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Comprehensiveevaluationofriskfactorsforlymph
nodemetastasiswithpapillarythyroidcarcinomain
SouthwestChinapatients
CURRENTSTATUS:POSTED
YanDong
ChongqingMedicalUniversityFirstAffiliatedHospital
DanWang
ChongqingMedicalUniversityFirstAffiliatedHospital
YishengLuo
ChongqingMedicalUniversityFirstAffiliatedHospital
LingChen
ChongqingMedicalUniversity
HuiliBai
ChongqingMedicalUniversityFirstAffiliatedHospital
YifanShen
ChongqingMedicalUniversityFirstAffiliatedHospital
YangliZhang
ChongqingMedicalUniversityFirstAffiliatedHospital
XuepingChen
ChongqingMedicalUniversityFirstAffiliatedHospital
XinliangSu
ChongqingMedicalUniversityFirstAffiliatedHospital
JinqiuZhao
ChongqingMedicalUniversityFirstAffiliatedHospital
HuandongLiu
TibetUniversityMedicalCollage
JungaoLu
2
GuizhouMedicalUniversity
ZuoyiYao
ChengduFirstPeople'sHospital
YajingZhao
ChongqingMedicalUniversityFirstAffiliatedHospital
ChanglongHe
ChongqingMedicalUniversityFirstAffiliatedHospital
XiaosongLi
ChongqingMedicalUniversityFirstAffiliatedHospital
250894831@qq.comCorrespondingAuthor
10.21203/rs.3.rs-26100/v1
SUBJECTAREAS
CancerBiology Oncology
KEYWORDS
Papillarythyroidcarcinoma(PTC);Papillarythyroidmicrocarcinoma(PTMC);non-
papillarythyroidmicrocarcinoma(non-PTMC);BRAFmutation;genetictesting
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Abstract
Background:Withtheincreasingincidencesofpapillarythyroidcancer(PTC),itisimportanttorisk-
stratifypatientswhomayhavemoreaggressivetumorbiology.Thisstudyaimedtoevaluatetherisk
factorsforlymphnodemetastasiswithPTCinSouthwestChinaPatientswhichmayprovidea
substantialreferenceforclinicaldiagnosisandtreatment.
Methods:1045PTCs(313PTMCand732non-PTMC)betweenAugust2016andAugust2019were
examinedtotally(includingoneTibetan).BRAFV600Emutationwastestedinallsamples.Theclinical
data(gender,age,tumorlocation,samplesourceandpathologicalfeatures)wereretrospectively
analyzed.LogisticregressionanalysiswasperformedtoevaluateindependentriskfactorsforLNM.
Results:181outof313PTMCcases(57.8%),145outof732non-PTMCcases(19.8%)hadBRAF
V600Emutation,theTibetanhadadoublemutationofBRAFL597QandV600Eintwoseparate
lesions.InPTMC,significantdifferenceingenderandsamplesourcewasfound(BRAFV600Emutation
vs.wild-type).Innon-PTMC,significantdifferenceingenderwasfound(BRAFV600Emutationvs.wild-
type).Thefemale(OR=1.952;95%CI=1.373-2.774;P=0.00),age(31-59years)anddiameterof
tumor≤1cm(OR=3.273;95%CI=2.417-4.432;P=0.000)weresignificantindependentpredictorsof
LNMinallPTCs.InPTMC,thefemale(OR=3.002;95%CI=1.654-5.446;P=0.00)wasasignificant
independentpredictorofLNM.Thetumorinleftandrightlobessimultaneouslywasanindependent
protectivefactorofLNMineachgroup(PTCs:OR=0.287;PTMC:OR=0.170;non-PTMC:OR=0.441,
respectively).TheBRAFV600EmutationrateofUS-FNACwasmuchhigherthanFFPEinPTMC
(P=0.018).
Conclusions:Unlikepreviousresearch,ourfindingssuggestedthatthefemalepatientsanddiameter
oftumor≤1cmwereriskfactorsforLNMandtheBRAFV600Ewild-typeofPTMCmightbemore
aggressivethanothers.Interestingly,thepositionoftumorinbilateralthyroidsimultaneouslywasan
independentprotectivefactorforLNM.TheUS-FNAshouldberecommendedforgeneanalysis(BRAF
V600E)inPTMC.TheBRAFL597QmutationmaybeanindependentaggressivefactorintheChinese
Tibetanpopulation.Hence,cliniciansshouldconsideranindividualizedtreatmentaccordingtogene
mutation,gender,age,tumorsizeandlocationoftumorinordertoachieveabettertherapeutic
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efficacy.
