Isabelle ! Aerts

Xavier ! Sastreggarau

François ! Doz

Nathalie ! ! Cassoux

! Je

Claude

Angel ! Montero

! Retinostop

Pierre ! Bey

Keywords: Caractérisation moléculaire et clinique complète du rétinoblastome Rétinoblastome, génomique, analyse en composantes indépendantes, méthylome, exome-seq, analyse de données Comprehensive molecular and clinical characterization of retinoblastoma Retinoblastoma, genomics, independent component analysis, methylome, exome-seq, data analysis

Le rétinoblastome est un cancer pédiatrique rare de la rétine en cours de développement. Si dans les pays développés, le taux de survie avoisine 100 %, une énucléation de l'oeil atteint est cependant nécessaire dans plus de 70 % des cas.

En 1971, Knudson émit l'hypothèse des deux "hits", qui permit de comprendre que le rétinoblastome s'initie généralement après une perte bi-allélique du gène RB1. Cependant, les autres mécanismes moléculaires qui régissent ce cancer restent depuis peu connus. Par exemple, peu d'études génomiques ont été conduites. Ainsi, la nature de la cellule d'origine, ainsi que la présence ou non d'une hétérogénéité intertumorale, font encore débat. Dans cette étude, nous avons dressé un portrait génomique et clinique complet du rétinoblastome; plusieurs observations ont montré qu'il s'agit bien d'une maladie hétérogène, avec deux sous-types distincts.

Nous avons d'abord identifié les deux sous-types grâce à une approche couplant une analyse en composantes indépendantes (ACI) de transcriptomes tumoraux avec des marquages immunohistochimiques. Les rétinoblastomes du premier sous-type, dits "cone-like" expriment uniformément des marqueurs de cônes, tandis que ceux du second sous-type, dits "bivalent-type", ont une forte hétérogénéité intratumorale, avec un enchevêtrement de zones de différenciation ganglionnaire ou cône. Grâce à une étude plus approfondie des transcriptomes et de données d'altérations génomiques, nous avons ensuite montré que les sous-types dépendent de voies de signalisation et d'oncogènes différents. Les bivalent-type ont notamment une présence quasisystématique de gains de MDM4 ou d'amplifications de MYCN. Nous nous sommes ensuite tournés vers les méthylomes des rétinoblastomes, et constaté une forte hétérogénéité entre les sous-types. Nous avons décomposé cette hétérogénéité grâce à une ACI, et constaté qu'elle n'était pas liée uniquement à la différenciation cône ou ganglion. Nous avons ensuite étudié les données cliniques de la cohorte, et constaté que les sous-types avaient des âges au diagnostic et des formes de croissance différents, les tumeurs cone-like se developpant généralement chez des patients jeunes avec des tumeurs exophytiques, et les bivalent-type chez des patients plus âgés avec des tumeurs endophytiques. De plus, les patients avec des inactivations constitutionnelles du gène RB1développent majoritairement des tumeurs cone-like; les cone-like s'initieraient donc plus tôt durant le développement de la rétine.

Nous avons finalement séquencé les exomes de 74 paires tumeur-normal. Les rétinoblastomes avaient un taux de mutations extrêmement faible (0.1 mutations par mégabase), comme beaucoup de cancers pédiatriques. Nous avons identifié des mutations somatiques récurrentes dans RB1, BCOR et ARID1A. Ces gènes se trouvaient de plus dans des régions minimales de pertes chromosomiques. Surtout, les inactivations de BCOR et ARID1A avaient souvent de fortes fréquences alléliques. Ceci indique que ces inactivations ont lieu précocément dans la tumorigénèse.

En conclusion, notre étude a permis de dresser un premier portrait génomique complet du rétinoblastome. Elle a révélé l'existence de deux sous-types distincts, ainsi que fourni des indices quant à leurs cellules d'origines et leurs mécanismes moléculaires.

Figure%I.3%Examples%of%epigenetic%marks% DNA-methylation-is-what-occurs-when-methyl-groups-can-tag-DNA.-This-canin-turn-activate-or-repress-genes.-Histones-are-proteins-around-which-DNAcan-wind-for-compaction.-Histone-modification-occurs-when-the-binding-ofepigenetic-factors-to-histone-"tails"-alters-the-extent-to-which-DNA-iswrapped-around-histones,-and-thus-changes-the-availability-of-genes-in-the-DNA-to-be-transcribed.-Figure-adapted-from-commonfund.nih.gov.-

I.1.2.3 Genetic* events* causing* widespread* epigenomic* alterations*

While! both! local! and! large5scale! epigenetic! alterations! have! been! described! in! cancer,!it!is!still!unclear!how!all!of!these!epigenetic!alterations!come!to!be.!Recent! studies!have!provided!some!clues!by!revealing!that!several!epigenetic!modifiers!are! mutated! in! human! cancers.! Some! examples! of! genetic! mutations! of! epigenetic! modifiers!are!shown!in!Figure!I.4.!! !

Figure%I.7%Intertumoral%and%intratumoral%heterogeneity%

Figure-adapted-from- [START_REF] Burrell | !The!causes!and! consequences!of!genetic!heterogeneity!in!cancer!evolution[END_REF].-! 16!

I.2.1.2.1 Cancer%stem%cells%

The! presence! of! cancer! stem! cell! is! one! potential! cause! for! intratumoral! heterogeneity.!The!cancer!stem5cell!model!suggests!that!some!cancers!are!organized! into!a!hierarchy!of!subpopulations!of!tumorigenic!cancer!stem!cells!and!their!non5 tumorigenic!progeny.!Furthermore,!it!is!also!generally!considered!that!cancer!stem! cells! represent! only! a! minority! of! all! tumor! cells,! while! the! majority! of! other! cells! have!little!or!no!to!capacity!to!contribute!to!tumor!growth.!In!vivo!experiments!have! demonstrated! the! existence! of! phenotypically! distinct! subpopulations! of! rare! tumorigenic! and! more! abundant! non5tumorigenic! cells! in! a! number! of! human! cancers,! including! acute! myeloid! leukaemia! (AML),! chronic! myeloid! leukaemia! (CML),! breast! cancer,! glioblastoma,! medulloblastoma,! colorectal! cancer,! pancreatic! cancer!and!ovarian!cancer.!Importantly,!these!cancer!stem!cells!are!thought!to!drive! tumor! growth! and! disease! progression.! It! is! therefore! hypothesized! that! cancer! stem! cells! need! to! be! first! and! foremost! targeted! by! anti5tumoral! treatments! (Meacham!and!Morrison,!2013)!(Figure!I.8).! ! The!initial!origins!of!cancer!stem!cells!can!be!varied.!Although!healthy!tissue!stem! cells! are! often! the! target! of! genetic! events! that! are! necessary! or! sufficient! for! malignant! transformation,! in! other! cases! restricted! progenitors! or! even! differentiated! cells! may! become! transformed.! These! transformed! progenitors! or! differentiated! cells! may! then! appropriate! the! machinery! for! self5renewing! cell! divisions!that!is!normally!expressed!in!stem!cells! [START_REF] Reya | !Stem!cells,!cancer,! and!cancer!stem!cells[END_REF].! ! Finally,! despite! the! evidence! that! cancer! stem! cells! exist! in! a! variety! of! cancers,! a! strong! inter5cancer! and! inter5patient! variability! has! been! observed! in! the! markers! expressed! by! cancer! stem! cells.! Several! elements! suggest! that! the! distinct! developmental!origins!of!tumors,!both!with!respect!to!regional!identity!and!position! in! the! normal! tissue! hierarchy,! contribute! to! differences! among! patients! in! tumor! stem!cell!cell!properties! [START_REF] Gibson | !Subtypes!of!medulloblastoma! have!distinct!developmental!origins[END_REF][START_REF] Krivtsov | !Cell!of!origin!determines! clinically!relevant!subtypes!of!MLL5rearranged!AML[END_REF].!

I.2.1.2.2 Genetic%intratumoral%heterogeneity%

Intratumoral! heterogeneity! can! also! be! attributed! to! genetically! distinct! subclonal! populations! of! cells! that! arise! through! intercellular! genetic! variation,! followed! by! selective! outgrowth! of! clones! that! have! a! phenotypic! advantage! within! a! given! tumor! micro5environmental! context! [START_REF] Cairns | !Mutation!selection!and!the!natural!history!of!cancer[END_REF][START_REF] Nowell | !The!clonal!evolution!of!tumor!cell!populations[END_REF].! Fluctuations! in! subclonal!architecture!can!occur,!for!example,!in!the!context!of!disease!progression! or! drug! treatment! [START_REF] Gerlinger | !Intratumor! Heterogeneity!and!Branched!Evolution!Revealed!by!Multiregion!Sequencing[END_REF][START_REF] Klein | !Selection!and!adaptation!during!metastatic!cancer!progression[END_REF].! A! tumor's! subclonal! architecture! can! be! reconstructed! from! sequencing! approaches ! or! multiplex! fluorescence!insitu!hybridization! (FISH),! providing! insight! into! cancer! evolution.! ! 17! Branched! intratumoral! evolution! has! now! been! documented! in! a! range! of! tumor! types,! including! adenoma5to5carcinoma! transition! of! the! colon,! childhood! acute! lymphoblastic!leukaemia!(ALL),!chronic!lymphoblastic!leukaemia!(CLL),!pancreatic! cancer,! renal! cancer! (Figure I.9),!and! breast! cancer! [START_REF] Burrell | !The!causes!and! consequences!of!genetic!heterogeneity!in!cancer!evolution[END_REF].! In! cases! of! ALL! that! exhibit! branched! evolutionary! trajectories,! heterogeneity! has! been! observed!between!leukaemia!stem!cells.!This!has!important!implications!because!it! suggests!that!each!subclonal!branch!was!probably!sustained!by!genetically!distinct! stem! cells.! It! is! conceivable! that! genetically! heterogeneous! subclonal! lineages! in! solid!tumors!could!also!be!maintained!by!distinct!stem!cells! [START_REF] Anderson | !Genetic!variegation!of!clonal! architecture!and!propagating!cells!in!leukaemia[END_REF][START_REF] Meacham | !Tumour!heterogeneity!and!cancer!cell! plasticity[END_REF].!

Figure%I.8%Cancer%stem%cells%

Figure-from- [START_REF] Succony | !Airway!stem!cells!and!lung!cancer[END_REF],--! 18! % Figure%I.9%Genetic%intratumor%heterogeneity%and%phylogeny% For-one-renal-carcinoma-patient.-(A)- Sites-of-core-biopsies-and-regionsharvested-from-nephrectomy-and-metastasectomy-specimens.-(B)-Phylogenetic-relationships-of-the-tumor-region.-Branch-lengths-areproportional-to-the-number-of-nonsynonymous-mutations-separatingthe-branching-points.--Figure-adapted-from-(Gerlinger-et-al.,-2012)

I.2.2 The%cancer%genomics%strategy%

The! main! goals! in! cancer! medicine! center! on! prevention,! detection! and! treatment.! The!extensive!intertumoral!and!intratumoral!heterogeneities!which!exist!in!cancer! represent! a! major! obstacle! to! achieving! these! goals.! It! is! widely! appreciated! that! cancer!genomics,!ie!the!study!of!comprehensive!catalogs!of!the!somatic!alterations! in!cancer!genomes!coupled!with!detailed!knowledge!and!modeling!of!the!epigenetic! and! transcriptional! states! of! these! genomes! could! inform! strategies! for! effective! prevention!and!early!detection,!as!well!as!guiding!the!development!of!therapeutics! in! the! appropriate! patient! subpopulation.! It! is! believed! that! cancer! care! will! thus! ultimately! become! increasingly! customized! to! the! unique! molecular! features! of! a! patient's!tumor.!

I.2.2.1 Exploring+ molecular+ features:+ from+ genotype+ to+ phenotype+

The! phenotype! of! a! eukaryotic! cell! results! from! the! expression! of! its! genotype! through! a! network! of! multiple! steps! (Figure! I.10).! The! process! is! first! initiated! through! the! DNA! sequence,! which! encodes! both! protein! coding! genes! and! non5 coding! RNAs.! Whether! these! RNAs! are! transcribed! in! a! given! cell! is! largely! dependent! upon! the! epigenetic! portrait! of! the! DNA! in! that! cell.! This! portrait! is! mainly!a!consequence!of!DNA!methylation!marks!and!histone!modifications.!! ! For! protein5coding! genes,! DNA! transcription! starts! with! the! synthesis! of! a! pre5 messenger!RNA,!which!is!spliced!to!form!a!messenger!RNA.!This!mRNA!is!decoded! by! the! translation! machinery! to! produce! a! protein.! Non5coding! RNAs,! such! as! microRNAs,! and! long! non! coding! RNAs,! are! transcribed! but! not! translated! into! proteins.! These! RNAs! act! as! gene! regulators;! they! can! for! instance! induce! mRNA! degradation! (in! the! case! of! microRNAs)! or! directly! regulate! gene! transcription! (in! the!case!of!long!non!coding!RNAs).!! ! Proteins! have! several! specific! functions! in! the! cell! life! cycle.! Furthermore,! their! functions! can! be! modified! through! post5translational! modifications! such! as! phosphorylation!of!a!particular!amino!acid!by!another!protein.!Finally,!endogenous! and!exogenous!sources!also!lead!to!the!presence!of!a!plethora!of!cellular!metabolites! whose!highly!dynamic!quantities!also!strongly!regulated!the!cellular!life!cycle.!! ! In! cancer,! almost! every! step! of! this! process! can! be! altered.! To! garner! full! tumoral! molecular! portraits,! genomic! studies! have! therefore! sought! to! comprehensively! assay!all!the!cellular!processes!that!lead!from!genotype!to!phenotype.! ! 20!

I.2.2.2 Steps+involved+in+cancer+genomics:+from+bedside+to+ bench+

• Gathering!the!tumoral!tissue! In! order! to! draw! a! genomic! portrait! of! a! patient's! tumor,! one! must! first! obtain! at! least!one!sample!of!said!tumor.!Tumor!biopsies!render!the!largest!amount!of!cells,! but! tumor! cells! can! also! be! extracted! from! blood! (circulating! tumor! cells)! or! body! fluids!(lymph,!ascites);!circulating!tumor!DNA!can!also!be!obtained!from!blood.!To!

Figure%I.10%From%genotype%to%phenotype%

Arrows-indicate-interactions-that-have-already-beenobserved.-! 21! fully!investigate!intratumoral!heterogeneity,!several!samples!of!the!same!tumor!are! required,!either!from!the!same!tumor!site!or!from!differing!sites.! !! Once!a!tumor!specimen!is!obtained,!it!can!be!preserved!in!a!number!of!ways;!snap! freezing!is!considered!to!be!the!most!efficient!method!for!maintaining!the!integrity! of!nucleic!acids!and!proteins.!Several!retrospective!studies!have!also!been!based!on! formalin5fixed! and! paraffin5embedded! (FFPE)! tissues.! This! is! indeed! the! preservation! method! that! has! been! the! most! routinely! used! by! pathologists,! as! it! optimizes! the! resolution! of! microscopic! histology.! While! nucleic! acids! from! FFPE! specimens!are!likely!to!have!undergone!crosslinking!and!may!also!be!degraded,!they! can!nevertheless!render!biologically!relevant!information.!!

