RecentadvancesinthemoleculardiagnosisofpaediatricsofttissuesarcomasBruceRPawelAbstractSarcomascompriseasignicant portionof solidtumoursdiagnosedinchildren. As they commonly are composed of primitive round or spindledcells, morphologicdiagnosiscanbedifcult, andancillarystudiesareoften necessary. In recent years, progress has been made in under-standing the cytogenetic and molecular changes underlying many ofthese tumours, andmolecular techniques have emergedfromstrictlybeingresearchtoolstobecomingstandarddiagnostictests. Paediatricsarcomasfall intotwocategories: thosewithcharacteristic underlyingtranslocations, andthose without. Translocationassociatedpaediatricsarcomas include, but arenot limitedtoalveolar rhabdomyosarcoma,Ewingsfamilyoftumours,desmoplasticsmallroundcellsarcoma,syno-vial sarcoma, infantile brosarcoma, clear cell sarcoma of soft tissue, andlow-grade bromyxoid sarcoma. Sarcomas without translocations tend tobe more pleomorphic in appearance, and include embryonal rhabdomyo-sarcoma, malignant rhabdoid tumour and epithelioid sarcoma. Thisreviewwill describethemolecular andimmunohistochemical methodsmostapplicableindiagnosingthesesofttissuemalignancies.Keywords molecular techniques; pathology; paediatrics; sarcoma;translocationsIntroductionPeadiatricsolidtumours canbechallengingtodiagnose, bothdue to their relatively rarity and frequently challengingmorphologic features. In contrast to adults, in whom themajorityof solidtumours are carcinomas of epithelial origin,solid malignancies occurring outside of the central nervoussysteminchildrenarealmost entirelycomposedof embryonaltumours andsoft tissuesarcomas, withcarcinomas appearingonly exceptionally. Nevertheless, as the overall incidence ofcancerismuchlowerinchildrenthaninadults,eventhemorecommonpeadiatricsarcomasarerelativelyrare, andmayonlybe sporadically encountered by most pathologists in generalpractice. Even in tertiary peadiatric referral centres, thesetumours are seen infrequently, with lack of experiencehampering both diagnosis and attempts to standardize andimprove treatment. The establishment of cooperative groups,suchas the Childrens Oncology Group(COG) inthe UnitedStates and the International Society of Peadiatric Oncology(SIOP) inEuropehas donemuchtoamelioratethis situation.These cooperative group studies and their predecessors haveclaried much of what we nowknowabout these tumours,throughcentralreview,biologystudiesandstandardizedmulti-disciplinary therapeutic trials spanning numerous memberinstitutions. In addition, they have demonstrated the pivotal roleof accuratepathologicdiagnosisinoptimizingtherapytowardsthebestpossibleclinicaloutcome.Thediagnosticspectrumof soft tissuetumoursisvast, andthis review of molecular diagnostic advances will focus upon softtissue malignancies that are prone to develop in children.Historically,paediatricsofttissuesarcomashavebeenclassiedaccordingtotheirdegreeandtypeofcellulardifferentiation;sofor example tumours that displayed histologic and ultrastructuralevidenceof skeletal myogenesis wereplacedintotherhabdo-myosarcomacategoryandtumours that displayedspindlecellmorphology were placed into the brosarcoma/myo-brosarcoma category. Small round cell tumours that displayed noevidenceof specic cytologic differentiationwererelegatedtothe categories of extraosseous Ewing sarcoma or undifferentiatedsarcoma. Earlyon, it wasclear that strictlymorphologicdiag-nosis, even when buttressed by histochemical staining andelectron microscopy was of limited value, as tumours withsimilar histologic featurescould have markedly different clinicalbehaviours and outcomes. Moreover, the form of cellulardifferentiation did not translate into an identiable cell oforigin, whichcontinues toremainelusiveinmost paediatricsarcomas. Even with immunohistochemistry many paediatricsarcomas remain diagnostic challenges, as different tumoursmayhaveonlysubtledifferences inproteinexpression, somehaveonlylimitedreactivitywithcommerciallyavailableanti-bodies,andthereisconsiderableoverlap,lackofspecicityandvariabilityinhowtumoursexpressproteins, aswell assigni-cant subjectivity in how immunohistochemical stains areinterpreted.Withtheadvent of karyotyping, it becameclearthat paedi-atricsarcomascouldbedividedintotwodistinctgroups: thosethathadrecurrentchromosomal translocations, andthosewithcomplexkaryotypes lackingrecurrent translocations. Morpho-logically, translocationassociatedsarcomaswerecharacterizedby more uniform cell populations, whereas those withouttranslocations were more likelyto be pleomorphic.Cytogeneticsbecame apowerful tool for diagnosis, andinsome cases forprognostication as well. This cytogenetic approach offered a newway to look at paediatric sarcomas, and was the foundation uponwhichmoleculardiagnosticpathologyhasbeenbuilt.However,classiccytogeneticanalysis, inwhichtumour cellsaregrown,metaphase chromosomes are stained, and banding patternsanalyzed, suffersfromsignicant