www.bjorl.org
Brazilian
Journal
of
OTORHINOLARYNGOLOGY
ORIGINAL
ARTICLE
Evaluation
of
circulating
cell
free
DNA
in
plasma
as
a
biomarker
of
different
thyroid
diseases
夽
Ozge
Caglar
a,∗,
Begum
Cilgin
b,
Mustafa
Eroglu
c,
Akin
Cayir
d,eaCanakkaleOnsekizMartUniversity,FacultyofMedicine,DepartmentofHeadandNeckSurgery,Canakkale,Turkey bCanakkaleOnsekizMartUniversity,InstituteofHealthSciences,Canakkale,Turkey
cCanakkale18MarchUniversityHospital,EndocrinDepartment,Canakkale,Turkey
dCanakkaleOnsekizMartUniversity,VocationalSchoolofHealthServices,Canakkale,Turkey
eHumanNutritionResearchCentre,InstituteofCellularMedicine,NewcastleUniversity,Newcastle-upon-Tyne,UnitedKingdom
Received13September2018;accepted15December2018 Availableonline18February2019
KEYWORDS
Thyroiddiseases; CellfreeDNA; Plasma; Biomarker
Abstract
Introduction:Many studies havebeen done onproteomics, genomics, epigenetic, immuno-genetics inmany body fluids. Among these,circulating cell-free DNA (ccfDNA)enteredthe literaturein1948,butithasnotbeenstudiedformanyyearsduetotechnologicaldeficiencies. Followingrecentadvances,geno-metastasishasbeenmentionedandnewresearchisneeded inthisarea.ccfDNAisknowntobeanimportantbiomoleculeinthisregard.
Objective: Thepresenceofcell-freeDNAinthecirculatorysystem mayofferatremendous opportunity toprovide novelbiomarkers for thyroiddiseases. This experimental study was conductedtodeterminetheamountofccfDNAindifferentthyroiddiseases,thentoevaluate whethertheccfDNAconcentrationvariedbetweenthediseasegroupsandcontrolgroup.
Methods:Intotal,weincluded121individualsinthepresentstudy.Wecollectedbloodsamples andthendeterminedtheccfDNAconcentrationinplasmaofcollectedbloodsamplesfromthree groups: thyroiditis(n=33),benign(n=37),andmalignant(n=30) andfromacontrolgroup (n=21).
Results:ThemedianvaluesoftheccfDNAgroupswerefoundas1610,1665,1685and576ng/mL forthethyroiditis,benign,malign,andcontrolgroups,respectively.Findingsshowedthatthe ccfDNAofthethreegroupswassignificantlyhigherthanthecontrol(p<0.0001).Eachgroup wascomparedintermsofccfDNAandthep-valuesofbenign-thyroiditis,benign-malign,and thyroiditis-malignwere0.09,0.65,and0.29,respectively.
夽 Pleasecitethisarticleas:CaglarO, CilginB,ErogluM,CayirA.EvaluationofcirculatingcellfreeDNAinplasmaasabiomarkerof
differentthyroiddiseases.BrazJOtorhinolaryngol.2020;86:321---6.
∗Correspondingauthor.
E-mail:ozgecaglar@comu.edu.tr(O.Caglar).
PeerReviewundertheresponsibilityofAssociac¸ãoBrasileiradeOtorrinolaringologiaeCirurgiaCérvico-Facial.
https://doi.org/10.1016/j.bjorl.2018.12.008
1808-8694/©2019Associac¸˜aoBrasileiradeOtorrinolaringologiaeCirurgiaC´ervico-Facial.PublishedbyElsevierEditoraLtda.Thisisanopen accessarticleundertheCCBYlicense(http://creativecommons.org/licenses/by/4.0/).
Conclusions:ThecleardifferencesbetweenthyroiddiseasesandcontrolssuggestthatccfDNAis worthyofattentionasabiomarkerforfurtherevaluationofdifferentthyroiddiseases.Likewise, itmightindicateacleartendencythatccfDNAcanalsobeusedtodistinguishdifferentthyroid diseases.
© 2019 Associac¸˜ao Brasileira de Otorrinolaringologia e Cirurgia C´ervico-Facial. Published by Elsevier Editora Ltda. This is an open access article under the CC BY license (http://
creativecommons.org/licenses/by/4.0/).
