Perfil genômico de um carcinoma espinocelular ex-adenoma pleomórfico comparado a um carcinoma espinocelular de cabeça e pescoço

www.bjorl.org

Brazilian

Journal

of

OTORHINOLARYNGOLOGY

CASE

REPORT

Genomic

profile

of

a

squamous

cell

carcinoma

ex

pleomorphic

adenoma

compared

to

a

head

and

neck

squamous

cell

carcinoma

夽

Perfil

genômico

de

um

carcinoma

espinocelular

ex-adenoma

pleomórfico

comparado

a

um

carcinoma

espinocelular

de

cabec

¸a

e

pescoc

¸o

Fernanda

Viviane

Mariano

_,

_Luis

_Fernando

_Vidal

_Saccomani

_,

_Karina

_Giovanetti

_,

André

Del

Negro

_,

_Luiz

_Paulo

_Kowalski

_,

_Ana

_Cristina

_Victorino

_Krepischi

_,

Albina

Altemani

a_{UniversidadeEstadualdeCampinas(UNICAMP),FaculdadedeCiênciasMédicas,DepartamentodePatologia,Campinas,SP,Brazil} b_{UniversidadeEstadualdeCampinas(UNICAMP),FaculdadedeCiênciasMédicas,CirurgiadeCabec¸aePescoc¸o,Campinas,}

SP,Brazil

c_{ACCamargoCancerCenter,DepartamentodeCirurgiadeCabec¸aePescoc¸o,SãoPaulo,SP,Brazil}

d_{UniversidadedeSãoPaulo(UNIFESP),InstitutodeBiociências,DepartamentodeGenéticaeBiologiaEvolucionária,SãoPaulo,}

SP,Brazil

Received6October2015;accepted13November2015 Availableonline24February2016

Introduction

Squamouscell carcinoma(SCC) isthe most frequent neo-plasm in the head and neck and in oral sites. Head and neck squamous cell carcinoma (HNSCC) is the sixth most common solid cancer, affecting more than 900,000 peo-ple worldwide each year. Oral squamous cell carcinoma (OSCC) is the largestsubgroup of HNSCC andcauses 7400

夽 _{Pleasecitethisarticleas:MarianoFV,SaccomaniLF,Giovanetti}

K,DelNegroA,KowalskiLP,KrepischiAC,etal.Genomicprofileof asquamouscellcarcinomaexpleomorphicadenomacomparedto aheadandnecksquamouscellcarcinoma.BrazJOtorhinolaryngol. 2018;84:393---7.

∗_{Correspondingauthor.}

E-mail:fevimariano@gmail.com(F.V.Mariano).

PeerReviewundertheresponsibilityofAssociac¸ãoBrasileirade OtorrinolaringologiaeCirurgiaCérvico-Facial.

deaths in the United States every year.1 _{Because of the}

highprevalenceandaggressiveness,HNSCChasbeenstudied byseveralmolecularandcytogenetictechniques.Frequent chromosomalimbalancesreportedinHNSCCare3p,4,5q, 8p, 9p, 11,13q,and 18q(losses)and3q25---26, 5p, 8q24, 9q22---34,11q13,14q24,16p,19p,20q24,and22q(gains).1

Thecarcinomatouscomponentofcarcinomaex pleomor-phicadenoma(CXPA)encompassesavarietyofhistological tumor types, such asadenocarcinoma, myoepithelial car-cinoma, epithelial---myoepithelial carcinoma, sarcomatoid carcinoma,and salivaryductcarcinoma.2 _Many

epidemio-logicalstudies about incidenceof CXPA showedfew or no caseswithanSCCcomponent.

Despitereportsofnumerouschromosomalalterationsin SCCfromothersites,noparticularchromosomalimbalances havebeendescribedforsquamouscellcarcinomaex pleo-morphic adenoma (SCC-CXPA). The aim of this study was toidentifychromosomalchangesassociatedwithSCC-CXPA, comparingwithHNSCCdatafromtheliterature.

https://doi.org/10.1016/j.bjorl.2015.11.015

1808-8694/©2016Associac¸˜aoBrasileiradeOtorrinolaringologiaeCirurgiaC´ervico-Facial.PublishedbyElsevierEditoraLtda.Thisisan openaccessarticleundertheCCBY-NC-NDlicense(http://creativecommons.org/licenses/by-nc-nd/4.0/).

