasvisible,withovalshapeandclearlydefinedborder.The“leftfrontallobeoccupancyand
skulltumorresection”surgicalplanwassuggestedforimplementationbythemulti-disciplinaryteam
(MDT).Intraoperativedisplayshowedthatthescalptissueandtheleftfrontalskullhadobvious
adhesion,andtheprotrudingbonetissuesurfacewasunevenandloose.Thehyperplasiaoftheinner
andouterplatesoftheskullwasobvious(Fig.1C).Thetumortissuewaswhiteandsolid,andthere
wasnoadhesionwiththesurroundingbraintissue(Fig.1D).Thepreoperativeconventionalcolor
ultrasounddiagnosisshowedthatthesizeofthebilateralthyroidwasnormal,andtheechointhe
rightlobewasuneven.Amass(0.7cm×0.5cm)wasvisible,withovalshapeandclearlydefined
border(Fig.2A).TheUS-FNAwasusedtobiopsythethyroidnodules,thetumorcellswererelatively
uniforminsize,withroundnuclei,smallnucleolivisibleinpart(Fig.2B).
SomenucleusofcancercellsshowedGroundGlassOpacity(GGO)byHEstaining.Thenuclear
groovesandpseudoinclusionsinnucleuswereobservedclearly,andpsammomabodieswasseenin
interstitialtissue(Fig.3A).Thecytokeratin(CK),thyroglobulin(Tg)andthyroidtransformingfactor-1
(TTF-1)wereimmunoreactive(Fig.3B,CandD)byIHC.TheARMS-PCR,NGSandSangersequencing
analysisshowedthatthepatienthadadoublemutationofBRAFL597QandV600Eintwoseparate
lesions(Fig.4).TheBRAFV600E(chr7:140453136c.1799T>A)mutationwaslocatedinsitu(Fig.4A
andC),buttheBRAFL597Q(chr7:140453145c.1790T>A)mutationwaslocatedintheintracranial
metastases(Fig.4BandD).TheabundancesofBRAFL597QandV600Ewere36.9%and8.1%
respectively,asdeterminedbyNGS,andwhichweresuccessfullyverifiedbySangersequencing(the
goldstandardofgenesequencing).
Discussion
Withtheincreasingincidencesofnon-PTMCandPTMC,itisimportanttorisk-stratifypatientswho
mayhavemoreaggressivetumorbiologyinwhatistraditionallythoughttobeamoreindolent
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disease,whichwillhavemanagementimplicationsincludingwhetherornottoobserve,theextentof
surgicalresection,theuseofRIAtherapyandthefrequencyoffollow-up[21].Li[22]havereportedthat
thePTMChadanindolentcourseandexcellentprognosis,whileourresultsdemonstratedthatthe
incidenceofLNMwasmorefrequentinPTMCthaninnon-PTMC.Thecorrelationanalysisrevealedthat
theincidenceofBRAFV600EmutationinmalePTMCwasmuchhigherthaninfemale.Lee[23]have
recommendedthatthemalecouldbeasanindependentprognosticfactorforrecurrenceinnon-
PTMC,butitwasnotaprognosticfactorinPTMC.Inourstudy,theincidenceofBRAFV600Emutation
inFNACwasmuchhigherthaninFFPEwhichwasconsistentwithsomepreviousstudies[24,25],
however,theLNMwasrelatedtoBRAFV600Ewild-typeinPTMCwhichwasinconsistentwithother
study[26].Thus,ourfindingsdemonstratedthattheBRAFV600Emutationwasmorelikelytomanifest
amongmalepatientsandmoreeasilytobedetectedinFNACofPTMC.
TofurtheranalyzethedifferencebetweenPTMCandnon-PTMCinthebiologicalbehavior,the
clinicopathologicalcharacteristicsandsamplesourceinBRAFV600Emutationpatientswerealso
compared.AlthoughseveralstudieshavereportedthatBRAFV600EmutationinPTCswasassociated
withaggressivepathologicalfeatures,negativeinfluenceon131Iavidity,reducedthyroperoxidase,
theincreasedriskoflymphnodemetastasisandrecurrenceaftertreatment[27-29],theclinical
implicationsandclearmechanismsinPTMCandnon-PTMCwascontradictory.Zheng[30]have
reportedthattumordiameter(>0.5cm)wasanindependentriskfactorcorrelatedwithLNMinPTCs.
