Laryngeal preneoplastic lesions and cancer: challenging diagnosis. Qualitative literature review and meta-analysis

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ContentslistsavailableatScienceDirect

Critical

Reviews

in

Oncology/Hematology

j ourna l h o m e pa g e :w w w . e l s e v i e r . c o m / l o c a t e / c r i t r e v o n c

Laryngeal

preneoplastic

lesions

and

cancer:

challenging

diagnosis.

Qualitative

literature

review

and

meta-analysis.

Giuditta

Mannelli

(MD)

a,∗

_,

_Lorenzo

_Cecconi

_(PhD)

b

_,

_Oreste

_Gallo

_(MD)

a

a_First_Clinic_of_{Otolaryngology,}_Head_and_Neck_Surgery,_Department_of_{Translational}_Surgery_and_Medicine,_University_of_Florence,_Italy b_Department_of_Statistics,_Informatics_and_Application_{“G.Parenti”,}_University_of_Florence,_Italy

Contents

1. INTRODUCTION...65

2. LARYNGEALDIAGNOSTICSYSTEMS...65

2.1. Endoscopy...66

2.2. Stroboscopy...66

2.3. Contactendoscopy...66

2.4. Autofluorescence...67

2.5. Narrowbandimaging(NBI)...68

2.6. Ultrasound...68

2.7. Computedaxialtomography(CAT)andMagneticresonanceimaging(MRI)...69

2.7.1. Relationshipofthetumortotheventricularcomplex...69

2.7.2. Involvementofthesubmucosalspaces...69

2.7.3. Anteriorandposteriorextension...69

2.7.4. CAT...70

2.7.5. MRI...70

2.8. Positronemissiontomography(PET,CT/PET)...70

3. OBJECTIVEOFTHEREVIEW...71

4. METHODS...71

4.1. Studycharacteristicsandqualityassessment...71

4.2. Statistics...72

5. RESULTS...72

5.1. grouparticles:cancerousvs.precancerouslesionsassessment...72

5.2. IIgrouparticles:advancedstagelaryngealtumorwithcartilageinvolvementvs.earlystagedtumorsassessment...78

6. DISCUSSION...81

6.1. Overviewinrelationtotheliterature...81

6.1.1. Whitelightendoscopyandstroboscopy...81

6.1.2. Contactendoscopy...82

6.1.3. Autofluorescence...83

6.1.4. NarrowBandImaging...83

6.1.5. Ultrasound...84

6.1.6. ComputedAxialTomographyandMagneticResonanceImaging...84

7. LIMITSOFTHESTUDY...85

8. CONCLUSIONS...85

9. CLINICALIMPLICATIONS...85

10. RESEARCHIMPLICATIONS...87

DISCLOSURE...87

CONFLICTINGOFINTERESTSTATEMENT...87

References...87

∗ Correspondingauthor.

E-mailaddress:mannelli.giuditta@gmail.com(G.Mannelli).

http://dx.doi.org/10.1016/j.critrevonc.2016.07.004

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Articlehistory:

Received20October2015 Receivedinrevisedform4June2016 Accepted11July2016

Keywords: laryngealcancer laryngeallesionsdiagnosis laryngealprecancerosis diagnosticimaging meta-analysis systematicreview

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Background:Multi-stepcancerogenesisguideslaryngealcanceronsetanditincludesawidevarietyof pre-cancerouslesionsmacroscopicallychallengingtoidentifyanddistinguishfrominitialcancerousfoci. Object:Differentmodalitiesofdiagnostictechniquesoflaryngealepitheliallesionsexistandtheydonot offerasinglesystemtomakeadifferentialdiagnosis.Hence,thismeta-analysisaimedtosynthesizethe validityofeachsinglediagnostictooltoimprovelaryngealpatientmanagement.

Methods:Asystematicreviewofliteraturewasledsearchingforarticlesmentioningthefollowingterms: larynx,laryngealprecancerouslesions,laryngealcancer,whitelight(WL)endoscopy,stroboscopy, con-tactendoscopy(CE),autofluorescence(AF),ultrasound(US),narrowbandimaging(NBI),computedaxial tomography(CAT),magneticresonanceimaging(MRI),positronemissiontomography(PET),CAT/PET. Then,aquantitativeanalysiswascarriedonforpaperpublishedafter2005onward.

Results:Thesearchidentified7215publications,ofwhich3616publishedafter2005,withafinalresultsof atotalof214articlesstratifiedandincludedbyourselectioncriteria.42outof214articleswereselected forquantitativesynthesis.25outof41studieshadagoodqualityscore,16werefair.

Conclusions:Acomprehensiveoverviewofthemostrecentadvancesinlaryngealimagingtechnology combinedwithalloftheinformationneededtointerpretfindingsandmanagepatientswithvoice dis-orderscanbefoundherein.Ourflow-chartallowscliniciansinrisk-stratifypatientsandselectproper examinationmodalitiestoprovideappropriatecare.Studylimitations,togetherwithpossibleclinical andresearchimplicationshavebeencounted.

1. INTRODUCTION

Inotolaryngology, hoarsenessisthemostcommonlaryngeal symptomforwhichpatientsseektreatmentandmaybecausedby adiversityofpotentialdisorders.Inthissetting,visualizationofthe structureandfunctionofthevocalfoldshasbecomeanessential componentoftheclinicalvoiceassessmentprotocol(Mehtaand Hillman,2008;DeliyskiandHillman,2010),becausethecapacityof historyandphysicalexaminationalonetoyieldadequate informa-tiontogettherightdiagnosisorevenrisk-stratifypatientsisonly 5%comparedwitha68.3%accuracyfollowinganinitialendoscopic laryngealevaluation(Pauletal.,2013).

Becauseofthewiderangeofpotentialcausesforthedysphonia, determiningthepreciseetiologyofthelaryngeal/voicedisorderis necessarytoplantreatment.Sincemalignanttransformationrate ofpremalignantlesionsrangesfrom6%to22%,anditincreases withtheseverityoftheprecancerousnature,theirearlydetection isof paramountimportance.Inthis setting,earlydetectionand preoperativeassessmentareimportanttoacurativeand function-preservingtherapy,becausethetreatmentoflaryngealcancerand itsprecursorlesionshasagreatimpactonimportantbasicfunctions ofdailylifesuch asbreathing,verbalcommunicationand swal-lowing.Furthermore,delayeddiagnosis,leadingtoloco-regional failure,andahighincidenceofsecondprimaryarethetwomain reasonsforpooroutcome.

Fromtheseobservations,itisobviousthatobtainingimagesof highqualityandresolution,revealingthedetailedmorphologyof theglottalstructures,isoneofthemaintasksinlaryngealimaging. Suchimagesareessentialinmakingcorrectdiagnosisand choos-ingthe treatmenttogain thebestresult.Attempts toexamine thehumanlarynxdatebackmorethan150years,infact,in1854 ManuelGarcia,a vocalmusicteacher,examinedhisownlarynx usingadentalmirrorandahandmirror,andpublishedhis obser-vationsin1855(Alberti,1996).Imagingtechnologiesapplicablein laryngologydevelopedenormouslysincethen.Nowadays,the diag-nosticprocedureoflaryngealdiseasesinclinicalpracticeisrather complexandisbasedontheevaluationofpatient’scomplaints, his-tory,anddataofinstrumentalaswellashistologicalexaminations. Duringthelasttwodecadesavarietyofimagingtechniquesforthe examinationofthelarynxandobtainingobjectivemeasurements ofvoicequalityhavebeendeveloped(Mafeeetal.,2005;Uloza etal.,2005).

Forinstance,theterm“laryngealimaging”typicallyreferstothe endoscopicimagingofvocalfoldtissueclinicalaspectandvibration viavideoendoscopyandvideostroboscopy;inaddition,evaluation oflarynxhasimprovedsignificantlywiththeestablishmentof com-puted axialtomography(CAT)andmagnetic resonanceimaging (MRI),asthesetechnologiesprovideinsightsintothe endoscopi-callyblindareasandrevealthedepthoftumorinfiltration(Gallivan andGallivan,2002).Thesetechnologiesmaybebeneficialin stag-inglaryngealcarcinomaandplanningthemostappropriatesurgical procedure(Rumboldtetal.,2006;Hoorwegetal.,2006).Recently, ultrasonographyhasbecomeusefulaswellincasesoflarger laryn-geal lesionsandmayhavesomerolein screeningforunilateral vocalfold pathologies. Atthesame time,further fine-tuningof thetechniquemaybenecessary(Rubinetal.,2004a;Schadeetal., 2003).

Despitealloftheseeffortsinachievingtheperfectdiagnostic tool, nowadays surgeonscannottrust one singleimaging tech-niquetoplanlaryngealpatientsmanagementandtreatment;in fact,recentarticles,publishedinliterature,emphasizedtheneed forfurthertechnological,methodologicalandclinicalresearchon laryngealimaging,includingthedevelopmentofclinicalnormsand objectiveimageprocessingandmeasurementmethods(Deliyski, 2007;Doellinger,2009;Verikasetal.,2009).

Thispaperprovidesa reviewofthelatestadvancesin laryn-gealimagingoverthelastdecadeinordertogiveacomprehensive overviewofthemostrecentprogressinlaryngealimaging tech-nologycombinedwithalloftheinformationneededtointerpret findingsandsuccessfullymanagepatientswithvoicedisorders. 2. LARYNGEALDIAGNOSTICSYSTEMS

Inpatientswithdysphonia,thediagnosticaccuracyfromhistory andphysicalexam,excludinglaryngoscopy,isonly5%compared witha 68.3%accuracy following aninitialendoscopiclaryngeal evaluation (Lichtenstein and Jaffee,1943).Because ofthe wide rangeofpotentialcausesforthedysphonia,determiningtheprecise etiologyofthelaryngeal/voicedisorderisnecessarytoplan treat-ment.Sincemalignanttransformationrateofpremalignantlesions rangesfrom6%to22%,anditincreaseswiththeseverityofthe precancerousnature,theirearlydetectionisofparamount impor-tance.Forinstance,survivalratesaresignificantlyhigherforearly stagecarcinomas,therefore,itisessentialtoconcentrateonthe

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initialstepsintumordevelopmentinordertofacilitateearly detec-tionandtimelyimplementationofsuitabletherapy(Ferlitoetal., 2000).Earlydetectionandpreoperativeassessmentareimportant toacurativeandfunction-preservingtherapy,becausethe treat-mentoflaryngealcanceranditsprecursorlesionshasagreatimpact onimportantbasicfunctionsofdailylifesuchasbreathing,verbal communicationandswallowing.

Fromtheseobservations,itisobviousthatobtainingimagesof highqualityandresolution,revealingthedetailedmorphologyof theglottalstructures,isoneofthemaintasksinlaryngealimaging. Suchimagesareessentialinmakingcorrectdiagnosisandchoosing thetreatmenttogainthebestresult.