Figure1 Squamouscellcarcinomaexpleomorphicadenoma.(A,B)Residualpleomorphicadenoma.(A)Pleomorphicadenoma areashowsextensivehyalinizationofthestroma.(B)Fewductalstructureslinedbycellswithoutatypicalfeaturescanbeseen.(C, D)Carcinomatouscomponent.(C)Afranklyinvasivesquamouscellcarcinomaisthemalignantcomponentofthetumor.(D)Detail ofthemalignantcomponentshowingislandsformedbycellswithsquamousdifferentiation.

Case

report

Clinicopathologicdata

Thecasewasreferredtothisdepartmentfor histopatholog-icalreview.Thegender andageareunknown.The patient presentedamassoftheparotidglandthathadbeen notice-ablefortenyears.Thetumorwasexcisedandhistological examinationshowedpleomorphic adenoma(PA)areasand SCCarisinginPA(Fig.1).Tumorcellspresentedkeratinpearl formationandinvadedtheadjacenttissuesofparotidgland. TheneoplasmwasclassifiedasafranklyinvasiveSCC-CXPA. Thepatientdeveloped distantmetastasis in thelungsbut thefollow-upinformationwasnotavailable.

Arraycomparativegenomichybridization(CGH) analysis

Somatic copy number alterations detected in the SCC-CXPA sample are detailed in Table 1 as well as affected known cancer genes according to the Sanger Cancer GeneCensus(https://www.sanger.ac.uk/research/projects/ cancergenome/census.html).

The current caseexhibiteda complexgenomicpattern withseveralcopynumberalterations.Lossesweredetected at1p,8p,17p,and18q,andgainswereidentifiedat1p,6, 8q,11q,17q,19q,and22q(Fig.2A).

Three regions exhibiting high copy number alter-ations (amplifications) were detected at 11q14.3q22.1,

11q22.1q22.3(Fig.2B),and19p13.2(Fig.2C).Amongother genes, these amplifications encompass the known cancer genesMAML2,BIRC3,andLYL1.

Discussion

Mariano et al.2 _{reported one SCC-CXPA out of 38 cases}

(2.6%)of CXPAfromaBrazilian population.To thebestof theauthors’knowledge,onlysporadiccasesweredescribed showingSCCasthemalignantcomponentofCXPA, empha-sizingtheclinicalandmorphologicalfeatures.

SCCoriginatesfromtheepithelialcomponentofthePA, and in lesions that arise from minorsalivary glands, it is important to demonstrate no continuity of the SCC nests withthebuccalmucosa.Inthecurrentcase,areasof resid-ualPAwereassociatedwithawidelyinvasiveSCCinthesoft tissues surroundingthe parotid gland. Distant metastases wereobservedinthiscase,suggestingthatSCCbelongsto the group of aggressiveCXPAs. It has been proposed that CXPA isactually acategoryof tumorsratherthanasingle tumortype,sincebothaggressiveandindolentcarcinomas canarisefromaPA.

Localaggressiveness,highrateofearlyrelapsetumors, andreducedfive-yearsurvivalratesnotexceeding50%are typical clinical features ofSCC. OSCCcauses 7400 deaths in theUSA everyyear.1 _{Forthese reasons,during thelast}

two decades, genome screening approaches like chromo-somalCGHwereappliedtodisclosethemolecularbasisof carcinogenesisofthistumor.

Table1 Somaticcopynumberalterationsdetectedbyarraycomparativegenomichybridizationinonesquamouscellcarcinoma expleomorphicadenomacase.