Interestingly,ourfindingsdefinitelydemonstratedthattheLNMratewasmuchhigherandwas
correlatedwithBRAFV600Ewild-typeinPTMC,whichpossiblyindicatedthatBRAFV600Emutationin
PTMCwaslessaggressivewhichwasdifferfromapreviousmeta-analysis[22].Astudy[31]have
reportedthatmostPTMCwithBRAFV600EmutationdidnotdisplayBRAFV600Eproteinexpression.
Correctpreoperativediagnosisisofimportance.Itisgenerallyagreedthatbetterknowledgeabout
predictiveriskfactorsforLNMmayguideclinicaldecisions,whilethegreaterriskofLNMremains
debatable.Afteradjustingforothersignificantpreoperativeclinicalfactors,univariateand
11
multivariateanalysiswasperformedtoidentifytheriskfactorsforLNM.Genderisaprominentpatient
backgroundparameterforPTC.Inrecentyears,theassociationbetweengenderandrecurrenceor
survivalofPTChasbeendebated.Apreviousstudyreportedthatthemalewasanindependent
clinicalprognosticfactorofpooroutcomeinPTC[32],butnotinPTMC[23].Recently,Roh[33]have
reportedthattherewasnoassociationbetweengenderandLNM.Inourstudy,althoughthefemale
hadalowerBRAFV600Emutationthanthemale,multivariateanalysisdemonstratedthatthefemale
wasariskfactorofLNMinPTMCandnon-PTMC,whichwasdifferentfromabovestudies.Aprevious
study[34]alsohaveexploredthatthefemalewasanindependentpredictiveriskfactorofCLNMin
PTC.Controversiesindifferentstudiescouldberelatedtodifferentsamplesource,samplesizeand
detectiontechniques.Ithasbeenreportedthatthefemalehadanearlierageofonset,butthemale
hadahighermortality[35,36].ItisrecommendedthatthemolecularmechanismsbetweenLNMand
genderinPTCpatientsshouldbeexploredinfuture.
Inourstudy,themiddleage(31-59)groupwasabout1.56timestheyoungage(≤30)groupforLNM
inPTCs,whichwasinconsistentwithotherstudy[37].Ourresultshaveshowedthatthelocationof
tumorinbilateralthyroidsimultaneouslywasaprotectivefactorforLNMinPTMCandnon-PTMC,
whichwasalsoinconsistentwitharetrospectivecohortstudy[38].
Thequestionistoclarifywhetherornot,aftercontrollingtheclinicalimportanceofBRAFV600E
mutation,totakeintoaccountthatsomeotherBRAFmutationsdeservethoughtfulanalysis.The
BRAFL597Qmutationincidencewaslessthan1%[39]suchasinchildhoodacutelymphoblastic
leukemia[18],whichmightbeassociatedwithaggressiveclinicopathologicfeatures,however,the
potentialroleofthepeculiarBRAFL597QmutationofPTCwasunclear.Tothebestofourknowledge,
wehavereportedthefirstcaseofsolitarybrainmetastasisfromoccultpapillarythyroidcarcinomain
theChineseTibetanpopulationindetail,asallknown,thecommonmetastaticsitesofthyroidcancer
arelungandbone,skullmetastasesareextremelyrare[40].Ourresultshavedemonstratedthatthe
rareBRAFL597Qpointmutationmightplayaspecificroleininducingthesolitaryintracranial
12
metastasisofoccultpapillarythyroidcarcinomaintheChineseTibetanpopulation.Inaddition,inthis
case,thebiopsyortraditionalgenesequencingtechnologies(suchastheARMS-PCR,solelyfor
detectingtheBRAFV600Emutation)hadcertainlimitations,thecombinationofNGSandSanger
sequencingshouldhelpindetectingtheraregenemutation(e.gBRAFL597Qmutation),whichwas
greatlyrecommendedtoimprovethediagnosticaccuracyandmolecularmechanismanalysisinrare
PTCcases.Itisconcludedthatthemultilevelgeneanalysismaybeagreatsubstituteforthe
traditionalgenetesting[41,42].