Nowadays,thereisawidechooseofdifferentdiagnostictools, buteachofthempresentsspecificpropertiesandlimitsthat influ-encetheirusageincommonpractice.Hereinasummaryoftheir characteristic.

2.1. Endoscopy

Over time, laryngeal photography was perfected, which enhanced diagnostic accuracy, documentation, and education. Shortly thereafter, in 1895, both stroboscopy and direct laryn-goscopy were introduced, which dramatically advanced laryn-goscopyandlaryngology(Oertel,1895).Intheearly20thcentury, direct laryngoscopy andendolaryngeal surgerymigrated tothe operating roomfor thepromise of improved surgicalprecision (Zeitels,2004).

Inlaryngologicalandphoniatricdiagnostics,whitelight laryn-goscopy(WL)isthekeyprocedureforfunctionalinvestigationand earlydetectionofneoplastictissue(Krausertetal.,2011).Infact, atpresent,whitelightlaryngoscopycombinedwithbiopsyisthe standarddiagnosticprocedureintheassessmentoflaryngeal can-cerandprecancerouslesions.However,whitelightlaryngoscopy providespoorqualityimagesandhasdifficultyidentifyingminute epithelialchangesanddirectlydifferentiatingbenignfrom malig-nanttumorsinvivo.

Furthermore,withimpreciselaryngealdiagnosticsandlackof clinicalexperience,veryearlystagesofmalignantlesions,suchas dysplasiaorcarcinomainsitu(CIS),canbeoverlooked,because theyoftenpresentthemselvesaslow-contrastmucosalchanges withsuperficialroughnessorreddeningandrarelyhavean obvi-ouslycharacteristicmalignantaspect(Gugatschkaetal.,2008). 2.2. Stroboscopy

Stroboscopyisconsideredtobeanimportantpartofdiagnosing patientswithlaryngealdysplasia.Nevertheless,wemustnotethat astrictcorrelationbetweenavocalfoldvibratorypatternanda cer-taintypeoflesiondoesnotexist.Vocalfoldpathologymayproduce changesintheappearanceandvibratorypatternsobservedduring stroboscopicexamination.Interpretingthestroboscopic examina-tion involvessystematic judgmentand describing thedifferent vibratorypattern signs.Thesesigns, whichwerefirst identified

byHiranoandBless(1993)includedthefundamentalfrequency

andperiodicity,amplitudeofhorizontalexcursion,glottalclosure, symmetryofbilateralmovement,mucosalwave,andnonvibrating portionsofthevocalfold.

Quantitativecharacterizationofthevibratorybehaviorofthe vocalfoldsisapivottasktoaddtothediagnosticstep.Awell-known drawbackof video-laryngo-stroboscopy(VLS) is therelianceon quasi-periodic voice signals (Kendall and Leonard, 2010) when producingareal-timeslow-motionstroboscopiceffect.Objective measurementsofthevocalfoldvibrationpatterndatebacktothe initialhigh-speedcinematographyrecordingsofvocalfold vibra-tionfromtheworksofMooreandvonLeden(1958),Timckeetal. (1958,1959)andTimcke(1960).

Nowadays,becauseofthemultiplefactorsrelatedtothe com-plexvocalfoldvibratorysystemtheactualclinicalapplicationof objectivemeasurementshasnotyetbeenachieved.

Severalindices,describing theglottalwaveform,areusually usedforthecharacterization.Theyprovidemainlythe“functional information”aboutthevibratoryfunctionofthevocalfoldsand theglottalclosure,andwhentheyarematchedwithdirect endo-scopicimages,theyprovidetogethermoredetailed information aboutstructuralandmorphologicalpeculiaritieswhichareboth fundamentalinmakingthecorrectdiagnosis.Infact,thistoolis abletorevealanumberofabnormalities,includingabnormalities oflaryngealstructure,absenceofvibration,andvibratory asym-metry.Flashinglightisusedtoilluminateanobjectinstroboscopy. Whentheflashesaresynchronizedwiththevocalfoldvibrations,a stationaryviewofthevocalfoldsisobtained.However,the single-flash-timinglaryngealvideostroboscopyhasalimitationthatitis effectiveonlywhenvocalfoldvibrationsexhibitonlyonesingle fundamentalfrequency.Multipletones(fundamentalfrequencies) mayberecordedinthepresenceofsomediseases,suchaspolyps, nodules,cystsorprecancerouslesions(Deguchietal.,2007).

Quantitativemeasuresofmotionandgeometryofvocalfolds canprovideobjectiveinformationandmaybeusefulinplanning medicaltreatmentandtracingprogressovertime.Fewstudieshave indicatedwhichstroboscopicsignsaremoresignificantthanothers inevaluatingthevibratorypatternofvocalfoldswithpremalignant lesions.Despitetheresomeknownfeaturesthatcannotbeimaged withstroboscope,suchasvoicebreaks,diplophonia,vocal func-tionduringvoiceonsetandvoiceoffset,vocaltremorandspasms, extremelyroughvoicequality,alternatelaryngealandpharyngeal sourcesofoscillation(Woo,2014),themainlimitationwiththe VLSremainsthesubjectivenatureoftheinterpretationof laryn-gealphonatoryfunctionexaminationresults,whichsignificantly reducesthereproducibilityandtheuseofVLSasaresearchtool orevenasaquantitativeinstrumentforcomparingoutcomesof treatmentofvoiceandlaryngealdisorders.Infact,thereisalack ofdataintheliteratureaboutthespecificityandsensitivityofVLS parametersdiscriminatingnormalandpathologicalvoices.

ThediscrepancyindiagnosiswithVLSseemstobelinkedto certainkeypoints:(1)duringofficeendoscopy,tangentialviewsof themedialsurfaceoftheglottislimitthediagnosticsensitivity;(2) sulciandmucosalbridgesaremostsubjecttothislimitation;(3) informedconsentshouldaddressthepotentialneedforachange inintraoperativemanagement.Itisadvisabletodiscussthe possi-bilityfordissectioninbothvocalfolds,evenifaunilaterallesion isobservedintheoffice;(4)suspensionmicrolaryngoscopy(SML) isthefinaldiagnosticstepintheevaluationofglotticpathology (Daileyetal.,2007).

2.3. Contactendoscopy

Contactendoscopy(CE),firstdescribedin1979byHamou,offers anadditionalinvivodiagnosticprocedurebasedonthestainingof thesuperficialmucosallayeranddirectinvivoandinsitu exam-inationoftheepithelialcells.ThebasictechniqueofCEinvolves stainingof the superficialcells of themucosa with1% of non-toxicmethylene bluebeforethemagnificationofthesuspected areasthroughthedirectcontactofthetipofanendoscopetothe mucosalsurfacetoobtaincytologicalimages(Puxedduetal.,2015). CEenablesvisualizationofthelaryngealmucosapathologythrough highmagnificationandthereforedetailedexaminationofcellsand bloodvesselswithoutrequiringtissuebiopsy.Thisnoninvasive methodallowsinvivovisualizationofvascularalterationsofthe mucosaandrateseitherasbenignormalignant.

The use of contact endoscopy in otolaryngology was first described in1995byAndrea etal.however, uptonow,only a few groups have evaluated this technique for invivo histology

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(Arensetal.,2003;Cikojevicetal.,2008;Andreaetal.,1995a,b). In fact, expansion ofsquamous epitheliumfrom thevocal fold edgestotheareasofcolumnarepitheliumcanbeclearly visual-izedbycontactendoscopy;hyperkeratosis(i.e.depositsofanuclear cellsontheepithelialsurface)orleukoplakiaareclearlyobserved oncontactendoscopy,andthegradeofdysplasiathatmayoccur inassociationwiththemisgenerallyindicated bytheimpaired nucleus/cytoplasmratio,nuclearhyperchromasia,andvariationin thenumberandappearanceofthenucleoli(Cikojevicetal.,2008). Falsenegativeresultsmayoccurincaseofincomplete penetra-tionofthestainthroughouttheepithelialthicknessthushindering theidentificationofthegradeofdysplasia;inthissettingthe pres-enceofsecretiondecreasesstainpenetration,whilethesecretion itself precludes anydirect contact between theendoscope and themucosaepithelium.Anothersituationthatmayinfluencethe incidenceoffalsenegativeisthediagnosisofcarcinomainsitu. Carcinomainsituischaracterizedbyheterogeneityofthecell pop-ulation;however,angiogenesisisnotpresentbecausethetumor processdoesnotcrossthebasementmembrane.Itistherefore diffi-culttodifferentiatecarcinomainsitufrominvasivecarcinoma.The findingofangioneogenesisdefinitelyindicatescarcinoma,whereas itsabsencedoesnotexcludethepossibilityofinvasivecarcinoma (i.e.:thediagnosisofcarcinomainsitucannotbemadewith cer-tainty).Finally,thepresenceofabundantnecrosisincaseoflarge tumormasscouldcausebleedwhentouchedbytheendoscopethus impairingtissuestainingandanalysisofcellpopulation.

Forallthesereasons,thereliabilityofcontactendoscopyin liter-atureisreportedwithin75%to88%(Wardropetal.,2000;Carriero etal.,2000).

Insummary,CEcanexamineonlylimitedcellulararchitecture oftheepithelium.Thisisduetothepoorpenetrationofmethylene blue,whichonlydyessuperficiallayersandpotentialoptical arti-factsathighmagnificationduetoglarefromlightreflectedfrom cellsthatarenotinfocus(thefocaldistanceoftheendoscopeis 80mm at603magnificationand 30mmat 1503magnification) (LeddaandPuxeddu,2006).

Inspecific,difficultiesofinterpretationofthevascularpatterns increaseinthefollow-upoftheupperaero-digestivetract(UADT) cancersduetothechangesofthesurroundingtissuesasa conse-quenceofprevioustreatmentssuchasradiotherapy.Inorderto improvetheselacks,StorzProfessionalImageEnhancement Sys-tem(SPIES)(KARLSTORZGmbH&Co.,Tuttlingen,Germany)has beenrecentlyproposedasanoveldigitaltechniqueproviding spe-cificcolorrenderingsthatpronouncethespectralseparationofthe recordedbroadvisiblespectrumwithinthehigh-definitioncamera systemanditdoesnotrequireanarrowbandlightsource.SPIES enhancestheappearanceofthemucosalsurface structuresand subepithelialvasculaturebyselectedwavelengthsoflight, provid-inginadditiontothestandardmodeusingwhitelight(WL),five differentdefinedspectralranges(Clara,Clara1-Chroma,Chroma, SpectraA,SpectraB).Themaintargetofanalysisofthesetechniques isneoangiogenesis,aprerequisitefortheprogressionof precancer-ousandcancerouslesionsoftheupperaero-digestivetract(UADT) (Puxedduetal.,2015).SPIES,aswellasnarrowbandimaging(NBI), allowfortherecognitionofthesuperficialchangesof neoangio-genesis,andtheyrequireacertaindegreeofexperiencetoavoid falsepositives,becausethetypicalmucosal“spots”arenot univo-calandarechallengingforclinicalinterpretation.Itappearstohave goodsensibility(79.6%–94.7%),specificity(81%–95.5%),and accu-racy(88%–94%)inthelarynx(Arensetal.,2003)fordistinguishing betweenbenignandmalignantmucosallesions.