limitations. Amongthesearedifcultiesingrowingcellsfrommanysolidtumours, potentialcontamination, prolongedturn-aroundtime, andlabour inten-sity. With the advent of standard molecular diagnostic tech-niquessuchasreversetranscriptase-polymerasechainreaction(RT-PCR) and uorescence in-situ hybridization (FISH), many ofthese problems inherent with classic cytogenetics have beenmitigated.BruceRPawel MDisAssistantProfessorofPathologyandLaboratoryMedicine at the University of Pennsylvania School of Medicine and TheChildrensHospitalofPhiladelphia,Philadelphia,Pennsylvania,USA.Conictsofinterest:nonedeclared.REVIEWDIAGNOSTICHISTOPATHOLOGY17:1 25 2010ElsevierLtd.Allrightsreserved.Molecular techniques applicable to paediatric sarcoma diagnosisReversetranscriptase-polymerasechainreaction(RT-PCR)isanexquisitelysensitiveRNAbasedassaycapableof copyinggenesequences of interest uptoabillion-fold, sothat theycanbedetectedinclinical material.1AstheDNAbreakpointsintrans-locationsoccurwithinintronsandnot inexons, RNAchimerictranscriptshaveamoreconsistentstructureandaremoresuit-able for probing.2In this methodology, RNAtranscripts arereversetranscribedintocomplimentaryDNA(cDNA), whichcan then be amplied. Since RNA is much more labile than DNA,thismethodgenerallyrequirestheavailabilityoffreshorfrozentumour tissue. Formalin xed parafn embedded tissue has beensuccessfully used; however it is less reliable, the methodology ismore tedious, and the probability of failure is much higher due tothepoorRNAqualityinherentinthismaterial.3Fluorescent in-situ hybridization (FISH) is a DNA basedmethod which can be applied to frozen tissue, xed tissue and toimprint preparations. There are a number of different variations ofFISH, and its amenability to use on parafn embedded tissue hasmade FISHa rst line molecular test modality for paediatricsarcoma diagnosis in many laboratories. Unlike routine kar-yotyping, which requires metaphase spreads, FISH can be appliedtointerphase, non-dividingcells, greatlyincreasingitsapplica-bility in archived tissue. Split signal, or break-apart FISH is widelyusedinpresent day practice, andemploys two probes, eachmarkedwithadifferent uorescent dye, andeachspecic toa anking regionof a translocationbreakpoint. Innormal cells, thetwo probes would overlap and give a fused signal, whereas if theprobes ank a translocation, the two signals would be separated.With this split-apart strategy, the necessity for primers specic toa chimeric fusion(as inRT-PCR) is obviated, as any rearrangementof that specicgenewouldbedetected, without regardtothespecictranslocationpartner.1For example, asplit-apart FISHprobe for the EWS gene would reveal an abnormal signal patterninany of the Ewings family of tumours that harbour an EWS trans-location,aswell asothernon-EwingstumourswithEWStrans-locations, such as desmoplastic round cell tumours and clear cellsarcomas.SinceFISHdoesnotemployanamplicationstep,itssensitivityisgenerallylessthanRT-PCR. Ingeneral, FISHcandetect fusions involving as fewas 5%of cells ina sample.1Anotherform of FISH involves fused signal probes, which are specic forthe two target genes involved in a given translocation.3RhabdomyosarcomaRhabdomyosarcomaisthemostcommonsofttissuesarcomaofchildhood. Under thecurrent WHOclassication, rhabdomyo-sarcomasareclassiedintodistinctsubgroupsthathavemark-edlydifferingclinical behaviour andrequiredifferent formsoftreatment.4The twomajor forms are alveolar rhabdomyosar-coma (ARMS), which is an aggressive tumour with a poorprognosis, and embryonal rhabdomyosarcoma (ERMS), which isgenerally more favourable. In their classic forms, these twotumours are histologically distinct; unfortunately many casespresent withambiguous histologic features, and morphologicdistinction may be difcult even for experienced paediatricpathologists. Since most ARMS are associated with specic genefusions,moleculargenetictestingisavaluableadjuncttorhab-domyosarcomadiagnosis.ARMSishistologicallytypiedbyamonotonouspopulationof primitive appearing tumour cells, andthe termalveolarreferstothetendencyoftheselooselycohesivecellstoseparatefromeachother, resultinginclusteringof tumour cells alongbrous septae, inafashionreminiscent of alveoli of thelung(Figure 1a,b). In many cases, this essentially artifactual change isnot evident, andARMSmaypresent histologicallyasformlesssheetsofcells. Pleomorphismisusuallyminimal, anddifferen-tiatedstrapcellsarenotseen.InERMS,tumourcellsdisplaya wider range of differentiation than in ARMS. Resembling stagesinembryonal skeletal muscledevelopment, cellsinERMSspantherangeof primitivestellatecells, spindledcells, cellsanalo-gous to myotubes, and cells with copious eosinophilic cytoplasm(so-calledstrapcellsandtadpolecells).Atlowpower,themosttypical nding inERMS is alternating zones of loose mesen-chyme and denser spindled regions (Figure 1c,d). In hollow visci,suchasthebladder, sometumoursshowamacroscopicgrape-likegrowthpattern, withahistologiccambium-layerformedby a condensation of tumour cells beneath the native epithelium;this is the botryoidvariant of