Nonetheless,ourretrospectivestudyhadseveralpotentiallimitations.Firstly,thenumberoftherare
cases(BRAFL597Qmutationwithskullmetastases)wasfew.Secondly,thedetailedmolecular
mechanismshouldbeconfirmedbyalargenumberoffunctionalexperimentsandclinicalresearches
whichmighthelptomanifestthattheBRAFL597Qmutationmightplayaspecificroleininducingthe
solitaryintracranialmetastasisofoccultPTC.Thirdly,ourstudydealtwithagroupofpatientswith
PTConlyinSouthwestofChina.
Conclusions
Inconclusion,wehavedemonstratedthatthefemale,middleage(31-59years)andPTMCwere
independentlycorrelatedwithLNMinPTCs,whilethetumorinleftandrightlobessimultaneouslywas
aprotectiveroleinLNM.Meanwhile,ourstudydemonstratedanegativeresultbetweenBRAFV600E
andLNM.Also,FNACfromtumorsampleshadahigherrateofBRAFV600EmutationthanFFPEin
PTMC,whichconfirmedthatFNACmightbeareliableinterventiontodetectBRAFV600Emutation.
Moreover,toourknowledge,apossibleassociationbetweentherareBRAFL597Qmutationand
intracranialmetastasisofoccultPTCintheChineseTibetanpopulationwasreportedfirstlywhichmay
beusedasatherapeutictargetinfuture.Therefore,wesuggestthatcliniciansshouldmakea
comprehensivelyconsiderationofclinicalfeatures:samplesource,BRAFmutation,tumorsize,
gender,locationofthyroidtumorandmultilevelgenesequencingtechnologiesandtherapeutic
scheduleinordertoachievearelativelygoodprognosis.
Abbreviations
PTC:papillarythyroidcarcinoma;PTMC:papillarythyroidmicrocarcinoma;NGS:nextgeneration
13
sequencing;US-FNA:ultrasound-guidedfine-needleaspiration;FFPE:formalin-fixedparaffin-
embedded;FNAC:fine-needleaspirationcytology;HE:hematoxylin-eosinstaining;IHC:immunohisto-
chemical(IHC)analysis;PCR:polymerasechainreaction;LNM:lymphnodemetastasis;OR:odds
ratios.
Declarations
DisclosureofPotentialConflflictsofInterest
Theauthorsdeclarethattheyhavenocompetinginterests.
Authors’Contributions
Conceptionanddesign:YanDong,DanWangandXiaosongLi.Developmentofmethodology:Huili
Bai,YifanShen,YangliZhang,XuepingChenandYajingZhao.Acquisitionofdata(acquiredand
managedpatients,providedfacilities,etc.):XinliangSu,JinqiuZhao,HuandongLiu,JungaoLu,Zuoyi
YaoandYajingZhao.Analysisandinterpretationofdata(e.g.,statisticalanalysis,biostatistics,
computationalanalysis):LingChen,DanWangandYishengLuo
Writing,review,and/orrevisionofthemanuscript:YanDong,DanWangandXiaosongLi.
Administrative,technical,ormaterialsupport(i.e.,reportingororganizingdata,constructing
databases):LingChen,DanWangandXiaosongLi.Studysupervision:XiaosongLi.Allauthors
contributedtothismanuscript.Allauthorsreadandapprovedthefinalmanuscript.
Acknowledgments
Theauthorswouldliketothankthepatientswhoparticipatedinthisstudy,andtheirfamilies,aswell
astheinvestigatorsandstudyteams.
Funding
ThisstudywassupportedbytheNationalNaturalScienceFoundationofChina(grantno.81871653)
andChongqingMedicalScienceProject(grantno.2018MSXM065).
Availabilityofdataandmaterials
Alldatageneratedoranalyzedduringthisstudyareincludedinthispublishedarticle.
Ethicsapprovalandconsenttoparticipate
ThisstudywasapprovedbytheEthicsCommitteeoftheFirstAffiliatedHospitalofChongqingMedical
14
UniversityEthicsReviewBoard.
Consentforpublication
Notapplicable.