Background
Papillarythyroidcarcinoma(PTC),isthemostcommonhistologicalsubtypeofthyroidcancer,which
occursinmorethan90%ofallthyroidmalignancies,hasincreasedincidenceatanalarmingpacein
recentyears[1-4].Thisrecentdramaticchangeisprimarilyattributabletotheincreasedusingofthe
fine-needleaspiration(FNA)orultrasonography-guidedbiopsyastheearlydiagnosismethodsin
patientswithoutpalpablethyroidnodes[5,6].AlthoughthemortalityrateofPTCisrelativelylow,20-
50%ofpatientsaccompanywiththeriskoftheworstclinicaloutcomes(e.g.distantmetastases[7],
thehighrateoflong-termpersistenceofthediseaseandpossibilityofrecurrence[8]).Papillarythyroid
microcarcinoma(PTMC,diameteroftumor≤1cm),accountsinmorethan50%ofallnew-onset
thyroidcancers,hasbeenincreasingrapidlyduringthelastseveraldecadesallaroundtheworld[9,10].
Clinically,manycaseshavedemonstratedthatPTMChadagoodprognosisinmostinstances
followingsurgicalinterventions.What'smore,PTMCtumorgrowthwasusuallyveryslowandsome
patientsdevelopedclinicallyproblematictumorgrowthaftermanyyearsofobservation[11,12].In
addition,mostPTMCalsohaveaveryindolentnatureandexcellentoutcomes,expertconsensus
recommendedthatPTMCshouldbeidentifiedandmanagedseparately[13,14].
TheB-typeRafkinase(BRAF)mutationhasfrequentlybeenthesubjectofintensiveresearchto
investigatethetumorigenicroleandclinicalimplicationsinthyroidcancers,particularlyPTCs.
Approximately,90%ofBRAFmutationswasT1799Atransversepointmutation,resultinginavalineto
glutamicacidswitchatcodon600(V600E)[15,16].ThekinaseactivityofBRAFV600Ewas460-fold
higherthanthewild-typeBRAF,andthisactiveconformationcanconstitutivelyactivateits
downstreameffectstotransformnormalcellsorinducecancerproliferationwithouttheneedofRAS
foractivation[17],whichsuggeststhatthemutationisaearlyeventduringPTCsdevelopment,and
thereisacomplexprocessthatmighteffecttumorigenesisandaggressiveness.TherareBRAFL597Q
(c.T1790A)pointmutation,whichisregardedasanoncogene,hasbeenpreviouslyreportedand
describedinchildhoodacutelymphoblasticleukemia[18].
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AsinPTCingeneral,lymphnodemetastasishasbeenreportedtobeariskfactorforincreasedtumor
recurrenceratesandalsobecloselyinconnectionwithreducedsurvivalrate[19].Inaddition,in2018,
Lutz.etal[20]reportedthatanimbalanceinDNArepairgeneexpressionisassociatedwithaggressive
clinicopathologicalfeaturesinPTCs.Giventhecontroversiesabove,atotalof1045PTCspatients
wereenrolledinthisstudy,including313patientswithPTMCand732patientsinnon-PTMC(diameter
oftumor1cm).Theaimofthisretrospectiveobservationalstudywastoverifytheassociationsof
BRAFV600EmutationswithclinicopathologicfeaturesandnexttoidentifytheriskfactorsforLNMof
PTCpatients.Onecaseofsolitarybrainmetastasisfromoccultpapillarythyroidcarcinomainthe
ChineseTibetanpopulationwasreportedfirstlyindetail.Thisinformationmaybringbenefitsfor
clinicianstomakecorrectclinicaldecisionsforPTCpatientsinfuture.
Methods
Patientpopulation
Inthisstudy,theclinicaldataof1045SouthwestChinapatients(includingoneTibetan)withPTCs
werecollectedforanalysisfromAugust2016toAugust2019.Accordingtotumordiameter,patients
werediagnosedwithPTMCornon-PTMC.Amongthesepatients,313werediagnosedwithPTMC,732
werediagnosedwithnon-PTMC.AllparticipantsofthisstudywereChinesewithoutbloodrelationship
witheachotherandhavesignedinformedconsent.
Allpatientsmettheinclusioncriteria,whichwerethefollowing:(1)underwenteitheraresectionora
diagnosticprocedure(biopsyorcytologicalspecimen);(2)confirmedasPTCbyintraoperativerapid
pathologyorpostoperativepathologydetection;(3)analysisofgenemutation.Differentlocationsof
thyroidtumorweredividedintosevenregions:leftlobe,rightlobe,bothleftandrightlobes,isthmus,
leftlobeandisthmus,rightlobeandisthmus,bilaterallobesandisthmusaccordingtoUSimaging
results.Gender,ageanddiagnosticdate,samplesourcewereavailablefor1045patients.