ERMS. Spindle forms of ERMSwhich resemble a broma or neurobroma can also beencountered.By immunohistochemical methods, bothARMS and ERMSwillshowexpressionofmusclerelatedantigenssuchasdesminand muscle specic actin. However, the most sensitive andspecic immunohistochemical test for RMS is for myogenin,askeletal muscletranscriptionfactor not expressedinmatureskeletal muscle.5The percentage of cells expressing myogenin issignicantlyhigher inARMSthaninERMS, whichmayhavediagnostic value, althoughoverlapexists.6e10Combiningdatafromseveralstudies,Morgensternetalfoundthatover80%ofcases diagnosed as ARMS displayed nuclear myogenin positivityinover50%oftumourcells,whereasonly25%ofERMSdid.11Diffusemyogeninpositivityisalsoamarkerofpoorprognosis,independent of fusion status and histology.12In addition tomyogenin, immunohistochemistry for another skeletal muscletranscription factor, MyoD1, has also been applied to RMS, but isless sensitive. Finally, immunohistochemistry for PAX-5,a member of the gene family involved in ARMS translocations, isalsoausefuldiscriminatorinthedifferentialdiagnosisbetweenARMSandERMS. Inarecent study, PAX-5nuclearexpressionwas not seen in any ERMS, and was detected in 67% of ARMS.13UnlikeARMS, ERMSisnot associatedwithaspecictrans-location. ERMStumours areassociatedwithmultiplenumericand structural chromosomal changes, most commonly involvingextra copies of chromosomes 2, 8 and 13. Loss of heterozygosityat 11p15 (the BeckwitheWiedemann region) has also beendescribed.4ARMS is associated with two gene fusions which are not seenin other tumours: PAX3eFOXO1 and PAX7eFOXO1 (FOXO1previouslyknownasFKHR); thesefusiongenescorrelatewithtranslocationst(2;13)(q35;q14)andt(1;13)(p36;q14)respective-ly14(Figure2).RareinstancesofvariantPAX3fusionshavealsobeendescribedinARMS, includingfusionswithAFX, NCOA1and NCOA2.15PAX3eFOX01 fusions are approximately threetimes more common than PAX7eFOX01 fusions, whereasPAX7eFOXO1fusionsappeartoconferabetterprognosis.Bothofthesechimericfusionproductsaremorepotentinactivatingtranscription than PAX3 or PAX7 alone. In approximatelyREVIEWDIAGNOSTICHISTOPATHOLOGY17:1 26 2010ElsevierLtd.Allrightsreserved.20e25%of cases withalveolar histologic features, neither ofthesetranslocationscanbeidentied.16,17Usingdatafromgeneexpression proling and loss of heterozygosity analysis, itappearsthat thesefusion-negativeARMS(i.e. tumours withalveolar histology) have genetic proles indistinguishable forERMS.18Whether or not they behave in a clinically similarmanner to ERMS is still open to question. In addition to RT-PCRusing specic probe sets, detectionof FOXO1 relatedfusionsusingbreak-apartFISHprobesonparafnembeddedspecimenshasbeenshowntobebothrelativelysensitiveandspecic.19EwingsfamilyoftumoursEwing sarcoma (ES) is the second most common primarymalignancyof boneandsoft tissueinchildhood, withapeakincidence in adolescence. Morphologically, Ewing sarcoma/primitive neuroectodermal tumour (PNET) can be considered theprototype of the small round blue cell tumour of childhood. Initsclassicform,Ewingsarcomamanifestsassheetsofundiffer-entiated small cells which formno particular architecturalpattern, except for the occasional production of neuroectodermaltyperosettes(Figure3). Althoughrst describedasaprimarybonetumour, this tumour alsooccurs insoft tissue, andhasvariabledegreesof neuroectodermal differentiation. Byexpres-sionprolingit appearsthat Ewingtumourcellsaresimilartomesenchymal progenitor cells.20,21The various modes ofpresentation, and occasional expression of neuroectodermalt(2;13)(q35;q14)der(2)der(1) Chrom 1Chrom 2Chrom 13Chrom 13der(13)der(13)t(1;13)(q36;q14)Chromosomal translocations in ARMSFigure2Chromosomaltranslocationsinalveolarrhabdomyosarcoma(CourtesyofDr.FredericBarr,UniversityofPennsylvaniaSchoolofMedicine).aAlveolarrhabdomyosarcoma.Notethediscohesivenatureoftheprimitiverhabdomyoblastsliningbrovascularseptae.Whenpresent,giantcells are a helpful feature in distinguishing ARMS from embryonal rhabdomyosarcoma (haematoxylin & eosin). b Typical myogenin staining patterninARMS;mosttumournucleiarepositive.cEmbryonalrhabdomyosarcoma,withalternatingcellularandmyxoidareas(haematoxylin&eosin).dtypicalmyogeninstainingpatterninERMS,onlyaminorityoftumourcellnucleistain.Figure1REVIEWDIAGNOSTICHISTOPATHOLOGY17:1 27 2010ElsevierLtd.Allrightsreserved.features led to a myriad of designations for this tumour, such asextraosseousEwingsarcomafor soft tissuetumours, Askintumour for tumours occurringin the thoracopulmonaryregion,and primitive neuroectodermal tumour for tumours displayingneuroectodermal differentiation, suchasformationof rosettes.With the advent of molecular genetic diagnostics, it became clearthat all of these entities shared recurrent translocations, and theyare no longer felt to be biologically distinct, and have beensubsumed into the category of Ewings family of tumours(EFTs).Giventheundifferentiatednatureofthistumour,differentialdiagnosis with undifferentiated examples of other