• Gathering!the!healthy!tissue!! Cancer! genomics! frequently! involves! comparing! the! tumor's! molecular! profile! to! that!of!a!healthy!tissue!of!reference.!This!is!rather!simple!for!genetic!comparisons,!as! the!cellular!DNA!is!identical!throughout!the!body.!Any!healthy!tissue!from!the!same! patient! can! thus! be! used,! and! blood! DNA! is! generally! chosen! owing! to! its! ease! of! access.! Comparisons! are! however! not! as! simple! for! epigenetic,! transcriptomic,! proteomic,!or!metabolomic!features,!as!these!change!between!tissues,!and!between! cell! types! of! the! same! tissue.! For! these! features,! the! appropriate! healthy! tissue! to! compare!a!tumor!to!is!therefore!one!that!closely!matches!the!tumor's!cell!of!origin.! This! can! constitute! a! significant! challenge! in! itself! for! several! reasons.! First,! the! precise! cell5type! of! origin! is! rarely! decisively! known;! it! is! furthermore! generally! considered! to! vary! within! a! particular! cancer! (as! is! for! example! thought! to! be! the! case!for!basal!and!luminal!bladder!cancers).!Second,!even!if!the!cell5type!or!origin!is! precisely!known,!obtaining!sufficient!amounts!of!healthy!cells!of!this!cell5type!raises! several!challenges.!This!is!for!instance!the!case!for!pediatric!cancers!stemming!from! developing! cells! that! no! longer! exist! in! adult! tissues.! This! is! also! the! case! for! epithelial!cancers!stemming!from!a!very!thin!layer!of!a!given!organ's!epithelium.!!

• Data%analysis%and%tumor%classification% %

Once! the! molecular! portraits! of! a! cohort! of! tumors! have! been! obtained,! computational!methods!must!be!employed!to!extract!as!much!biologically!relevant! information!as!possible,!such!as!the!presence!of!any!tumor!subgroups,!characterized! by! specifically! altered! genes! and! pathways.! Several! time5consuming! steps! are! involved!in!this!analytical!process.!! !

Figure%I.11%TCGA%breast%cancer%classification%

To-reveal-higherZorder-structure-in-breast-tumours-based-on-multiple-data-types,significant-clusters/subtypes-from-each-of-five-platforms-(miRNAs,-DNA-methylation,copy-number-(CN),-PAM50-mRNA-expression,-and-RPPA-expression)-were-analysedusing-a-multiplatform-data-matrix-subjected-to-unsupervised-consensus-clustering:---a,-Consensus-clustering-of-the-subtypes-identifies-four-major-groups.-b,-HeatZmap-displayof-the-subtypes-defined-independently-by-each-platform.--This-'cluster-of-clusters'-approach-illustrated-that-basalZlike-cancers-had-the-mostdistinct-multiplatform-signature-as-all-the-different-platforms-for-the-basalZlike-groupsclustered-together.-The-three-other-subgroups-did-not-cluster-as-clearly-together.- Figure-from-(Cancer-Genome-Atlas-Research-Network,-2012).--

! 23!
Next,! a! large! variety! of! statistical,! informatic! and! mathematical! methods! are! employed! to! study! processed! genomic! data.! This! step! may! be! the! most! time5 consuming,! as! it! is! fueled! by! open5ended! biological! questions.! For! instance,! while! tumor! classification! seems! relatively! straightforward,! it! hides! a! wide! variety! of! obstacles! stemming! from! biological! unknowns.! For! example,! while! Luminal! A,! Luminal!B,!and!Luminal!C!breast!cancers!are!often!considered!to!represent!separate! disease!subgroups,!some!studies!have!suggested!that!they!may!actually!represent!a! continuum! in! luminal! tumors.! Moreover,! classifications! performed! using! different! molecular!profiles!(epigenomic,!transcriptomic,!genomic)!generally!lead!to!different! subgroups,! thus! revealing! all! the! layers! of! phenotypic! heterogeneity! between! tumors,!and!adding!complexity!to!the!classification!issue! (Figure!I.11).!! ! Finally,!all!computational!investigations!require!a!perpetual!back!and!forth!feedback! between!analysis!outputs,!and!biological!interpretation.!Taken!together,!these!steps! render! the! analysis! of! cancer! genomic! data! a! major! bottleneck! of! the! research! process.!

• Validation!

In! addition! to! computational! analysis,! identifying! pertinent! cancer! genes! and! pathways!requires!functional!validation,!which!in!turn!will!provide!'ground5truth'!to! refine! the! analytical! approaches.! A! wide! variety! of! biological! hypotheses! can! be! functionally!validated,!such!as!for!example!showing!experimentally!that!a!candidate! gene! mutation! or! altered! pathway! makes! a! substantial! contribution! to! driving! cancer! predisposition,! initiation,! maintenance! or! progression.! Every! functional! assay!(in-vitro!and!in-vivo-alike)!is!limited!in!its!ability!to!report!on!any!given!cancer5 relevant!activity,!so!it!is!necessary!to!reinforce!data!from!multiple!assay!systems!to! establish!the!biological!weight!of!evidence!for!a!particular!hypothesis.!!

I.2.2.3 Cancer*genomics*centers*consortiums*

Despite! the! falling! costs! of! sequencing! and! array5based! technologies,! cancer! genomics! remains! a! relatively! costly! endeavour! for! one! single! laboratory! to! carry.! Beyond! costs,! the! acquisition! of! large! and! representative! cohorts! of! tumor! and! healthy! tissue! samples,! especially! in! the! case! of! rare! cancers,! can! constitute! an! obstacle!in!itself.!Finally,!the!diversity!of!genomic!data!available!today!has!lead!to!a! diversity!of!analysis!methods,!and!developing!expertise!in!all!of!these!methods!can! also! prove! challenging! for! one! single! laboratory.! For! these! resasons,! the! field! of! cancer!genomics!research!is!becoming!more!and!more!centralized,!concentrating!in! each! country! in! large! cancer! or! bioinformatics! institutes.! Furthermore,! two! large! ! 24! international! consortia,! the! Tumor! Cancer! Genome! Atlas! (TCGA)! as! well! as! the! International!Cancer!Genome!Consortium!(ICGC)!have!also!been!organized!to!launch! and!coordinate!a!large!number!of!cancer!genomics!research!projects!(Figure!I.12).! These!consortia!facilitate!communication!among!the!members!and!provide!a!forum! for! coordination! with! the! objective! of! maximizing! efficiency! among! the! scientists! working!to!understand,!treat,!and!prevent!cancer.! !

Figure%I.12%ICGC%cancer%genome%project%map%

April-2015-! 25!

I.3 Methodologies%for%genomics% I.3.1 Technology%overview%

Genomics! for! the! study! of! cancer! was! made! possible! by! the! development! of! high5 throughput! genomic! technologies,! coupled! to! a! precipitous! fall! in! the! cost! of! these! technologies.!Two!main!biotechnologies!have!shaped!genomic!reseach!in!the!past!20! years.!The!first!was!microarrays,!which!appeared!in!1995! [START_REF] Schena | !Quantitative!Monitoring! of!Gene!Expression!Patterns!with!a!Complementary!DNA!Microarray[END_REF].!The! second! was! whole! genome! sequencing.! The! first! human! genome! was! fully! sequenced! in! 2003.! Sequencing! technologies! then! rapidly! evolved,! and! next! generation!sequencing!started!to!materialize!in!2005.!These!changes!increased!the! technical! and! economic! feasibility! of! complete! molecular! profiling! of! a! human! genome,!and!ultimately!of!a!cancer!genome.!! ! High5throughput!genomic!technologies!all!share!the!common!endpoint!of!studying! entire! tumor! genomes! through! the! simultaneous! dosage! of! millions! of! DNA! sequences.!The!data!thus!obtained!leads!to!non!hypothesis5driven!investigations,!as! there!is!no!a!priori!selection!of!a!particular!set!of!genes.!Genomic!technologies!have! thus! set! in! motion! a! transition! from! a! morphology5based! to! a! genetics5based! taxonomy!of!cancer.! !

I.3.1.1 DNA+microarray+technology+

Microarrays! first! appeared! in! 1995! in! the! field! of! transcriptomics,! but! have! since! been! widely! transposed! for! every! kind! of! genome5wide! approach! (genomics,! proteomics,!epigenomics,!etc).!While!a!large!variety!of!microarray!techniques!have! been! developed! [START_REF] Hoheisel | !Microarray!technology:!beyond!transcript!profiling!and! genotype!analysis[END_REF],! those! that! study! RNA! or! DNA! all! share! one! core! concept.! They! all! rely! on! the! ability! of! two! complementary! DNA! sequences! to! hybridize,! forming! a! two5stranded! DNA! molecule.! This! principle,! described! by! E.! Southern! in! 1975! [START_REF] Southern | !Detection!of!specific!sequences!among!DNA!fragments! separated!by!gel!electrophoresis[END_REF],! gave! rise! to! the! techniques! of! Southern! and! Northern! blot,! from! which! the! first! DNA! microarrays! were! derived! [START_REF] Lander | !Array!of!hope[END_REF][START_REF] Schena | !Quantitative!Monitoring! of!Gene!Expression!Patterns!with!a!Complementary!DNA!Microarray[END_REF]

Figure%I.13%Hybridization%with%gene%elements%on%a%microarray%

Central-to-microarray-analysis-is-the-hybridization-of-fluorescently-labeled-RNA-or- DNA-with-ashort-strand-of-complementary-DNA-that-is-tethered-to-a-solid-surface,-such-as-glass.-The-naturalaffinity-of-two-complementary-strands-of-nucleic-acid-for-each-other-drives-the-reaction.-Becausethe-position-and-sequence-of-the-stationary-probe-are-known,-hybridization-between-probe-andlabeled-target-provides-an-assay-of-the-level-of-target-in-solution.--Figure-from-(Quackenbush,-2006) [START_REF] Kuhn | !A!novel,!high5performance!random!array! platform!for!quantitative!gene!expression!profiling[END_REF].! This! technology! is! based! on! 35micron! silica! beads! that! self! assemble! in! microwells! on! either! of! two! substrates:! fiber! optic! bundles! or! planar!

- ! 27! result! in! false! differences! due! to! array5to5array! variation.! However,! a! two5color! approach!in!which!the!ratio!of!signals!on!the!same!array!is!measured!is!much!more! reproducible.! • InZsitu-

Figure%I.14%Masking%systems%for%selectively%unprotecting%the%desired%spots%

In-the-Affymetrix-manufacturing,-a-different-photo-mask-is-applied-at-each-step-ofsynthesis-to-unmask-the-selected-feature.-Figure-from-Affymetrix-website.-! 28! silica!slides.!Each!bead!is!covered!with!hundreds!of!thousands!of!copies!of!a!specific! oligonucleotide.! There! are! 30! beads! with! the! same! oligonucleotide! on! average.! Interestingly,! the! beads! are! randomly! assembled! on! the! substrate,! so! that! the! location! of! each! bead! is! specific! to! each! array.! Each! bead! contains! an! etched! bar! code! to! enable! its! localization! by! the! scanner.! The! fact! that! each! probe! is! represented! by! 30! beads! randomly! localized! on! the! array! has! the! advantage! of! considerably!reducing!the!impact!of!spatial!biases.!

I.3.1.2 Next+generation+DNA+sequencing+

• History!

In!1977!Fred!Sanger!and!Alan!R.!Coulson!published!two!methodological!papers!on! the! rapid! determination! of! DNA! sequences,! that! is! to! say! the! precise! order! of! nucleotides! within! a! DNA! molecule! (Sanger! et! al.,! 1977a(Sanger! et! al.,! ,! 1977b)) -image-of-the-base-extension-is-captured.-This-cycle-and-isrepeated-a-100-times-from-1-each-of-the-molecule.-Figure-from-(Johnsen-et-al.,-2013)-! 31! repeated.! Typically,! 100! bp! sequence! reads! are! obtained! from! each! end! of! the! cluster,! although! read! lengths! from! 50! to! 200! bp! are! possible.! An! entire! run! from! multiple!channels!can!generate!600!gigabases!(Gb)!of!sequence!in!an!115day!period! [START_REF] Johnsen | !Massively!parallel! sequencing:!The!new!frontier!of!hematologic!genomics[END_REF].!

I.3.1.3 Genomic+endpoints+investigated+

The!aforementioned!technological!leaps!have!allowed!several!genomic!endpoints!to! be! investigated! in! a! high5throughput! manner,! whether! through! array5based! methods! or! sequencing5based! methods.! Here! we! present! some! of! the! genomic! endpoints! that! are! commonly! studied! in! cancer,! and! the! methods! used! to! assay! them.! -is-decreased-for-losses,-and-increased-for-gains.-Red:-gain,-green:-loss,-purple:-amplicon,-blue:-homozygous-deletion,-yellow:-normal.-(B)+Copy-number-profile-of-a-bladder-tumor-obtained-with-a-SNP-array.-Log-R-ratioinformation-is-available-as-for-BACZCGH-arrays.-However,-SNP-arrays-also-inform-

I.3.1.3.1 Somatic%DNA%alterations% • Array5based!methods! While!array5based!methods!are!not!able!to!detect!

onthe-B-allele-frequency-(BAF),-which-in-normal-regions-can-take-one-of-three-values:-0-(AA),-0.5-(AB)-or-1-(BB).-The-BAF-signal-is-affected-by-chromosome-aberrations,-butonly-for-heterozygous-SNPs.-For-example,-a-homozygous-SNP-of-genotype-AA-(BAF=0)-will-not-see-its-

Figure%I.19%Illumina%Infinium%methylation%BeadChip%assay%

The-Infinium-Methylation- Assay-uses-two-different-bead-types-to-detect-CpGmethylation.-The-U-bead-type-matches-the-unmethylated-CpG-site;-the-M-beadtype-matches-the-methylated-site.-In-the-top-figure,-the-unmethylated-CpG-targetsite-matches-with-the-U-probe,-enabling-singleZbase-extension-and-detection.-Ithas-a-singleZbase-mismatch-to-the-M-probe,-which-inhibits-extension.-If-the-CpGlocus-of-interest-is-methylated-(bottom-figure),-the-reverse-occurs.-fromwww.illumina.com--

! 35! • Sequencing5based!methods!
Bisulfite! conversion! of! genomic! DNA! combined! with! whole! genome! sequencing! is! also!a!method!that!is!starting!to!be!widely!used!to!measure!the!methylation!state!of! a! whole! genome.! Whole! genome! bisulfite! sequencing! (WGBS)! has! the! strong! advantage! of! providing! single5nucleotide! resolution! and! whole5genome! coverage.! However,! several! obstacles! make! WGBS! less! accessible! today.! First.! it! is! still! quite! costly,! and! typically! requires! relatively! large! quantities! of! DNA.! Second,! standard! whole! genome! bisulfite! sequencing! protocols! or! library! preparation! methods! are! just! beginning! to! emerge.! Finally,! the! bioinformatics! requirements! for! data! interpretation!present!additional!challenges! [START_REF] Krueger | !DNA!methylome!analysis! using!short!bisulfite!sequencing!data[END_REF].!However,!public! tools!for!the!analysis!of!WGBS!data!are!being!developed!as!the!technique!becomes! more!accessible.!!