Pathologicalexamination
PTCtissueswereembeddedinparaffinandweresectionedat4umaccordingtostandardprocedures.
ThesectionswereprocessedforHEstainingandwereusedforobservationbylightmicroscopy.
DifferenttypeofPTCsandpresenceoflymphnodemetastasiswerereviewedbytwopathologists
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independentlyinablindedmanner.Theinconsistentdiagnosticcaseshavebeendiscussedwitha
thirdpathologist.
PreoperativeskullCT/MRIscanandmolecularpathologydiagnosis
TheCT,MRI,colorultrasounddiagnosis,ultrasound-guidedfine-needleaspiration(US-FNA)andHE
stainingwereusedformorphologicaldetection.Thespecificexpressionofthyroidcancer-related
proteins(cytokeratin,CK;thyroglobulin,Tgandthyroidtransformingfactor-1,TTF-1)weredetected
bytheimmunohistochemical(IHC)analysis.
Samplecollection,DNAextractionandMutationScreening
DNAwereextractedfromformalin-fixedparaffin-embedded(FFPE)orFNACbyTRIzolreagent
(InvitrogenU.S.Cat.No.15596-026)accordingtothemanufacturer’sprotocols.DNAconcentrationsof
allsamplesweredeterminedbytheNanoDropND-1000spectrophotometerat280nm(Thermo
Scientific,Waltham,andMass).Thegenemutationwasdetectedbythreedifferenttechnology
platforms(ARMS-PCR,NGSandSangersequencing).TheARMS-PCRreagentswereprovidedbyAmoy
DiagnosticsCo.,Ltd(P215101901X,Xiamen,China).TheNGSandSangersequencingwereperformed
bylaboratorydevelopedtests(LDTs),andsomehigh-frequencymutationandtargeteddrug-related
genesofthyroidcancerhavebeendetectedbyNGS(Table7),theforwardprimerofBRAFinSanger
sequencing:5'-GCTTGCTCTGATAGGAAAATGAG-3',thereverseprimerofBRAFinSangersequencing:
5'-GGGCCAAAAATTTAATCAGTGG-3'andtheprimersweresynthesizedbyInvitrogenBio-TechCo.,Ltd.
(Shanghai,China).
Statisticalanalysis
StatisticalanalysiswasperformedusingIBMSPSS22.0software(IBMCorp.,Version22.0,Armonk,
NY,USA).Quantitativedatawereexpressedasmean±SD.Qualitativedatawererepresentedasa
percentageorfrequency.TheChisquaretestorFisher’sexacttestwasusedtoevaluatethe
differenceinclinicalfeaturesbetweentwodifferentgroups.Theunivariateandmultivariatelogistic
regressionanalysiswereperformedtoassessindependentriskfactorsforpresenceofLNMinPTCs,
resultsarereportedasoddsratios(OR)with95%confdenceintervals(CI).Ap-valuelessthan0.05
wasconsideredstatisticallysignificant.
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Results
ClinicopathologicalCharacteristicsof1045PatientswithPTMCornon-PTMC
Aretrospectivestudyof732patientswithnon-PTMCand313patientswithPTMCduringtheperiodof
2016.08-2019.08wasperformedtoassesstheclinicopathologicalcharacteristicsatdiagnosis,
includinggender,ageatdiagnosis,samplesource(FFPEtissuesorFNAC),lymphnodemetastasis,
differentlocationsofthyroidtumor(leftlobe,rightlobe,bothleftandrightlobes,isthmus,leftlobe
andisthmus,rightlobeandisthmus,bilaterallobesandisthmus)andBRAFV600Emutationstatus,
etc(Table1).TheBRAFV600EmutationratesofPTCshavebeenincreasingduring2016.08-2019.08
(Figure5),inaddition,themutationrateofPTMCwassignificanthigherthannon-PTMC(P<0.05).
298maleand747femalepatientshavebeenanalyzedinthisstudy.Themeanagewas41.97±
12.94.Thepatientsweredividedintothreesubgroupsaccordingtoage:youngsubgroup(<30years,
n=206),middlesubgroup(31-59years,n=735),andoldsubgroup(≥60years,n=104).Sample
sourcewasconsistofFFPE(n=742)andFNAC(n=303).Lymphnodemetastasiswaspresentin181
cases(57.8%)ofPTMCand145cases(19.8%)ofnon-PTMC.Thelymphnodemetastasiswaspresentin
31.2%patients.Forlocationofthyroidtumor:382PTCpatientsinleftlobe,480inrightlobe,6in
isthmus,150inbothleftandrightlobes,7inleftlobeandIsthmus,15inrightlobeandIsthmus,5in
bilaterallobesandisthmus.TheBRAFV600Emutationoccurredin273cases(87.2%)ofPTMCand
566cases(77.3%)ofnon-PTMC.ThetotalmutationrateofBRAFV600Ewas80.3%.The
clinicopathologicalcharacteristicsandsamplesourcebetweenPTMCandnon-PTMCwascomparedin
thisstudy(Table1).TheBRAFV600EmutationrateinPTMCgroupwasmuchhigherthanthenon-
PTMCgroup(P=0.00).ThefrequencyoflymphnodemetastasisinPTMCgroupwasalsosignificantly
higherthanthenon-PTMCgroup(P=0.00).Otherclinicalparametersshowednosignificant
differencesbetweenthetwogroups.