2.4. Autofluorescence

Autofluorescence(AF)isdefinedasnaturalfluorescence emis-sionoftissuearisingfromendogenousfluorophoresafterexposure

andactivationbyradiationofasuitablewavelength.Initsresting state,afluorophoreisatastableenergylevelatwhichitdoesnot fluoresce.Whenafluorophoreisilluminated,itselectronsare pro-motedtoahigherenergylevel.Inthisexcitedstate,thefluorophore isunstableandwillquicklyreverttoaslightlymorestablelower energylevelbyreleasingheat.Toreturntoitsbaselinethe fluo-rophoreemitslight.Sincesomeenergyhasalreadybeenreleased as heat,theemitted light is oflower energy and longer wave-lengththan thatof theilluminating light(Hugheset al.,2010). Fluorophores arepresent at differentconcentrations in healthy andneoplasticlaryngealmucosa;forexample,nicotinamide ade-nine dinucleotide(NADH) predominates within neoplasticcells initsdehydrogenatednonfluorescentform(Richards-Kortumand Sevick-Muraca,1996;Schwartzetal.,1974).Severalfluorophores arenormallyfoundwithinlaryngealmucosaincludingprophyrins, elastin,collagen,andNADH(Gillenwateretal.,1998;Malzahnetal., 2002).

Becauseeachfluorophorehasaspecificwavelengthatwhich itselectronsaremaximallyexcited,it ispossibletotarget spe-cificfluorophores,suchasNADH,usinganilluminatinglightofa singlewavelength(monochromatic). Theunderlying fundamen-talprincipleofdifferentfluorescenceemissioninAFisrelatedto neoplasia-inducedchangesinterms oftissuemorphology, opti-cal properties, and concentration of endogenous fluorophores (Wagnieresetal.,1998).Autofluorescencediagnosisisbasedon theabilityof oxidisedflavinmononucleotide(FMN)in the nor-malcellstoemitgreenfluorescencewhenexposedtobluelight. Nicotinamide adenine dinucleotide plus hydrogen (NADH) and flavinadeninedinucleotide(FAD)areimportantintracellular fluo-rophoresfoundinalltissuelayers;theirconcentrationisnearly100 timeslowerinmalignanttissuethaninbenigntissue(Uppaland Gupta,2003),therefore,malignantcellsdonothavefluorescence tothesamedegreeasbenigncells(Baleticetal.,2004).

Atpresent, autofluorescence endoscopy(AFE)is increasingly being used for early detection of malignant mucosal changes in diverse areas of localization and various medical specialties (Jacobsonet al., 2012; Sieron-Stoltny et al., 2012).It is argued thattheadvantageofAFEcomparedtoconventionalwhitelight endoscopyisbasedonthefactthatpremalignantandmalignant lesionsmightbedifferentiatedclearlyfromnormaltissuebecause ofdecreasedAF.

In1924,Policardobservedtheabilityoftissuetofluoresceunder certainconditions.Alfanoetal.,in1984,reportedthepossibility ofdifferentiatingbetweenhealthyandmalignanttissuebymeans oftheirfluorescentcharacteristics.In1933,SutroandBurmann describedthephenomenonofthedifferentfluorescencesofnormal andtumortissue.

Harriesetal.(1995)firstusedautofluorescencetoidentify neo-plastic cellswithinthelaryngealmucosa,hedemonstratedin a pilotstudyofeightcaseswithcarcinomaofthevocalfolds con-cludedthatthetechniquecanincreasetheaccuracyofstagingof cancerofthelarynxandallowsearlierdiagnosisoftumorsandtheir recurrence.InthesameyearChissovetal.(1995)reachedthesame conclusion.Malzahnetal.(2002)reportedasensitivityof97.3%and specificityof83.8%forthistechniqueindetectingprecancerousand cancerouslesionsin127patients.BeneficialroleforAFhasbeenask reportedbyotherworks(Zargietal.,2000;Arensetal.,1999),even ifithasbeenidentifiedahighrateoffalsepositiveresults,aswell (Delanketal.,2000).

In summary, despite promising literature evidence for this technique,thereare,however,severallimitationsto autofluores-cenceexamination.First,theilluminatinglightdoesnotpenetrate throughdiseasedepithelium(Baleticetal.,2004;Mostafaetal., 2007).As a result, epithelial hyperkeratosismay hide neoplas-ticchangeswithinthebasalmucosallayer(Arensetal.,2006a). Second, granulation tissue and teleangiectasia produce a

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sim-ilar reduction in bright-green fluorescence, attributed to the absorbativepropertiesof a heme molecule,which makes them indistinguishable from neoplasia by autofluorescence (Malzahn et al., 2002). Third,scar tissue, necrosis,and inflammationcan unpredictablyalter mucosalfluorescence(Malzahn et al.,2002; Zargietal.,2000;Mostafaetal.,2007).Thus,severalconditionsin thelarynxmayproducefalsepositiveandfalsenegativefindings.

In conclusion, furtherstudies toincrease thesensitivity and specificityandinvestigationoftechnicalsolutionstoreducethe numberoffalsenegativeandfalsepositivecasesarerequired. 2.5. Narrowbandimaging(NBI)

Narrowbandimaging(NBI)isanopticalimageenhancement technologythatenhancesvesselsinthesurfaceofmucosausing thecharacteristicsofthelightspectrum(Sanoetal.,2001).TheNBI systemconsistsofthesamecomponentsasconventional videoen-doscopic systems:a light source,a camera unit, and a camera headorchip-equippedvideoendoscope.Additionally,theNBI sys-temcontains aspecialimageprocessorandalightingunitwith specialfiltersthatnarrowthefrequencyrangeofemittedlightto 400–430nm(centeredat415nm,blue)and525–555nm(centered at540nm,green)bands.Sincethebluelightwavelength(415nm) isabsorbed by hemoglobinthecapillaryblood vesselsare seen browninthesummarypicture.Currently,availableevidence indi-catesthatNBImaybeapromising approachinthediagnosisof laryngealcancer.Itallowstovisualizethestructureofthe intraep-ithelialbloodvasculaturewhichcannotbeseenwithconventional whitelight(Irjalaetal.,2011).Infact,theemittedlighthasless pen-etrationandlessscatteringandishighlyabsorbedinhemoglobin, thusenhancingtheimageresolution.Thereflectioniscapturedbya charge-coupleddevicechip(CCD),andanimageprocessorcreates acompositepseudocolorimage,whichisdisplayedonamonitor, enablingNBItoenhancemucosalcontrastwithouttheuseofdyes (Piazzaetal.,2008).Thedetectionofsurfacemucosalchangesthat arecharacteristicofneoplasticlesions(e.g.,dysplasia,insitu car-cinoma,andcarcinoma),epithelialabnormalities(thickeningand changesin thesurface layer),andvascular changescanbebest achievedwithNBI.

IntheNBImode,normallaryngealmucosaconsistsof submu-cosalvessels (appearinggreen)connecting withanarborescent vascularnetwork(appearingdarkbrown).Abnormalitiesofthese intraepithelialpapillarycapillaryloop(IPCL),locatedbeneaththe basementmembraneofepithelium,areusuallyclassifiedin accor-dancewiththeirshapechanges;suchchangeshavebeenfoundto predictthedepthofsuperficialcancerinvasionandareclassified intofivedifferentcategories(Kumagaietal.,2002;Nietal.,2011).In typeI,theintraepithelialpapillarycapillaryloopsarealmost invis-ible,andobliqueandarborescentvesselsofsmalldiametercanbe clearlyseen.IntypeII,theintraepithelialpapillarycapillaryloops arealsoalmostinvisible,butthediameteroftheclearlyobserved obliqueandarborescentvesselsisenlarged.IntypeIII,themucosa iswhiteandtheintraepithelialpapillarycapillaryloopscannotbe seen;ifthewhitepatchisthin,theobliqueandarborescent ves-selsmaybeseenindistinctly,butifthewhitepatchisthickthe vesselswill beobscured.In type IV,themucosal intraepithe-lialpapillarycapillary loopsarevisiblewitha relativelyregular arrangementand lowdensity, thecapillaryterminalsare bifur-catedorslightlydilated,andtheintraepithelialpapillarycapillary loopsappearasscattered,small,darkbrownspots;theoblique andarborescentvessels areusuallynot visible.TypeV changes aresubdividedintotypesVa,VbandVcaccordingtotheshape, regularityand distributionofvessels. Intype Va,intraepithelial papillarycapillaryloopsaresignificantlydilatedandofrelatively highdensity,andappeartobesolidortohavehollow,brownish, speckledfeaturesandvariousshapes.IntypeVb,the

intraepithe-lialpapillarycapillaryloopitselfisdestroyed,withitsremnants presentinginasnake-,earthworm-,tadpole-orbranch-likeshape, andthemicrovesselsaredilated,elongatedand‘woven’in appear-ance.IntypeVc,thelesionsurfaceiscoveredwithnecrotictissue, andtheintraepithelialpapillarycapillaryloopspresentasbrownish specklesortortuousshapesofunevendensitywhichareirregularly scatteredonthetumorsurface.LesionsviewedunderNBIare usu-allyrecordedas:(1)malignant(i.e.typeV);(2)suspectedmalignant (i.e.protuberantorulcerativelesionscoveredwithnecrotictissue, orleukoplakiaofunknowntype);or(3)benign(typesItoIV)(Ni etal.,2011).Intheliterature,thereexistonlyafewpublicationson theexclusiveuseofNBIinlaryngology(Nietal.,2011;Watanabe etal.,2009;Piazzaetal.,2010a;Bertinoetal.,2015a;Masakietal., 2009;Piazzaetal.,2012;TjonPianGietal.,2012;Imaizumietal., 2012).However,severalstudiesassessedthevalueofNBIinthe headandneckalsoincludingthelarynx(Irjalaetal.,2011;Piazza etal.,2010a;Watanabeetal.,2008;Ugumorietal.,2009;Linetal., 2010;Piazzaetal.,2011;Shinozakietal.,2012;Linetal.,2012). NBIhasshowninthelarynx,overlastyears,improved sensitiv-ity61-91%andspecificity87-92%(Piazzaetal.,2011;Yangetal., 2012)butfalsepositivesandfalsenegativeshavebeenreported, especiallyinthelarynx,andincreasewithashortlearningcurve. Thisapproachispromisingforbetterdiscriminationofmalignant andbenignlesionsaspartof“prehistologicdiagnosis”or“optical biopsy”(68).Nevertheless,thereisnoultrathin,zooming,flexible videoendoscopeavailableforENTpurposes;therefore,a combina-tionofrigidtelescopesandanHDTVcameraheadmustbeused toachievesufficientresolutionandultrahighmagnification(Lukes etal.,2014).AlthoughmostauthorsconcurthatNBIwithorwithout HDTVisavaliddiagnostictool,thismethodhassomelimitations. Themostrelevantiscertainlythepossibilityofgenerating,atleast intheearlyphaseofthelearningcurve,anincreasednumberoffalse positiveswith consequentlyunjustifiedbiopsies. Thisis mostly relatedtoacuteinflammationandchronicpost-RTchanges. There-fore,webelievethattheincidenceoffalsepositivefindingswas mainlyrelatedtotheexperienceoftheexaminersmorethanto specificmucosalchanges.