paediatricmalignanciescanbechallenging. Inparticular, it isdifcult, ifnot impossible to distinguish Ewing sarcoma from lymphoblasticlymphoma, solid variant ARMS, and undifferentiated neuro-blastoma, without the judicious application of immunohisto-chemistry. In addition to vimentin, Ewing sarcomacharacteristicallydisplaysstrongperimembranousstainingwithCD99;tumours thatlack suchstaining shouldnotbe consideredEwingsarcomaintheabsenceof conrmatorymoleculardata.Althoughsensitive,CD99stainingisunfortunatelynon-specic,asother tumourssuchaslymphoblasticlymphomas, rhabdoidtumours, and synovial sarcomas also may express CD99. Also tobeborneinmindisthat Ewingsarcomamayexpresscytoker-atins. Incasesof suspectedEwingsarcoma, areasonableanti-body panel might include LCA and Tdt to rule out lymphoblasticlymphoma, as well as a muscle marker such as myogenin to ruleoutrhabdomyosarcoma.Ewingsarcomaischaracterizedbytranslocations involvingthe EWS gene on chromosome 22, and these can be interrogatedwith both FISHand RT-PCR assays. Amongst the paediatricsarcomas, Ewing sarcoma appears to have the greatest variety offusionpatterns, resultinginconsiderablecomplexityintestingandinterpretation. Themost commontranslocation, present inapproximately85e90%ofESis11;22,involvingtheEWSgeneon chromosome 22 and the FLI1 gene on chromosome 11(Figure 4). EWS is a widely expressed normal proteinFigure3aEwingsarcoma.Primitivecellsarrangedinformlesssheets(haematoxylin & eosin). b CD99 strong perimembranous staining in Ewingsarcoma.cDualcolourFISHwithbreak-apartEWSprobe,performedoninterphasenucleiinacaseofEwingsarcoma.Noteonenormalfusionsignalandseparationoftheother greenandredprobes(CourtesyofDr.PaulJ.Zhang,UniversityofPennsylvaniaSchoolofMedicine).1514131211111213131211111213142122232425Der 11Normal 11Der 22Normal 22t(11;22) Translocation in Ewings SarcomaFigure4Thet(11;22)translocation,themostcommontranslocationinEwings sarcoma (Courtesy of Dr. Frederic Barr, University of PennsylvaniaSchoolofMedicine).DIAGNOSTICHISTOPATHOLOGY17:1 28 2010ElsevierLtd.Allrightsreserved.REVIEWtranscriptionfactor whoseexact roleis unclear22, andFLI1isa highly regulated lineage restricted member of the ETS family oftranscriptionfactors.23Breakpointsvaryamongthefourintronsof EWSandthesixintronsof FLI1, andasaresult thefusiontranscriptsarealsovariableinsize, whichmust beconsideredwhen interpreting diagnostic RT-PCR.24The most commonfusion is EWS exon 7 to FLI1 exon 6, designated Type 1, followedby fusion of EWS exon 7 to FLI1 exon 5, designated Type 2. Type1 fusions may have a better prognosis than type 2 fusions,although this is still a matter of some debate.25,26Approximately5e10%ofEScarryanalternatetranslocation, that of21;22, inwhichthefusionpartnerforEWSistheERGgeneonchromo-some21. ERGisalsointheETSfamilyoftranscriptionfactorsand is closely relatedto FLI1. Inadditionto EWSeFLI1 andEWSeERG, there are a number of less common EWS trans-locationstoother ETStranscriptionfamilymembersthat havebeendescribed: t(2;22)whichtranslocatesEWStoFEV, t(7;22)which translocates EWS to ETV1, and t(17;22) which translocatesEWS to E1AF. In addition to fusions involving the EWS gene, rarecasesof EShavebeendescribedthat involvetheFUSgeneonchromosome 16 (FUSeERG and FUSeFEV).27,28Finally, it isapparent that rare small cell undifferentiated tumours that do notexpress CD99 may also have gene fusions, which may or may notinvolve the EWS gene. EWS has been described as a translocationpartner with non-ETS genes such as SP3, ZNF278, andPOU5F1.23,24TwocasesofEwings-liketumourshavealsobeendescribed with CICeDUX4 fusions.24All told, variant fusions (i.e.neitherEWSeFLI1norEWSeERG)areestimatedtoaccount for4e9%ofcaseshistologicallydenedasES.24Fromapracticalstandpoint,itmaybeimpractibletotestforall possible fusion combinations by RT-PCR. If parafnembedded tissue is tested, this becomes evenmore problematic,asdegradationfromprocessingprecludesamplicationoflargeRNAsegments. FISHusingbreak-apartprobes for EWScanpartially circumvent this, as described previously inthis review.AnyfusioninvolvingEWSshouldbepickedupbyFISH, andsimilarbreak-apartprobesforFUScanbeappliedtocasesthatlacktheEWSfusion.28Positivebreak-apartFISHforEWSmustbe interpreted cautiously and in the context of other clinical andpathologicdata, assplittingcanalsobeseeninothertumours,such as desmoplastic small round cell tumour, clear cellsarcoma, extraskeletal myxoid chondrosarcoma, polyphenotypicroundcell sarcomaof bone29, myxoidliposarcoma, andangio-matoidbroushistiocytoma.DesmoplasticsmallroundcelltumourDesmoplastic small round cell tumour (DSRCT) is a highlyaggressiveand lethal tumourwhichmainly afictsyoungadultsand adolescents, and has a strong male predilection. Firstdescribed20yearsago,30thistumour typicallypresentsasanintrabdominal masswithperitoneal seeding, andinitsclassichistologicformiscomposedof sheetsof undifferentiatedsmallbluecellsinterruptedbybroadswathsof paucicellularbrosis(Figure5a). It displays acharacteristic polyphenotypic immu-nohistochemical