PALAVRAS-CHAVE
Doenc¸asdatireoide; DNAlivredecélulas; Plasma;
Biomarcador
Avaliac¸ãodoDNAcirculantelivredecélulasnoplasmacomoumbiomarcadorde diferentesdoenc¸asdatireoide
Resumo
Introduc¸ão: Muitosestudosforamrealizadosemproteômica,genômica,epigenéticae imuno-genéticaem vários fluidoscorporais. Entreesses,oDNA circulantelivre de células(cfDNA) despontounaliteraturaem 1948, mas nãofoi estudadopormuitos anosdevidoa deficiên-ciastecnológicas.Apósrecentesavanc¸os, agenometástase émencionadaenovaspesquisas tornam-senecessáriasnessaárea.Nessesentido,ocfDNAéconhecidoporserumaimportante biomolécula.
Objetivo:Apresenc¸adeDNAlivredecélulasnosistemacirculatóriopodeofereceruma exce-lenteoportunidadeparafornecernovosbiomarcadoresparadoenc¸asdatireoide.Esteestudo experimentalfoiconduzidoparadeterminaraquantidadedecfDNAemdiferentesdoenc¸asda tireoideeentãoavaliarseaconcentrac¸ãodecfDNAvariou entreosgruposcomdoenc¸aeo grupocontrole.
Método: Nototal,121indivíduosforamincluídosnoestudo.Coletamosamostrasdesanguee, ementão,determinamosaconcentrac¸ãodecfDNAnoplasmadeamostrasdesanguedetrês grupos:tireoidite(n=33),benigno(n=37)emaligno(n=30)edeumgrupocontrole(n=21).
Resultados: Asmedianas dos valores dos grupos de cfDNA foram de 1.610, 1.665, 1.685 e 576ng/mLparaosgrupostireoidite,benigno,malignoecontrole,respectivamente.Osachados mostraramqueocfDNAdostrêsgruposcomdoenc¸aerasignificativamentemaiordoqueodo grupocontrole(p<0,0001).CadagrupofoicomparadoemtermosdecfDNAeosp-valoresde benigno-tireoidite,benigno-malignoetireoidite-malignoforamde0,09,0,65e0,29, respecti-vamente.
Conclusões:Comoresultado,asóbviasdiferenc¸asentreasdoenc¸asdatireoideeoscontroles sugeremqueocfDNAédignodeatenc¸ãocomoumbiomarcadorparaavaliac¸ãoadicionaldas diferentesdoenc¸asdatireoide.Damesmaforma,issopodeindicarumaclaratendênciadeque ocfDNAtambémpodeserutilizadoparadistinc¸ãodasdiferentesdoenc¸asdatireoide. © 2019 Associac¸˜ao Brasileira de Otorrinolaringologia e Cirurgia C´ervico-Facial. Publicado por Elsevier Editora Ltda. Este ´e um artigo Open Access sob uma licenc¸a CC BY (http://
creativecommons.org/licenses/by/4.0/).
Introduction
Ithasbeen estimatedthat6.71% oftheEuropean popula-tionand4.78%oftheUnitedStates(U.S.)populationwith thyroiddisordershasnotbeendiagnosed,1whichrepresents
halfthenumberofpeoplewiththedisease.2Astudy
per-formedintheU.S.showedthat4.6%and1.3%ofindividuals werediagnosedwithhypothyroidism andhyperthyroidism, respectively, in the screening of 13,344 individuals with previously-unrecognized thyroid disease.3 Similar findings
observedin a meta-analysisstudy showed that 4.94%and 1.72% of individuals were diagnosed with hypothyroidism andhyperthyroidismrespectively.4
Thyroid dysfunctions and hypothyroidism have become more common in recent years in the middle age group and in women as a result of increased thyroid
inflammation. Thyroiditis is evaluatedin three categories according toitshealingtime:acutethyroiditis(fastonset and short duration), delayed thyroiditis (lastingless than a year), and chronic thyroiditis (life-long). Among these, Hashimato thyroiditis is a chronic thyroid pathology that is the most common life-threatening inflammation of the thyroid.Althoughthecauseisnotcompletelyknown,some factorsincludinguseofiodizedsalts,infections,and expo-sure to radiation are considered to be effective in the formationofHashimoto’sthyroiditis.