2.6. Ultrasound

Overthepast15years,therehasbeenaneffortbyheadandneck surgeonstobringopticalimagingtechnologyfromtheresearch laboratoryintotheoperatingtheatertoimprovetheirabilityto identifythetumormargininvivotoguidesurgicalexcision(Hughes etal.,2010).

Sonographyisregardedasthefirstimagingmethodroutinely usedfordetectingcervicallymphnodemetastasesfromheadand necktumors(Ahujaetal.,2008;Chevallieretal.,2002)butithas rarelybeenusedasanimagingtechniqueforinvestigationofthe larynx.Previousstudiesintheliteraturehaveyieldedpromising resultsfor high-frequencysonography indiagnosisof laryngeal carcinoma(Kuribayashietal.,2009;Desaietal.,2004;Gritzmann etal.,1989).However,theuseofsonographyintumorstagingis stilllimited.

Sincethe1970s,B-modeUSimaginghasbeenusedwithsome successinthelaryngealareatoidentifymassandcysticlesions atthevocalfolds(Rubinetal.,2004b)and todetectvocalfold paralysis(Vatsetal.,2004;Ooietal.,1995);moreover, ultrasonog-raphyhasbeenalsousedtoevaluatelaryngealtumorsina few studies,andsomeofthesehaveprovedthatultrasonographycould assisttumorstaginginpatients withadvanced laryngealcancer (Gritzmannetal.,1989;Lovedayetal.,1994;Huetal.,2012).

Ithasbeendemonstratedthatultrasoundhasasimilarrolein thevisualizationofhypopharyngealtumortoCAT(Loveday,2003), thus whether ultrasonographyhas similar ability in evaluating laryngealcancerisworthytobeinvestigated.Thyroidcartilage,

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pre-epiglotticspace,paraglotticspace,thyroidandcervicalsofttissues, lyinginthelarynxanteriorlyorsuperficially,areeasytobeimaged withahigh-frequencyprobe.Commonly,thesestructuresare isoe-choicorhyperechoic,whichprovideacontrastwithahypoechoic invadingtumor(Lovedayetal.,1994).Hence,ahighsensitivityand specificityhasbeenachievedbyultrasonographyintheevaluation ofinvolvementofthesestructuresandreportedinliterature(Hu etal.,2012;Xiaetal.,2013;Huetal.,2011).

For instance,Kraftetal.(2010) establishedanatomical land-marksfor laryngealendosonographyinordertoallowacorrect interpretation of sonographic images of its structures, and its known capacity to visualize critical regions such as the pre-epiglottic and paraglottic space endoscopically. Therefore, endolaryngealultrasonographyseems tobeable topredict the exactextensionofalaryngealtumorbeforesurgeryisperformed, andtoassistinfindingthebesttherapeuticsolutionforthepatient (ArensandGlanz,1999).Furthermore,givenitsnoninvasivenature andminimaldisturbancetonormalvoiceproduction,medicalUS imaging shouldbe an ideal tool for studying vocal fold vibra-tion.However,onlyafewstudieshaveuseddynamicUSimaging toinvestigatethevibratingvocalfolds(Hsiaoetal.,2001;Hsiao etal., 2002).Thebody motionofthevocalfold, whichis ruled bymechanicalprocessdescribedbyHirano(1974,1975),presents a vibrationfrequency (f)>70Hz whichis higherthan theframe rate(fs)ofdynamic B-modeUS (<50Hz).Under this condition, real-timedynamicmotionpicturesofvocalfoldvibrationcannot beobtainedusingB-modeimaging.Ifthephonationfrequencyis tunedtobeclosetoanintegermultipleoffs,aslowmotionmontage ofvocalfoldvibrationmaybeobtainedwithB-modeUSimaging. Suchmotionpicturesmayprovidevaluabledataforanalyzingvocal foldvibrationespeciallyinthebody.Thisuniquemethodhasbeen describedforthefirsttimebyTsaietal.(2009)whofirstattempted torepresentadynamicB-modeimageofvocalfoldvibration.

Inconclusion,endosonographyofthelarynxproduces horizon-talsliceimagescomparablewithcomputedaxialtomography(CAT) ormagneticresonanceimaging(MRI)scansbutwithahigher reso-lution(Tsaietal.,2009).Duringtheimagingprocess,itisessential toposition theprobe as centrally as possible,avoiding contact withthelaryngealwall,itallowstodirectlymeasurethe antero-posteriorandmedio-lateraldiametersofthelaryngealneoplasm. Theorotrachealtube,togetherwithlaryngealstructuressuchas cricoidand thyroidcartilages,are usefulanatomical landmarks. Hence,directobservationofsonographicimageshelpin orienta-tion,too.

2.7. Computedaxialtomography(CAT)andMagneticresonance imaging(MRI)

Theradiologist makesa valuablecontributiontothestaging oflaryngealcancerandthis hasadirectinfluenceontreatment planning.Sinceapproacheswithbothcomputedaxialtomography (CAT)andmagneticresonanceimaging(MRI)areacceptable,the choiceinpracticedependsonmachineavailabilityandlocal exper-tise aswellastheability ofthepatienttotoleratea prolonged MRIexamination.Crosssectionalimagingprovidesinformationon tumorvolume,extensiontoandacrossthelaryngealventricle, infil-trationofpharynx,para-glottic,pre-epiglotticandextra-laryngeal spaces,allofwhichimpactonthepotentialforvoiceconserving partiallaryngectomiesandresponsetoradiotherapy.Togetherwith anassessmentofnodalandsystemicmetastases,thisenablesthe radiologisttoplayanintegralroleinthemultidisciplinaryselection oftreatmentoptions.

Inspecific,radiologistshavetodescribeiftheclinicallaryngeal lesionspresentsadeeperextensionandifthisextensionaffectmain

laryngealstructures.Theydothisbyanalyzingdifferentparameters asfollowing:

2.7.1. Relationshipofthetumortotheventricularcomplex

Inordertodefinethetumorassupraglottic,glotticorsubglottic theradiologistmustdeterminetheleveloftheventricularcomplex (comprisingthetruecord,falsecordandtheintervening ventri-cle).Theselandmarksareessentiallymucosal,andusuallytheyare assessedendoscopically,however,sometimesabulkytumorcould hideitsdistalvisualization.Thesuperioraspectoftheventricular complexisdefinedbythesuperiormarginofthearytenoids carti-lages.Theinferioraspectoftheventricularcomplexisdefinedby thetruecords;radiologicallyidentifiedbythetransitionof para-glotticfattosofttissueinthewallofthelarynx.Thissofttissue representsthethyroarytenoidmuscle.Inaddition,thetruecord liesattheuppermarginofthecricoidcartilage(atthelevelofthe anteriorlypointingvocalprocessesofthearytenoidcartilages).The posteriorcommissureisseenbetweenthearytenoidscartilages. Thesubglotticregionhasa characteristicappearancewhere the cricoidcartilageisseenasacompleteringprovidingthe founda-tionofthelaryngealskeleton.Anysofttissueatthesubglotticlevel isabnormal.Thepresenceofanenlargeddelphiannodeanteriorto thetracheaalsoindicatessubglotticinvolvement.

2.7.2. Involvementofthesubmucosalspaces

Anunderstandingofthepre-epiglotticandpara-glotticspaces allowstheradiologist topredictandidentifypatternsoftumor spreadwhichhaveacriticalimpactontherapeuticdecisions. Sub-mucosalinvolvementofthepre-epiglotticandpara-glotticspaces is difficult to evaluate clinically and endoscopically. The fatty C-shaped pre-epiglotticspaceisboundedanteriorlybythe thy-roidcartilage andthyrohyoidmembraneand posteriorlyby the epiglottisandquadrangularmembrane.Thepre-epiglotticspace iscontinuouslaterallywiththepairedpara-glotticspaces.

CTand MRIare88-93%accurateintheassessmentoftumor extensionwithinthesespaces(Yeageretal.,1982).

2.7.3. Anteriorandposteriorextension

Inadditiontodefiningthecraniocaudalextensionwithinthe submucosalspacesrelativetotheventricularcomplexlandmarks, it isimportanttoevaluatetheanteriorand posteriorextension oftumor.Thisisparticularlyimportantatthelevelofthe glot-tis,whereusuallyglotticcarcinoma,growstowardstheanterior commissurewhichisfrequentlyassociatedwiththyroidcartilage involvementofthebaseofepiglottis.

Thecartilageispronetoinvasionatthissitebecausethe inter-nalperichondriumisdeficientandtheexternalperichondriumis thinner.Theperichondriumcoveringthecartilageactsasa resis-tantbarriertoinvasionbytumor(Yeageretal.,1982).Ithasbeen shownthatcartilageinvasionmainlyoccurswheretheattachment ofthecollagenbundlesinterruptstheperichondrialbarrier.Asthe cancer cellsmultiply,theyseparatethecollagenbundles, form-inglinearpassagewaysthroughtheperichondrium.Thus,thesites ofattachmentofthestrongestmembranes,suchastheanterior commissuretendon,arealsothemostfrequentsitesofinvasion. Invasionofthecartilageframeworkinvariablytakesplaceatthe siteofossifiedorcalcifiedcartilage(GregorandHammond,1987). Non-ossifiedcartilageisresistanttotumorinfiltrationduetoits capacitytoreleaseproteinsthatinhibittumorangiogeneticfactor andcollagenases(Galloetal.,1992).Recentpublishedreportshave shownthatCTmayyieldacceptablesensitivityfordetecting neo-plasticinvasionoflaryngealcartilageifthediagnosticcriteriaare selectedandcombinedappropriately(Becker,2000).

However,cartilageinvasionisstillsometimesoverestimated, resulting in unnecessary total laryngectomies in some patients (Kunoetal.,2014).

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Previousstudiesusingsingle-slicespiralCTscannershave con-cludedthattheCTcriteriausedfordeterminingneoplasticinvasion ofthethyroidcartilageincludeerosion,lysis,andtransmural extra-laryngealtumorspread(Sulfaroetal.,1989a;Zbarenetal.,1996; Beckeretal.,1995;Beckeretal.,1997;Beitleretal.,2010).