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19
Characteristics Total
(N=1045) PTMC(N=313) non-PTMC
(N=732) P
value
Gender
Male/Female
298/747
85/228
213/519
0.524
Age(years)
≤30
31–59
≥60
41.97±12.94
206
735
104
42.57±11.40
58(18.5%)
236(75.4%)
19(6.1%)
41.60±13.78
148(20.2%)
499(68.2%)
85(11.6%)
0.130
Lymphnodemetastasis 326 181(57.8%) 145(19.8%) 0.000
BRAFV600Emutation 839 273(87.2%) 566(77.3%) 0.000
Differentlocationsofthyroidtumor
Leftlobe
Rightlobe
Isthmus
Leftandrightlobes
Leftlobeandisthmus
Rightlobeandisthmus
Bilaterallobesandisthmus
382
480
6
150
7
15
5
104(33.2%)
147(47.0%)
3(1.0%)
52(16.6%)
1(0.3%)
6(1.9%)
0(0.0%)
278(38.0%)
333(45.5%)
3(0.4%)
98(13.4%)
6(0.8%)
9(1.2%)
5(0.7%)
0.254
Samplesource
FNAC
FFPE
303
742
78(24.9%)
235(75.1%)
225(30.7%)
507(69.3%)
0.058
Tables
Table1.Characteristicsofpatients
Note: Quantitative data were showed as Mean±SD or N (%);P value of 0.05 or less was
consideredsignificantFisherexacttest.FNAC:fine-needleaspirationcytology;FFPE:formalin-fixed,
paraffin-embedded
Table2.CorrelationbetweenclinicopathologicalcharacteristicsandBRAFV600Emutation
NoteFisherexacttest
20
Characteristics
PTMC P
value
non-PTMC
BRAFV600E
mutation BRAFV600Ewild-
type BRAFV600E
mutation
Gender
Male/Female
80/193
5/35
0.026
180/386
Age(years)
≤30
31–59
≥60
42.02±11.31
52
206
15
43.07±11.48
6
30
4
0.478
43.32±14.06
105
387
74
Lymphnodemetastasis
Yes(+)
No(-)
158
115
23
17
0.964
449
117
Differentlocationsofthyroidtumor
Leftlobe
Rightlobe
Isthmus
Leftandrightlobes
Leftlobeandisthmus
Rightlobeandisthmus
Bilaterallobesandisthmus
88
127
3
49
1
5
0
16
20
0
3
0
1
0
0.475
211
259
3
79
3
8
3
Samplesource
FNAC
FFPE
75
198
4
36
0.018
176
390
Table3.ComparisonofprogressionbetweenPTMCandnon-PTMCwithBRAFV600Emutation
21
Characteristics BRAFV600Emutation χ2
P
PTMC non-PTMC
Gender
Male/Female
80/193
180/386
0.537
0.464
Age(years)
≤30
31–59
≥60
42.02±11.31
52
206
15
43.32±14.06
105
387
74
11.306
0.004
Lymphnodemetastasis
Yes(+)
No(-)
158
115
449
117
42.369
0.000
Differentlocationsofthyroidtumor
Leftlobe
Rightlobe
Isthmus
Leftandrightlobes
Leftlobeandisthmus
Rightlobeandisthmus
Bilaterallobesandisthmus
88
127
3
49
1
5
0
211
259
3
79
3
8
3
5.644
0.445
Samplesource
FNAC
FFPE
75
198
176
390
1.153
0.283
NoteFisherexacttest
Table4.UnivariateandmultivariateanalysisofriskfactorsforLNMinPTC(n=1045)
22
Variable
Univariateanalysis 
OR95%
CI POR95%
Gender(Maleasreference)  
Female 1.868(1.340-
2.604) 0.000  1.95(1.373-
2.774)
Age(≤30asreference)  
31–59 1.48(1.024-
2.157) 0.037  1.56(1.050-
2.318)
≥60 1.32(0.765-
2.309) 0.313  1.53(0853-
2.74)
BRAFV600E(Yesasreference)  
No(-) 0.72(0.505-
1.043) 0.084 
Tumortype(non-PTMCasreference)  
PTMC 2.93(2.198-
3.910) 0.000  3.27(2.417-
4.432)
Differentlocationsofthyroidtumor(Leftlobeasreference)  
Rightlobe
Leftandrightlobes
Isthmus
Bilaterallobesandisthmus
Rightlobeandisthmus
Leftlobeandisthmus
0.82(0.611-
1.103)
0.32(0.189-
0.543)
1.11(0.200-