Chromosomecoordinates (Hg19)

Eventtype Size (Mb)

Cytoband Genes (n)

Knowcancergenes(CGCS) chr1:783,108-10,814,886 Gain 10.0 1p36.33---p36.22 162 TNFRSF14,PRDM16,RPL22,

CAMTA1 chr1:55,751,323-74,536,392 Loss 18.7 1p32.3---p31.1 94 JUN,JAK1

chr1:106,671,417-121,354,529 Loss 14.6 1p21.1---p11.2 171 RBM15,TRIM33,NRAS,FAM46C, NOTCH2

chr6:122,468-21,138,924 Gain 21.0 6p25.3---p22.3 126 IRF4,DEK

chr6:147,333,160-170,923,962 Gain 23.5 6q24.3---q27 154 EZR,FGFR1OP,MLLT4

chr8:239,745-43,440,193 Loss 43.0 8p23.3---p11.1 364 PCM1,WRN,WHSC1L1,FGFR1, HOOK3 chr8:93,473,949-109,349,316 Gain 15.8 8q22.1---q23.1 94 COX6C chr11:84,971,351-92,399,403 Gain 7.4 11q14.1---q14.3 43 PICALM chr11:92,410,361-99,634,930 Amplificationa _{7.0 11q14.3---q22.1 48 MAML2} chr11:99,645,163-104,334,763 Amplificationa _{4.6 11q22.1---q22.3 29 BIRC3}

chr17:9,134,319-22,224,426 Loss 13.0 17p13.1---p11.1 165 GAS7,MAP2K4

chr17:46,160,506-59,942,017 Gain 13.7 17q21.32---q23.2 177 COL1A1,HLF,MSI2,CLTC,BRIP1 chr18:18,440,032-78,062,375 Loss 59.6 18q11.1---q23 278 ZNF521,SS18,MALT1,BCL2 chr19:12,951,065-13,396,186 Amplificationa _{0.4 19p13.2 19 LYL1}

chr19:28,039,051-59,128,983 Gain 31.0 19q11---q13.43 1123 CCNE1,CEBPA,AKT2,CD79A, CIC,BCL3,CBLC,ERCC2,KLK2, PPP2R1A,ZNF331,TFPT chr22:31,865,770-43,582,078 Gain 11.7 22q12.2---q13.2 205 MYH9,PDGFB,MKL1,EP300

a _{Highcopynumbergains.}

Genomic profile of SCNA in case 1

Chromosome 1p to Xq

A

B

C

Figure2 Copynumberalterationsdetectedbyarraycomparativegenomichybridization(CGH)inthesquamouscellcarcinoma

expleomorphicadenomacase.(A)Array-CGHgenomicprofileexhibitingthealltheidentifiedcopynumberalterationsofthiscase. Thex-axisrepresentsprobesorderedaccordingtotheirgenomicpositionfromchromosomes1ptoXq(eachchromosomeislabeled withadifferentcolor).They-axisdenotesthelog2test/referencevalues(genomicgainsandlossesareplottedaboveorbelow

the0baseline,respectively;images adaptedfromthe softwareNexus CopyNumber7.0,Biodiscovery).The redbox indicates chromosomesthatharborgenomicrearrangementsoflossesorgains:1,6,8,11,17,18,19and22.Thearrowsshowtheregionsof genomicamplificationsatchromosomes11and19.(B)Array-CGHprofileofthechromosome11showingindetailthetworegions withhighcopynumbergain(amplifications):at11q14q22.1(7Mb)andat11q22.1-q22.3(4.6Mb)(bluearrowinA).(C)Array-CGH profileofthechromosome19exhibitingoneregionofgenomicamplificationat19p13.2(0.4Mb)(redarrowinA).