I.3.1.3.3 Transcriptome%

The!transcriptome!is!the!complete!set!of!transcripts!in!a!cell,!and!their!quantity,!for! a! specific! developmental! stage! or! physiological! condition.! Understanding! the! transcriptome! is! essential! for! interpreting! the! functional! elements! of! the! genome! and! revealing! the! molecular! constituents! of! cells! and! tissues,! and! as! such! for! understanding!tumoral!tissues.!The!key!aims!of!transcriptomics!are:!to!catalogue!all! species! of! transcripts,! including! mRNAs,! non5coding! RNAs! and! small! RNAs,! to! determine!the!transcriptional!structure!of!genes,!in!terms!of!their!start!sites,!5'!and! 3'!ends,!splicing!patterns!and!other!post5transcriptional!modifications,!and!finally!to! detect!and!characterize!any!aberrant!gene!fusions.!Various!technologies!have!been! developed!to!deduce!and!quantify!the!transcriptome,!including!array5!or!sequence5 based!approaches.!

• Array5based!methods! The! general! principle! of! array5based! expression! analyses! is! to! extract! the! RNA! molecules! of! interest! from! a! sample,! produce! the! matching! dyed! cDNA! by! reverse! transcription!and!hybridize!the!cDNA!to!the!array.!In!the!case!of!Affymetrix!arrays,! the! cDNA! is! retranscribed! into! RNA! before! hybridization.! By! complementarity,! if! a! piece!of!cDNA!(or!retranscribed!RNA)!is!derived!from!a!
gene,!it!will!be!hybridized!at! the!location!of!the!corresponding!probe.!Intensity!of!the!hybridization!at!each!probe! can! then! be! optically! measured! and! ultimately! renders! an! estimate! of! the! relative! quantity! of! the! mRNA! of! interest! in! the! sample.! In! an! error5free! scenario,! this! intensity! is! proportional! to! the! true! number! of! transcripts! present! in! the! sample.! The! most! commonly! studied! RNAs! are! the! messenger! RNAs.! The! standard! type! of! array5based! method! for! analyzing! these! mRNAs! are! the! so! called! 3'! microarrays.! ! 36! These! microarrays! are! 3'5end! biased! because! they! rely! on! primers! targeting! the! poly5A! tail! of! mRNAs! (at! the! 3'! end! of! the! transcript),! and! contain! probes! that! are! clustered! for! detection! of! sequences! at! the! 3'5end! of! the! target.! A! commonly! used! version! of! these! arrays! is! the! Affymetrix! GeneChip! Human! Genome! U133! Plus! 2.0! Array.!This!array!enables!the!quantification!of!about!47,000!transcripts.!Despite!the! technology! behind! this! array! being! almost! 20! years! old! [START_REF] Lockhart | !Expression!monitoring!by! hybridization!to!high5density!oligonucleotide!arrays[END_REF]

,! it! remains! one! of! the! most! commonly! used! techniques! to! investigate! human! transcriptomes.!! • Sequencing5based!methods!
In!contrast!to!microarray!methods,!sequence5based!approaches!directly!determine! the!cDNA!sequence.!Initially,!Sanger!sequencing!of!cDNA!or!EST!libraries!was!used! [START_REF] Gerhard | !The!status,!quality,!and! expansion!of!the!NIH!full5length!cDNA!project:!The!Mammalian!Gene!Collection! (MGC)[END_REF],! but! this! approach! was! relatively! low! throughput,! expensive! and! generally! not! quantitative.! Tag5based! methods! were! developed! to! overcome! these! limitations,! including!serial! analysis! of! gene! expression!(SAGE)! [START_REF] Harbers | !Tag5based!approaches!for!transcriptome! research!and!genome!annotation[END_REF].!These!tag5based!sequencing!approaches!were!high!throughput!and! could!provide!precise,!'digital'!gene!expression!levels.!However,!a!significant!portion! of! the! short! tags! used! in! these! methods! could! not! be! uniquely! mapped! to! the! reference! genome.! Moreover,! only! a! portion! of! the! transcript! were! analyzed! and! isoforms!were!generally!indistinguishable!from!each!other.!The!recent!development! of! novel! NGS! DNA! sequencing! methods! has! provided! a! new! method! for! both! mapping! and! quantifying! transcriptomes.! This! method,! termed! RNA5Seq! (RNA! sequencing),!has!clear!advantages!over!existing!approaches.!In!general,!a!population! of! RNA! (total! or! fractionated,! such! as! poly(A)+,! or! ribosomal! RNA! depleted)! is! converted! to! a! library! of! cDNA! fragments! with! adaptors! attached! to! one! or! both! ends.! Each! molecule,! with! or! without! amplification,! is! then! sequenced! in! a! high5 throughput! manner! to! obtain! short! sequences! from! one! end! (single5end! sequencing)!or!both!ends!(pair5end!sequencing).!The!reads!are!typically!30-400!bp,! depending! on! the! DNA5sequencing! technology! used.! Following! sequencing,! the! resulting!reads!are!either!aligned!to!a!reference!genome!or!reference!transcripts,!or! assembled!de-novo!without! the! genomic! sequence! to! produce! a! genome5scale! transcription!map!that!consists!of!both!the!transcriptional!structure!and/or!level!of! expression!for!each!gene! (Wang!et!al.,!2009).!

I.3.1.3.4 Other%«%omics%»%

The! aforementionned! genomic! endpoints! and! technologies! constitute! a! non! comprehensive! list.! Other! commonly! studied! endpoints! are! proteomics,! often! through! reverse! phase! protein! arrays! or! mass! spectrometry,! as! well! as! ! 37! metabolomics,! studied! through! a! combination! of! chromatography! methods! and! mass!spectrometry.!!

I.3.2 Analysis% methods% for% tumor% subgroup% identification%and%characterization%

Capturing!and!understanding!the!diversity!of!cancers!from!omics!data!can!be!done! from!a!variety!of!viewpoints,!and!with!different!computational!tools.!In!this!section,! we!will!give!a!brief!overview!of!these!tools,!with!a!particular!focus!on!unsupervised! methods.! Unsupervised! methods! are! analysis! approaches! that! use! no! a! priori! knowledge!on!the!data!structure.!These!are!often!used!for!identifying!new!disease! subtypes!based!on!similarity!of!RNA!expression!profiles,!or!less!often!epigenomic!or! genomic!profiles.!!

I.3.2.1 Clustering+

Genomic clustering! algorithm! (linkage! function).! Given! the! choice! of! these! parameters,! the! resulting!clusters!will!often!be!different.!Given!this!subjectivity,!how!can!one!be!sure! that! a! cluster! identified! through! hierarchical! clustering! actually! reflects! a! biologically!meaningful!group?!! ! The! concept! of! consensus! clustering! was! developped! as! an! answer! to! the! stability! issue!of!clusters! [START_REF] Monti | !Consensus!clustering:!A! resampling5based!method!for!class!discovery!and!visualization!of!gene!expression! microarray!data[END_REF].!The!basic!goal!of!consensus!clustering!is,!for!a! given! dataset,! to! reconcile! the! different! clustering! outputs! coming! from! runs! of! different!algorithms.!! ! There! is! no! single! method! to! perform! a! consensus! clustering! but! the! approach! is! basically!divided!into!the!following!steps:!! ! 1.!Select!a!subset!of!the!p!features!that!will!be!used!to!classify!the!n!samples,!choose! a!distance!between!samples,!choose!a!linkage!function!to!agglomerate!the!clusters,! and!perform!a!hierarchical!clustering!using!these!defined!parameters.!! ! 2.!Repeat!the!first!step!by!changing!at!least!one!of!the!parameters.!! ! 3.!Agglomerate!the!results!from!the!different!hierarchical!clusters!obtained.!Samples! that! remain! co5clustered! regardless! of! the! chosen! parameters! will! define! stable! clusters!which!are!likely!to!be!biologically!relevant.!Consensus!clustering!has!been! widely! used! on! microarray! data! [START_REF] Deodhar | !Consensus!Clustering!for!Detection!of! Overlapping!Clusters!in!Microarray!Data[END_REF][START_REF] Filkov | !Integrating!microarray!data!by!consensus! clustering[END_REF].!

I.3.2.2 Reducing+the+complexity+

Analysis

×!p! gene! features)! to! obtain! a! centered! matrix! X! where! each! feature! average! is! 0.! Projection! of! the! data! in! a! lower! dimensional! space! with! K! dimensions! (K! <<! p)! is! achieved!through!factorization!of!the!centered!matrix!X!while!solving!the!following! optimisation!problem!:!! ! Find!CK-and!MK!such!as!:! ! -! ! ! ! != ! min !∈! !×! ,!∈!! !×! ! ! -!" ! ! ! ! ! ! ! This! problem! can! be! easily! solved! with! a! singular! value! decomposition! of! X.! The! optimal!matrix!M!contains!a!component!in!each!row,!which!defines!the!directions!of! projections,! while! the! optimal! C-matrix! contains! the! coordinates! of! the! samples! in! the!subspace!defined!by!the!K!component.!! ! 40!

I.3.2.2.2 Independent%component%analysis%(ICA)%

ICA!was!originally!developed!to!solve!the!so5called!blind!source!separation!problem,! its! classical! illustration! being! the! "cocktail! party! problem"! [START_REF] Cardoso | !High5order!contrasts!for!independent!component!analysis[END_REF][START_REF] Chiappetta | !Blind!source!separation!and! the!analysis!of!microarray!data[END_REF][START_REF] Comon | !Independent!component!analysis,!A!new!concept?![END_REF][START_REF] Hyvarinen | !Survey!on!independent!component!analysis[END_REF][START_REF] Hyvarinen | !Independent!component!analysis:!algorithms!and! applications[END_REF].!In! this! problem,! two! conversations! are! occurring! simultaneously! in! a! room,! and! one! person!is!trying!to!follow!one!of!the!two!conversations.!For!the!human!brain,!it!is!an! easy! task,! but! in! the! signal5processing! domain! it! is! a! very! tricky! problem.! Two! microphones,! held! at! different! positions,! record! the! speeches! and! return! two! recorded! signals!! ! and!! ! !(for! simplicity,! we! will

! omit! time! in! this! model).! The! objective!is!then!to!estimate!the!two!original!speech!signals!! ! and!! ! ,!using!only!the! recorded! signals!! ! and!! ! .! This! is! equivalent! to! estimating! a! linear! combination! such!that:!! ! ! = ! !! ! ! + ! ! !" ! ! ! ! ! = ! !" ! ! + ! ! !! ! ! ! ! Neither!the!! !" nor!the!! ! are!known.!To!solve!this!problem,!it!is!sufficient!to!assume! that!! ! and!! ! are! statistically! independent.! This! is! not! an! unrealistic!
assumption! in! many! cases,! and! it! need! not! to! be! exactly! true! in! practice.! In! the! cocktail! party! problem,!this!independence!assumption!can!be!illustrated!by!the!fact!that!if!all!the! speeches!were!equal!and!simultaneous!(i.e!dependent),!like!in!a!chorus,!it!would!be! impossible! to! distinguish! between! both! conversations.! ICA! has! proved! very! successful! for! the! treatment! of! multi5sensor! signal! in! many! areas,! including! gene! expression!data.! ! More!formally,!ICA!is!generally!defined!as!a!generative!model,!which!describes!how! the! n! observed! variables!

! = !1, … , !" !are! generated! by! a! process! of! mixing! the! components!! = !1, … , !" .!This!is!modeled!as!a!linear!combination:! ! ! ! = ! ! ! ! ! !!! ! !! which!is!equivalent!in!matrix!notation!to!have:! ! ! = !"! !!
The!!!matrix! is! called! the! mixing! matrix! whose! elements!! !"! denote! how! the! i th! individual! is! influenced! by! the! j th ! source.! For! simplicity,! we! consider! here! a! basic! unnoised!model!(no!noise!term),!where!the!number!of!ICs!is!assumed!to!be!equal!to! ! 41! the!dimension!of!the!observations!(the!mixing!matrix!is!square!of!dimension!!×!).! In! addition,! we! assume! that! both! the! independent! components! and! the! mixture! variables! have! zero! means! (the! observed! variables! must! be! centered! during! the! preprocessing!if!it!is!not!the!case).!! ! The!starting!point!for!ICA!is!the!very!simple!assumption!that!the!components!si!are! statistically! independent.! The! objective! is! to! jointly! estimate! the! matrix!!!and!!!by! determining!a!separation!matrix!!,!the!inverse!of!!: ICA! looks! for! components! which! are! not! necessarily! geometrically! orthogonal! but! are! statistically! independent.! This! enables! the! ICs! to! be! closer! to! the! physical/biological!reality.!However,!ICA!does!not!provide!a!unique!decomposition,!! and!there!is!no!"universal"!ICA!algorithm.!Indeed,!as!ICA!is!based!on!independence,! whose! empirical! estimation! is! not! trivial,! many! algorithms! have! been! proposed.! Despite! their! similarities,! each! algorithm! will! lead! to! a! slightly! different! solution! [START_REF] Cardoso | !The!three!easy!routes!to!independent!component!analysis;! contrasts!and!geometry[END_REF].!While!PCA!provides!components!ranked!by!decreasing!energy,!ICA! furnishes!nor!ranking!neither!scaling!of!the!variables!(Figure!I.20).! ! a-group-of-genes-which-are-coexpressed-on-a-subset-of-samples.-For-examplethe-IC1-is-triggered-by-a-group-of-genes-that-are-overZexpressed-in-samples-1and-2-relative-to-sample-3.-From-A.-Biton-thesis-(2011)

!! = ! !! ,!such!that:! ! ! = !"! ! From!