6.117)
0.55(0.061-
4.996)
1.11(0.370-
3.304)
0.37(0.044-
3.094)
0.191
0.000
0.909
0.597
0.858
0.358
 0.77(0.564-
1.050)
0.28(0.166-
0.497)
0.83(0.146-
4.740)
0.40(0.041-
4.024)
0.85(0.270-
2.725)
0.40(0.048-
3.462)
Note:LNM:lymphnodemetastasis;OR:oddsratio;CI:confidenceinterval
Table5.UnivariateandmultivariateanalysisofriskfactorsforLNMinPTMC
23
Variable
Univariateanalysis 
OR95%
CI POR95%
Gender(Maleasreference)  
Female 2.91(1.671-
5.077) 0.000  3.00(1.654-
5.446)
Age(≤30asreference)   
31–59 1.38(0.764-
2.494) 0.285  1.76(0.941-
3.327)
≥60 1.48(0.529-
4.145) 0.455  1.45(0.496-
4.257)
BRAFV600E(Yesasreference)   
No(-) 0.93(0.481-
1.805) 0.835  0.72(0.356-
1.454)
Differentlocationsofthyroidtumor(Leftlobeasreference)   
Rightlobe
Leftandrightlobes
Isthmus
Bilaterallobesandisthmus
Rightlobeandisthmus
Leftlobeandisthmus
0.70(0.427-
1.169)
0.15(0.068-
0.369)
1.78(0.157-
20.263)
0.44(0.039-
5.066)
0.44(0.078-
2.539)
0.00
0.176
0.000
0.641
0.514
0.362
1.000
 0.71(0.425-
1.203)
0.17(0.071-
0.405)
2.78(0.223-
34.698)
0.47(0.038-
5.791)
0.40(0.068-
2.417)
0.00
Note:LNM:lymphnodemetastasis;OR:oddsratio;CI:confidenceinterval
Table6.UnivariateandmultivariateanalysisofriskfactorsforLNMinnon-PTMC
24
Variable
Univariateanalysis 
OR95%
CI POR95%
CI
Gender(Maleasreference)  
Female 1.42(0.929-
2.165) 0.105  1.52(0.980-
2.346)
Age(≤30asreference)   
31–59 1.50(0.910-
2.484) 0.111  1.46(0.873-
2.437)
≥60 1.55(0.777-
3.091) 0.214  1.48(0.729-
3.002)
BRAFV600E(Yesasreference)   
No(-) 0.79(0.500-
1.241) 0.304  0.77(0.482-
1.224)
Differentlocationsofthyroidtumor(Leftlobeasreference)   
Rightlobe
Leftandrightlobes
Isthmus
Bilaterallobesandisthmus
Rightlobeandisthmus
Leftlobeandisthmus
0.81(0.550-
1.197)
0.43(0.215-
0.850)
0.0
0.0
1.67(0.407-
6.874)
0.67(0.077-
5.829)
0.291
0.015
0.999
0.999
0.476
0.716
 0.79(0.533-
1.175)
0.44(0.220-
0.882)
0.0
0.0
1.52(0.361-
6.419)
0.64(0.72-
5.707)
Note:LNM:lymphnodemetastasis;OR:oddsratio;CI:confidenceinterval
Table7.TheNGSpanelforthyroidcancer
25
List Genes
1 AKT1(NM_001014432.1)
2 ALK(NM_004304.4)
3 BRAF(NM_004333.4)
4 CTNNB1(NM_001904.3)
5 EIF1AX(NM_001412.3)
6 ETV6(NM_001987.4)
7 GNAS(NM_080425.3)
8 HRAS(NM_005343.3)
9 KRAS(NM_033360.3)
10 NRAS(NM_002524.4)
11 NTRK1(NM_001007792.1)
12 PIK3CA(NM_006218.3)
13 PPARG(NM_015869.4)
14 PTEN(NM_000314.6)
15 RET(NM_020975.4)
16 TERT(NM_198253.2)
17 TP53(NM_000546.5)
18 TSHR(NM_000369.2)
Figures
26
Figure1
PreoperativeskullCT/MRIscanandintraoperativephotographsduringresectionand
debulking.(A:Irregularbonedestructionoftheleftfrontalbone,therangeisabout5.7
cm×6.3cm.B:Irregularmassesareseenintheleftfrontalcranialandsubscalp,thesizeis
about4.9cm×5.4cm,itisunevenandobviouslystrengthened,andthecenterline
structureisskewedtotheright.C:Theouterplateofskull,raisedbone(4.8cm×5.2cm)
surfaceoftheskull’souterplatewasuneven,showingastateofcancellousbone.D:The
tumorisolatedfromtheoperationwashardintexture,about5.7cm×6.3cminsize.)