Tumormayalsoextendfromtheanteriorcommissuretoan extra-laryngeallocation caudal tothethyroid cartilage(viathe cricothyroidmembrane)andinferiorlytothesubglottis.CATis75% accurateinpredictinganteriorcommissureinvolvement(Barbosa etal.,2005).Predictingthepresenceoflaryngealcartilageinvasion isakeyaspectoftheimagingassessment.

CATand MRI are generally usedto supplement microlaryn-goscopy(MLS)instaginglaryngealcancer.Ideally,imagingshould beabletopredictthetumorsize, theexact extension,a possi-ble midline crossing, and cartilage infiltration in theselesions. AlthoughCTprovedtobehelpfulintheinvestigationofadvanced malignancies (T2–T4), it often fails to show early cancer (Tis, T1<10mm)duetothealmostidenticaldensityoftumortissueand thevocalmuscle.WithMRI,thesamedifficultiesareencountered indemonstratingsmallertumors(Tis,T1<9mm),butthelattercan betterdifferentiatecancerfrommuscletissueanddepictinvasion ofthelaryngealframework(Hermans,2006).However,CTandMRI areexpensiveandcannotbeperformedduringMLS.Further disad-vantagesaretheradiationloadofCT,thelongacquisitiontimesfor MRI,andtheneedforcontrastagentsinbothmethods.

2.7.4. CAT

CATisthepreferredimagingmethodforstagingoflaryngeal andhypopharyngealcancer.MultisliceCATallowstheradiologistto evaluatealmostalltherelevantimagingissues.Voltageof120-140 kVp,tubecurrentofatleast180mAs,displaymatrixof512×512 pixelsandacollimationofapproximately1mmshouldbeused. Asinglebolusofcontrastmediumiseffective,howevera bipha-siccontrastenhancementprotocolmaybeemployed.Theimages areobtainedwiththepatientsupineandduringquietrespiration (notwhileholdingthebreath).Theneckshouldbeinslight exten-sion,andtheheadisalignedalongthecephalocaudalaxistoallow comparisonofsymmetricalstructures.Alongitudinalfieldofview extendsfromtheskullbasetothesterno-clavicularjointswiththe patientbreathingquietlyandnotswallowing.Malpositioningmay createanappearancethatsimulatesdisease.Everyeffortshould bemadetomakethepatientfeelcomfortable.Itmaybehelpful toperformanadditionalexaminationfocusedonthetumorwith e-phonation(forbetterassessmentofthelaryngealventricle, ante-riorcommissureandaryepiglotticfolds)ormodifiedValsalva(for betterassessmentofthepriformsinusandpostcricoidregions) manoeuvres(Kunoetal.,2014).Inadditiontosofttissuewindows, thebonewindows(reconstructedwithabonyalgorithm)should beroutinelyevaluatedifthetumorcontactsossifiedlaryngeal car-tilage.Itshouldbeensuredthataxialimagesarereformattedinthe planeofthelarynx.

SpiralCATisentirelydependentontheemergenceof“slipring” technology,whichallowselectricalenergytobetransmittedtothe gantryasitpassesalongthepatient,allowingacquisitionofCAT datainahelicalfashion.Itisalsoafasterprocess,withless discom-forttothepatientanditisdynamic,withhighresolutiongiving slicesincontinuity(Baumetal.,2000).

CATprovidesaclearerillustrationofbonystructuresand cal-cificationthandoesMRI.Moreover,itislessexpensive,fasterand lesssusceptibletomotionartifacts.Sclerosisisthemostsensitive criterionbutthisoftencorrespondstoreactiveinflammation, par-ticularlywithrespecttothethyroidcartilage.For instance,CAT signsoferosionandextra-laryngealtumorofthethyroid,cricoid andarytenoidcartilagestogetherwithsclerosisofthecricoidand arytenoidcartilages resultin a sensitivityof 64-72%and speci-ficityof86-94%forcartilageinvolvement,withanaccuracyvalueof

approximately80%(Zbarenetal.,1996;Zbarenetal.,1997a;Zbaren etal.,1997b).

2.7.5. MRI

MRIisbestperformedwithahighfieldMRIscanner.TheMR examinationrequiresaneckcoiltoobtainadequateresolution. Sec-tionthicknessshouldbe4–5mmfortheneckcoverageand2–3mm (0.1-0.2mminterslicegap)forthefocusedstudyofthelarynx.The displaymatrixsizeis170×256pixelsminimum.Acombinationof T2-wsequenceswithfatsaturation,T1-wsequencesandT1-wfat saturatedsequenceswithgadolinium,shouldbeusedinaxialand coronalplanes.Thesagittalplaneisalsousefultoassesspotential tonguebaseinvolvement.Patientsareaskedtorefrainfrom cough-ingandswallowingduringtheacquisitionandMRImayproduce poorresultsinthebreathlessorrestlesspatientwhois compro-misedbythetumor.

MRimagingismostfrequentlyusedifthereisuncertaintyin assessingcartilageinvolvement,whenthisiscriticaltotherapeutic decisions.Itmayalsobetterdefinethemarginbetweenthetumor andthyroarytenoidmuscleandinvolvementofthetonguebase.

MRIscanninghasbecomeincreasinglyinvolvedasasensitive imagingmodalityfordetectingneoplasticinvolvementofthe thy-roid cartilage, primarilybecauseof its highnegative-predictive value.However,recentlyhasbeendemonstratedMRIlow positive-predictivevalue(68-71%),duetoitsfailureindistinguishedema andinflammationsurroundingthetumorfromthetruecartilage invasion(Becker,2000;Zbarenetal.,1996;Beckeretal.,1995).In addition,itcouldoverestimatecartilageinvasion(Declercqetal., 1998).MRIcriteria forcartilageinvolvement bytumorare high signal onfat suppressed T2-w imagesand/or enhancement on postgadoliniumfatsuppressedT1-wimagesinthecartilage adja-centtothetumor,orthepresenceofextra-laryngealtumor.The mostrecentstudiesdescribing radiological-pathological correla-tion demonstrated slightly improved sensitivity of 89-95% and reduced specificityof74-84%relative toCAT;butaccuracy still demonstratesvalueofabout80%asCAT(Zbarenetal.,1996;Becker etal.,1995;Zbarenetal.,1997a;Zbarenetal.,1997b).Moreover, MRIcanbealengthyandexpensiveprocedure.Anapatientmotion assimpleasswallowingmaygeneratesignificantartifacts.

Moreover,high-resolution MRIhadbeenusedtoconstructa three-dimensionalanatomicalmodelofthecartilagesofthehuman larynx(Selbieetal.,2002),providingananatomicalframeworkfor registeringdifferentlaryngestothesamecoordinatespace.Lately,a three-dimensionaleducationalcomputermodelofthelarynxwas developedfromMRIscans(Huetal.,2009).Tworecentstudies openednewdirectionsforapplyingMRIforlaryngealimaging:MR microimagingofexcisedlaryngesconfirmedvalidhigh-resolution imagingoflaryngealtissuemicrostructure(Herreraetal.,2009), anddynamicMRIoflaryngealandvocalfoldvibrationswas real-izedforthefirsttimefor measuringoflaryngealstructuresand glottalparametersindynamicfunction(Ahmadetal.,2009). 2.8. Positronemissiontomography(PET,CT/PET)

Fluorine-18 fluorodeoxyglucose (FDG) positron emission tomography (PET)playsan increasingrollintheassessment of head and neck cancer, both for primary staging and for post-therapy management (Gordinet al.,2006a).PETis a functional imagingmodalityassessingthemetabolicstatusoftumorsandhas provedsuperiortoCTandMRIindifferentiatingrecurrencefrom post-radiationeffects or scarin patients withcarcinomaofthe larynx(Gordinetal.,2006a;Kresniketal.,2001).AnegativePET scanexcludesrecurrencewithahighcertainty.PositivePETscan findingsshouldbeassessedwithbiopsyandfollowupPETimaging ifthis is negative.It shouldbenoted that physiological uptake of[18F]fluorodeoxyglucose([18F]FDG) isobserveddue tovocal

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cord activity,soa“silentprotocol” shouldbeemployed.Recent advancesintechnologymakeitpossibletofuseanatomicalimages with functional images. The use of combined 18F-FDG PET/CT (PET/CT) fusion images has beenshown toimprove diagnostic accuracy.Forheadand neckcancers,thereportedsensitivityof PET/CTis98%,specificityof92%,withanaccuracyof94%forthe identificationofamalignancyoftheheadandneck;thisisahigher accuracythanwithPETorCTalone(Jeongetal.,2008).Duetoits highcostsitsuseisadvocatedincaseofuncertainresultsofCAT orMRI,andtocompletepretreatmentneckstagingorincaseof suspectforpost-radiotherapyrecurrence.

3. OBJECTIVEOFTHEREVIEW

Theclinicaldiagnosisoflaryngealpathologyisimportant,asthis primarilydeterminesthenextsteptowardsthetreatment. Unfor-tunately,itisnotuncommonfordifferentclinicianstousedifferent nomenclatureortoidentifydifferentstageforthesamelaryngeal lesion.Thisobviouslymakes evaluationandcomparison among specialistsindicationsdifficult,anditdoesaffectthetreatment.

Inordertomakethisinter-observervariabilityasmuchloweras possible,attemptsshouldbemadetoimprovediagnostic technol-ogy,stricter/moreuniversallyaccepteddefinitionsandsupervised trainingofjuniordoctorsinavoiceclinicenvironment.Thisshould inturnleadtousefuloutcomedataandtreatment recommenda-tions.

Theobjectivesofthissystematicreviewandmeta-analysiswere tosynthesizethekeydiagnosticmanagementforlaryngeallesions throughareviewofthelatestadvancesinlaryngealimaging modal-itiespublishedinthepast10years.

4. METHODS

ThesystematicreviewwaswritteninaccordancewithPRISMA Statement (www.prisma-statement.org) (Moher et al., 2009; Shamseeretal.,2014),inordertoguaranteeascientificstrategy ofresearchtolimitbiasbyasystematicassembly,criticalappraisal andsynthesisofallthemostrelevantstudiespublishedonthistopic (Sedgwick,2015a;GoodmanandMetabias,2011).

ThedatabasesinterrogatedincludedPubMedClinicalQueries www.ncbi.nlm.nih.gov.Referencelistsfromidentifiedarticleswere searchedandcross-referencedtoidentifyadditionalrelevant arti-cles,which wealsoincludeinthisreview,andrelevantopinion leaderswerecontacted.