BRAFV600EmutationalstatusandclinicalcharacteristicsinpatientswithPTMCornon-
PTMC
TherelationshipofBRAFmutationstatusandclinicalcharacteristicsof313PTMCpatientswere
analyzedinthisstudy.TheBRAFV600Emutationshowedsignificantassociationwithmalegender
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(P=0.026)andsamplesourcefromFFPEtissues(P=0.018)comparedwiththeBRAFV600Ewild-type
inPTMCpatients.However,therewasnodifferenceinlymphnodemetastases,ageandlocationof
thyroidtumorbetweentheBRAFV600EmutationandtheBRAFV600Ewild-type(Table2).
TheBRAFV600Emutationshowedsignificantassociationwithmalegender(P=0.003)comparedwith
theBRAFV600Ewild-typeinnon-PTMCpatients.However,therewasnodifferenceinotherclinical
featuresbetweentheBRAFV600EmutationandtheBRAFV600Ewild-type(Table2).
TheclinicopathologicalcharacteristicsandsamplesourceinallBRAFV600Emutationpatientswere
comparedadditionally(Table3).Alowerrateoflymphnodemetastasis(P=0.00,χ2=42.369)was
presentedinPTMCthaninnon-PTMC.Thenumberofmiddlesubgroup(31-59,P=0.004,χ2=11.306)
hadstatisticalsignificancebetweenPTMCandnon-PTMC.However,therewasnodifferenceinother
clinicalfeaturesbetweenPTMCandnon-PTMC.
UnivariateandmultivariateanalysisofriskfactorsforLNMinPTCs,PTMCandnon-PTMC
InPTCs(Table4),thefemale(OR=1.952;95%CI=1.373-2.774;P=0.00),middlesubgroup(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)
werecharacterizedasindependentriskyfactorsforLNM.Moreover,thetumorinleftandrightlobes
simultaneously(OR=0.287;95%CI=0.166-0.497;P=0.000)wascharacterizedasaprotectivefactor
forLNM.However,therewasnodifferencebetweenBRAFV600EmutationandLNM(P>0.05).
InPTMC(Table5),thefemale(OR=3.002;95%CI=1.654-5.446;P=0.00)wascharacterizedas
independentriskyfactor,thetumorinleftandrightlobessimultaneously(OR=0.170;95%CI=
0.071-0.405;P=0.00)wascharacterizedasaprotectivefactor.Theage,BRAFV600Emutationdid
notshowstatisticaldifferenceswithLNM(P>0.05).
Innon-PTMC(Table6),thetumorinleftandrightlobessimultaneously(OR=0.441;95%CI=0.220-
0.882;P=0.00)wascharacterizedasaprotectivefactorforLNM.Thegender,ageandBRAFV600E
mutationdidnotshowstatisticaldifferenceswithLNM(P>0.05).
TheBRAFL597QmutationofPTCinoneTibetan
A57-year-oldTibetanmalepatient(withaprotrudingmassontheleftforehead)cametohospital
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withnumbnessintherightlimbforthreeweeks.ThepreoperativeskullCTandMRIscanshoweda
9.3cm×8.1cmmassintheleftfrontal(Fig.1AandB).Theskinonthesurfaceofthemasswas
normalandhardintexture.Thepreoperativeconventionalcolorultrasounddiagnosisshowedthatthe
sizeofthebilateralthyroidwasnormal,andtheechointherightlobewasuneven.Amass(1.2cm×
0.7cm)wasvisible,withovalshapeandclearlydefinedborder.The“leftfrontallobeoccupancyand
skulltumorresection”surgicalplanwassuggestedforimplementationbythemulti-disciplinaryteam
(MDT).Intraoperativedisplayshowedthatthescalptissueandtheleftfrontalskullhadobvious
adhesion,andtheprotrudingbonetissuesurfacewasunevenandloose.Thehyperplasiaoftheinner
andouterplatesoftheskullwasobvious(Fig.1C).Thetumortissuewaswhiteandsolid,andthere
wasnoadhesionwiththesurroundingbraintissue(Fig.1D).Thepreoperativeconventionalcolor
ultrasounddiagnosisshowedthatthesizeofthebilateralthyroidwasnormal,andtheechointhe
rightlobewasuneven.Amass(0.7cm×0.5cm)wasvisible,withovalshapeandclearlydefined
border(Fig.2A).TheUS-FNAwasusedtobiopsythethyroidnodules,thetumorcellswererelatively
uniforminsize,withroundnuclei,smallnucleolivisibleinpart(Fig.2B).