Figure%I.20%Comparison%of%PCA%and%ICA%

At-the-top-of-the-figure,-two-schemes-of-an-expression-data-in-three-dimensions.-Each-red-point-is-a-gene-and-each-axis-denotes-a-sample.-In-the-middle-the-redcircle-denote-the-noise,-i.e.-the-genes-whose-expression-do-not-vary-between-thesamples.-On-the-left,-the-blue-directions-correspond-to-the-two-first-principalcomponents-(PCs)-that-would-be-obtained-on-such-data-configuration.-On-theright,-the-yellow-directions-correspond-to-three-independent-components-(ICs)that-would-be-obtained.-The-representation-of-the-ICs-on-the-right-highlightthat-ICs-can-be-interpreted-as-directions-in-the-gene-space-that-are-triggered-by-

Figure%I.22%Kundson's%two%hits% (A)-These-semilog-plots-of-the-fraction-of-23-bilateral-(heritable)-cases-and-25unilateral-(most-expected-to-be-nonZheritable)-cases-that-were-still-notdiagnosed-at-plotted-ages-(data-were-analysed-retrospectively)-show-that-thebilateral-cases-match-the-expected-shape-of-a-oneZhit-curve,-whereas-theunilateral-cases-match-the-shape-of-a-twoZhit-curve.-As-the-bilateral-cases-inheritone-genetic-hit,-both-heritable-and-spontaneous-retinoblastoma-are-due-to-twohits.-(B)-A-'oneZhit'-clone-is-a-precursor-to-the-tumor-in-nonhereditaryretinoblastomas,-whereas-all-retinoblasts-(indeed,-all-cells)-are-oneZhit-clones-inhereditary

-retinoblastoma.-From-(Knudson,-2001)- ! 45! patients! that! inherit! one! mutation! develop! tumors! earlier! than! do! those! who! develop! the! nonhereditary! form! of! the! disease.! Importantly,! he! hypothesized! that! the! majority! or! patients! who! inherit! a! constitutional! mutation! develop! more! than! one!tumor.!On!the!other!hand,!he!estimated!that!the!probability!that!an!individual! without! a! constitutional! mutation! would! develop! more! than! one! tumor! was! vanishingly! small,! so! that! nonhereditary! cases! were! invariably! unilateral! and! unifocal.! The! discovery! of! the! RB1! gene! at! chromosome! 13q14! in! the! 1980s! (Cavenee!et!al.;!Friend!et!al.,!1986)!confirmed!these!hypotheses.!RB1!was!thus!the! first!tumor5suppressor!gene!ever!identified.!! ! In!both!heritable!and!non5heritable!retinoblastoma,!biallelic!inactivations!of!the!RB1! tumor5suppressor! gene! generally! initiate! tumor! growth.! In! heritable! retinoblastoma,! the! first! RB1! mutation! is! constitutional,! predisposing! the! child! to! retinal!tumors.!Somatic!mutations!in!one!or!more!retinal!cells,!the!second!hits,!then! initiate!the!growth!of!one!or!several!tumors.!Consistent!with!Knudson's!hypotheses,! it!is!now!known!that!the!majority!of!heritable!retinoblastomas!are!bilateral!(~80%).! For! non5heritable! retinoblastoma,! two! somatic! mutations! in! one! retinal! cell! generally!initiate!the!growth!of!a!unifocal!tumor.!Non5heritable!retinoblastomas!are! always!unilateral.!! • Exceptions!to!the!two!hits!rule?! While! the! majority! of! retinoblastoma! patients! have! a! bi5allelic! inactivation! of! RB1,! this! is! not! systematically! the! case.! 10%! of! non5heritable! retinoblastomas! display! only! a! mono5allelic! loss! of! RB1,! with! no! identifiable! "second! hit".! This! raises! questions! regarding! the! phenotypic! impact! of! a! mono5allelic! loss! of! RB1! on! proliferation! regulation.! A! recent! study! also! reported! a! total! absence! of!RB1!mutations!in!1%!of!retinoblastomas.!These!tumors!however!displayed!high5 level!MYCN!oncogene!amplifications!(28-121!copies)!(Rushlow!et!al.,!2013).!

I.4.2 Medical%management%of%retinoblastoma%

The

!most!important!treatment!goal!of!retinoblastoma!is!to!save!the!child's!life.!The! next! step! is! to! save! as! much! of! the! child's! vision! as! possible.! If! detected! early,! the! prognosis! is! usually! very! good.! If! left! untreated,! children! usually! die! within! 254! years! from! the! onset! of! symptoms.! Retinoblastoma! survival! now! nears! 100%! in! developed! countries.! This! is! first! due! to! the! advent! of! retinoblastoma! referral! centers!where!expertise!in!the!treatment!of!this!very!rare!disease!can!be!acquired.! This! is! also! thanks! to! a! a! multidisciplinary! effort! between! primary! eye! care! ! 46!
providers!and!specialists!from!various!fields,!such!as!pediatric!oncology,!pathology,! radiology!and!genetics.!!

I.4.2.1 Diagnosis+

An

I.4.2.2 Treatment+

The! therapeutic! approaches! used! today! for! retinoblastoma! are! eye! enculeation,! or! conservative! treatments! such! as! chemotherapy! and! focal! treatment! methods! (transpupillary!thermotherapy,!photocoagulation!and!cryotherapy).!

• Classification! Classification!of!the!extent!of!cancer!at!presentation!is!fundamental!for!assessment! of! prognosis,! prediction! of! outcomes,! and! initial! treatment.! Two! classifications! are! currently! used! for! grouping! intraocular! retinoblastoma.! The! first! is! the! Reese! Ellsworth!classification,!which!groups!tumors!according!to!the!chance!of!preserving! the! eye! using! external! beam! radiotherapy.! In! the! 1980s,! radiotherapy! was! discovered! to! induce! a! high! risk! of! secondary! malignancy! in! children! with! constitutional! RB1! mutations! (including! all! bilateral! cases)
,! and! its! use! was! progressively!abandonned.!The!new!ABC!classification!groups!tumors!according!to! the!chance!of!preserving!the!eye!using!all!modern!therapeutic!approaches.!

• Enucleation! The!most!frequent!cure!for!intraocular!retinoblastoma!is!achieved!by!enucleation,!ie! removal! of! the! affected! eye.! This! method! is! employed! for! high5risk! eyes! showing! signs!of!potential!tumor!spread!such!as!:!poor!view!of!the!inside!of!the!eye,!bleeding! inside!the!eye,!neovascular!glaucoma,!tumor!anterior!to!the!retina,!suspicious!optic! nerve,!or!suspected!extraocular!disease!on!imaging.!Timely!enucleation!reduces!risk! of! metastatic! spread!

Figure%I.24%Intra\arterial%chemotherapy%for%retinoblastoma%

From- [START_REF] Friedrich | !Retinoblastoma!therapy!delivers!power!of!chemotherapy! with!surgical!precision[END_REF])-! 49! Table% I.1% Estimated% worldwide% incidence,% mortality% and% prevalence% of% metastatic% disease% at% diagnosis% and% families% opting% out% of% treatment% for% retinoblastoma%

I.4.2.3 Retinoblastoma+ prognosis+ in+ lowerKincome+ countries+

Retinoblastoma! has! no! validated! geographic! or! population! hotspots.! The! greatest! disease!burden!is!recorded!in!large!populations!that!have!high!birth!rates,!such!as!in! Asia!and!Africa.!Therefore,!90%!of!children!affected!with!retinoblastoma!do!not!live! in! high5income! countries.! In! low! income! and! middle5income! countries,! retinoblastoma! outcome! is! poorer.! The! percentage! of! patients! presenting! with! metastatic!disease!is!of!35%!in!low5income!countries,!15%!in!lower!middle5income! countries! and! 10! %! in! upper! middle5income! countries.! Estimated! survival! is! estimated! to! be! of! 30%! in! low5income! countries,! 60%! in! lower! middle5income! countries!and!75%!in!upper!middle5income!countries!(Chantada!et!al.,!2011)(Table! I.1).!! ! The! reasons! for! such! differences! in! outcome! are! multiple.! As! reported! for! other! pediatric! malignancies,! socioeconomic! and! health5related! indicators! are! significantly!correlated!with!outcome.!However,!while!most!pediatric!malignancies! require! a! rather! sophisticated! and! expensive! facility! to! be! successfully! treated,! retinoblastoma!can!be!cured!by!enucleation!of!the!affected!eye(s),!a!simple!surgical! procedure! if! the! diagnosis! is! timely! and! the! families! accept! it.! It! is! speculated! that! factors!related!to!maternal!health!and!societal!development!substantially!influence! the!likelihood!of!early!diagnosis!before!the!onset!of!metastatic!disease!and,!hence,! outcome! regardless! of! the! facilities! available.! Treatment! refusal! is! a! common! problem,! occurring! in! up! to! 50%! in! low!
Adapted-from- [START_REF] Chantada | !Strategies!to!manage! retinoblastoma!in!developing!countries[END_REF])-! 50!

I.4.3 Retinoblastoma%morphology%

I.4.3.1 Retinoblastoma+

I.4.4 Genomic%events%in%retinoblastoma%

Beyond! a! bi5allelic! loss! of! RB1,! other! genomic! events! have! been! described! in! retinoblastoma.!!

I.4.4.1 Recurrent+gains+and+losses+

Array! CGH! studies! have! lead! to! the! identification! of! frequent! regions! of! chromosomal!alterations!in!retinoblastomas.!Beyond!losses!at!13q!(driven!by!RB1),!

Figure%I.26%Retinoma%

From-retinagallery.com-! 52! these! alterations! include! gains! at! 1q,! 2p,! 6p,! and! 13q,! and! losses! at! 16q.! New! technologies! such! as! SNP! arrays! have! subsequently! validated! these! initial! discoveries! (Corson!and!Gallie,!2007)

(Table!I.2).!! !!
somal changes in retinoblastoma. To date, five CGH (Mairal et al., 2000;Chen et al., 2001;[START_REF] Herzog | !Marked!differences!in!unilateral!isolated!retinoblastomas!from! young!and!older!children!studied!by!comparative!genomic!hybridization[END_REF]Lillington et al., 2003;van der Wal et al., 2003) and one aCGH [START_REF] Zielinski | !Detection!of!chromosomal!imbalances!in! retinoblastoma!by!matrix5based!comparative!genomic!hybridization[END_REF] series of retinoblastoma have been reported; a recently described second, small (seven-patient) aCGH series has not been presented in full [START_REF] Laurie | !Inactivation!of!the!p53!pathway!in! retinoblastoma[END_REF]. All but two (Mairal et al., 2000;van der Wal et al., 2003) of the six published studies are included in the Progenetix CGH database (Baudis and Cleary, 2001) at www.progenetix.net.

Taken together, these studies confirm and expand the earlier karyotype studies, identifying recurrent gains of 1q, 2p, and 6p, gains and losses at 13q, and losses at 16q, as well as other changes occurring at lower frequencies. However, the limited resolution of CGH and the aCGH methodologies used by [START_REF] Zielinski | !Detection!of!chromosomal!imbalances!in! retinoblastoma!by!matrix5based!comparative!genomic!hybridization[END_REF] and [START_REF] Laurie | !Inactivation!of!the!p53!pathway!in! retinoblastoma[END_REF] (*1 Mbp) is an important caveat for consideration in interpreting these studies, as is the inability of CGH methods to detect balanced translocations.

Recently, the first aCGH study of murine retinal tumors, caused by retinal-specific Rb1 knockout in a Rbl2 À/À [p130 À/À ] background was presented (Macpherson et al., 2007). These results overlap with the human findings: recurrent gains of mouse chromosome 1 (which includes large regions of synteny to human chromosomes 1q, 2q, 6, and 8) and 12 (with synteny to human 2p, 7, and 14q).

Key results of the six human studies are summarized in Table 1. We will discuss each commonly aberrant chromosomal region in numerical order, summarizing the CGH data, and subsequent efforts to identify the candidate genes in these regions, which as target genes of genomic changes, may have wide importance in cancer biology.

RECURRENT GAINS ON 1q:

KIF14, MDM4, AND MORE Gain of chromosome arm 1q is the second most common chromosomal change in retinoblastoma overall, seen in 53% of 179 tumors analyzed by CGH (Table 1). Interestingly, þ1q is a very common genomic change in many cancer types (discussed in Corson et al., 2005), arguing for the presence of oncogene(s) that confer selective advantages to tumor cells with gains. The 1q gains seen by CGH in retinoblastoma are usually low-level, sometimes spanning the whole arm, but more localized or multiple in other cases (Fig. 1A). Amplification has been observed by CGH in rare cases (Fig. 1A), spanning 1q11-q24, 1q (Lillington et al., 2003) and1q21 (Mairal et al., 2000). Unfortunately, the definition of amplification in CGH is somewhat arbitrary, and these putative amplicons have not been confirmed by other techniques. Karyotyping identified portions of this chromosome translocated to numerous locales in the retinoblastoma genome (Squire et al., 1985).

The high frequency of þ1q in retinoblastoma prompted fruitful searches for candidate oncogenes. The minimal common region of gain (MRG), based on comparison of all CGH results, spans 1q31-q32 (seen in 90 of 179 tumors; Fig. 1A). This same MRG was implicated by karyotype Average losses per tumor

1.4 [0-9] 1.5 [0-5] 0.6 [0-5] 1.2 [0-6] 1.9 [0-7] 2.4 [0-9] 1.7 [0-7]
No changes n 25 ( 14) b 1 (4) 9 ( 18) 9 ( 35) 2 ( 4) 2 ( 15) 2 (12) þ1q n 95 ( 53) 12 ( 50) 28 ( 56) 10 ( 38) 28 ( 57) 5 ( 38) 12 (71) þ2p n 60 (34) 9 ( 38) 17 ( 34) 4 ( 15) 21 ( 43) 4 ( 30) 5 (29) þ6p n 97 ( 54) 13 ( 54) 23 ( 46) 11 ( 42) 34 ( 69) 6 ( 46) 10 (59) þ13q n 28 ( 16) 4 ( 18) 6 ( 12) 3 ( 12) 10 ( 20) 3 ( 23) 2 (12) À13q n 22 ( 12) 7 ( 29) 2 ( 4) 2 ( 8) 6 ( 12) 3 ( 23

Table%I.2%Summary%of%recurrent%changes%in%six%CGH%studies%of%retinoblastoma%

From- (Corson-and-Gallie,-2007)-Table% I.3% Candidate% oncogenes% and% tumor% suppressor% genes% in% retinoblastoma%

From- [START_REF] Thériault | !The!genomic! landscape!of!retinoblastoma:!A!review[END_REF]-

! 53!
Minimal! region! analyses! have! identified! candidate! oncogenes! driving! these! alterations.! These! include! MDM4-(also! known! as!MDMX)! and! KIF14for! 1q! gain,! MYCN-for!2p!gain,!DEK!and!E2F3-for!6p!gain,!RBL2,!and!CDH11-for!16q!loss,!as!well! as! the! miR517~92! cluster! for! 13q! gain.! There! is! mounting! evidence! implicating! these!genes!as!drivers!in!retinoblastoma!progression! [START_REF] Thériault | !The!genomic! landscape!of!retinoblastoma:!A!review[END_REF]