Thesearchtermsincludedthefollowingvariouscombinations to maximize the yield: larynx, laryngeal precancerous lesions, laryngealcancer,whitelightendoscopy(WLE),stroboscopy, con-tactendoscopy(CE),autofluorescenceendoscopy(AFE),ultrasound (US), narrow band imaging (NBI), computed axial tomography (CAT), magnetic resonance imaging (MRI), positron emission tomography(PET-CAT/PET).

ThesearchwasperformedforthefirsttimeonJanuary2015and wassettoautomaticallyupdateperiodicallyuntilMay2015.

First,duplicateswereremovedelectronically.Then, abstracts were reviewed toexclude obviously irrelevant articles. Experi-mental studiesand papers dealing withpathologies otherthan precancerousandcancerouslesionsofthelarynxwereexcluded.

Theinclusioncriteriaweredeliberatelykeptwidetoencompass asmanyarticlesaspossiblewithoutcompromisingthevalidityof theresults,andtheyarelistedinTable1.

Wefilteredthestudiestoensurethatonlydatafromcentersthat hadpublishedonatleast10patientswereincludedinthereview; thiswasdoneasaqualityassurancemeasureasthereareseveral caseseriesintheliterature,whichhavepublishedtheresultsof

Table1

Inclusioncriterialist.

Inclusioncriteriaforpaperselectedforthereview

1 Articlespublishedfrom2005onward;

2 Publishedseriesof≥10patients;

3 Sensitivityandspecifityclearlyshownordetachable;

4 Cleardescriptionofselectioncriteria;

5 Diagnosticmethodsincluding:ultrasoundanalysis,

autofluorescenceandfluorescentanalysis,contactendoscopy, endoscopy,stroboscopy,narrowbandimagind(NBI), computedaxialtomography(CAT),magneticresonantimaging (MRI),positronemissiontomography(PET);

smallnumbersofcasesspanningseveralyears.Non-English lan-guagepapersandduplicateswereexcluded.

Thesearchexcludedarticlesthatwerepublishedbefore2005 becausewebelievethatitwaspragmatictoincludearticlesthat werepublishedoneachdiagnostictechniqueatleastinthelast decade, allowingtime for thelearning curve tobeclimbed for methodsuchasnarrowbandimaging(NBI),cameintoclinical prac-ticesince1997,andinordertotrytocomparedataasmuchmore recentaspossible,duetothelatesttechnologyprogressionandthe entranceinusageofmoreadvancedinstrumentsasmagnetic res-onanceimaging(MRI)withhigh-resolutionordiffusion-weighted imaging(DWI)MRI.

Alldatawereindependentlyextractedbytwoauthorsand qual-ityassessed.Eligibilityforinclusionwasseparatelyassessedand whenindoubtdiscussedanddecidedbyconsensus.

Afirstqualitativeanddescriptivereview-analysisofselected articleswascarriedon;whilst,exclusively,publicationscomparing diagnosticimagingfindingswithdefinitivehistopathologyresults wereincludedinourmeta-analysis.Forarticlesnotreportingraw data,lettersweresent tothecorrespondingauthorsrequesting them,otherwisetheywereexcludedfromthequantitativeanalysis. Raw data from the meta-analysis were entered into the appropriatecontingencytablestoallowcalculationofsensitivity, specificity,accuracy,Jounden’sindex,positiveandnegative predic-tivevaluesforeachdiagnosticimagingtechnique.

Inaccordancewiththeliterature,malignant(i.e.,carcinomain situ,invasivecancer)wereclassifiedaspositivewhereas prema-lignantlesions(i.e.,moderateandseveredysplasia)wereclassified asnegative;benignlesions(inclusiveofsimplehyperplasiaand/or milddysplasia)werecalculatedasnegative,aswell.

Similaranalyseswereperformedfortheproceduretype.Each diagnostictechniquewasevaluatedforitscapacityindistinguish premalignant from cancerous laryngeallesions; when possible, everymethodwasanalyzedforitsaccuracyinidentifyingadvanced stagecancersfromearlystages.

Forthisreason,inordertofulfilltheneedtoidentifythebest diagnostictechniqueforeverydiagnosticstep,wemadetwo dif-ferentmeta-analysis:

1.Onarticlesreportingdiagnostictechniquesabletomakea differ-entialdiagnosisbetweenpreneoplasticandcancerouslesions:I group;

2.Onthosediagnosticmethodsthatallowamoreaccurateanalysis oftumor’svolumeandextensioninordertorefinethestaging phase:IIgroup.

4.1. Studycharacteristicsandqualityassessment

To our knowledge, no widely accepted measures of qual-ity assessment of case series exist; here, all included papers were graded using the NICE scoring scale for retrospective case series (Available at:http://www.nice.org.uk/nicemedia/pdf/ Appendix04qualityofcaseseriesformpreop.pdf).Thisisa

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scor-Table2

Qualityassessmentscale.

Qualityassessmentforcaseseries

1 Caseseriescollectedinmorethan1center(i.e.:amulticentric study)

2 Isthehypothesis/aim/objectiveofthestudyclearlydescribed?

3 Aretheinclusionandexclusioncriteria(casedefinition)

celarlyreported?

4 Isthereacleardefinitionoftheoutcomesreported?

5 Weredatacollectedprospectively?

6 Isthereanexplicitstatementthatpatientswererecruited

consecutively?

7 Arethemainfindingsofthestudyclearlydescribed?

8 Areoutcomesstratified(i.e.:bydiseasestage,abnormaltest results,patientcharacteristics)?

ingscalewitheightitems,witheachitemscoringzerooronebased onthestudymethods(Yes=1;No=0).Scoresof≥6areconsidered toindicateagoodqualitystudy,scoresbetweenfourandfiveasfair andthosestudieswithascoreofthreearetreatedaspoorquality (Table2).

4.2. Statistics

Fisher’sexacttestwasusedforstatisticalanalysisofcategorical dataforthedescriptivereview,andavalueofp<.05wasconsidered significant.

Then,alltheincludedstudiesforthemeta-analysis,provided sufficientdata(true-positive[TP],false-positive[FP],true-negative [TN],andfalse-negative[FN])topermitcalculationofsensitivity (Se)andspecificity(Sp).Sensitivityandspecificityata95% confi-denceinterval(CI)werethenpooledusingabivariateregression approach(Reitsmaetal.,2005).

We constructed hierarchical summary receiver operating characteristic(SROC)curvestoassesstheinteractionbetween sen-sitivityandspecificity.TheareasundertheROCcurves(AUCs)were usedtoanalyzethediagnosticprecisionofeachdiagnostic tech-niquefordifferentiationofprecancerouslesionsfromcancersand fordistinguishadvancedstartumorsfromearlystagecases.

Heterogeneity (or absence of homogeneity) of the results betweenthestudiesisassessedgraphicallybyforestplotsand sta-tisticallyusingthequantityI2_that_describes_the_percentage_of_total variationacrossstudiesthatisattributabletoheterogeneityrather thanchance(Higginsetal.,2003;Sedgwick,2015b).Thisisa mea-sureofheterogeneitybetweenthestudiesandrangesfrom0%to 100%;highfiguresindicategreaterheterogeneityinthedata.

Whenstudieshavelowheterogeneity(pragmatically,I2_<_25%), thedifferencesbetweenreportedoutcomescanbeexplained sim-plybytheobservednaturaldifferencesbetweenpatients.Inthis casewecanconsiderthatallpatientsarepartofthesamelarger pool.Afixed-effectsmeta-analysisis appropriateinwhich each patientisgivenapproximatelyequalweight.However,withhigh heterogeneity,thestudiesdifferbymorethancanbeexplained byintrapatientseffects.Thisimpliesthatthereweredifferences inthepatients studies,inthetreatmentinterventions,orinthe outcomemeasures.Inthiscase,arandom-effectsmeta-analysisis appropriateinwhicheachstudyisgivenmoreequalweight.

All statistical analyses were performed using STATA soft-ware,version12.1.Inparticular,“midas”routine(Dwamena,Ben A.(2007)midas:AprogramforMeta-analyticalIntegrationof Diag-nostic Accuracy Studies in Stata. Division of Nuclear Medicine, DepartmentofRadiology,UniversityofMichiganMedicalSchool, AnnArbor,Michigan.)wasusedtoundertakingmeta-analysis.

5. RESULTS

Thesearch strategyidentified 7215articlesin Medlinefrom 1950stothesearchdate.3616articlespublishedafterthe2005 wereselected,imported into Endnote,and theduplicateswere removed.Theremovalofduplicates,non-Englishlanguageworks and ofarticlesaboutotheritems ratherthan precancerousand cancerouslaryngeallesionsyieldedatotalof214publications.

These214publicationswereconsideredforthequalitative sys-tematicreviewoftheliterature,ontheotherhand,only41outof 241wereselectedforthemeta-analysis.

Thevariousstagesofsystematicallyassessingtheabstractsand reasonsforexclusionfromthereviewaredescribedinFig.1.

23articles(Igroup)outof41wereaddressedtowardsthe differ-entialdiagnosisamongprecancerousandcancerouslesions,whilst theremaining18articles(IIgroup)wereanalyzedforthe assess-ment of the identification of advanced stage cancers reporting cartilageinvolvementagainstthediagnosisofearlystaged laryn-gealtumors.

Qualityscoreassessmentandeacharticleclinicalcharacteristics selectedforthemeta-analysisaresummarizedinTables3and4. 5.1. grouparticles:cancerousvs.precancerouslesionsassessment

TheIgroupofarticlesreportedanequalnumberofgood(12) andfair(11)quality,intermsofdeterminationoflaryngeal can-cerouslesionsagainstprecancerouslesions,ameanvalueforSeof 84.425±19.098SD(95%CI78.640-90.210,median90.5%)together withasimilarmeanvalueof83.031±17.559SD forSp(95%CI 77.089-88.972,median88.5%).Theseresultsarereferringtoagood pool of laryngeal lesionsanalyzed (3301) witha total amount of1386cancerouslesionsidentifiedoverallthestudied popula-tions.Anystatisticalsignificancewasfoundamongcontinuosdata (p=0.7818).

Inspecific,resultsaboutWLEprecisiononidentifyand distin-guishsuperficialcancerouslesionsfromsuperficialprecancerous lesionsreportedaveryhighI2_value_for_both_Se_and_Sp_(81.38%_and 70.82%,respectively),butwithaAUCvalueof91%(Fig.2).