SomenucleusofcancercellsshowedGroundGlassOpacity(GGO)byHEstaining.Thenuclear
groovesandpseudoinclusionsinnucleuswereobservedclearly,andpsammomabodieswasseenin
interstitialtissue(Fig.3A).Thecytokeratin(CK),thyroglobulin(Tg)andthyroidtransformingfactor-1
(TTF-1)wereimmunoreactive(Fig.3B,CandD)byIHC.TheARMS-PCR,NGSandSangersequencing
analysisshowedthatthepatienthadadoublemutationofBRAFL597QandV600Eintwoseparate
lesions(Fig.4).TheBRAFV600E(chr7:140453136c.1799T>A)mutationwaslocatedinsitu(Fig.4A
andC),buttheBRAFL597Q(chr7:140453145c.1790T>A)mutationwaslocatedintheintracranial
metastases(Fig.4BandD).TheabundancesofBRAFL597QandV600Ewere36.9%and8.1%
respectively,asdeterminedbyNGS,andwhichweresuccessfullyverifiedbySangersequencing(the
goldstandardofgenesequencing).
Discussion
Withtheincreasingincidencesofnon-PTMCandPTMC,itisimportanttorisk-stratifypatientswho
mayhavemoreaggressivetumorbiologyinwhatistraditionallythoughttobeamoreindolent
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disease,whichwillhavemanagementimplicationsincludingwhetherornottoobserve,theextentof
surgicalresection,theuseofRIAtherapyandthefrequencyoffollow-up[21].Li[22]havereportedthat
thePTMChadanindolentcourseandexcellentprognosis,whileourresultsdemonstratedthatthe
incidenceofLNMwasmorefrequentinPTMCthaninnon-PTMC.Thecorrelationanalysisrevealedthat
theincidenceofBRAFV600EmutationinmalePTMCwasmuchhigherthaninfemale.Lee[23]have
recommendedthatthemalecouldbeasanindependentprognosticfactorforrecurrenceinnon-
PTMC,butitwasnotaprognosticfactorinPTMC.Inourstudy,theincidenceofBRAFV600Emutation
inFNACwasmuchhigherthaninFFPEwhichwasconsistentwithsomepreviousstudies[24,25],
however,theLNMwasrelatedtoBRAFV600Ewild-typeinPTMCwhichwasinconsistentwithother
study[26].Thus,ourfindingsdemonstratedthattheBRAFV600Emutationwasmorelikelytomanifest
amongmalepatientsandmoreeasilytobedetectedinFNACofPTMC.
TofurtheranalyzethedifferencebetweenPTMCandnon-PTMCinthebiologicalbehavior,the
clinicopathologicalcharacteristicsandsamplesourceinBRAFV600Emutationpatientswerealso
compared.AlthoughseveralstudieshavereportedthatBRAFV600EmutationinPTCswasassociated
withaggressivepathologicalfeatures,negativeinfluenceon131Iavidity,reducedthyroperoxidase,
theincreasedriskoflymphnodemetastasisandrecurrenceaftertreatment[27-29],theclinical
implicationsandclearmechanismsinPTMCandnon-PTMCwascontradictory.Zheng[30]have
reportedthattumordiameter(>0.5cm)wasanindependentriskfactorcorrelatedwithLNMinPTCs.
Interestingly,ourfindingsdefinitelydemonstratedthattheLNMratewasmuchhigherandwas
correlatedwithBRAFV600Ewild-typeinPTMC,whichpossiblyindicatedthatBRAFV600Emutationin
PTMCwaslessaggressivewhichwasdifferfromapreviousmeta-analysis[22].Astudy[31]have
reportedthatmostPTMCwithBRAFV600EmutationdidnotdisplayBRAFV600Eproteinexpression.