Amongthyroiddiseases,3---7%ofpeoplewiththyroid nod-uleshasathyroiddisorder.Accordingtoresearch,mostof these nodules are benign tumors and 5---8% of them can be converted to malignant tumors5 which are the most
commontumorsoftheendocrinesystem. Theclinical sig-nificanceofthyroidnodulesisbasedontheneedtoexclude
thyroidcancer,whichisseenin7---15%ofcases,dependingon age,gender,radiationexposurehistory,familyhistory,and otherfactors.6,7Therearefourmajortypesofcancerinthe
thyroidgland.Theseare,inorderoffrequency: papillary, follicular,medullary,andanaplasticcarcinomas.A consider-ablenumberofcancersarefollicularcells.5Differentiated
ThyroidCancer(DTC)includingpapillaryandfollicular carci-nomaconstitutesthemajority(>90%)ofallthyroidcancers.8
Currently,FineNeedleAspirationBiopsy(FNAB) isused to diagnose and treat malignant tumors, but a 20% fail-urerateoccursduetothelack ofadequatespecimenand specifictissue,cytologictechniquesanddifferentmethods used by technicians. Therefore, there is a need for new, more reliable biomarkersable todiagnose thyroid cancer early and definitively, which can be measured in a non-invasive,easyandaccuratemanner.Forthispurpose,many studies have been carried out on proteomics, genomics, epigenetics, immunogenetics and other molecules in var-ious bodily fluids. Among these, circulating cell-free DNA (ccfDNA) entered theliterature in 1948 but hasnot been studiedfor manyyearsduetotechnologicalinadequacies. Following recent technologicaldevelopments, the mecha-nism of metastasis has begun to be discussed again, and mentionof geno-metastasis, leading tothe need for new researchinthisarea.ccfDNAisanimportantbimolecularin thisregard.Thismoleculegainsattentioninmanydiseases, mainly in cancer studies at the genomic and epigenomic level,inrelation tocancer type,development,amountof bloodplasma,andothermoleculesintheplasma.For exam-ple,suchdataisverypromisingindeterminingwhetheror notapersonhascancerandifthecancerhasmetastasized inthe body,whether successhasbeen achievedfollowing treatment,andwhethersurgeryisnecessary.
Inthepresent study,we aimedtomeasuretheamount ofccfDNAinplasmasamplescollectedfromindividualswith thyroiddiseasesandhealthyindividuals,andthento com-pare the amount of each groups. The realization of this studywillmakeanimportantcontributiontoward determin-ingwhetherthelevelofccfDNAcanbeusedasamolecular biomarkerfordifferentthyroiddiseases.
Methods
Thepresent studywasconductedontheblood samplesof 100 thyroid patients. Of the 100 patients included in our study, 33 were patients withthyroid disease, 37 patients hada benign thyroid nodule,and30 patients had thyroid cancer. Forthe controlgroup, 21 patients withnoknown additionaldiseaseswereincluded.Weperformedadditional examinationofpatientswithnodulesormassesinthe thy-roid.PatientswerediagnosedwiththyroidUSG,followedby FNAB,however,somecancerpatientsweredefinitively diag-nosedafterundergoinganoperation.Onlythepatientswho had thyroid disease were evaluatedin the present study. Before taking peripheral blood samples,the patients and controlswereinformedaboutthestudyanditsaimsandthen each gave their consentby signing theappropriate form. The characteristics of the patients and the controls were collectedby means ofa questionnaire.Ethical permission wasgrantedbythe Facultyof Medicine EthicsCommittee (27/2016-E.70096).
Forthe ccfDNA, 5mL blood wascollected in sterilized tubescontainingK3-EDTA.Aftertakingthebloodsamples, theywereimmediatelycentrifugedat3000gfor10min.The plasmasamplesweretransferredtoanothersterile, DNAse-freetubeandthesampleswerecentrifugedat16,000gfor 10min.Then,theplasmasampleswereseparatedandstored at−80◦Cforfurtherstudy.TheDNAcontentoftheplasma sampleswasmeasured directly withafluorescence-based Quant-iTTM high-sensitivityDNAassaykitandaQubit®
flu-orometer(Invitrogen, Carlsbad,CA, USA).The Qubit® 2.0 FluorometerisusedforquantitationofDNA,RNA,and pro-tein.Itusesspecificdyesforeach typeofmoleculewhich haveextremelylowfluorescenceuntiltheybindtotheir tar-gets.Afterbinding, theygiveoff an intenselyfluorescent signalwhichisdirectlyproportionaltotheDNA concentra-tionof any solution. In the present study, the ccfDNA of eachindividualwasmeasuredusinga DNAcurveobtained from the DNA standard of known concentrations. Plasma sampleswere analyzed in duplicate andthe mean of the twovalueswasusedasthefinalDNAamount.The between-measurements and coefficients of variation of this assay werelessthan1.00%.