Acomplexgenomicprofileofthecopynumberalterations of HNSCC has been described in the literature, including 3p, 4,5q, 8p, 9p, 11, 13q,and 18qlosses,and 3q25q26, 5p, 8q24, 9q22q34, 11q13, 14q24, 16p, 19q, 20q24, and 22qgains.1_{Thecurrentcasealsoshowsacomplexgenomic}

profile with several copy number alterations, but with a different pattern compared to SCC from head and neck, probablydue to distinct cellular lineage.Interestingly, in HNSCCtherearemultiplediscreteregionsoflossat3p.1,3

Eventhoughcytogeneticinvestigationshavesuggestedthat 3p loss is a key genomic change in HNSCC, the present case didnot showthis alteration.However,some similar-itiesbetweenHNSCCandthiscasewerefound,suchas8p23 and18q11 losses, and 8q22.1,19q,and 22q gains. These alterationscanbespecifictosquamouscelldifferentiation andcontributetoaggressivebehavior.3

IthasbeensuggestedthatinHNSCC,gainson3q26and 11q13 and deletions on 8p23 and 22q could be valuable markers of aggressive disease.1 _{The loss at 8p23 in the}

currentcaseencompassesimportantcancer-relatedgenes, suchasPCM1,WRN,WHSCILI,FGFR1,andHOOK3.However, chromosomalgains on1qand16qaswellaschromosomal loss on 18q have been suggested torepresent prognostic markersinHNSCC.1,3 _{Thepresentreportalsofoundlossat}

18qinvolvingthecancerrelatedgenesZNF521,SS18,MALT1, andBCL2.Againat8q22.1q23.1wasalsoobserved.Inthis genomic interval, the YWHAZ gene is mapped, a HNSCC proto-oncogenecandidate.

New findings not previously reported for HNSCC were alsoidentified,suchaslossesat1p32,1p21,and17p13.1, as well as gains at 1p36.33, 6p25.3, 6q24, 11q14, and 17q21.32. The 1p32 loss is associated with the develop-mentofadenoidcysticcarcinoma,1_{and1p36isfrequently}

deletedin human cancers.Amongthese regionsof imbal-ance,1p21.1lossseemsparticularlyinteresting,becauseit comprisesseveralcancerrelatedgenes.1_{Overexpressionof}

theDEKgene(6p25.3)isassociatedwithgastricandbreast adenocarcinoma,4_{aswellastheroleEZRINhasincolorectal,}

pancreatic,gastric,andotheradenocarcinomas.5

It was noteworthy that three genomic amplifications weredetected encompassing the region at 11q14.3q22.1, 11q22.1q22.3,and 19p13.2.Theseamplifications can har-borrelevantgenesdrivingthecarcinogenesis,andMAML2, BIRC3, and LYL1 are known cancer genes mapped within theseregions.

MAML2(11q14.3q22.1)(mastermind-like2)hasaroleas a transcriptional coactivator for the Notch receptor, and wasreported tobe rearranged with CRTC1 and CRTC3 in mucoepidermoidcarcinomas.6_{Inthepresentcase,the}

diag-nosisofmucoepidermoidcarcinomaarisinginPAwasruled outinthehistologicexaminationsincethemalignant com-ponent did not contain mucous cells. MAML2-CRTC1 and MAML2-CRTC3havenotbeenfoundinadenosquamous carci-noma,SCC,oranyothersalivaryglandcarcinoma.6_MAML2

amplified induces the transcription of the HES-1 gene (a Notchtargetgene)thatregulatestheexpressionof tissue-specific transcription factors that influence lineage and otherevents.Thisamplificationisdescribedwithalow fre-quencyincancersofseveralsites,butnotinHNSCC.

Another amplified gene, BIRC3 (baculoviral IAP repeat containing3),encodesamemberoftheIAPfamilyof pro-teins that inhibit apoptosis. Amplificationinvolving BIRC3

has been detected in pancreatic cancer, osteosarcoma, acute myeloid leukemia, mammary carcinoma, lung can-cer, cervical cancer, and SCC.7 _{It has been found that}

BIRC3mayenhancecancercellsurvivalandcancer progres-sion,suggestingthattreatmentstargetingthisgeneproduct mayprovideusefultherapeuticoptions.LYL1(lymphoblastic leukemia-associated hematopoiesisregulator 1)gene rep-resents abasic helix-loop-helixtranscription factor, which encodesaproteinwithrolesinbloodvesselmaturationand hematopoiesis.8_{Itisbelievedthatinsalivaryglandtumors}