Correctpreoperativediagnosisisofimportance.Itisgenerallyagreedthatbetterknowledgeabout
predictiveriskfactorsforLNMmayguideclinicaldecisions,whilethegreaterriskofLNMremains
debatable.Afteradjustingforothersignificantpreoperativeclinicalfactors,univariateand
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multivariateanalysiswasperformedtoidentifytheriskfactorsforLNM.Genderisaprominentpatient
backgroundparameterforPTC.Inrecentyears,theassociationbetweengenderandrecurrenceor
survivalofPTChasbeendebated.Apreviousstudyreportedthatthemalewasanindependent
clinicalprognosticfactorofpooroutcomeinPTC[32],butnotinPTMC[23].Recently,Roh[33]have
reportedthattherewasnoassociationbetweengenderandLNM.Inourstudy,althoughthefemale
hadalowerBRAFV600Emutationthanthemale,multivariateanalysisdemonstratedthatthefemale
wasariskfactorofLNMinPTMCandnon-PTMC,whichwasdifferentfromabovestudies.Aprevious
study[34]alsohaveexploredthatthefemalewasanindependentpredictiveriskfactorofCLNMin
PTC.Controversiesindifferentstudiescouldberelatedtodifferentsamplesource,samplesizeand
detectiontechniques.Ithasbeenreportedthatthefemalehadanearlierageofonset,butthemale
hadahighermortality[35,36].ItisrecommendedthatthemolecularmechanismsbetweenLNMand
genderinPTCpatientsshouldbeexploredinfuture.
Inourstudy,themiddleage(31-59)groupwasabout1.56timestheyoungage(≤30)groupforLNM
inPTCs,whichwasinconsistentwithotherstudy[37].Ourresultshaveshowedthatthelocationof
tumorinbilateralthyroidsimultaneouslywasaprotectivefactorforLNMinPTMCandnon-PTMC,
whichwasalsoinconsistentwitharetrospectivecohortstudy[38].
Thequestionistoclarifywhetherornot,aftercontrollingtheclinicalimportanceofBRAFV600E
mutation,totakeintoaccountthatsomeotherBRAFmutationsdeservethoughtfulanalysis.The
BRAFL597Qmutationincidencewaslessthan1%[39]suchasinchildhoodacutelymphoblastic
leukemia[18],whichmightbeassociatedwithaggressiveclinicopathologicfeatures,however,the
potentialroleofthepeculiarBRAFL597QmutationofPTCwasunclear.Tothebestofourknowledge,
wehavereportedthefirstcaseofsolitarybrainmetastasisfromoccultpapillarythyroidcarcinomain
theChineseTibetanpopulationindetail,asallknown,thecommonmetastaticsitesofthyroidcancer
arelungandbone,skullmetastasesareextremelyrare[40].Ourresultshavedemonstratedthatthe
rareBRAFL597Qpointmutationmightplayaspecificroleininducingthesolitaryintracranial
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metastasisofoccultpapillarythyroidcarcinomaintheChineseTibetanpopulation.Inaddition,inthis
case,thebiopsyortraditionalgenesequencingtechnologies(suchastheARMS-PCR,solelyfor
detectingtheBRAFV600Emutation)hadcertainlimitations,thecombinationofNGSandSanger
sequencingshouldhelpindetectingtheraregenemutation(e.gBRAFL597Qmutation),whichwas
greatlyrecommendedtoimprovethediagnosticaccuracyandmolecularmechanismanalysisinrare
PTCcases.Itisconcludedthatthemultilevelgeneanalysismaybeagreatsubstituteforthe
traditionalgenetesting[41,42].
Nonetheless,ourretrospectivestudyhadseveralpotentiallimitations.Firstly,thenumberoftherare
cases(BRAFL597Qmutationwithskullmetastases)wasfew.Secondly,thedetailedmolecular
mechanismshouldbeconfirmedbyalargenumberoffunctionalexperimentsandclinicalresearches
whichmighthelptomanifestthattheBRAFL597Qmutationmightplayaspecificroleininducingthe
solitaryintracranialmetastasisofoccultPTC.Thirdly,ourstudydealtwithagroupofpatientswith
PTConlyinSouthwestofChina.
Conclusions
Inconclusion,wehavedemonstratedthatthefemale,middleage(31-59years)andPTMCwere
independentlycorrelatedwithLNMinPTCs,whilethetumorinleftandrightlobessimultaneouslywas
aprotectiveroleinLNM.Meanwhile,ourstudydemonstratedanegativeresultbetweenBRAFV600E
andLNM.Also,FNACfromtumorsampleshadahigherrateofBRAFV600EmutationthanFFPEin
PTMC,whichconfirmedthatFNACmightbeareliableinterventiontodetectBRAFV600Emutation.
Moreover,toourknowledge,apossibleassociationbetweentherareBRAFL597Qmutationand
intracranialmetastasisofoccultPTCintheChineseTibetanpopulationwasreportedfirstlywhichmay
beusedasatherapeutictargetinfuture.Therefore,wesuggestthatcliniciansshouldmakea
comprehensivelyconsiderationofclinicalfeatures:samplesource,BRAFmutation,tumorsize,
gender,locationofthyroidtumorandmultilevelgenesequencingtechnologiesandtherapeutic
scheduleinordertoachievearelativelygoodprognosis.