prole, co-expressing neural, muscular andepidermal markers, including desmin, cytokeratins, epithelialmembraneantigen,vimentin,andneuronspecicenolase.4Theoverall prognosisisdismal, withmost patientsdyingwithin2years despite extensive multimodalitytherapy.31Inits classicpresentation, this tumour usuallydoes not present diagnosticdifculties. However, cases occasionally arise outside of theabdomen and at a younger age; the brous stroma may be poorlyrepresented in small biopsies, and sometimes immunohisto-chemistrymaybeinconclusive.32Adeningmolecular featureof DSRCTis the reciprocal t(11;22)(p13;q12) translocation, which rearranges the EWS genetothe WT-1gene onchromosome 11, producing achimerictranscript. In most cases, the rst seven exons of EWS are fusedtothe last three exons of WT-1. As this is the C-terminal regionof WT-1, immunohistochemistryfor the carboxyterminus ofWT-1isusuallypositive(nuclear)inDSRCT, whereasimmu-nohistochemistry for the N-terminus is usually negative.33VariantshavebeenreportedwithalternativeEWSbreakpointscontaining additional EWS exons and conservation of the WT-1portion, as well as rare variants withnon-typical transcriptsfromboth EWS and WT-1.34These rare transcripts may beassociatedwithaberrant(negative)stainingfortheC-terminusendofWT-1. Giventhesmall numberofcases, itisunclearifthesemoreuncommontranslocationvariantscarryanyprog-nosticimplications. Interestingly, thesevariant fusionsappearmore commoninDSRCTs thatarisinginunusualsites.34Also,the EWSeWT-1 fusion is not entirely specic, as there isarecent report of twocasesof EWSeWT-1positivepaediatrictumours withspindlecell morphology, expressionof smoothmuscleantigensandafavourableclinical course, quiteunlikeDSRCT.35InfantilebrosarcomaInfantile brosarcoma is a tumour of very young childhood, andisthemost commonsoft tissuesarcomaseenduringtherstyear of life, withhalf of the cases presenting as congenitaltumours. Althoughcharacterizedbyrapidgrowth, it seldommetastasizes. Itmostcommonlyoccursintheextremities, butmay also be seeninaxial locations. Histologically, infantilebrosarcoma is similar to adult brosarcoma, composed offasciclesof variablymitoticallyactivespindledcells, ofteninaherringbone-typepattern(Figure5b). However, unlikeadultbrosarcoma, infantile brosarcoma is often inltrated byinammatorycells, andtends tohave less pleomorphism. Aprominent hemangiopericytoma-like vascular pattern isa common feature. Immunohistochemistry tends to be non-specic, withnearuniversal reactivityforvimentin, andvari-able positivity for neuron specic enolase, smooth muscle actinandmusclespecicactin.4Mostinfantilebrosarcomascarryat(12;15)(p13;q26)trans-locationwhichfusesthegenesETV6(TEL) andNTRK3.36Thistranslocation is identical to that seen in cellular mesoblasticnephroma, a rare kidney tumour of infants with a similarhistology. Importantly, this fusion has not been described in adultbrosarcomas, which have complex karyotypes and carry a graverprognosis. The fusion may be difcult to recognize with standardcytogeneticbandingtechniques,andeitherFISHorRT-PCRper-formed on frozen or parafn embedded tissue are recommendedtechniques to evaluate for it.31The fusion protein leads to activa-tionoftheP13-Aktpathwaywhichmediatescellsurvival,mostlikely through the intermediate activation of c-Src.37Trisomies forREVIEWDIAGNOSTICHISTOPATHOLOGY17:1 29 2010ElsevierLtd.Allrightsreserved.chromosomes 11, 20, 17 and 8 are also typical for infantile bro-sarcoma, and have been described in over 90% of cases.38ClearcellsarcomaofsofttissueThis tumour is another example of a translocationassociatedsarcoma involving the EWS gene. Previously referred to asmalignant melanoma of soft parts, it has morphologic andimmunophenotypicsimilaritytomalignantmelanoma.However,clearcell sarcomaofsoft tissue (CCS) is adistinctive clinicalandbiologic entity, as melanoma does not carry the translocation, andCCSalmost alwaysarisesindeepsoft tissues, closelyrelatedtoaponeuroses andtendons. CCSalsolacks melanoma-associatedBRAFmutations.39CCSisprimarilyatumourofadolescentsandyoung adults, and is unusual in children younger than 10. It is themostcommonmalignancyof theankleandfoot inadolescents,withupto75%arisinginthelowerextremity. It isaggressive,with5- yearsurvival ratesrangingfrom47to63%.40e43Earlydiagnosiswithcompletewideexcisionoffersthebest chanceofcure.Histologically, thetumour typicallydisplaysanestedarchi-tecture, with groups of tumour cells subdivided by brous septaeofvariablethickness(Figure5c).Tumourcellsrangefromovaltospindled, witheither clear or palecytoplasm, impartedbyahighglycogencontent. Nuclei arevesicular, withprominentnucleoli, andmultinucleatedgiant cells maybe seen. Mitoticactivityis usuallylowandpleomorphismcanbeminimal; inthese cases distinction from benign tumours such as cellular bluenevi canbechallenging. Immunohistochemically, CCSwill bepositive at least focally for melanoma related antigens such as S-100,Melan-AandHMB-45inalmostallcases.4Typically, CCSis