27
Figure2
Preoperativeconventionalcolorultrasounddiagnosis(A)andUS-FNAdiagnosis(B)(A:The
echointherightlobewasuneven,anda0.7cm×0.5cmmasswasvisible.B:Alesional
biopsyspecimenofthethyroidnoduleswasobtained(hematoxylin-eosin,400×.)
28
Figure3
TheHEandIHCstainingoftheintracranialmetastases(400×)(A:Tumorcellsarerelatively
uniforminsize,withroundnuclei,smallnucleolivisibleinpart,cytoplasmicstainingand
mitoticdivisionsthatarenoteasilyseen.B:CK19stainingshowedbrownishyellowparticles
onthecellmembrane(+++).C:TGstainingshowedbrownishyellowparticlesonthecell
membrane(+).D:TTF1stainingshowednucleusshowingbrownishyellowparticles(+++).)
29
Figure4
Geneticdiagnosisresults(A:BRAFV600E(c.1799T>A)mutationtestedbySanger
sequencinginsitu.B:BRAFL597Q(c.1790T>A)mutationtestedbySangersequencingin
metastases.C:BRAFV600E(c.1799T>A)mutationtestedbyNGSinsitu,andD:BRAF
L597Q(c.1790T>A)mutationtestedbyNGSinmetastases.)
30
Figure5
ComparisonofmutationratesbetweenPTMCandnon-PTMC(Comparisonofmutationrates
betweenPTMCandnon-PTMCover3years;DatawereshowedasN(%);Thedifferencein
mutationratebetweenPTMCgroupsandnon-PTMCgroupswerehighlysignificant.)
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Purpose: Activating BRAF mutations, most commonly BRAFV600E, are a major oncogenic driver of many cancers. We explored whether BRAFV600E promotes radiation resistance and whether selectively targeting BRAFV600E with a BRAF inhibitor (vemurafenib, BRAFi) sensitizes BRAFV600E thyroid cancer cells to radiotherapy. Experimental design: Immunoblotting, neutral comet, immunocytochemistry, functional reporter, and clonogenic assays were used to analyze the outcome and molecular characteristics following radiotherapy with or without BRAFV600E or vemurafenib in thyroid cancer cells. Results: BRAFV600E thyroid cancer cell lines were associated with resistance to ionizing radiation (IR), and expression of BRAFV600E into wild-type BRAF thyroid cancer cells led to IR resistance. BRAFi inhibited ERK signaling in BRAFV600E mutants, but not BRAF wild-type thyroid cancer cell lines. BRAFi selectively radiosensitized and delayed resolution of IR-induced γH2AX nuclear foci in BRAFV600E cells. Moreover, BRAFi impaired global DNA repair and altered the resolution of 53BP1 and RAD51 nuclear foci in BRAFV600E cells following IR. BRAFV600E mutants displayed enhanced nonhomologous end-joining (NHEJ) repair activity, which was abolished by BRAFi. Intriguingly, BRAFV600E mutation led to upregulation of XLF, a component of NHEJ, which was prevented by BRAFi. Importantly, BRAFi in combination with radiotherapy resulted in marked and sustained tumor regression of BRAFV600E thyroid tumor xenografts. Conclusions: BRAFV600E mutation promotes NHEJ activity leading to radioresistance and BRAFi selectively radiosensitizes BRAFV600E thyroid cancer cells through inhibiting NHEJ. Our findings suggest that combining BRAFi and radiation may improve the therapeutic outcome of patients with BRAFV600E-mutant thyroid cancer.