Abbreviations
PTC:papillarythyroidcarcinoma;PTMC:papillarythyroidmicrocarcinoma;NGS:nextgeneration
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sequencing;US-FNA:ultrasound-guidedfine-needleaspiration;FFPE:formalin-fixedparaffin-
embedded;FNAC:fine-needleaspirationcytology;HE:hematoxylin-eosinstaining;IHC:immunohisto-
chemical(IHC)analysis;PCR:polymerasechainreaction;LNM:lymphnodemetastasis;OR:odds
ratios.
Declarations
DisclosureofPotentialConflflictsofInterest
Theauthorsdeclarethattheyhavenocompetinginterests.
Authors’Contributions
Conceptionanddesign:YanDong,DanWangandXiaosongLi.Developmentofmethodology:Huili
Bai,YifanShen,YangliZhang,XuepingChenandYajingZhao.Acquisitionofdata(acquiredand
managedpatients,providedfacilities,etc.):XinliangSu,JinqiuZhao,HuandongLiu,JungaoLu,Zuoyi
YaoandYajingZhao.Analysisandinterpretationofdata(e.g.,statisticalanalysis,biostatistics,
computationalanalysis):LingChen,DanWangandYishengLuo
Writing,review,and/orrevisionofthemanuscript:YanDong,DanWangandXiaosongLi.
Administrative,technical,ormaterialsupport(i.e.,reportingororganizingdata,constructing
databases):LingChen,DanWangandXiaosongLi.Studysupervision:XiaosongLi.Allauthors
contributedtothismanuscript.Allauthorsreadandapprovedthefinalmanuscript.
Acknowledgments
Theauthorswouldliketothankthepatientswhoparticipatedinthisstudy,andtheirfamilies,aswell
astheinvestigatorsandstudyteams.
Funding
ThisstudywassupportedbytheNationalNaturalScienceFoundationofChina(grantno.81871653)
andChongqingMedicalScienceProject(grantno.2018MSXM065).
Availabilityofdataandmaterials
Alldatageneratedoranalyzedduringthisstudyareincludedinthispublishedarticle.
Ethicsapprovalandconsenttoparticipate
ThisstudywasapprovedbytheEthicsCommitteeoftheFirstAffiliatedHospitalofChongqingMedical
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UniversityEthicsReviewBoard.
Consentforpublication
Notapplicable.
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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
Lymphnodemetastasis 326 181(57.8%) 145(19.8%) 0.000
BRAFV600Emutation 839 273(87.2%) 566(77.3%) 0.000
Differentlocationsofthyroidtumor
Leftlobe
Rightlobe
Isthmus
Leftandrightlobes
Leftlobeandisthmus
Rightlobeandisthmus
Bilaterallobesandisthmus
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▲
Samplesource
FNAC
FFPE
303
742
78(24.9%)
235(75.1%)
225(30.7%)
507(69.3%)
0.058
Tables
Table1.Characteristicsofpatients
Note: Quantitative data were showed as Mean±SD or N (%);P value of 0.05 or less was
consideredsignificant▲Fisherexacttest.FNAC:fine-needleaspirationcytology;FFPE:formalin-fixed,
paraffin-embedded
Table2.CorrelationbetweenclinicopathologicalcharacteristicsandBRAFV600Emutation
Note▲Fisherexacttest
20
Characteristics
PTMC P
value
non-PTMC
BRAFV600E
mutation BRAFV600Ewild-
type BRAFV600E
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
Lymphnodemetastasis
Yes(+)
No(-)
158
115
23
17
0.964
449
117
Differentlocationsofthyroidtumor
Leftlobe
Rightlobe
Isthmus
Leftandrightlobes
Leftlobeandisthmus
Rightlobeandisthmus
Bilaterallobesandisthmus
88
127
3
49
1
5
0
16
20
0
3
0
1
0
0.475▲
211
259
3
79
3
8
3
Samplesource
FNAC
FFPE
75
198
4
36
0.018
176
390
Table3.ComparisonofprogressionbetweenPTMCandnon-PTMCwithBRAFV600Emutation
21
Characteristics BRAFV600Emutation 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
Lymphnodemetastasis
Yes(+)
No(-)
158
115
449
117
42.369
0.000
Differentlocationsofthyroidtumor
Leftlobe
Rightlobe
Isthmus
Leftandrightlobes
Leftlobeandisthmus
Rightlobeandisthmus
Bilaterallobesandisthmus
88
127
3
49
1
5
0
211
259
3
79
3
8
3
5.644
0.445
Samplesource
FNAC
FFPE
75
198
176
390
1.153
0.283
