Universite Paris Xi Faculte De Medecine Paris-sud These

Background:!!Children!!with!ependymoma!may!experience!a!relapse!!in!up!to!50%!of!cases!depending!!on!the!extent!!of!resection.!Key!biological!events!associated!with!recurrence!are!unknown.!Methodology/Principal!!Findings:!!To!!discover!!the!!biology!!behind!!the!!recurrence!!of!!ependymomas,!!we!!performed!CGHarray!!and!!a!!dual-color!!gene!!expression!!microarray!!analysis!!of!!17!!tumors!!at!!diagnosis!!co-hybridized!!with!!the!corresponding!27!first!or!subsequent!relapses!from!the!same!patient.!As!treatment!and!location!had!only!limited!influence!on!specific!gene!expression!changes!at!relapse,!we!established!a!common!signature!for!relapse.!Eighty-seven!genes!showed!an!absolute!fold!change!$2!in!at!least!50%!of!relapses!and!were!defined!as!the!gene!expression!signature!of!ependymoma!recurrence.!The!most!frequently!upregulated!genes!are!involved!in!the!kinetochore!(ASPM,!KIF11)!or!in!neural!development!(CD133,!!Wnt!!and!!Notch!!pathways).!!Metallothionein!!(MT)!!genes!!were!!downregulated!!in!!up!!to!!80%!!of!!the!!recurrences.!Quantitative!PCR!for!ASPM,!KIF11!and!MT3!plus!immunohistochemistry!for!ASPM!and!MT3!confirmed!the!microarray!results.!Immunohistochemistry!on!an!independent!series!of!24!tumor!pairs!at!diagnosis!and!at!relapse!confirmed!the!decrease!of!MT3!expression!at!recurrence!in!17/24!tumor!pairs!(p!=!0.002).!Conversely,!ASPM!expression!was!more!frequently!positive!at!relapse!!(87.5%!!vs!!37.5%,!!p!=!0.03).!!Loss!!or!!deletion!!of!!the!!MT!!genes!!cluster!!was!!never!!observed!!at!!relapse.!!Promoter!sequencing!!after!!bisulfite!!treatment!!of!!DNA!!from!!primary!!tumors!!and!!recurrences!!as!!well!!as!!treatment!!of!!short-term!ependymoma!cells!cultures!with!a!demethylating!agent!showed!that!methylation!was!not!involved!in!MT3!downregulation.!However,!in!vitro!treatment!with!a!histone!deacetylase!inhibitor!or!zinc!restored!MT3!expression.!Conclusions/Significance:!!The!!most!!frequent!!molecular!!events!!associated!!with!!ependymoma!!recurrence!!were!!over-!expression!of!kinetochore!proteins!and!down-regulation!of!metallothioneins.!Metallothionein-3!expression!is!epigenetically!controlled!and!can!be!restored!in!vitro!by!histone!deacetylase!inhibitors.!!Citation:!!!Peyre!!!M,!!!Commo!!!F,!!!Dantas-Barbosa!!!C,!!!Andreiuolo!!!F,!!!Puget!!!S,!!!et!!!al.!!!(2010)!!!Portrait!!!of!!!Ependymoma!!!Recurrence!!!in!!!Children:!!!Biomarkers!!!of!!!Tumor!Progression!!Identified!!by!!Dual-Color!!Microarray-Based!!Gene!!Expression!!Analysis.!!PLoS!!ONE!!5(9):!!e12932.!!doi:10.1371/journal.pone.0012932!Editor:!!Chris!!Jones,!!Institute!!of!!Cancer!!Research,!!United!!Kingdom!Received!!January!!14,!!2010;!!Accepted!!July!!14,!!2010;!!Published!!September!!24,!!2010!Copyright:!!!ß!!!2010!!!Peyre!!!et!!!al.!!!This!!!is!!!an!!!open-access!!!article!!!distributed!!!under!!!the!!!terms!!!of!!!the!!!Creative!!!Commons!!!Attribution!!!License,!!!which!!!permits!unrestricted!!use,!!distribution,!!and!!reproduction!!in!!any!!medium,!!provided!!the!!original!!author!!and!!source!!are!!credited.!Funding:!!The!!authors!!thank!!the!!Socie ́te ́!!de!!Neurochirurgie!!de!!Langue!!Franc ̧aise!!for!!the!!fellowship!!of!!MP!!and!!the!!Institut!!National!!du!!Cancer!!and!!the!!Region!!Ile!de!!France!!for!!the!!Fellowship!!of!!CDB!!and!!FA,!!as!!well!!as!!the!!grant!!support!!from!!the!!Socie ́te ́!!Franc ̧aise!!des!!Cancers!!de!!l’Enfant/Enfants!!et!!Sante ́!!2006!!(http://www.!enfants-sante.asso.fr/)!!and!!from!!the!!charity!!‘‘l’Etoile!!de!!Martin’’!!(http://www.letoiledemartin.org/)!!for!!FA!!and!!CDB.!!The!!funders!!had!!no!!role!!in!!study!!design,!!data!collection!!and!!analysis,!!decision!!to!!publish,!!or!!preparation!!of!!the!!manuscript.!Competing!!Interests:!!The!!authors!!have!!declared!!that!!no!!competing!!interests!!exist.!*!!E-mail:[email protected]!.!!These!!authors!!contributed!!equally!!to!!this!!work.!Introduction! Ependymomas!are!tumours!thought!to!derive!from!radial!glial!cells!!![1]!!!and!!!display!!!morphological!!!characteristics!!!of!!!normal!ependyma![2].!They!represent!the!third!most!common!intracra-!nial!tumour!in!children!and!intracranial!location!account!for!more!than!90%!of!cases![3].!The!incidence!is!higher!in!young!children!as!!more!!than!!fifty!!percent!!occur!!before!!the!!age!!of!!5!![4].!!The!!overall!prognosis!of!this!tumour!remains!poor,!especially!in!young!children!![5]!!with!a!!10-year!!survival!!between!!30!!and!!70%!![6,7].!Extent!!of!!initial!!surgery!!remains!!the!!only!!consensual!!prognostic!factor!!across!!studies!![6,8,9].!!Recurrences!!are!!most!!of!!the!!time!local,!!at!!least!!at!!the!!beginning!!of!!the!!natural!!history;!!distant!metastases!!!become!!!more!!!frequent!!!with!!!more!!!effective!!!local!treatment![7].!Treatment!strategy!is!actually!based!on!surgery!at!diagnosis!and!!at!each!relapse!completed!!with!local!!radiotherapy!!PLoS!ONE!!|!!www.plosone.org! September!2010!!|!!Volume!!5!!|!!Issue!!9!!|!!e12932!tel-00913042,version1-3Dec2013