associatedwithat(12;22)(q13;q12) trans-location, inwhichEWS is fusedwiththe ATF1 gene onchromosome12.44Using FISH, rearrangements of the EWSR1 locus canbedemonstratedingreater than 90%of CCS, and it has beensuggestedthat the cases that lacksucha EWSR1 rearrangement mayhave beentruemelanomas.45Todate, four variants of theEWSR1eATF1chimeric transcript have been documented, with the most commonbeing fusions of the EWSR1 exon 8 with ATF1 exon4 (type 1 fusion)and EWSR1exon 7 and ATF1 exon5 (type 2fusion). There isnoapparent prognosticsignicanceimpartedbythefusiontype.46Other EWSR1eATF1fusiontypeshavebeendescribed, but arequite rare.45Inadditiontothe EWSR1eATF1fusion, Antonescuet aldescribedavariant fusionEWSeCREB1inthreecasesofCCS.47Thesecaseswereunusualinthattheyhadalackofmelanocyticdifferentiation,andallwerelocatedwithintheabdomen.Subse-quently, three additional cases of CCS with the EWSeCREB1 weredescribed, all of which were soft tissue based.42,45Molecular diagnosis of CCS can be accomplished by break-apartFISHforEWSR1rearrangements,aswellasRT-PCRassayusingaDesmoplasticsmallroundcelltumour.bInfantilebrosarcoma.cClearcellsarcoma.dLow-gradebromyxoidsarcoma,withlargebrousrosette(allhaematoxylin&eosin).Figure5REVIEWDIAGNOSTICHISTOPATHOLOGY17:1 30 2010ElsevierLtd.Allrightsreserved.both CREB1 and ATF1 primers. Both methods have been shown tobe highly sensitive in parafn embedded tissue, with a recent studyshowing detection of the EWS rearrangement by FISHin all 15 casestested, and detection of a CCS related fusiontranscript by RT-PCRin13/13 cases in which sufcient RNA was available.45Curiously, the two fusions associated with CCS are notspecicforthat entity, asbothhavealsobeenassociatedwithanother soft tissue tumour, angiomatoid brous histiocytoma(AFH), whichhasadistinct morphology, clinical presentation,andmost importantlyamuchmorefavourableprognosisthanCCS.48AFHisararetumourofsofttissuesthatpredominantlyaffects children and young adults. Previously referred to asmalignant angiomatoidbroushistiocytomabasedonmalig-nant behaviour in some of the earliest described cases, the wordmalignant was subsequentlydropped, as larger series haveshown an overall excellent prognosis, with less than 1%metastasizing.49In addition to the EWSReCREB1 andEWSR1eATF1fusions, casesof AFHhavealsobeenshowntoharbour a third fusion; FUSeATF1. As it is only of low malignantpotential,AFHwillnotbefurtherdiscussedhere.Low-gradebromyxoidsarcomaLow-grade bromyxoid sarcoma (LGFMS) is a neoplasmthatdisplays a deceptively bland microscopic appearance, and yetcarries a signicant risk of metastases, with a propensity for a longlatentperiod before metastases develop. First described by Evansin 1987,50it hasmultiplehistologicappearances,andcytogeneticand molecular data have played a key role inreconciling itsdiverse histologies into a single diagnostic entity. Although a veryhighrateofmetastaseswasdocumentedininitialreports(upto68%), subsequent studies showed lower rates (4e27%).51,52Thismay reect the adoption of more aggressive surgery as the biologicbehaviour of this tumour became better understood. Howevergiventhelonglatencyseeninsomemetastaticcases, thetrueincidence of metastatic disease has yet to be determined. It occursprimarily in young adults, but 19% of cases are seen in children.51Histologically, the tumour typically shows a mixture of myxoidzonesandhypocellularbrousareas, oftenwith anabrupttran-sition between zones. Branching, curvilinear small blood vesselsare characteristic. Cytology is generally bland, and mitotic activityis low. Inupto40%of tumours, strikingly large brous rosettes arepresent (Figure 5d). Originally designated as hyalinizing spindlecell tumour with giant collagen rosettes tumours with thishistology are nowsubsumed into the category of LGFMS.53Immunohistochemically, these tumours have a broblasticimmunophenotype and may express smooth muscle actin inaddition to vimentin; other antigens being only rarely expressed.LGFMSis associatedwithat(7;16)(q34;p11) translocation,which is seen in examples of the tumour with and without giantcollagen rosettes.54This translocation fuses the FUS gene toCREB3L2in95%of cases, withasmall subset displayingat(11;16)(p11;p11) fusionof FUStoCREB3L1.55,56Thesefusionshave not been described in any other neoplasms, with theexceptionof asmall number of sclerosingepithelioidbrosar-comas whichwere showntoharbour FUSeCREB3L2 fusions,raising the possibility that at least some of those tumours may beclosely related to LGFMS.52The fusions can be demonstrated byFISHandRT-PCRinparafnembeddedtissues.57,58SynovialsarcomaDespite its name, synovial sarcoma (SS) is not a tumour ofsynovial origin, andindeedfewexamplesoccur withinjoints.The most common locations are in the soft tissues of theextremities, especiallyaroundtheknee, but synovial sarcomashave been reported in a wide variety of anatomic sites, includingvisceral organs andthe headandneck. Inthe paediatric agegroup, the tumour most commonlyaffects adolescents. Initsclassic histologic