Note▲Fisherexacttest
Table4.UnivariateandmultivariateanalysisofriskfactorsforLNMinPTC(n=1045)
22
Variable
Univariateanalysis
OR95%
CI POR95%
Gender(Maleasreference)
Female 1.868(1.340-
2.604) 0.000 1.95(1.373-
2.774)
Age(≤30asreference)
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)
BRAFV600E(Yesasreference)
No(-) 0.72(0.505-
1.043) 0.084
Tumortype(non-PTMCasreference)
PTMC 2.93(2.198-
3.910) 0.000 3.27(2.417-
4.432)
Differentlocationsofthyroidtumor(Leftlobeasreference)
Rightlobe
Leftandrightlobes
Isthmus
Bilaterallobesandisthmus
Rightlobeandisthmus
Leftlobeandisthmus
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:lymphnodemetastasis;OR:oddsratio;CI:confidenceinterval
Table5.UnivariateandmultivariateanalysisofriskfactorsforLNMinPTMC
23
Variable
Univariateanalysis
OR95%
CI POR95%
Gender(Maleasreference)
Female 2.91(1.671-
5.077) 0.000 3.00(1.654-
5.446)
Age(≤30asreference)
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)
BRAFV600E(Yesasreference)
No(-) 0.93(0.481-
1.805) 0.835 0.72(0.356-
1.454)
Differentlocationsofthyroidtumor(Leftlobeasreference)
Rightlobe
Leftandrightlobes
Isthmus
Bilaterallobesandisthmus
Rightlobeandisthmus
Leftlobeandisthmus
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:lymphnodemetastasis;OR:oddsratio;CI:confidenceinterval
Table6.UnivariateandmultivariateanalysisofriskfactorsforLNMinnon-PTMC
24
Variable
Univariateanalysis
OR95%
CI POR95%
CI
Gender(Maleasreference)
Female 1.42(0.929-
2.165) 0.105 1.52(0.980-
2.346)
Age(≤30asreference)
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)
BRAFV600E(Yesasreference)
No(-) 0.79(0.500-
1.241) 0.304 0.77(0.482-
1.224)
Differentlocationsofthyroidtumor(Leftlobeasreference)
Rightlobe
Leftandrightlobes
Isthmus
Bilaterallobesandisthmus
Rightlobeandisthmus
Leftlobeandisthmus
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:lymphnodemetastasis;OR:oddsratio;CI:confidenceinterval
Table7.TheNGSpanelforthyroidcancer
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
Figure1
PreoperativeskullCT/MRIscanandintraoperativephotographsduringresectionand
debulking.(A:Irregularbonedestructionoftheleftfrontalbone,therangeisabout5.7
cm×6.3cm.B:Irregularmassesareseenintheleftfrontalcranialandsubscalp,thesizeis
about4.9cm×5.4cm,itisunevenandobviouslystrengthened,andthecenterline
structureisskewedtotheright.C:Theouterplateofskull,raisedbone(4.8cm×5.2cm)
surfaceoftheskull’souterplatewasuneven,showingastateofcancellousbone.D:The
tumorisolatedfromtheoperationwashardintexture,about5.7cm×6.3cminsize.)
27
Figure2
Preoperativeconventionalcolorultrasounddiagnosis(A)andUS-FNAdiagnosis(B)(A:The
echointherightlobewasuneven,anda0.7cm×0.5cmmasswasvisible.B:Alesional
biopsyspecimenofthethyroidnoduleswasobtained(hematoxylin-eosin,400×.)
28
Figure3
TheHEandIHCstainingoftheintracranialmetastases(400×)(A:Tumorcellsarerelatively
uniforminsize,withroundnuclei,smallnucleolivisibleinpart,cytoplasmicstainingand
mitoticdivisionsthatarenoteasilyseen.B:CK19stainingshowedbrownishyellowparticles
onthecellmembrane(+++).C:TGstainingshowedbrownishyellowparticlesonthecell
membrane(+).D:TTF1stainingshowednucleusshowingbrownishyellowparticles(+++).)
29
Figure4
Geneticdiagnosisresults(A:BRAFV600E(c.1799T>A)mutationtestedbySanger
sequencinginsitu.B:BRAFL597Q(c.1790T>A)mutationtestedbySangersequencingin
metastases.C:BRAFV600E(c.1799T>A)mutationtestedbyNGSinsitu,andD:BRAF
L597Q(c.1790T>A)mutationtestedbyNGSinmetastases.)
30
Figure5
ComparisonofmutationratesbetweenPTMCandnon-PTMC(Comparisonofmutationrates
betweenPTMCandnon-PTMCover3years;DatawereshowedasN(%);Thedifferencein
mutationratebetweenPTMCgroupsandnon-PTMCgroupswerehighlysignificant.)