Differences between groups were detected using one-way ANOVA then Bonferroni as a post hoc test. The differencesbetweenmaleandfemaleswereanalyzedusing Mann---WhitneyU test. Significance levels were indicated. The effects of confounding factors (age, gender, smok-ing, habits, etc.) were also evaluated statistically. The contribution of each factor taken into consideration was evaluatedusingmultipleregressionanalysis.Possible associ-ationswereinvestigatedwithPearsoncorrelation.SPSS18.0 programwasusedforstatisticalcalculations.
Results
Theageandgenderdistributionofeachgrouparepresented inTable1.Themajorityofpatientsinourstudywerefemale. Thirty(90.9%)hadthyroiditisand64.9(24%)hadbenign nod-ules.Twenty-three(76.7%)of themalignantpatientswere females.Themeanagewas37.6forthosewiththyroid dis-eases,54.1inbenignnodules,and47.8forcancerpatients. Inthecaseofsmokingandalcoholuse,8(23.5%)ofthe thyroidpatientswereactiveusers,6 (17.8%)wereformer users,and7(20.5%)reportedactivealcoholuse.Amongthe malignantpatients,13(43.3%)patientswereactiveusers,3 (10%)hadusedalcoholbefore,and7(23.3%)patientsused alcoholcurrently.Inthebenignnodulepatients,6(15.7%) patientsreported activecigarette use, 6(15.7%) patients hadthe habit previously, and 4 (10.5%) patients reported continuing alcohol use. The control group consisted of 9 (42.85%)malesand12(57.14%)females.Themeanagewas 30.1inthecontrolgroup.
Theresults obtainedforccfDNA ineachgroupare pre-sented in Table 2 and Fig 1. According to the results obtained,theminimumandmaximum valuesofccfDNAin the patients were found to be 908---2280ng/mL for thy-roiditis,1344---2560ng/mLforbenign,1212---2840ng/mLfor malign,and454---1018ng/mLfor thecontrols.The median valuesoftheccfDNAgroupswerefoundas1610,1665,1685, and576ng/mLforthethyroiditis,benign,malign,and con-trolgroups,respectively.
Table1 Characteristicsofthepopulations.
Control(n=21) Thyroiditis(n=33) Benign(n=37) Malign(n=30)
Age 30.1±15.2 37.6±10.9 54.1±13.1 47.8±11.9
Male 29.8±15.8(43%) 47.3±7.5(10%) 58.9±10.1(33%) 46.1±13.4(23%) Female 30.4±15.4(47%) 36.7±10.8(90%) 51.7±13.9(67%) 48.3±11.8(77%)
Table2 ccfDNAindiseasedandcontrolgroups.
ccfDNA Control(n=21) Thyroiditis(n=33) Benign(n=37) Malign(n=30)
Totalmean 623.3±172.9 1613±284 1721.3±243 1691±292 Minimum 454 908 1344 1212 Maximum 1081 2280 2560 2840 Male 608.0±120 1667±217 1825±310 1687±251 Female 634.8±208 1608±292 1664±181 1692±309 ccFDNA Groups 1900.00 2400.00 650.00 400.00 900.00 1150.00 1400.00 1650.00 2150.00 Control Group Malign Thyroiditis Benign
Figure1 ccfDNAamountineachgroup.
Four groups including control, malign, benign, and thyroiditis were compared using one-way ANOVA then Bonferroni as a post hoc test. According to the obtained results,ccfDNAinthethreepatientgroupswassignificantly higher than the controls (p<0.0001 for all groups). Each groupwascomparedin terms of ccfDNAand thep-values
ofbenign-thyroiditis,benign-malign,andthyroiditis-malign were0.09,0.65,and0.29,respectively.