form, synovial sarcoma displays a biphasicpattern of spindled cells and epithelial gland components; whichis very useful diagnostically (Figure 6). However, the morecommonforminchildrenismonophasic,withoutglandforma-tion.Documentationofbiphenotypicexpressionofbothmesen-chymal and epithelial markers by immunohistochemistry isuseful inthesecases.59ThemajorityofSSexpresscytokeratinsandepithelialmembraneantigen,aswellasvimentin.Approxi-matelyone-thirdexpressS-100,andmorethanhalfwillexpressSynovialsarcoma.aBiphasicpatternwithglandsandspindledcells(haematoxylin&eosin).bImmunohistochemistryforINI1insynovialsarcoma,withendothelialcellsservingaspositiveinternalcontrols.TumourcellsdemonstrateweaktoabsentnuclearexpressionofINI1.Figure6REVIEWDIAGNOSTICHISTOPATHOLOGY17:1 31 2010ElsevierLtd.Allrightsreserved.CD99. Less differentiated forms of SS may assume a small roundcell appearance or a predominantly epithelioid appearance,creating diagnostic challenges, particularly with malignantperipheral nerve sheathtumours andEwingsarcomas, whichmay be difcult to resolve by immunohistochemistry, butamenable to clarication by cytogenetic and moleculartechniques.Inat least 95%of SS, acharacteristic t(X;18)(p11.2;q11.2)translocationis demonstrable, inwhichtheSS18gene(previ-ously known as SYT) on chromosome 18 is juxtaposed with oneoftheSSXgenesonchromosomeX.OfthenineSSXgenetran-scripts, the majority of fusions involve SSX1 and SSX2, with rareexamples of fusions involving SSX4.60This translocation appearsspecic, and has not been associated with other tumourtypes.59,61Demonstration of the fusion by either cytogeneticanalysis, FISH or RT-PCR is considered to be the diagnostic goldstandardfor SS. The latter two modalities canbe appliedtoformalinxedparafnembeddedtissuesuccessfully, withonestudy showing sensitivities of 96% for quantitative RT-PCR, 92%for conventional RT-PCR, and86%for FISH.62The resultingfusionproteinisbelievedtodysregulatetranscriptional activityandthroughepigenetic mechanisms alters tumour suppressorgene expression. One of the pathways implicatedinsynovialsarcoma development is the Wnt/b-catenin pathway, andacomponentofthatpathway,TLE(transducer-likeenhancerofsplit 1), hasrecentlybecomeafocus of interest asapossibleadditional immunohistochemical marker. TLE1nuclear expres-sionhasbeenfoundtobeasensitivemarkerforSS,presentinover 90e100% of cases.63e66The specicity of TLE1 for synovialsarcomaisstillamatterofdebate.InthestudyofKosemehme-togluet al, 37%of non-synovial sarcoma cases stainedwithTLE1,including30%oftheirmalignantperipheralnervesheathtumours. However, Jagdis et al found that TLE1 was veryspecic, with rare to absent staining in 73 various non-SStumours tested, and Knosel et al also felt that TLE1 was relativelyspecicintheproperclinicalcontext,with83%oftheirnon-SScontrols displayingnostainingat all, withtheremainder dis-playingonlyweak(1)staining.RecentlyithasbeenshownthatSStumoursdisplayabnormalexpressionof SMARCB1/INI1, a ubiquitous proteininvolvedinpost-transcriptional chromatin remodelling. Hoot et al rst demonstratedvariable INI1staining inthree cases of synovial sarcoma, andsubsequentstudieshavedemonstratedpartialtocompletelossofINI1, without evidenceof reducedINI1RNA.67e69ThesestudiessuggestthatthereducedINI1expressionisanepigeneticndinginvolving interaction of the fusion protein and INI1.MalignantrhabdoidtumourMalignant rhabdoid tumour(MRT)is a rare neoplasmoriginallydescribedinthekidneysandcentralnervoussystembutsubse-quentlyidentiedinotherlocations,includingsofttissues.70e72Mosttumoursoccurininfantsandyoungchildren,andbehaveaggressively, demonstratingapoor responsetotreatment andrapidfataloutcome.73Histologically, these tumours tend to grow in sheets entirely orpartially composed of rhabdoid cells. These are large cells witheccentricallyplacednuclei, vesicular chromatinandprominenteosinophilic nucleoli. They characteristically have abundanteosinophilic cytoplasm andglobularpinkcytoplasmicinclusions,whichcorrespondtowhorledintermediatelamentsultrastruc-turally(Figure7).74Theyhaveapolyphenotypicimmunohisto-chemical prole,withalmost allbeingpositivefor vimentin,andmost expressing EMA, CD99, and cytokeratin, with manyexpressing neural markers as well. Of interest, desminandCD34 arenot expressed.75Throughmolecular genetic investigations, a characteristic loss ormutationof the INI1(SNF5, SMARCB1, BAF47) gene was foundto becharacteristic of MRT, detectable by FISH and sequence analysis inat least 95% of tumours. This gene is located in chromosome band22q11.2.76,77, andispart of theSWI/SNFchromatinremodellingcomplex. SWI/SNFacts asaclassictumour suppressor geneinpatients withMRT, as germlinemutations or deletions of INI1predispose patients to the development of rhabdoid tumours, withinactivationof bothalleles leading to malignancy.77Deletionand/ormutation of both copies of the INI1 gene results in loss of expressionof the INI1 protein, which can be evaluated by immunohistochem-istry.INI1immunohistochemistryissensitiveandspecicfortheMalignant