InregardtothediagnosticutilityofWLE,Caffieretal.(2013) showed a higher accuracy for WL (97%vs. 81%) than AFE and AFEdata analysisprovided a reduction in specificity of 25% in comparison to WLS, together with a high false positive value (31%).Thesefindingswereinaccordancewithliteraturereports (Arens et al., 2007a; Kraft et al., 2011). On the other hand,

even if Crosetti et al. (2012) confirmed that sensitivity and

specificity of AFE in distinguish between benign and dysplas-tic/neoplasticlesionsarenegativelyimpactedbydifferentfeatures (i.e.: hypertkeratosis-leucoplakia, abnormal hyperplasia, hyper-vascularizedlesions,chroniclaryngitisandlesionswithbacterial infectionsorpreviouslytreatedbysurgeryorradiotherapy),their resultswerecompletelydifferentfromthosereportedbyCaffier et al. (2013), indicating a very low accuracy in diagnostics for boththesesingleusedmethods.Theyconcludedsupportingtheir hypothesisthata combinationofboth diagnostictoolsallows a riseinsensitivityandspecificityofaboutmorethan35%and25%, respectively. Thus, theyproposed tocombine endoscopic tools throughamultistepsystem,inordertoobtainagreatersensitivity andspecificity,toimprovedefinitionindirectmicrolaryngoscopy andinfollow-upsearchingforsynchronous/metachronoustumors. Only one full text article were selected for the qualitative andquantitativeanalysisofthestroboscopediagnosticmethods, confirmingthelackofa statisticaland accurateanalysisof this techniqueinliterature.Ulozaetal.(2013)foundmoderate-to-high ratesofsensitivity(55.9–85.3%)andmoderateratesofspecificity (51.1–60.0%)ofthebasicVLSparametersdiscriminatinglaryngeal carcinomaandothermasslesionsofvocalfolds.However,

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opti-Fig.1.Thisflowchartillustratestheprocessthatwasusedtoselectarticlesforthereview.

mumsystemoffourfollowingVLSvariables—regularity,mucosal waveontheaffectedside,symmetryofvibration,andsymmetryof glottalimage—increasedthesensitivityupto85.3%andspecificity upto74.4%,andafinalJindexof0.60.

FewstudiesonCEwereselected,aswell.Arecentarticle pub-lishedbyPuxedduetal.(2015)observedanaccuracyofCEinthe differentialdiagnosisbetweennormaltissueanddysplasiaversus carcinomaof97.6%.Warneckeetal.,2010declareda sensibility andspecificityachievedbyCEof90%and93.75%,respectively.But amongthese,onlyonestudycomparedcontact endoscopywith

frozensectionhistopathologyin142patientswithlesionsofthe larynx,whereCEyieldedasensitivityof79.59%andaspecificityof 100%,withaJindexof0.79(Cikojevicetal.,2008).Insummarywe obtaineda meanvalueof89.0633±9.0415SD(95%CI 66.6031-111.5236,median 90%)forSeand of97.200±3.020SD (95%CI 89.689-104.702,median97.6%)forSp,withapvalueof0.3992, showinganywayahigherNPV(97%)thanPPV(82%)forCE.

About10yearslatertheintroductionofCEintotheclinical prac-tice,fewpublicationsaboutautofluorescenceinlaryngologycame out;itwas1995whenHarriesetal.demonstratedthroughtheir

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pilotinvivostudytheautofluorescencepropertiesof the laryn-gealtissueandthepotentialaccuracyofthissystemofperforming earlierdiagnosisandstagingoflaryngealtumors.Consideringat leastonedecadetoallowtheflattingofthelearningcurve,our selectedarticlesshowedvaluesofSeandSpof92.550±3.611SD (95%CI89.531-95.569,median93%)andof78.850±17.624SD(95% CI64.116-93.584,median83%),respectively;withanotquite stat-icallysignificantp valueof0.0583.Weconstructedhierarchical

SROCcurvetoassesstheinteractionbetweenSeandSpandthese were93%and80%,respectivelywithaAUCof94%.(Fig.3).

OurresultsconfirmeddatapreviouslyreportedbyKraftetal.in theirmeta-analysisin2011(Kraftetal.,2011).Weperformedan up-dateofKraft’sresultsandweobtainedfromtheircombination afinalSeof93%(95%CI0.86-0.97)andavalueof80%(95%CI 0.68-0.89)forSp,reportingalowerI2_value_for_Se_(67%)_than_for_Sp_(77%). (Fig.4).

Table3

Variouscontributionsofthearticleselectedforthemeta-analysisaddressedtowardsthedefinitionofthediagnosticprecisionofwhitelightendoscopy(WLE)(yellow), stroboscope(white),contactendoscopy(CE)(lightblue),autofluorescenceendoscopy(AFE)(grey),narrowbandimaging(NBI)(red),computedaxialtomography(CTA) (orange),magneticresonanceimaging(MRI)(green)forlaryngealcanceragainstprecancerouslesions.

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Table3(Continued)

Inspecific,Succoetal.(2014)highlightedevidenceof advan-tagesin allowingprecise calibrationoftheentity of superficial resectionduringtrans-orallasersurgery(TLS)asalreadyreported byLucionietal.(2012).Kraftetal.(2011)throughtheirreview ofliteraturefoundalowerJvalueforAF(0.75)incomparisonto thelatestpublications,buttheyconcludedthatFAEishighly effec-tiveintheearlydiagnosisof laryngealcancerand itsprecursor

lesions,andthatcanbeappliedintheprimarydetectionofthese lesionsprovidingabetterevaluationoftheirhorizontalextension anddelineation.However,itisequallyrecognizedthatthistoolis notasyetqualifiedtosubstituteforhistologicalwork-up.Baletic etal.(2010)evidencedmanyconditionsimpactingAFfeaturesof laryngealmucosa;whileRydelletal.(2008)confirmedahighvalue ofJindexforAF(0.89).Saettietal.(2007)revealedahigh

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sensitiv-Table4

Variouscontributionsofthearticleselectedforthemeta-analysisaddressedtowardsthedefinitionofthediagnosticprecisionofultrasound(US)(yellow),computedaxial tomography(CTA)(orange),magneticresonanceimaging(MRI)(green)andpositionemissiontomography(PET-CAT/PET)(purple)foradvancedlaryngealtumorswith cartilageinvolvementagainstearlystagedcancers.(Somearticlesanalyzedmorethanonesystemandtheyareindicatedby*or◦_;).₍_Amilibia_et_al.,_2001;_Celebi_et_al.,_2012;

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Fig.2.SROCcurvefrombivariatemodelofWLEforcancerouslesionsvs. precan-cerouslesionsidentification.

ityoftheAFbythepresenceofdemarcationofthecancerlimits, withanincreaseincontrastbetweenthenormalandpathological mucosa.Here,thegapinsensitivity,betweenWLandAFE,resulted

Fig.3. SROCcurvefrombivariatemodelofAFEforcancerouslesionsvs. precancer-ouslesionsidentification.

evenmoreevident,about37%,thanasreportedinliterature;infact, comparingconfidentintervals,AFErevealedasignificantlyhigher sensitivitythanstandardWL(p<0.05).Nevertheless,theyinsisted

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inspecifyingtheneedofalonglearningcurveandthatrisksare greatlyoperator-dependent.

Themostrecentdiagnostictechniquethathasbeenintroduced intheclinicalpracticeisrepresentedbytheNarrowBand Imag-ing(NBI),infact,firstpublicationsaboutthismethodaredated 2007.NBIhasprogressivelyshowedverypromisingresultsofboth SeandSpforthedifferentialdiagnosisoflaryngealprecancerous andcancerouslesionsinthelarynxthankstoitsschematic clas-sificationofabnormalitiesoftheintraepithelialpapillarycapillary loop(IPCL),thatseemstohaveadirectmatchingwith histologi-calfeatures,sofar.Infact,Nietal.(2011)throughtheirproposal ofclassificationofICPL,obtainedaJindexforNBImuch higher thanWLE(0.82vs.0.59),reachingstatisticalsignificanceinterms ofaccuracy(p=0.028),sensitivity(p=0.020),andnegative predic-tivevalue(p=0.048),incomparisontoWLmode.Greaterresults,in termsofsensitivity,specificityandaccuracyofNBIwereobtained byBertinoetal.(2015b),whodemonstratedthevalidity ofthis techniquebasedonlesions’vascularpatternproposedbyNiand colleagues.AlsoLukesandcolleaguesconfirmedhighvaluesof sen-sitivityandspecificityforsquamouscellcarcinoma(100%and82%, respectively)whenIPCLwerevisible(Lukesetal.2014);andKraft etal.(2014)confirmedthecapacityofNBItoeasilydetectand dis-tinguishmalignantandpremalignanttumorsfrombenignlesions, withafinalaccuracyof97%,withaJvalueof0.93.Watanabeetal. (2009)achievedintheircohortofpatientsahighsensitivityforthe NBI,almostequaltoAFE(91.3%),butshowedahigherspecificity (91.6%)comparedwithAFmethod.Inaddition,theydemonstrated thatNBIsystemcanevaluaterecurrentlaryngealcancersaswell asnewlydevelopedsuperficialcancers,whichareoneofthe draw-backstousingautofluorescenceendoscopy(Arensetal.,2007b). TheseresultswereconfirmedbyZabrodskyetal.recently,in2014 (Zobrodskyetal.,2014).

Irjalaetal.(2011)supportedsameresultsaboutbenefitsofNBI accuracybytheadditionofhigh-definitiontelevision(HDTV).In thisrespect,Piazzaetal.(2010a)showedthattheuseofhigh defi-nitiontelevision(HDTV)NBIsystem,helpedinincreasingtherateof diagnosissignificantly,from20.8%(withoutHDTV)to42.7%,where thesensitivityraseupto98%starting from61%, andspecificity increasedby3%,withavalueofaccuracyof92%andaglobalJindex of0.88,almostdoubledincomparisontoNBIalone.Thus,itseems thatHDTV-NBIprovesasignificantaccuracyinrecognizingtrue positivesandindistinguishing,atthesametime,truenegatives.

From our study we can affirm that values of Se and Sp of 91.44±13.279SD (95% CI81.941-100.939, median 97%)and of 89.470±7.529SD(95%CI84.084-94.856,median90.8%), respec-tively;withadifferencebetweenSeandSpnotstaticallysignificant (p=0.5527).WeconstructedhierarchicalSROCcurvetoassessthe interactionbetweenSeandSpandthesewere94%and93%, respec-tivelywithaAUCof97%.(Fig.5).

Then,weobtainedfromtheircombinationafinal95%CI 0.83-0.98forSeandavalueof95%CI0.88-0.96forSp,reportingalower I2 _value_for _Sp_(61.3%) _than_for _Sp_(87.48%)._Here_the hypothe-sisofhegemonyamongthesedifferentstudieswasabouttobe statisticallysignificant(p=0.04)(Fig.6).

BycomparingAFEandNBIvalueswecanaffirmthatthereis nostatisticalsignificancyintermsofbothSe(p=0.7268)andSp (p=0.1543),eveniftheAUCvaluesrevealedaslightlyhigher per-centageforNBI(97%)thanforAFE(94%).