!(Table! I.3).! • MDM4!-!mouse!double!minute!4,!human!homologue!(1q32.1)! MDM4-is!a!nuclear!protein!that!binds!through!its!transcriptional!activation!domain! to! inhibit! p53! activity! (Marine! and! Jochemsen,! 2004).! Amplification! and! overexpression! of!MDM4-has! been! observed! in! 65%! of! retinoblastomas! in! comparison! with! foetal! retina!and! could! explain! inactivation! of! the! p53! pathway! without! any! genetic! alteration! of! the! TP53-gene! in! retinoblastoma.! Functional! analyses! of! MDM4! have! shown! that! in! mice! lacking! RB1! and! RBL1- (p107),!MDM4!could! promote! tumorigenesis,! and! that! treatment! of! retinoblastoma! cell! line! xenografts! with! the! small! molecule! nutlin53,! which! targets! the! p535 MDM4!interaction,!can!reduce!tumor!growth!(Laurie!et!al.,!2006).! • MYCN!-! v5Myc! myelocytomatosis! viral5related! oncogene,! neuroblastoma! derived!(2p24.3)!
MYCN-encodes! N5Myc,! a! basic! helix5loop5helix! protein! that! binds! with! other! basic! helix5loop5helix! protein! proteins,! acting! as! a! transcription! factor! to! control! the! expression! of! cell! cycle! genes! that! promote! proliferation! [START_REF] Woo | !Use!of!RNA! interference!to!elucidate!the!effect!of!MYCN!on!cell!cycle!in!neuroblastoma[END_REF].!It! is! frequently!amplified!in!tumors!of!neuroectodermal!origin,!including!neuroblastoma,! retinoblastoma,! glioblastoma,! medulloblastoma,! rhabdomyosarcoma! and! small! cell! lung!carcinoma! [START_REF] Barr | !Genomic!and!clinical!analyses!of!2p24!and!12q135q14! amplification!in!alveolar!rhabdomyosarcoma:!a!report!from!the!Children's!Oncology! Group.!Genes[END_REF][START_REF] Bayani | !Molecular!cytogenetic!analysis!of!medulloblastomas!and! supratentorial!primitive!neuroectodermal!tumors!by!using!conventional!banding,! comparative!genomic!hybridization,!and!spectral!karyotyping[END_REF][START_REF] Dietzsch | ! Characterization!of!homogeneously!staining!regions!in!a!small!cell!lung!cancer!cell! line,!using!in!situ!hybridization!with!an!MYCN!probe.![END_REF][START_REF] Fix | !Characterization!of!amplicons!in!neuroblastoma:! high5resolution!mapping!using!DNA!microarrays,!relationship!with!outcome,!and! identification!of!overexpressed!genes.!Genes[END_REF][START_REF] Hui | !Detection!of!multiple! gene!amplifications!in!glioblastoma!multiforme!using!array5based!comparative! genomic!hybridization[END_REF][START_REF] Nau | !Human!small5cell!lung!cancers!show!amplification!and! expression!of!the!N5myc!gene[END_REF][START_REF] Salido | !Cytogenetic!characterization!of!NCI5H69!and! NCI5H69AR!small!cell!lung!cancer!cell!lines!by!spectral!karyotyping[END_REF],! and! is! associated! with! poor! prognosis! in! neuroblastoma! [START_REF] Moreau | !Does!MYCN!amplification!manifested!as! homogeneously!staining!regions!at!diagnosis!predict!a!worse!outcome!in!children! with!neuroblastoma?!A!Children's!Oncology!Group!study[END_REF].

! MYCN-is! also! amplified! in! the! archetypal! retinoblastoma! cell! line! Y79! (Schwab! et! al.)!and! has! been! reported! amplified!in!approximately!3%!of!primary!retinoblastomas!(Bowles!et!al.,!2007).! • RBL2!-!retinoblastoma!like52!(16q12.2)!
The!RBL2-locus!(encoding!RB!family!member!p130)!lies!at!16q12.2.!Its!expression!is! commonly!lost!in!retinoblastoma! [START_REF] Bellan | !Missing!expression!of!pRb2/p130!in! Bibliography ! 144! human!retinoblastomas!is!associated!with!reduced!apoptosis!and!lesser! differentiation[END_REF][START_REF] Tosi | !Genetic!and!epigenetic! alterations!of!RB2/p130!tumor!suppressor!gene!in!human!sporadic!retinoblastoma:! implications!for!pathogenesis!and!therapeutic!approach[END_REF],!and!it!is!one! of!the!genes!that!can!be!ablated!along!with!RB1!to!cause!retinal!tumor!formation!in! mice! [START_REF] Macpherson | !Cell! type5specific!effects!of!Rb!deletion!in!the!murine!retina[END_REF].!! ! 54!

I.4.4.2 Mutations+

The!first!whole!genome!sequencing!study!of!retinoblastomas!was!recently!pulished! (Zhang! et! al.,! 2012)

I.4.4.3 Methylation+analyses+

The!first!epigenetic!event!to!be!described!in!retinoblastoma!was!methylation!of!the! RB1-promoter!itself.!This!was!first!reported!in!1989!by!Greger!et al,!who!identified! CpG!106,!an!island!overlapping!the!promoter!and!exon!1,!to!be!methylated!in!some! retinoblastomas,!thus! silencing! gene! expression! [START_REF] Greger | ! Epigenetic!changes!may!contribute!to!the!formation!and!spontaneous!regression!of! retinoblastoma[END_REF].! Since! then,!

Table%I.4%Differentially%methylated%genes%in%retinoblastoma%

From- [START_REF] Thériault | !The!genomic! landscape!of!retinoblastoma:!A!review[END_REF]

Table%I.5%Single%nucleotide%polymorphisms%associated%with%retinoblastoma%

Adapted-from- [START_REF] Thériault | !The!genomic! landscape!of!retinoblastoma:!A!review[END_REF])-! 56! overexpression! of! key! inhibitors! of! p53! activity,!MDM2-and!MDM4.!MDM2-was! the! first!modifier!gene!identified!in!retinoblastoma,!when!Castera!et al.!identified!a!T > G! transversion!SNP!at!nucleotide!309!in!the!MDM2-promoter!(rs2279744)!to!be!highly! associated! with! the! incidence! of! bilateral! and! unilateral! retinoblastoma! in!RB1-mutation!carrier!families! [START_REF] Castéra | !MDM2!as!a! modifier!gene!in!retinoblastoma[END_REF] The!cell!type!composition!of!the!retina!varies!not!only!between!its!different!layers,! but!also!between!its!center!and!its!edges.!For!instance,!the!ratio!of!cones!to!rods!is! not! the! same! across! the! retina.! A! slightly! elongated! visual! streak! in! the! retina! contains!higher!densities!of!cones!relative!to!rods.!At!the!"center"!of!this!streak!and! of!the!retina!(the!point!directly!behind!the!lens)!lies!the!fovea,!which!contains!only! cone!cells;!this!region!is!capable!of!producing!the!highest!visual!acuity.!Blue!cones! are!however!absent!from!the!fovea!(Figure!I.30).!!

I.4.5.2.2 Light%processing%

The! only! neurons! that! are! directly! sensitive! to! light! are! the! photoreceptor! cells,! the!rods!and!cones.!Rods!function!mainly!in!dim!light!and!provide!black5and5white! vision,! while! cones! support! daytime! vision! and! the! perception! of! color.! Light! processing! can! be! simplified! into! four! main! stages.! First! photoreception! occurs! in! rod! and! cone! cells.! Second,! nerve! impulses! are! transferred! from! these! photoreceptors! to! bipolar! cells.! Next,! nerve! impulses! reach! the! retinal! ganglion! cells;! these! are! the! final! actors! in! the! light! processing! system.! Ganglion! cells! have! long!axons!that! extend! into! the! brain! and! form! the! optic! nerve,! optic! chiasm,! and! optic!tract.!The!last!stage!of!light!processing!is!therefore!the!synaptic!transmission! of! information! from! ganglion! cells! along! the! optic! nerve.! At! each! synaptic! stage! there! are! also! laterally! connecting!horizontal!and!amacrine! cells.! Amacrine! and! horizontal!cells!have!several!different!functions,!many!of!them!unknown.!One!of!the! leading!theories!on!the!function!of!horizontal!cells!is!that!they!increase!the!contrast! between! the! light! and! dark! regions.! Amacrine! cells! are! thought! to! allow! ganglion! cells! to! fire! in! coordination! with! one! another,! which! increases! the! amount! of! information!that!can!be!transferred!by!the!optic!nerve! [START_REF] Masland | !The!fundamental!plan!of!the!retina[END_REF] individual!stages!of!retinal!development!in!mice!and!rats.!For!example,!at!embryonic! day!(E)!14.5!a!subset!of!retinal!progenitor!cells!expresses!SYNTAXIN,!and!these!cells! are! biased! towards! the! amacrine! cell! fate! [START_REF] Cepko | !Intrinsically!different!retinal!progenitor!cells!produce!specific! types!of!progeny[END_REF][START_REF] Livesey | !Vertebrate!neural!cell5fate!determination:! lessons!from!the!retina[END_REF][START_REF] Mu | !A!gene! network!downstream!of!transcription!factor!Math5!regulates!retinal!progenitor!cell! competence!and!ganglion!cell!fate[END_REF].!! ! As! retinal! progenitor! cells! make! the! transition! through! various! stages! of! competence,!cell5cycle!exit!must!be!carefully!regulated!to!ensure!that!the!final!tissue! is! the! appropriate! size! and! that! the! numerous! cell! types! are! produced! in! the! appropriate! ratios! for! visual! signal! processing.! Early5stage! retinal! progenitor! cells! are! more! likely! to! give! rise! to! two! mitotic! daughter! cells,! whereas! late5stage! progenitor!cells!are!more!likely!to!give!rise!to!two!postmitotic!daughter!cells.! !

.!! ! 60! ! ! Figure%I.30%Photoreceptor%distributions%in%the%human%retina% (

I.4.6 The%search%for%the%retinoblastoma%cell%of%origin%

To!begin!to!understand!the!relative!contribution!of!individual!genetic!and!epigenetic! factors! to! tumorigenesis,! the! cell! or! cells! of! origin! of! a! particular! cancer! must! be! defined.!Unlike!adult!cancers,!which!are!believed!to!arise!from!relatively!stable!cells! of! origin,! childhood! cancers! of! the! nervous! system! such! as! retinoblastoma,! medulloblastoma! and! neuroblastoma! arise! from! developing! neural! progenitors.! This! poses! a! significant! challenge! for! the! study! of! these! cancers,! as! neural! progenitor! cells! and! their! environment! are! constantly! changing! because! of! their! dynamic! developmental! programs.! This! renders! precise! identification! of! a! these! cancers'!cell!or!cells!of!origin!particularly!difficult.!! !

I.4.6.1 The+cone+precursor+cell+of+origin+hypothesis+

Retinal! photoreceptors,! and! cones! in! particular,! have! for! long! been! thought! to! initiate!retinoblastoma.!

• [START_REF] Xu | !Rb!suppresses!human!cone5precursor5derived! retinoblastoma!tumours[END_REF].!This! has! been! hypothesized! to! be! due! to! the! fact! that! cone! precursors! prominently! express! MDM2! and! MYCN,! and! in-vitro! studies! indicated! that! retinoblastoma! cells! require! both! of! these! proteins! for! proliferation! and!survival.!MDM2!has!been!hypothesized!to!be!needed!to!suppress!ARF5induced! apoptosis! in! cultured! retinoblastoma! cells.! These! findings! provide! support! for! a! cone! precursor! origin! of! retinoblastoma! and! suggest! that! human! cone5specific! signaling!circuitry!sensitizes!to!the!oncogenic!effects!of!RB1!mutations.!! !

I.4.6.2 The+retinal+progenitor+cell+of+origin+hypothesis+

Recent! findings! have! however! shed! doubt! on! the! cone! precursor! cell! of! origin! hypothesis,!and!suggested!that!retinoblastomas!may!arise!from!multipotent!retinal! progenitor!cells.!!

II.2 Rationale%

!Knudson's! 1971! two5hit! hypothesis! led! to! the! discovery! that! retinoblastoma! most! often! initiates! after! a! bi5allelic! loss! of! the! RB1tumor! suppressor! gene.! Since,! this! cancer! had! often! been! described! as! a! homogeneous! disease,! despite! the! genomic! portraits! of! retinoblastomas! having! rarely! been! comprehensively! studied.! Few$ articles$ had$ described$ the$ genomic$ portraits$ of$ retinoblastoma$ tumors,$ and$ even$ less$ had$jointly$studied$clinical$data.$For! instance,! while! genomic! events! such! as! 1q! gain,! 16q! loss,! 2p! gain! and! 6p! gain! had! been! observed! in! retinoblastomas,! it! remained! unclear! whether! the! tumors! carrying! these! alterations! had! additional! molecular! specificities.!Furthermore,!while!epigenomic!events!such!as!aberrant!methylation!of! several! individual! genes! had! been! reported,! the! epigenomic! landscape! of! retinoblastoma!as!a!whole!had!never!been!described.!! $ Furthermore,!while!the!retinal!differentiation!marks!expressed!by!retinoblastomas! had!often!been!investigated,!different!approaches!had!led!to!to!seemingly!conflicting! conclusions.! While! several! studies! had! described! cone5like! characteristics! in! retinoblastomas,! transcriptome5based! studies! had! reported! the! co5expression! of! a! seemingly! senseless! mix! of! markers! of! several! retinal! cell! types.! In! 2010,! the! retinoblastoma!PIC!working!group!therefore!decided!to!focus!on!the!joint!genomic! and! clinical! study! of! retinoblastoma.! The! general! goal! of! my! PhD! was! therefore! to! coordinate!and!perform!all!bioinformatic!analyses!relative!to!this!project.! (Xuet-al.,-2009)-! 75! authors!detected!no!tumor!cells!expressing!the!amacrine!and!horizontal!cell5specific! syntaxin,!the!ganglion!cell5specific!Brn53b,!or!the!horizontal,!amacrine,!bipolar,!and! progenitor! cell5specific! Prox1.! They! reported! occasional! clusters! of! cells! that! expressed! the! retinal! progenitor! cell5! and! bipolar! cell5specific! Chx10! or! the! progenitor5specific! Pax6,! as! well! as! one! cluster! in! one! tumor! expressing! the! rod5 Table% III.1% Retinal% cell% type% markers% detected% in% retinoblastoma% tumors% From- [START_REF] Xu | !Retinoblastoma!has! properties!of!a!cone!precursor!tumor!and!depends!upon!cone5specific!MDM2! signaling[END_REF]

II.3 Timeline%

When!I!started!my!doctoral!research!in!2012,!two!retinoblastoma!genomic!datasets! had! already! been! established,! largely! in! part! thanks! to! Dr.! Jérôme! Coututrier,! a! cytogeneticist!at!the!Curie!Institute!Hospital:!! • A!transcriptomic!dataset!derived!from!37!primary!untreated!retinoblastoma! samples! • A!copy!number!alteration!dataset!derived!from!~70!samples!studied!through! BAC!array!CGH.!Some!of!these!samples!had!also!been!studied!through!high5 resolution!Illumina!SNP!370K!arrays.!! • A!matching!clinical!dataset.! ! The!initial!