TheccfDNAofmaleswiththyroiditiswasslightlyhigher than the females; however, this difference did not reach asignificantlevel(p=0.7).Thesmokerthyroiditispatients (1658ng/mL)hadhigherccfDNAwhencomparedwith non-smokerpatients(1599ng/mL).However,thedifferencewas notstatisticallysignificant (p>0.05). Analysisrevealed no correlationbetweenccfDNAandage.Linearregression anal-ysisshowed thatccfDNAwasnot affectedby confounding factorssuchasage,gender,smoking,andthealcoholhabit (p>0.05).
Inthisgroup,thereisaclearsignificanttendencytoward males(1825ng/mL)thaninfemales (1664ng/mL)interms of ccfDNA (Mann Whitney U, p=0.054). The amount of ccfDNAinsmokersandnonsmokersissimilarandthusthere is no relationship between smoking and ccfDNA. It was observedthattheccfDNAinbenignindividualsdidnot corre-latewithage(p>0.05).Linearregressionanalysisindicated
thatgender might affectccfDNAin benignpatientsin the modeladjustedbyage,gender,smoking,andalcoholhabit (p=0.08).
Intermsofgender,thereisnodifferencebetweenmales andfemales.Similarly,itwasobservedthattheccfDNAof smokers and non-smokers is similar. In the malign nodule groupwedidnotobserveanycorrelation betweenccfDNA andage.Intheregressionanalysis,nopotentialfactorswere determined which affected the amount of ccfDNA in the plasmasamples.
As a group containing three different diseases, the ccfDNA of males (1762ng/mL) was higher than females (1651ng/mL), which had a tendency to be significant (p=0.08). In terms of the smokinghabit, the amount for smokersandnon-smokers’ccfDNAwasveryclose(p>0.05). AccordingtoSpearmancorrelation,theccfDNAispositively correlatedwithageatasignificantlevel(r=0.21,p<0.05). Inmultivariateregressionmodelsadjustedbyage,gender, smoking, and alcohol habit, the ccfDNA did notassociate withanyfactorsinthemodels.Afterbackwardmethodwas appliedtothesameanalysis,agewasthesolefactor that affectedccfDNAinthyroiddisease.
Discussion
In many diseases, especially for cancer, it is crucial to find surrogate samples due to the invasiveness of the samples. Today, it is well-known that ccfDNA can be measured in healthy individuals in body fluids including plasma, serum, sputum, and urine.9 On the other hand,
ccfDNAcan alsobemeasuredat higherlevelsinother ill-nessesincludingautoimmunediseases,severalcancersand cardiovasculardiseases.9---11Morespecifically,the
character-ization of ccfDNA in respect of its mutations, epigenetic alterations, andfragment size anddistributionhave been associatedwithseveraltypesofcancer.12Thus,non-invasive
biomarkersenableustofollowthediagnosis,prognosisand monitoringofdiseases.
Although the origin of ccfDNA is not clear, there are several mechanismsthatcontribute tothe amountof cir-culatingDNA.ThemechanismscontributingtoccfDNAmay
changedependingonthetypeand/orstageofthedisease. Ithasbeenacceptedthatdeadordyingcellsarethemain source of ccfDNA. However,it has also been proven that living cells can releaseccfDNA. Furthermore,the amount in circulation may vary depending on the disease stage. Above all, cell breakdown, apoptosis, necrosis, exosome, andvirtosomesareacceptedasthemainmechanisms.Ina suggestedmodel,itissupposedthattherearethreeevents responsiblefortheccfDNAinthebloodofacancerpatient, includingnecroptosis,apoptosis,andsecretionbytumorand macrophages.13Duetotheseevents,ccfDNAcanbereleased
single-stranded ordouble-stranded, anddepending onthe tumortype,differentformsofccfDNAmaybereleased.13
In the present study, we determined that the ccfDNA measuredingroupshavingthreedifferenttypesofthyroid diseasewasconsistentlysignificantlyhigherthanthecontrol group.WefoundthattheccfDNAinthyroiditis,benign,and maligngroupswere2.59,2.76,and2.72timeshigherthan the control group.Therefore, we present ccfDNA amount inplasmasamplesasanovelbiomarkerofthyroiddiseases. Amongthethyroiddiseases,therewerenocleardifferences. However,thehigherccfDNAofbenigncomparedwith thy-roiditismightindicatethatccfDNAcanbeusedtoshowthe differencebetweenthyroiddiseases.