IfwemakeacomparisonbetweenAFEandWL,NBIandWL, weobtaininterestingresults.Infact,intermsofSeAFEshoweda highermeanvalueof91.914±3.383SDthanWL(71.133±21.579 SD)withasignificantpvalueof0.0229;whileSpdidnotshowany statisticalsignificantdifference(p=0.8699),withameanvalueof 78.400±18.987SDforAFEandof80.100±22.750SDforWL.If wemakethesamecomparisonbetweenNBIandWL,therewasa significantdifferenceintermsofSe(p=0.0283),referringto

val-Fig.5. SROCcurvefrombivariatemodelofNBIforcancerouslesionsvs. precancer-ouslesionsidentification.

uesof88.422±7.171SDforNBIandof71.133±21.579SDofWL; but,Spdidnotshowanystatisticalsignificanceaswell(p=0.3056) whereSpofNBI(89.470±7.529SD)washigherthanthatoneof WL(80.100±22.750SD).

5.2. IIgrouparticles:advancedstagelaryngealtumorwith cartilageinvolvementvs.earlystagedtumorsassessment.

Thisgroupcountedahigher numberofgoodqualityarticles (13vs.5),whereSeandSpfordeterminationofadvanced laryn-gealcancerswithcartilageinvolvementpresentedameanvalue of 78.268±20.611SD (95% CI69.760-86.776, median84%) and 82.140±17.335SD(95%CI74.984-89.296,median89%), respec-tively,calculatedonaglobalnumberofpatientsof1136withan amountof497diseasedpopulations(peoplewithcartilage involve-ment).Anystatisticalsignificancewasfoundamongcontinuosdata (p=0.3855).

Inthesearchforalternativeimagingtechniques,whichcouldbe performedbythetreatingphysician,endosonographyhasbecome animportantdiagnostictoolfromthe1960s’.

Preliminaryresultshavereportedasensitivityofultrasoundin theassessmentoflaryngealcancerof84%,withaspecificityof93% andanaccuracyof89%;thesevalueswerestatisticalsignificantin comparisontoCATandMRI(p<0.0001)inaccordancewithKraft’s results(Kraftetal.2013).

Recent investigations on human laryngeal endosonography, havedemonstratedapenetratingdepthof10to25mm (depend-ingontheapplyingfrequency)andahightissueresolution.Infact, in1994,Zechetal.werefirsttoreportonthissubjecton cadav-ers;someyearslater, Arensetal.(1998,1999) andArensetal.

(1998)publishedanexperimentalstudyonsonographicanatomy

ofthelarynxandthefirstclinicalexperienceswiththispromising methodinavarietyoflaryngeallesionsthroughtheuseofa flexi-bleminiprobes,withafrequencyof10–30MHzanda360fieldof view.Independently,Tamuraetal.(2001,2002)publishedsimilar clinicalandexperimentalstudieswiththesameprobe.Tsuietal.in 2011firstcharacterizedthelaminapropriaandthevocalmuscleby ultrasoundinordertodescribebiochemicalpropertiesofthevocal folds.

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Fig.6. Meta-analysisofsensitivityandspecificityofNBI.

Thusthepromisingpropertiesof ultrasonographyofthe lar-ynx,someauthorsmadecomparisonamongthisemergenttooland standarddiagnosticimagesacquisition.Infact,Huetal.compared sonographytoMRI(91)andCAT(88)intheevaluationandstaging ofglotticcarcinoma,andtheirresultsrevealedthattherewasno statisticaldifferenceamongthesetwotechniqueinpre-therapeutic staging accuracy(80%vs.76.7%;p>0.99), and(83.3%vs. 88.8%; p=0.735)respectively,suggestingultrasonographyasa noninva-sivecomplementarymodalityfordetectionandinitialstagingof glotticcarcinoma.Infact,whenlandmarks,i.e.ventricularbands andvocalcords,forlocalizationareclearlyidentifiedby ultrasonog-raphy,laryngealtumorscouldbecorrectlylocated(p=0.392)(Xia etal.,2013).Moreover,theyshowedtheutilityof ultrasonogra-phyindiagnosingtheneoplasticspreadtotheparaglotticspace, evidencingitssignificativehigherspecificityincomparisontoCAT (94.9%vs.66.7%;p=0.001).WithafinalJvalueforsonographyof 0.907againstavalueof0.667forCAT.

Fromourfewselectedarticleswecanaffirmthatweobtained aSeandSpforUSof88.067±3.828SDandof91.367±5.244SD, respectively,accordinglytorecentreports.

AboutthepredictabilityofCATinthedeterminationof laryn-gealcartilageinvasionitvariesconsiderablyamongthemostrecent publishedreports(Table5).Withalowmeanvalueforsensitivity of64.194±20.862SD(95%CI53.468-74-921,median66%)in con-trasttoahighermeanvalueof82.782±22.289SDforspecificity (95%CI72.322-95.242,median91%).Thiscomparisonpresentsa significantpvalueof0.0199.

We can see how different reports are in contradiction; for instance,Gordinetal.(2006b)showedaCATsensitivityin

identi-fyingcartilageinvolvementof88%,withaverylowspecificity(8%); resultsthathavebeendisprovedbyAllegraetal.(2014)whofound CATvaluesof50%(sensitivity)and100%(specificity),respectively foridentificationofcartilageinvasion.Despitethisconflictingfinal results,ifwecomparereportspublishedbefore2005tothemost recentworks,wedonotfindanystatisticalsignificancesamong thesetwogroupsintermsofsensitivity(p=0.3367)andspecificity (p=0.2574)fortumorcartilageinvolvement.

Fromouranalyzedstudies(seeTable4)wecanaffirmthatvalues ofSeandSpof71.750±23.428SD(95%CI58.223-85.277,median 81.25%)andof83.029±18.474SD(95%CI72.362-93.695,median 91.8%),respectively;withadifferencebetweenSeandSpnot stat-icallysignificant(p=0.1691).

The pooled sensitivity and specificity of CAT for advanced tumorswithcartilageinvolvementratewere74%and84%, respec-tively,withaAUCof85%.(Fig.7).

TheFig.8providestheforestplotofstudiesthatcontributedto theseresultsandthespreaddatawith95%CIforeachstudy repre-sentedbyhorizontallines.Weobtainedfromtheircombinationa final95%CI0.51-0.88forSeandavalueof95%CI0.55-0.96forSp, reportingaveryhighI2_value_for_both_Se_(87.7%)_and_Sp_(95.91%), wherethelowestratesofSewerereportedbyHartletal.(2013) andAllegraetal.(2014),Beseretal.(2009),whilstGordinetal. (2006b),showedthelowestrateforSp.

Herethehypothesisofhegemonyamongthesedifferentstudies wasnotstatisticallysignificant(Fig.8).

ItisobviousthatoverthelastdecadethesensitivityofCAThas improvedconsiderably,risingitsmedianvaluebyabout10%and

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Table5

SensitivityandspecificityofCATandMRIinidentifyinglaryngealcartilageinvasion,variousreports.(Hanetal.2013;Justetal.2010;Sulfaroetal.1989b)

Fig.7. SROCcurvefrombivariatemodelofCATforadvancedstagecancerswith cartilageinvolvementvs.earlystagedcancersidentification.

thuscancelingthepreviousreportedsignificancybetweenSeand SpofCATinidentifyinglaryngealcartilageinvolvement.

Fromthe1980s’themagneticresonanceimaging(MRI)sawit enterinclinicalpractice.

Table6

SensitivityandspecificityofMRIinidentifyingthyroidcartilageinvasions,various reports.

Author Year Sensitivity(%) Specificity(%)

Beckeretal.(2008) 2008 96 75 Limetal.(2011) 2011 84.8 89 Bankoetal.(2011) 2011 100 93 Kinshucketal.(2012) 2012 64 67 Kraftetal.(2013) 2013 63 89 Shangetal.(2013) 2013 90.9 76.5 Bankoetal.(2014) 2014 79 47 Allegraetal.(2014) 2014 100 100

Its predictability in the determination of laryngeal cartilage invasionvaries considerablyamongpublishedreports, asit has beenshownofCATaswell(Table6).Withameanvaluefor sensitiv-ityof84.713±14.958SD(95%CI72.207-97.218,median87.85%)in contrasttoameanvalueof79.563±17.007SDforspecificity(95% CI63.344-93.781,median82.75%)Thiscomparisondidnotpresent asignificantpvalue(0.3321).

Thosereportsthatwereanalyzedforthemeta-analysisshowed throughtheSROCcurveaninteractionbetweenSeandSpof86% and83%,respectivelywithaAUCof91%.(Fig.9).

TheFig.10providestheforestplotofstudiesthatcontributed totheseresultsandthespreaddatawith95%CIforeachstudy rep-resentedbyhorizontallines.Thepooledsensitivityandspecificity ofMRIforadvancedtumorswithcartilageinvolvementratewere 86%and83%,respectively,withafinal95%CI0.62-0.96forSeand avalueof95%CI0.64-0.93forSp.Moreover,heterogeneityofthe studiesincludedinthisanalysiswasveryhighforbothsensitivity (83.29%)andspecificity(83.79%),wherethelowestratesofSewere reportedbyKraftetal.(2013),whilstBankoetal.(2014)showed

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Fig.8.Meta-analysisofsensitivityandspecificityofCAT.

Fig.9. SROCcurvefrombivariatemodelofMRIforadvancedstagecancerswith cartilageinvolvementvs.earlystagedcancersidentification.

thelowestrateforSp.Herethehypothesisofhegemonyamong thesedifferentstudieswasnotstatisticallysignificant,too.

Inthe1990s’PETandCAT/PETenteredintotheclinicalpractice andGordinetal.(2006b)showedhowthecombinationofCATand PETcanreachabettersensitivityandspecificityincomparisonto PETstudybyitself(92%and96%vs.92%and73%,respectively).But, forthefirsttime,Jeongetal.(2008)showedthatPET/CTfindings donotaddtotheconventionalwork-upfortheinitialevaluation ofglotticcancer anyfurther advantagesthatwouldsupportthe additionalcostofthisexam.

BycomparingCATandMRIvalueswecanaffirmthatthereis nostatisticalsignificancyintermsofbothSe(p=0.1708)andSp (p=0.6274),eveniftheAUCvaluesrevealedahigherpercentage forMRI(91%)thanforCAT(85%).

6. DISCUSSION

Advancedimageanalysisproceduresareusedinlaryngeal diag-nosticswithincreasingfrequency.Nonetheless,theimageanalysis proceduresusedarequiteoftenlimitedtoimagevisualization,for example,inindirectautofluorescencelaryngoscopy.Quantification ofcolor,texture,andshapeoflesionsandnormaltissuecouldhelp inamoreaccuratecategorizationoflesionsaswellasinfollow-up procedures.

6.1. Overviewinrelationtotheliterature 6.1.1. Whitelightendoscopyandstroboscopy.

WLEtogetherwithstroboscopyremaintheclinicalkeyelement for detecting and assessing vocalfold lesions,representing the essentialdiagnostictechniqueforevaluationoflaryngealmucosa