Figure% III.1% Cone% precursor% markers% in% the% developing% retina% and% retinoblastomas% (A-D)-Photoreceptor-precursor-markers-(red)-and-RB1-(green)-in-gestationalweek-16-human-retina.-Note-coexpression-of-RB1-with-cone-markers-CRX,-RXRγ,and-TRβ2-but-not-with-the-rod-marker-NRL-(arrows).-(E-L)-Cone-but-not-rod-precursor-markers-(red)-in-retinoblastomas-without-(EZH)or-with-(IZL)-Ki67-costaining.-The-specificity-of-TRβ2-staining-was-confirmed-byknockdown-analyses.-(M)-Coexpression-of-L/M-opsin-(red)-and-Ki67-(green)-in-retinoblastoma.-(N-and-O)-Coexpression-of-L/M-opsin-and-S-opsin-(arrows)-in-retinoblastoma-(N)and-in-week-18-perifoveal-L/M-cone-precursors,-but-not-in-S-cone-precursors-(arrowheads,-O).-(P)-Mean-percentage-of-cells-expressing-each-marker-in-representative-sections-often-tumors.-Error-bars-indicate-the-standard-deviation.-Scale-bars-represent-50-μm-in-(A)-(N)-and-20-μm-in-(O).-Figure-adaped-from-

Figure%III.2%Molecular%characterization%of%human%retinoblastomas% (A)-Principal-component-analysis-(PCA)-of-gene-expression-array-data-fromprimary-human-retinoblastoms,-human-retinoblastoma-cell-lines-and-xenografts.-(B)-Hierarchical-cluster-of-gene-expression-array-data.-(C)-TEM-of-a-primarytumor.-Arrows-indicate-invaginations-in-the-nucleus-(n)-adjacent-to-a-mitoticfigure-(m).-(D)-Overview-of-retinal-development-from-multipotent-progenitor-cells;representative-cell-typeZrestricted-genes-appear-in-the-boxes.(E)-Box-plots-of-themicroarray-expression-values-for-a-series-of-genes-found-in-retinal-progenitor-cells,rod-and-cone-photoreceptors,-amacrine-/ganglion-/horizontal-cells,-orbipolar/Müller-cells.-Each-box-includes-the-data-for-all-52-human-gene-expressionarrays.-(F)-RealZtime-RTZPCR-data-using-TaqMan-probes-of-genes-selected-from-(E).-Abbreviations:-Adult,-adult-human-retina;-FW9,-human-fetal-week-9.-Figurefrom-(McEvoy-et-al.,-2011)-

vascular endothelial cells (CD34), and macrophages (Mac2, F4/80) on paraffin-embedded sections from five mice per genotype and scored the proportion of immunopositive cells (Figures S2G-S2I). At the stage when these tumors were isolated (moribund status), there were no dramatic differences across strains in the expression of these markers by genotype. In addition, TEM analysis of another 30 tumors across the six genotypes indicated that there were no significant differences in cellular morphology, including differentiation features such as processes and junctions (Figure S2J).

To develop a molecular profile of our retinoblastoma mice, we performed gene expression array analysis on 120 tumors (20 per strain). When combined on a PCA plot, the six retinoblastoma mouse strains exhibited extensive overlap and intermixing of their gene expression profiles (Figure 2K). The gene expression profiles were highly correlated (95%-99%) across all strains but the most distinct were p53 TKO and Rb TKO (Figures S2K and S2L and Table S2). The orthologs of the 28 genes used to characterize the retinal differentiation properties of human retinoblastomas (Figure 1) were also coexpressed in the mouse (E) Box plots of the Log2 expression from the microarrays for a series of genes found in retinal progenitor cells, rod and cone photoreceptors, amacrine/ganglion/ horizontal cells, or bipolar/Mu ¨ller cells. Each box includes the data for all 52 human gene expression arrays. The bottom and top of the boxes are the 25th and 75th percentile and the line in the middle is the 50th percentile. The lines represent one standard deviation above and below the mean and data points outside of those lines represent outliers. (F) Real-time RT-PCR data using TaqMan probes of genes selected from (E). Each bar is the mean and standard deviation of triplicate samples. Abbreviations: Adult, adult human retina; FW9, human fetal week 9; m, mitotic figure; n, nucleus. See also Figure S1 and Table S1.

Cancer Cell

Differentiation of Retinoblastoma

III.1.3 Results-

III.1.3.1 Independent* component* analysis* isolated* three* main* sources*of*gene*expression*variation*in*retinoblastoma.*

We! first! focused! on! the! trasncriptomes! of! an! initial! set! of! 37! retinoblastoma! samples,! queried! through! Affymetrix! U133! plus! 2.0! arrays.! We! analyzed! these! arrays! using! independent! component! analysis! (ICA),! an! unsupervised! method! specifically! designed! to! identify! the! original! contributing! sources! in! a! mixture! of! signals.!Three!independent!components!(ICs)!were!calculated!( ).!Among!the!genes!overexpressed!by!the! first!subtype,!were!genes!that!included!known!cone!photoreceptor!markers:!ARR3,! GNAT2,!GNGT2,!GUCA1C,!PDE6H,!PDE6C,!and!OPN1SW- [START_REF] Craft | !Cone!arrestin!identified!by! targeting!expression!of!a!functional!family[END_REF][START_REF] Imanishi | !Characterization!of!retinal!guanylate!cyclase5 activating!protein!3!(GCAP3)!from!zebrafish!to!man.![END_REF][START_REF] Kubo | !Molecular!cloning!and!sequence! analysis!of!cDNA!and!genomic!DNA!for!the!human!cone!transducin!alpha!subunit[END_REF][START_REF] Nathans | !Molecular!genetics!of!human!color! vision:!the!genes!encoding!blue,!green,!and!red!pigments[END_REF][START_REF] Ong | !Gene!structure!and! chromosome!localization!of!the!G!gamma!c!subunit!of!human!cone!G5protein! (GNGT2)[END_REF]! Shimizu5 Matsumoto! et! al.,! 1996;[START_REF] Viczian | !Isolation!and!characterization!of!a! cDNA!encoding!the!alpha'!subunit!of!human!cone!cGMP5phosphodiesterase[END_REF].! This! was! confirmed! through! Gene! ontology!(GO)!analysis,!as!the!genes!most!characteristic!of!this!first!subtype,!ie!the! 322! genes! with! projections! lower! than! 52.5,! were! enriched! in! GO! processes! that! included! "retinal! cone! cell! development",! "visual! perception",! and! "phototransduction".! The! second! potential! subtype! strongly! expressed! genes! that! included!markers!characteristic!of!retinal!ganglion!cells:!EBF3,!GAP43,!ISL1,!NEFM,-NEFL,-POU4F2,! NRN1,! DCX,! RAB33A,! STMN2,! BASP1,-D4S234E,-and! SOX1- [START_REF] Ivanov | !Microarray!analysis!of!gene!expression!in!adult!retinal!ganglion!cells[END_REF][START_REF] Mcevoy | !Coexpression!of!normally! incompatible!developmental!pathways!in!retinoblastoma!genesis[END_REF][START_REF] Mu | !A!gene! network!downstream!of!transcription!factor!Math5!regulates!retinal!progenitor!cell! competence!and!ganglion!cell!fate[END_REF][START_REF] Trimarchi | !Molecular!heterogeneity!of!developing!retinal!ganglion!and!amacrine!cells! revealed!through!single!cell!gene!expression!profiling[END_REF].-The! genes! most!characteristic!of!this!subtype!were!defined!as!the!260!genes!with!projections! higher!than!2.5!on!the!IC.!They!were!enriched!in!GO!processes!that!included!"neuron! projection",!"axonogenesis",!and"axon!guidance";!this!is!consistent!with!a!ganglion5 like! differentiation.! TFF1,a! gene! not! known! to! be! expressed! in! the! normal! adult! retina,! but! often! implicated! in! cancer! progression,! was! also! among! the! genes! strongly!overexpressed!by!this!subtype! [START_REF] Emami | !Trefoil!factor!family!(TFF)!peptides!and!cancer!progression[END_REF][START_REF] Rodrigues | !Induction!of!the!adenoma5carcinoma!progression!and! Cdc25A5B!phosphatases!by!the!trefoil!factor!TFF1!in!human!colon!epithelial!cells[END_REF][START_REF] Smid | !Genes!associated!with!breast!cancer!metastatic!to!bone[END_REF].!! ! To!formally!attribute!each!tumor!to!a!subtype,!we!used!consensus!clustering!of!the! 582! genes! most! characteristic! of! both! subtypes! (ie! with! the! strongest! projections)! (Figure! III.5).! In! total,! differential! expression! analysis! revealed! that,! beyond! the! differentiation!marks!described!previously,!a!total!of!4287!genes!were!significantly! differentially!expressed!between!both!subtypes.! ! We!next!studied!an!independent!gene!expression!dataset!to!validate!the!existence!of! both! subtypes.! We! used! the! NanoString! nCounter! Assay! System! to! measure! the! ! 82! expression!of!genes!characteristic!of!each!subtype!(33!and!26!genes,!respectively)!in! 68!tumors:!33!of!the!initial!samples!and!35!additional!validation!samples.! !

Figure%III.4%Transcriptomic%IC1%sets%apart%two%retinoblastoma%subtypes% (A)-Left:-Three-dimensional-plot-of-the-contributions-of-the-initial-set-of-samples-onthe-three-transcriptomic-independent-components-(ICZt)-calculated.-Samples

Figure%III.6%Classification%of%initial%set%of%samples%

For-the-initial-set-of-samples-(n=37),-coneZlike-and-bivalentZtype-tumors-wereseparated-through-consensus-clustering-of-the-Affymetrix-U133-plus-2. .-Samples-are-colored-based-on-cluster-assignment.-Figure% III.5% NanoString% expression% values% correlate% with% Affymetrix% expression%values% .-

33-tumor-samples-were-studied-both-with-Affymetrix-U133-plus-2.0-transcriptomicarrays-and-with-the-NanoString-nCounter-Assay-System.-Barplot-representation-of-Z log-(pZvalues)-of-Pearson-correlations-between-the-U133-plus-2.0-expression-valuesand-NanoString-expression-values-of-each-of-the-99-genes-measured-using-bothtechnologies.-Only-one-gene-(OTX1)-was-not-significantly-correlated,-and-was

Heatmap

Figure%III.8%Classification%of%validation%samples%

For - The! 37! "intial"! samples! were! uniformly! distributed! along! this! IC,! signifying! that! there!was!no!strong!heterogeneity!among!the!samples!for!this!signal.!Furthermore,! -! 90! the!IC!was!almost!exclusively!characterized!by!genes!with!high!positive!projections,! or! "overexpressed"! genes! (only! 28! of! 462! of! genes! with! absolute! projections! >2.5! had! negative! projections).! Finally,! while! this! signal! was! expressed! slightly! more! strongly!by!the!cone5like!tumors,!only!a!few!samples!expressed!it!very!strongly.! ! The!genes!with!the!highest!projections!included!several!Müller!glia!markers-DKK3,! RLBP1,!GPR37,!SYNPR,!CLU,!CAV1,!CAV2,!VIM,!ABCA8,!APOE!and!GFAP-as!well!as!rod! photoreceptor! markers-RHO,! SAG,! NRL,! PDE6A,! PDE6G,! GNGT1,! GNAT1,! and! CNGA1 [START_REF] Roesch | !The!transcriptome!of!retinal!Müller!glial!cells[END_REF][START_REF] Trimarchi | !Molecular!heterogeneity!of!developing!retinal!ganglion!and!amacrine!cells! revealed!through!single!cell!gene!expression!profiling[END_REF].!Accordingly,!gene!ontology!(GO)! analyses!indicated!that!the!genes!with!high!projections!were!enriched!in!the!"glial! cell!differentiation"!and!"retinal!rod!cell!development"!processes!(a!projection!cutoff! of! 2.5! was! chosen! for! this! analysis).! However,! they! were! also! enriched! in! the! processes! of! "locomotion"! and! "response! to! wounding".! Moreover,! PAX6! was! also! among!the!genes!with!high!projections.!While!PAX6!is!not!expressed!in!rod!cells,!it!is! expressed!in!activated!Müller!glia.!Müller!glia!are!usually!activated!in!the!context!of! a!retinal!wound,!during!which!they!exit!their!quiescent!state!and!start!to!proliferate! and!migrate! [START_REF] Bernardos | !Late5stage! neuronal!progenitors!in!the!retina!are!radial!Müller!glia!that!function!as!retinal!stem! cells[END_REF][START_REF] Dyer | !Control!of!Müller!glial!cell!proliferation!and! activation!following!retinal!injury[END_REF][START_REF] Giannelli | !Adult! human!Müller!glia!cells!are!a!highly!efficient!source!of!rod!photoreceptors[END_REF].! Taken! together,! these! observations! showed! that! this! IC! seemed! to! recapitulate! expression! patterns! reminiscent! of! two! cell! types:! activated! Müller! glia,! and! rod! cells.! !! To! study! this! signal! further,! we! measured! the! expression! of! 8! of! these! overexpressed! genes! in! 35! additional! validation! samples! using! the! NanoString! nCounter! Assay! System.! This! confirmed! that! Müller! and! rod! genes! were! co5 expressed!(Figure!III.15).!! Figure% III.15% Müller\rod% signal% present% in% validation% samples% [START_REF] Popova | !Genome!Alteration!Print!(GAP):!a!tool!to!visualize!and!mine!complex!cancer! genomic!profiles!obtained!by!SNP!arrays[END_REF].!Loss!of!one!copy!of!RB1!or!a!copy!number! neutral! loss! of! heterozygosity! of! RB1! are! two! early! alterations! in! retinoblastoma! We! hypothesized! that! reactive! gliosis! was! responsible! for! both! the! Müller! and! the! rod! cells! present! in! some! tumor! areas.! Indeed,! among! the! genes! reflecting! their! presence!were!genes!characteristic!of!activated!Müller!glia,!such!as!PAX6.!Reactive! ! 99! gliosis!usually!takes!place!in!the!context!of!a!retinal!wound,!and!it!would!therefore! not!be!surprising!that!it!take!place!in!retinal!tumors.!Moreover,!if!the!reactive!glial! cells! originate! from! the! healthy! retina,! it! is! reasonable! to! assume! that! they! could! drag!along!other!healthy!retinal!cells!when!growing!into!the!tumor.!Given!that!rods! make!up!70%!of!the!healthy!retina,!this!would!explain!the!presence!of!healthy!rod! cells! surrounding! the! activated! glia.! Rod! cells! could! also! be! present! due! to! a! transdifferentiation! of! the! glial! cells! themselves.! This! has! been! shown! to! occur! during! reactive! gliosis! in! other! species! and! in-vitro! in! humans! [START_REF] Bernardos | !Late5stage! neuronal!progenitors!in!the!retina!are!radial!Müller!glia!that!function!as!retinal!stem! cells[END_REF][START_REF] Dyer | !Control!of!Müller!glial!cell!proliferation!and! activation!following!retinal!injury[END_REF][START_REF] Giannelli | !Adult! human!Müller!glia!cells!are!a!highly!efficient!source!of!rod!photoreceptors[END_REF][START_REF] Jadhav | !Development!and!neurogenic! potential!of!Müller!glial!cells!in!the!vertebrate!retina[END_REF][START_REF] Wan | ! Preferential!regeneration!of!photoreceptor!from!Müller!glia!after!retinal! degeneration!in!adult!rat[END_REF].! III.2 Oncogenic% pathways% and% genes% involved% in% cone\like%and%bivalent\type%retinoblastomas%