rhabdoid tumour. a Typical rhabdoid cells with eccentriceosinophilicglobularcytoplasmicinclusions,vesicularnucleiandprominentnucleoli(haematoxylin&eosin).bImmunohistochem-istry for INI1, showing absence of nuclear staining in tumour cells.Figure7REVIEWDIAGNOSTICHISTOPATHOLOGY17:1 32 2010ElsevierLtd.Allrightsreserved.diagnosis of malignant rhabdoidtumour, andINI1proteinlossappears to be quite rare in other tumours.69,78,79Inaddition to MRT,tumours that display complete or partial loss of INI1 proteinexpression include the very rare renal medullary carcinoma,epithelioid sarcomas, synovial sarcomas, and a subset of paediatricundifferentiated sarcomas. To date, there is no convincing evidencethat INI1genemutationsor deletionsunderliethelossof INI1proteinexpressioninnon-rhabdoidtumours, andthepossibilityexiststhatsomeoftheundifferentiatedsarcomasandepithelioidsarcomas with INI1 loss may actually be examples of MRT that lackclassic morphologic and/or immunohistochemical features.EpithelioidsarcomaEpithelioid sarcoma is a rare soft tissue tumour that primarily affectsyoung adults and adolescents. It most commonly occurs asa supercial lesioninthe distal extremities, particularlyinthe hands,and although typically indolent in behaviour, carries a high risk ofrecurrence anda highriskof late metastases. Prognosis of epithelioidsarcoma is poor; in a recent study of 106 patients, 54% developedmetastases and 31%succumbedto the disease.80The classic formofthetumour is characterizedhistologicallybyanodular growthpattern, and commonly displays granuloma like features and centralnecrosis. The tumour cells are epithelioid and spindled and tend togrow in an inltrative pattern along fascial planes. Akin to synovialsarcoma, this is another example of a tumour with a biphenotypicimmunohistochemical prole, typically expressing epithelialmarkers suchas EMAandcytokeratins inadditiontovimentin. Morerecently, Guillou et al described a deep-seated proximal form ofepithelioidsarcoma, whichwas axiallylocated, witha propensityforoccurrenceintheperineum, pelvisandgenitourinarytract. Thisproximal form of epithelioid sarcoma shares some histological andimmunohistochemicalfeatureswithMRT,andismoreaggressivethan the classic form of epithelioid sarcoma.81Cytogenetically, epithelioid sarcomas have shown non-specic chromosomal gains and deletions. In 2009, three studiesindependentlydescribedlackofINI1proteinexpressioninboththe majority (85e93%) of both proximal and classic forms of thetumour.80,82,83Although still a matter of some debate, it appearsthat lossof INI1 expression inepithelioidsarcomas (particularlytheclassicforms) maybeanacquiredchangeassociatedwithcomplexkaryotypicabnormalities, anddistinct fromtheinacti-vationsecondarytoINI1deletionor mutation. Several recentstudies have shown that INI1 alterations at the DNA level are rarein epithelioid sarcoma, and more likely to be found in proximal-type tumorus.82,84These ndings raise the question as towhether proximal-type epithelioid sarcomas bear a closer kinshiptoMRTthantoclassicepithelioidsarcoma. AREFERENCES1 GulleyML,Kaiser-RogersKA.Arationalapproachtogenetictestingforsarcoma.DiagnMolPathol2009;18:1e10.2 LadanyiM,BridgeJA.Contributionofmoleculargeneticdatatotheclassicationofsarcomas.HumPathol2000;31:532e8.3 Mertens F, Antonescu CR,Hohenberger P,et al. 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Am JSurg Pathol 1997; 21: 130e46.82 KohashiK,IzumiT,OdaY,etal.InfrequentSMARCB1/INI1genealterationinepithelioidsarcoma:ausefultoolindistinguishingepithelioidsarcomafrommalignantrhabdoidtumor.HumPathol2009;40:349e55.83 HornickJL,DalCinP,FletcherCD.LossofINI1expressionischar-acteristicofbothconventionalandproximal-typeepithelioidsarcoma.AmJSurgPathol2009;33:542e50.84 FluckeU,SlootwegPJ,MentzelT,PauwelsP,HulsebosTJ.Re:infre-quent SMARCB1/INI1 gene alteration in epithelioid sarcoma: a usefultoolindistinguishingepithelioidsarcomafrommalignantrhabdoidtumor: direct evidence of mutational inactivation of SMARCB1/INI1 inepithelioidsarcoma.HumPathol2009;40:1361e2.authorreply1362e1364.PracticepointsCAlthough it is optimal practice to allocate fresh or frozen tissueformolecular/cytogeneticstudies,somemolecularstudies,mostnotablyFISH,canbesuccessfullyappliedtoformalinxedparafnembeddedtissue.CTranslocation associated sarcomas tend to have simplekaryotypesandoftenarelativelyuniformhistologicappear-ance; whereas those without translocations tend to have morecomplexkaryotypesandmorepleomorphism.CBreak-apart FISHand RT-PCR are complimentary; althoughbreak-apartFISHcandemonstrateaninterruptionofagenelocus,RT-PCRwithspecicprimerscanidentifythespecictranslocationpartner.CImmunohistochemistry for myogenin and PAX-5 can be helpfulin distinguishing alveolar rhabdomyosarcoma from embryonalrhabdomyosarcoma.CInadditiontoits lackof expressioninmalignant rhabdoidtumour,INI1expression isalsoaltered inepithelioidsarcomaandsynovialsarcoma.REVIEWDIAGNOSTICHISTOPATHOLOGY17:1 35 2010ElsevierLtd.Allrightsreserved.