The rationale behind thyroid cancer is not yet fully understood.However,ithasbeensuggestedthatthereare multiple factors including radiation, environmental pollu-tion, and nutrition that cause the individual to be prone tothyroid cancer. All multifactorial causes have the abil-itytoinduceapoptoticmechanisms.14 As indicatedabove,
environmentalexposurecanaffectthemechanisms associ-atedwiththeccfDNAamount.15Themechanismsregarding
ccfDNA release arealso associated withthyroid diseases. Forexample,ithasbeenstatedthatapoptosisisassociated withhomoeostasisofthethyroidgland.Italsoplaysarole in the mechanisms of autoimmune thyroiditisand in thy-roidcâncer.16Inthisregard,thehigherquantityofccfDNA
inthepresentstudy canbeattributedtoapoptotic mech-anisms.Inanotherstudy,therewasasignificantdifference intheccfDNAintegrityindexbetweenpatientswithbenign nodulesandpatientswithcytologicallythyroidcarcinoma. This findingsupportsccfDNAasa promisingbiomarker for thediagnosisofthyroidnodules.17Inourstudy,weincluded
these patients in addition to follow-up patients with the causeofthyroiddisease(thyroiditis).
TheabnormalincreaseintheamountofccfDNAinserum and plasma in cancer patients wasdetected for the first timein1977.18 However,theuseof prognosisand
diagno-sis as a marker has only recently been addressed.19 In a
largegroupofcancersincludingcolorectal,pancreas,lung, bladder, bass-neck, and liver cancer, free DNA mutations weredetected intheccfDNA.20---25 SeveralDNAalterations
havebeenreportedinfreeDNA,includingpointmutations, DNA hypermethylation,Microsatellite Instability (MI), and HeterozygousLoss(LOH)(A).Inmanycases,thesechanges arethesameasthosefoundinthepatient’sprimarytumor siteandsupportthetumoraloriginofthealteredfreeDNA. ChangesinccfDNAarenotconfinedtoanyparticulartumor region,speciesorgrade.However,patientswithlatestage diseaseandmetastasistendtohavesignificantlyhigher lev-elsofccfDNA.Thus,inthepresent study,ccfDNAcanalso
provideavaluableresourceofgeneticmaterialasa surro-gateformolecularanalysisinthyroiddiseases.
It hasbeen well knownthat larger datacould provide morepreciseresultsin suchastudy.Thesmallnumberof participantsis a limitingaspect of ourstudy. Despitethis situation,the statistically significant differences between thediseasesgroupsandthecontrolssupportedtheccfDNA asadistinctfeatureforthyroiddiseaseswhichcouldbeused fordiagnosis,prognosisandmonitoringofdiseases.
Conclusion
The differencesbetween thyroiddiseases and thecontrol groupsuggestthatcfDNAshouldbenotedasabiomarkerfor furtherevaluationofthyroiddiseases.Inaddition,weoffer severalsuggestions.First,itisimportanttodevelop quanti-tativeapproachestodeterminingtheburdenofDNAchanges inccfDNAandtoestablish alowerdetectionlimitfor the methodologiesused.Thiswillbecrucialfordeterminingthe cutoffvaluesfor the significanceof the determination of DNAchanges. Second, futurework shouldconsider incor-poratingdifferentgenesandmodificationtypestoincrease sensitivityandspecificityfor cancerdetection.Third, sys-tematiccomparisonsbetween differentcancersshouldbe madetodeterminewhethersometypesofcanceraremore prone to releasing modified ccfDNA than others. Fourth, it is very important to develop large-scale studies based on case-control and/or prospective study designs instead ofanalyzingsequentially-recruitedclinicalcases.Fifth,the transformation of experimental ccfDNA studies into epi-demiologicalandclinicalpracticerequiresthedevelopment ofinstrumentationandmethodsadaptedtolarge-scale, low-costanalysisofmutationsinDNAinverysmallquantities. GiventhepotentialforcancermanagementofccfDNA,these developmentsmayquicklyleadtoimportantapplicationsin cancerdetection,diagnosis,prognosis,andfollow-up treat-ment.
Funding
Thisworkwassupportedby C¸anakkaleOnsekiz Mart Uni-versity The Scientific Research Coordination Unit ((BAP), Projectnumber:TYL-2017-1114).
Conflicts
of
interest
Theauthorsdeclarenoconflictsofinterest.
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