Article
Objective: The role of routine central lymph node dissection (CLND) for clinically central lymph node negative (CN0) papillary thyroid microcarcinoma (PTMC) remains uncertain. We aim to determine the predictive factors for central lymph node metastasis (CLNM) in papillary thyroid microcarcinoma. Patients and methods: A total of 273 patients diagnosed with clinically central lymph node negative PTMC from 2014 to 2016 were included. The predictive risk factors for CLNM were analyzed with respect to age, sex, tumor size, tumor multifocal, lymphadenectasis of lateral neck, capsular invasion, extra capsular spread (ECS), coexistence of chronic lymphocytic thyroiditis (Hashimoto thyroiditis, HT) and nodular goiter (NG), BRAFV600E mutation and subtype of papillary thyroid carcinoma (PTC). Univariate and multivariate analyses were performed to identify the risk factors for CLNM. Results: Among the 273 patients, the CLNM occurred in 80 patients (29.3%). By univariate and multivariate analyses, tumor size (OR 2.07; p<0.001), multifocal (OR 2.67; p<0.004), lymphadenectasis of lateral neck (OR 9.28; p<0.001), tumor extent (OR 42.01; p<0.001) were independently correlated with CLNM. In further study, dorsal part of solitary lesion (OR: 16.312, 95%CI: 3.349-79.455, p=0.001), capsular invasion (OR: 42.012, 95% CI: 5.209-338.861, p<0.001), 6<D≤9 (OR: 8.400, 95% CI: 1.866-37.807, p=0.006) and D=1 (OR: 11.455, 95% CI: 2.500-52.480, p=0.002) were more tended to have CLNM. Conclusions: A prophylactic central lymph node dissection should be considered particularly to PTMC patients with each of tumor size > 6 mm, dorsal part of solitary lesion, multifocal, lymphadenectasis of lateral neck and capsular invasion.
Article
Purpose: To define the genetic landscape of advanced differentiated and anaplastic thyroid cancer and identify genetic alterations of potential diagnostic, prognostic and therapeutic significance. Experimental design: The genetic profiles of 583 advanced differentiated and 196 anaplastic thyroid cancers (ATC) generated with targeted next-generation sequencing cancer-associated gene panels MSK-IMPACT and FoundationOne were analyzed. Results: ATC had more genetic alterations per tumor, and pediatric papillary thyroid cancer had fewer genetic alterations per tumor when compared to other thyroid cancer types. DNA mismatch repair deficit and activity of APOBEC cytidine deaminases were identified as mechanisms associated with high mutational burden in a subset of differentiated and anaplastic thyroid cancers. Copy number losses and mutations ofCDKN2AandCDKN2B, amplification ofCCNE1, amplification of receptor tyrosine kinase genesKDR, KITandPDGFRA, amplification of immune evasion genesCD274, PDCD1LG2andJAK2and activating point mutations in small GTPaseRAC1were associated with ATC. An association ofKDR, KITandPDGFRAamplification with the sensitivity of thyroid cancer cells to lenvatinib was shownin vitroThree genetically distinct types of ATC are proposed. Conclusions: This large-scale analysis describes genetic alterations in a cohort of thyroid cancers enriched in advanced cases. Many novel genetic events previously not seen in thyroid cancer were found. Genetic alterations associated with anaplastic transformation were identified. An updated schematic of thyroid cancer genetic evolution is proposed.
Article
The involvement of alterations in MLH1, an essential mismatch repair component, in BRAFV600E mutated papillary thyroid carcinoma (PTC) has been suggested to be associated with features of tumor aggressiveness. Thirty-two PTC and surrounding normal thyroid tissues were evaluated for 11 representative DNA repair genes expression. BRAFV600E mutational status assessment and clinicopathological correlations were evaluated for their gene and protein expression. BRAFV600E PTC is associated with lower levels of XPD and MLH1 gene expression. Decrease in MLH1 and XPD mRNA levels in BRAFV600E PTC (but not their protein products) are associated with predictors of poor patient outcomes. Considering the complete subset of patients, MGMT and XRCC2 genes were shown down and upregulated, respectively, in PTC tissues. Low expression of MGMT gene and weak XRCC2 protein expression were correlated with characteristics of tumor aggressiveness. These results suggest that an imbalance in DNA repair gene expression in PTC is associated with aggressive clinicopathological features and BRAFV600E mutation.