LYL1amplificationcouldcontributewithoncogenesis. PA as well as CXPA has been reported to harbor highlyspecificchromosomeabnormalitiesrelatedtoPLAG1 and HMGA2 genes. Rearrangements of these genes (8q12 rearrangements, target gene PLAG1 and 12q14-15 rear-rangements, targetgeneHMGA2) havebeen implicated in thegenesisofPAandCXPA.However,althoughthese chro-mosomeabnormalities arefrequentin thesetumors,they arenotdetectedinallofthem.9_{Inthecurrentcase,}

ampli-fiedPLAG1orHMGA2geneswerenotfound,suggestingthat thetumorprobablybelongedtothegroupwithoutsuch kary-oticalteration.

Conclusion

In summary, SCC-CXPA showeda complexpattern of copy numberalterations thathassomesimilarities withthat of HSCCN. It is likelythat these chromosomalabnormalities maybeinvolvedinthedevelopmentofthesquamous phe-notypeandaggressivebehavioroftheSCC-CXPA.However, genomicamplificationsofknowncancergenes,suchasLYL1, BIRC3, and MAML2 detected only in SCC-CXPA couldhave importancefortheprocessofmalignanttransformationof PA.Theselatteralterationsmayrepresentclinicallyrelevant markersforSCC-CXPAdiagnosis.

Funding

FAPESPProcessNos.2011/23204-5and2011/23366-5.

Conflicts

of

interest

Theauthorsdeclarenoconflictsofinterest.

References

1.BockmühlU,SchlünsK,KüchlerI,PetersenS,PetersenI.Genetic imbalances withimpact on survival in head and neck cancer patients.AmJPathol.2000;57:369---75.

2.MarianoFV,NoronhaAL,GondakRO,AltemaniAM,deAlmeidaOP, KowaslkiLP.CarcinomaexpleomorphicadenomainaBrazilian population:clinico-pathologicalanalysisof38cases.IntJOral MaxillofacSurg.2013;42:685---92.

3.AshmanJN,PatmoreHS,CondonLT,CawkwellL,StaffordND, GreenmanJ.Prognosticvalueofgenomicalterationsinheadand necksquamouscellcarcinomadetectedbycomparativegenomic hybridization.BrJCancer.2003;89:864---9.

4.PiaoJ,ShangY,LiuS,PiaoY,CuiX,LiY,etal.Highexpression ofDEKpredictspoorprognosisofgastricadenocarcinoma.Diagn Pathol.2014;9:67.

5.ZhouJ,FengY,TaoK,SuZ,YuX,ZhengJ,etal.The expres-sionandphosphorylationofezrinandmerlininhumanpancreatic cancer.IntJOncol.2014;44:2059---67.

6.Okumura Y, Miyabe S, Nakayama T, Fujiyoshi Y, Hattori H, Shimozako K, et al. Impact of CRTC1/3-MAML2 fusions on histologicalclassificationandprognosisofmucoepidermoid car-cinoma.Histopathology.2011;59:90---7.

7.ChengL,ZhouZ,Flesken-NikitinA,ToshkovIA,WangW, Cam-posJ,etal.Rbinactivationacceleratesneoplasticgrowthand

substitutesforrecurrentamplificationofcIAP1,cIAP2andYap1 insporadicmammarycarcinomaassociatedwithp53deficiency. Oncogene.2010;29:5700---11.

8.NagelS,VenturiniL,MeyerC,KaufmannM,ScherrM,DrexlerHG, etal.MultiplemechanismsinduceectopicexpressionofLYL1in subsetsofT-ALLcelllines.LeukRes.2010;34:521---8.

9.RotelliniM,PalombaA,BaroniG, FranchiA.Diagnosticutility ofPLAG1immunohistochemicaldeterminationinsalivarygland tumors.ApplImmunohistochemicalMolMorphol.2014;22:390---4.