(A)-Boxplots-of-the-percent-of-ARR3-(left)-or-TFF1-(right)-positive-tumorareas-in-tumor-sections-from-34-initial-samples-(17-coneZlike-and-17-bivalentZtype).-The-pZvalues-indicated-are-of-tZtests-between-the-twosubtypes.-(B)-The

! 88! For! the! samples! without! transcriptomic! data! available,! we! used! the! NanoString! nCounter TM ! expression! data! to! calculate! their! estimated! contributions! on! the! first! transcritpomic!IC,!ie!estimated!cone5relative5to!ganglion!differentiation!signatures:! ! contribution(sample!x) ! = ! !"#"!!"#$!%%&'( * !"#"!!"#$%&'(#)!!"!!" !"#$%&!!"!!"#"$ ! Equation% III.1%

Figure%III.14%Müller%and%rod%markers%characterize%second%transcriptomic%IC% (A)-Left:-Three-dimensional-plot-of-the-contributions-of-the-initial-set-of-samples-on-thethree-transcriptomic-independent-components-(ICZt)-calculated.-Right:-Histogram-ofthese-samples'-contributions-on-the-second-IC.-(B)-

Figure%III.16%NanoString%estimated%Müller\rod%signatures%

Plot-of-the-NanoStringZestimated-sample-contributions-on-transcriptomic-IC2-(MüllerZrod-signatures)-versus-the-U133-plus-2.0Zestimated-sample-contributionson-this-IC-(for-the-33-samples-for-which-both-datasets-were-available

BAF!=! !"#$%&!!"!!!!""#"#$ !"!#!!!"#$%&!!"!!"!!!!"!!!"#$%& ! ! BAF!= !!! . !"#$%&!!"!!!!""#"#$!!"!!"#$%!!"#$%" !!.(!"#$%&!!"!!!!"""#$!!"!!"#$%&!!"#$%") !!! . !"#$%&!!"!!"!!!""#"#$!!"!!!"#$! !!!! ! ! BAF!= !!! . !"#$%&!!"!!"!!!""#"#$!!"!!"#$% !!!! !!! !"#$%&!!"!!"!!!""#"#$!!"!!"#$% !!! !=! !!! .!" !"# !!"! !!! .!" !"# !!!" ! ! and!therefore! % p=% !" !"# !!"#.!! !"# !!!!".!! !"# !!.!"#!!!! !"# !!! %% % % (Equation%III.2)%
! Unlogged!Müller5rod!score!=! !!"#$!%%&'(%! !"#$%%&', !"#$%&!!"#!!"#$"%"& !"!!"#"$!!"#ℎ!!"#$%&!!"#$%&'(#)!!"!!"2 !! (
To! understand! the! gene! expression! patterns! of! retinoblastomas,! we! used! a! novel! approach! combining! an! independent! component! analysis! of! transcriptomic! data! with! immunohistochemical! stainings.! While! ICA! is! less! commonly! used! for! exploratory!data!analysis!than!principal!component!analysis!(PCA),!we!found!that!it! rendered!components!whose!biological!meaning!was!simpler!to!interpret!than!PCA! (data! no! shown).! We! thus! isolated! the! independent! sources! of! gene! expression! variation! in! retinoblastoma,! which! were! characterized! in! part! by! a! strong! expression!of!retinal!differentiation!markers.!The!first!source!reflected!the!mutually! exclusive! presence! of! cone5like! or! ganglion5like! differentiation! in!

III.2.1 Introduction-

A!question!that!naturally!arose!early!on!in!our!investigations!was!whether!the!cone5 like! and! bivalent5type! subtypes! represented! consecutive! steps! in! retinoblastoma! development,! with! cone5like! tumors! evolving! to! bivalent5type! tumors,! or! were! rather! the! reflection! of! two! distinct! progression! pathways.! Concurrently! with! the! initial!transcriptomic!identification!of!these!subtypes,!the!first!part!of!our!study!was! therefore! also! focused! on! identifying! oncogenes! and! pathways! involved! in! their! tumorigenesis!and!progression!mecanisms.!More!specifically,!we!searched!for!genes! and!pathways!whose!expression!was!specific!to!either!subtype!as!a!whole,!and!not! merely! a! consequence! of! cone5like! or! ganglion5like! tumoral! differentiation.! To! achieve! these! goals,! we! initially! analyzed! transcriptomic! data! as! well! as! copy! number!alteration!data!for!the!tumors!of!our!cohort.! ! !In! 2012,! recurrent! chromosomal! alterations,! including! gains! of! 1q,! 2p! and! 6p! as! well! as! losses! of! 16q,! had! already! been! described! in! retinoblastoma.! However,! the! significance!of!these!alterations!with!regards!to!the!oncogenic!pathways!involved!in! retinoblastoma! tumorigenesis! remained! an! active! field! of! research.! More! specifically,! the! role! of! the! TP53! pathway! in! retinoblastoma! tumorigenesis! had! for! long! been! debated.! Initially,! transgenic! and! chimeric! studies! [START_REF] Howes | !Apoptosis!or!retinoblastoma:!Alternative!fates!of!photoreceptors! expressing!the!HPV516!E7!gene!in!the!presence!or!absence!of!p53[END_REF][START_REF] Maandag | !Developmental!rescue!of!an! embryonic5lethal!mutation!in!the!retinoblastoma!gene!in!chimeric!mice[END_REF][START_REF] Robanus5maandag | !p107!is!a!suppressor!of! retinoblastoma!development!in!pRB5deficient!mice[END_REF])!provided!indirect!evidence!for! the! idea! that! the! retinoblastoma! cell5of5origin! must! overcome! a! propensity! to! undergo! apoptosis.! The! hypothesis! was! therefore! that! retinoblastoma! ! 100! tumorigenesis!requires!inactivation!of!both!the!RB1-pathway!and!the!TP53!pathway.! However,! it! was! later! observed! that! TP53! mutations! are! not! seen! in! human! retinoblastoma! or! in! mouse! retinoblastoma! derived! from! double! knock! out! cells! [START_REF] Gallie | ! Developmental!basis!of!retinal5specific!induction!of!cancer!by!RB!mutation.!In! Cancer!Research[END_REF][START_REF] Robanus5maandag | !p107!is!a!suppressor!of! retinoblastoma!development!in!pRB5deficient!mice[END_REF] ).! This! study! therefore! provided! evidence! that! the! TP53! pathway! may! be! inactivated! in! retinoblastoma! and! that! this! cancer! may! not! originate!from!intrinsically!death5resistant!cells,!as!previously!thought.!! ! It!is!in!this!context,!and!with!the!knowledge!of!the!existence!of!two!retinoblastoma! subtypes!-!the!cone5like!and!the!bivalent5type,!that!we!next!sought!to!further!study! the!oncogenes!and!pathways!involved!in!the!subtypes'!tumorigenesis.!! Figure% III.25% MDM4% rescues% cell% death% in% RB1\deficient% human% retinoblasts%

FW14-human-retinae-were-squareZwave-electroporated-with-a-GFPreporter-gene-along-with-the-indicated-expression-vectors-and-culturedfor-10d-as-explants.-After-5d-in-culture,-human-fetal-retinaeelectroporated-with-the-indicated-plasmids-were-labelled-with-[3H]thymidine-for-24-h,-and-48-h-later-were-labelled-with-BrdU-for-1-h.-(A)-The-proportions-of-BrdU+,-[3H]thymidine+-and-doubleZpositive-cellswere-scored-in-duplicate.-(B)-

The-proportions-of-activated-caspaseZ3+and-TUNEL+-cells-were-also-scored.-Adapted-from- [START_REF] Laurie | !Inactivation!of!the!p53!pathway!in! retinoblastoma[END_REF]

- ! ! 102!

III.2.2 Materials-and-Methods-

III.2.2.1 BAC@CGH*array*dataset*and*pre@processing*

64!tumor!samples!were!analyzed!on!BAC!CGH!microarrays.!These!arrays!consisted! of! 3511! clones! covering! the! human! genome! with! an! average! resolution! of! 850Kb;! they! were! designed! by! the! CIT5CGH! Consortium! (INSERM! U830,! Institut! Curie,! Paris)! and! IntegraGen TM! [START_REF] Idbaih | !Two!types!of!chromosome!1p!losses! with!opposite!significance!in!gliomas[END_REF].! Hybridized! slides! were! scanned! and! the! scan! data! was! pre5processed! as! described! in! [START_REF] Trolet | !Genomic!profiling!and!identification! of!high5risk!uveal!melanoma!by!array!CGH!analysis!of!primary!tumors!and!liver! metastases[END_REF].! Each! array! CGH! profile! was! centered! on! the! median! log2! ratio! and! then! analyzed! using! the! GLAD!algorithm! [START_REF] Hupé | !Analysis!of! array!CGH!data:!from!signal!ratio!to!gain!and!loss!of!DNA!regions[END_REF].!This!BAC!CGH!GLAD5smoothed!log2!ratio!data! was!used!for!all!other!log2!ratio!data!analysis.!The!BAC!log2!ratio!data!was!preferred! to!the!SNP!array!log2!ratio!data!as!it!had!a!stronger!dynamic!range!(data!not!shown).!

III.2.3 Results-

III.2.3.1 Several* genes* and* pathways* are* differentially* expressed* between* both* subtypes* in* a* differentiation@independent* manner.*

The! first! transcriptomic! independent! component! isolated! two! potentially! distinct! tumor! subtypes:! the! cone5like! tumors! presenting! mainly! areas! of! cone5like! differentiation,!and!the!bivalent5type!characterized!by!both!cone5like!and!ganglion5 like!areas!of!tumor!differentiation.!To!elucidate!whether!both!subtypes!were!driven! by!different!genes!and!pathways,!we!sought!to!isolate!the!genes!whose!expression! were!specific!to!either!subtype!as!a!whole,!and!not!merely!a!consequence!of!cone5 like!or!ganglion5like!tumoral!differentiation.!! ! We!first!studied!the!4287!genes!significantly!differentially!expressed!between!both! subtypes,! and! sought! to! filter! any! genes! whose! expression! could! potentially! be! specific! to! only! one! of! the! two! observed! types! of! areas! of! differentiation! (Figure! III.26,! A).! These! genes! were! identified! as! those! whose! expression! values! in! the! bivalent5type! subtype! were! correlated! to! the! samples'! cone5relative5to5ganglion! expression!signatures.!Through!this!step,!we!for!instance!removed!the!known!cone! markers,!as!well!as!the!genes!specific!to!the!ganglion5like!areas,!such!as!TFF1!and! EBF3.!We!also!filtered!the!genes!strongly!associated!to!the!Müller5rod!signal,!as!the! cone5like! subtype! tended! to! express! these! slightly! more! in! average.! 3081! ! 103! differentially! expressed! genes! remained,! supposably! not! linked! to! cone5like! or! ganglion5like!differentiation;!we!investigated!these!further.! ! At!the!individual!gene!level,!we!first!observed!that!MDM4!and!MYCN!had!a!stronger! expression!in!the!bivalent5type!samples!(Figure!III.26,!B).!MDM4!is!though!to!inhibit! TP53! at! the! protein! level,! and! we! interestingly! found! TP53's! mRNA! levels! to! be! higher! in! the! bivalent5type! subtype.! Notable! differentially! expressed! genes! also! included! RBL2,! less! expressed! in! the! bivalent5type! subtype.! Moreover,! E2F1! and! E2F2!were!up5regulated!in!the!bivalent5type!subtype.!Interestingly,!E2F3!and!other! members!of!the!E2F!family!were!not!differentially!expressed.!Finally,!we!noted!a!25 fold!stronger!expression!of!MKI67!in!the!bivalent5type!subtype.!! ! At! a! more! global! level,! Functional! Annotation! revealed! that! the! cone5like! subtype! was!notably!enriched!in!the!GO!processes!of!"regulation!of!neurotransmitter!levels",! "regulation! of! intracellular! pH",! "regulation! of! membrane! potential",! "ATP! Figure% III.26% Differentially% expressed% genes,% not% associated% to% tumor% differentiation% (A)-Strategy-used-to-study-the-genes-differentially-expressed-between-the-coneZ like-and-the-bivalentZtype-subtypes-that-were-not-specifically-associated-to-the-coneZlike,-or-the-ganglionZlike-areas.-(B)- Boxplot-of-expression-values-of-genessignificantly-differentially-expressed-between-the-subtypes-without-beingassociated-to-a-particular-coneZlike-or-ganglionZlike-area.-The-pZvaluesindicated-are-of-tZtests-between-the-two-subtypes,-corrected-for-multiple-testing.-! 104! biosynthetic! process",! "ATP! synthesis! coupled! transport",! "neurotransmitter! metabolic!process",!and!"cilium!assembly",!as!well!as!in!the!"Jak5STAT!signaling"!and! the! "glycolysis/gluconeogenesis! pathways.! The! bivalent5type! subtype! was! on! the! other!hand!enriched!in!the!GO!processes!of!"mitosis",!"rRNA!processing",!"chromatin! organization",! "regulation! of! transcription,! and! "homophilic! cell! adhesion"! as! well! the!"spliceosome"!and!"cyclins!and!cell!cycle!regulation"!pathways.!These!processes! and!pathways!were!differentially!expressed!between!the!subtypes!all!the!while!not! being!associated!to!a!particular!tumor!area!of!differentiation,!cone5like!or!ganglion5 like.!! ! In! conclusion,! these! observations! indicated! that! both! tumor! subtypes! may! rely! on! different! oncogenes! and! pathways,! and! that! they! may! thus! be! the! result! of! two! separate!progression!pathways.!

III.2.3.2 Cone

Figure%III.27%Genomic%alterations%in%retinoblastomas%

Plot We! studied! the! methylomes! of! 40! retinoblastoma! samples! using! Infinium! HumanMethylation450!BeadChip!arrays.!34!of!these!samples!were!of!the!initial!set! of! tumors! characterized! through! Affymetrix! U133! plus! 2.0! arrays,! and! 6! samples! were! of! the! validation! set! of! tumors! characterized! through! the! NanoString! nCounter TM! system.! DNA! samples! were! hybridized! on! Infinium! HumanMethylation450!BeadChip!arrays!(Illumina,!San!Diego,!CA).!4!μl!of!bisulfite5 converted! DNA! were! used! for! hybridization,! following! the! Illumina! Infinium! HD! Methylation! protocol! [START_REF] Fan | !High!density!DNA!methylation!array!with!single! CpG!site!resolution[END_REF].! Data! was! normalized! using! GenomeStudio! (Illumina,!Inc.)!and!R!statistical!software.!All!CpGs!were!further!analyzed,!with!the! exception!of!those!located!on!chromosomes!X!and!Y.!!

Figure%III.28%Genomic%alterations%of%chromosomes%1,%2,%16%and%6%for% both%subtypes%

Schematic

III.3.2.2 ICA,* CpG* annotation* and* expression/methylation* correlation*calculation*pipeline*

Methylome!independent!component!analysis!was!performed!in!the!same!manner!as! transcriptome!independent!component!analysis:!using!the!JADE!algorithm,!with!the! help!of!the!Bioconductor!package!MineICA.!Three!components!were!calculated.!For! each!methylomic!IC,!we!considered!that!the!CpGs!with!an!absolute!projection!higher! than!2.5!"strongly!contributed"!to!the!component.!These!CpGs!were!annotated!using! data! from! the! hg19! version! of! the! UCSC! Genome! Browser! database.! CpGs! were! considered!to!be!associated!to!a!gene!if!they!were!located!between!1500!base!pairs! upstream!of!the!transcriptional!start!site!and!the!end!of!the!3'UTR.!The!correlations! p5values!between!expression!and!methylation!were!corrected!through!a!Bonferroni! correction!that!took!into!account!the!number!of!CpGs!associated!to!the!same!gene.! We! chose! a! significance! cutoff! of! 0.05! (Figure! III.32).! Each! significant! CpG! methylation/gene! expression! correlation! could! be! positive! or! negative,! and! the! CpG's! projection! on! its! IC! could! itself! be! positive! or! negative.! Therefore,! to! determine!how!the!gene's!expression!varied!with!the!IC,!we!multipied!the!sign!of!the! CpG's!projection!on!the!IC!with!the!sign!of!the!expression/methylation!coefficient!of! the!correlation.!For!example,!if!a!CpG's!methylation!decresases!with!the!IC,!but!this!

Figure%III.33%Strong%inter\subtype%epigenomic%heterogeneity%

(A)-Three-dimensional-plot-of-retinoblastomas .-(B)-

Figure%III.33%Correlation%of%methylomic%ICs%to%expression%%

(A)- .-(B)- )-whosemethylation-correlates-to-expression,-for-the-different-ICZm's.-! 113! Methylomic!IC1!strongly!separated!the!cone5like!tumors!from!the!bivalent5type.!9%! of! the! IC1! CpGs! had! their! methylations! correlated! to! gene! expression.! These! correlations! (positive! or! negative)! were! associated! to! an! overexpression! in! the! cone5like!samples!of!genes!enriched!in!the!GO!processes!of!"phototransduction"!and! "retinal! cone! cell! development";! two! examples! of! such! genes! were! GNAT2! and! GUCA1C,! known! cone! markers! (Figure! III.34).! These! correlations! were! also! associated! to! an! overexpression! in! the! bivalent5type! samples! of! genes! enriched! in! the! GO! processes! of! "neuron! projection! guidance"! and! "axon! guidance";! two! examples!of!such!genes!were!EBF3,!a!ganglion!marker,!and!TFF1,!overexpressed!in! the! ganglion5like! areas! of! bivalent5type! tumors.! Taken! together,! these! findings! indicated!that!the!first!methylomic!IC!was!a!reflection!of!varying!ratios!of!cone5like! to!ganglion5like!areas!of!differentiation!across!samples.!! ! We! next! turned! to! the! second! and! third! methylomic! ICs,! two! other! independent! sources! of! epigenomic! variation! across! samples.! While! the! second! IC! strongly! separated! both! subgroups,! it! was! very! difficult! to! interpret,! given! its! low! rate! of! correlation!to!expression.!Nevertheless,!cone!or!ganglion!markers!were!not!among! the! genes! that! IC2! CpGs! were! correlated! to.! The! second! IC! therefore! reflected! a! strong!inter5subtype!epigenomic!heterogeneity!that!was!independent!of!cone5like!or! Figure% III.34% Examples% of% correlations% between% CpG% methylation% and%gene%expression% Four-examples-of-correlation-between-a-gene's-expression-and-one-of-itsassociated-CpGs'-methylation-(the-most-correlated) --! 120! patients!with!germline!inactivations!of!RB1-without!prior!attempts!at!conservative! treatments,! as! these! patients! are! highly! likely! to! develop! tumors! in! both! eyes.! Therefore,! while! RB1! germline5inactivated! patients! represent! 40%! of! all! retinoblastmoma!patients,!they!only!represented!25%!of!our!cohort,!ie!19!of!72.! ! 10! of! the! 19! RB1! germline5inactivated! patients! developed! bilateral! disease.! Two! bilateral!patients!were!so!at!diagnosis,!while!the!eight!others!presented!as!unilateral! at! diagnosis,! and! then! developed! tumors! in! their! second! eye! while! undergoing! treatment.!For!these!patients,!only!one!of!the!two!tumors!was!included!our!cohort:! this!corresponded!to!the!largest!of!the!two,!enucleated!prior!to!any!treatment,!and! also!the!first!to!be!diagnosed.!Generally,!conservative!treaments!were!attempted!on! the!second!eye.!This!again!created!a!bias!in!the!characteristics!of!the!tumors!of!our! cohort.!! ! The! median! age! at! enucleation!was! 21! months,! the! youngest! patient! to! be! enucleated! was! so! at! 51! days,! and! the! oldest! at! 6! years! of! age! (72! months).! Enucleation! was! usually! performed! within! one! week! of! diagnosis! at! the! Curie! Hospital.!Therefore,!age!at!enucleation!was!used!as!a!surrogate!for!age!at!diagnosis.! Results! were! strictly! similar! with! age! at! diagnosis! (data! not! shown). We! found! that! the! cone5like! subtype! was! significantly! enriched! in! tumors! from! patients! carrying! a! germline! inactivation! of! RB1:! 14! of! 30! cone5like! tumors! were! from! such! patients,! while! this! was! the! case! for! only! 5! of! 42! bivalent5type! tumors.! Furthermore,! most! RB1germline! inactivated! patients! (9! of! 14)! with! cone5like! tumors!ultimately!developped!bilateral!retinoblastoma,!while!this!was!the!case!for! only! one! of! the! RB1! germline! inactivated! bivalent5type! patients.! RB1! germline! inactivated! patients! develop! bilateral! disease! when! their! mutation! is! highly! penetrant.!Retinoblastomas!initiate!sooner!in!patients!with!germline!inactivations!of! RB1,! and! it! is! reasonable! to! assume! that! this! is! even! more! the! case! when! these! inactivations! are! highly! penetrant.! This! signifies! that! the! cone5like! subtype! was! significantly!enriched!in!tumors!that!initiated!earlier!during!retinal!development.! ! Consistent! with! these! observations,! we! noted! that! cone5like! tumors! were! found! in! patients! that! were! younger! at! diagnosis! than! those! with! bivalent5type! tumors! (Figure!III.39,!B).!This!was!the!case!both!for!the!patients!with!a!germline!mutation,!

Figure%III.40%Histopathologic%features%of%cone\like%and%bivalent\type%tumors%

Fisher-tests-(necrosis),-tZtests-(tumor-diameter),-or-chiZsquare-tests-(optic-nerve-orchoroid-and-scleral-invasion)-were-performed-between-the-two

III.5 Search%for%new%cancer%genes%in%retinoblastoma%

III.5.1 Introduction-

In!the!past!657!years,!major!international!cancer!genomic!consortiums!such!as!the! TCGA!and!the!ICGC!have!sought!to!create!a!comprehensive!catalogue!of!significantly! mutated! genes! across! all! major! cancer! types.! These! systematic! studies! have! revealed!many!new!cancer!genes,!as!well!as!new!classes!of!cancer!genes.!They!have! also!made!clear!that,!although!some!cancer!genes!are!mutated!at!high!frequencies,! most!cancer!genes!occur!at!intermediate!frequencies!(2-20%)!or!lower! [START_REF] Lawrence | !Discovery!and! saturation!analysis!of!cancer!genes!across!21!tumour!types[END_REF].!! ! The! technical! challenge! of! identifying! these! intermediate! frequency! cancer! genes,! which! drive! tumorigenesis,! it! to! distinguish! then! from! passenger! genes.! Passenger! genes! are! mutated! in! cancers,! but! their! mutation! does! not! confer! a! selective! advantage!to!the!tumor!cell.!The!power!to!identify!a!gene!as!a!significantly!mutated! driver!depends!on!the!properties!of!the!tumor!type,!namely!the!average!background! somatic!mutation!frequency!along!the!genome!for!the!tumor!type!('noise'),!and!the! target!frequency!(across!patients)-above!the!background!rate-that!one!wishes!to! detect! ('signal').! It! also! depends! on! the! properties! of! the! gene,! namely! its! background! mutation! frequency! relative! to! other! genes! (which! depends! on! length! and! local! mutation! rate).! As! a! result,! the! number! of! samples! which! need! to! be! sequenced! to! identify! cancer! genes! varies! across! tumor! types.! For! instance,! while! approximately!150!tumor5normal!pairs!would!be!necessary!to!identify!90%!of!genes! mutated!in!5%!or!more!of!medulloblastoma!patients,!1000!pairs!would!be!needed!to! accomplish!the!same!goal!in!melanoma!(Figure!III.41).!! ! Until! 2012,! no! disease! causing! genes! other! than! RB1! had! been! described! for! retinoblastoma.! A! first! study! (Zhang! et! al.,! 2012)! then! performed! whole! genome! sequencing!on!a!discovery!cohort!of!4!tumors.!These!tumors!allowed!the!authors!to! identify!a!new!retinoblastoma!cancer!gene,!BCOR,-which!they!found!mutated!in!13%! of! retinoblastomas! of! their! validation! cohort.! They! further! calculated! that! the! background!mutation!rate!of!retinoblastomas,!like!many!pediatric!cancers,!was!very! low,! with! an! estimated! background! mutation! rate! of! 0. .4-copy-number-for-72-samples-(positions-38,000,000-to-44,000,000).

-(B)-BCOR-log2Zration-versus-BCOR-expression-(U133-plus-2.0-data,-n=37

).-Each-pointrepresents-a-sample;-red-points-represent-samples-with-a-mutation-in-BCOR.--! !7! out! of! 9! of! the! identified! mutations! were! indels! (7! out! of! 9),! while! 2! of! 9! were! SNVs!leading!to!premature!stop!codons.!Three!mutations!were!located!in!exon!4!(2! indels! and! one! SNV),! the! gene's! longest! exon.! Two! deletions! were! located! at! the! same! location! on! exon! 7.! Interestingly,! allelic! frequencies! for! these! BCORinactivations!varied!from!10%!to!98%!(we!multiplied!girls'!allelic!frequencies!by!2! to!account!for!the!inactivation!of!one!X!chromosome),!but!were!higher!than!80%!in! 5!of!9!samples.!! ! We! analyzed! our! available! copy! number! alteration! data! (72! samples,! 25! of! which! had!been!sequenced)!in!the!BCOR-gene!area!(Figure!III.43).!We!identified!a!minimal! loss! region! which! was! limited! to! BCOR.-Four! samples! had! lost! one! gene! copy,! and! one! sample! (a! girl)! had! lost! both! BCORcopies.-These! losses! were! moreover! Through!whole5exome!sequencing!of!74!retinoblastomas,!we!gained!several!insights! into!retinoblastoma!tumorigenesis.!First,!we!confirmed!that!retinoblastoma!has!an! overall!very!low!mutation!rate:!beyond!RB1!mutations,!we!identified!an!average!of!2! non5synonymous!coding!mutations!per!tumor.!The!BCOR-gene!was!confirmed!to!be! recurrently!mutated!or!lost!in!at!least!18%!of!retinoblastomas.!We!also!identified!a! novel!cancer!gene!for!retinoblastomas:!ARID1A!was!mutated!or!lost!in!at!least!18%! of! samples.! Interestingly,! most! BCORinactivations! were! indels,! and! two! of! these! were!located!at!the!same!position!on!exon!7.!This!suggests!that!BCOR!may!be!located! in! an! area! of! the! genome! that! is! sensitive! to! base! insertions! of! deletions.! ARID1A! losses! were! on! the! other! hand! mostly! due! to! copy! number! losses,! suggesting! that! this!may!be!a!more!likely!means!of!inactivation!of!ARID1A!for!retinoblastomas.! ! Recent! studies! have! uncovered! several! new! loss5of5function! mutations! of! both! ARID1A- [START_REF] Cornen | !Mutations!and!deletions!of!ARID1A!in!breast!tumors[END_REF][START_REF] Huang | !Genomic!and!functional! evidence!for!an!ARID1A!tumor!suppressor!role.!Genes!Chromosom[END_REF]!Jones!et!al.,!2010!Jones!et!al.,! ,!2012;;[START_REF] Wang | !Exome!sequencing!identifies!frequent!mutation!of! ARID1A!in!molecular!subtypes!of!gastric!cancer[END_REF]and!BCOR! [START_REF] Damm | !BCOR!and!BCORL1!mutations! in!myelodysplastic!syndromes!and!related!disorders[END_REF][START_REF] Grossmann | !Whole5exome! sequencing!identifies!somatic!mutations!of!BCOR!in!acute!myeloid!leukemia!with! normal!karyotype[END_REF][START_REF] Pugh | ! Medulloblastoma!exome!sequencing!uncovers!subtype5specific!somatic!mutations[END_REF]!in!a! wide! range! of! malignancies,! suggesting! that! these! genes! may! be! common! tumor! suppressors.! Furthermore,! these! genes! have! both! been! implicated! in! chromatin! regulation.!This!suggests!that!disruption!of!epigenomic!structure!is!a!crucial!step!in! retinoblastoma! tumorigenesis.! Importantly,! the! ARID1A! mutations! in! our! retinoblastoma! samples! had! allelic! frequencies! nearing! the! 50%! of! a! fully! clonal! heterozygote,! and! BCOR,! located! on! chromosome! X,! often! had! allelic! frequencies! nearing!100%.!This!indicates!that!the!disruptions!of!either!of!these!genes!are!often! major! events! in! retinoblastoma! tumorigenesis,! and! not! consequences! of! tumor! progression.!! ! Finally,! it! is! worth! pointing! out! that! 20%! of! all! RB1-somatic! mutations,! which! had! previously! been! characterized! through! in! depth! Sanger! analyses,! were! missed! by! whole! exome! sequencing! (mainly! due! to! lack! of! coverage,! data! not! shown).! It! is!

Table%III.3%BCOR%mutations%

• !ARID1A! This! gene! is! located! on! chromosome! 1p36.11! and! codes! for! the! protein! AT! Rich! Interactive! Domain! 1A.! ! The! ARID1A! protein! is! part! of! the! large! ATP5dependent! chromatin! remodeling! complex! SNF/SWI,! which! is! required! for! transcriptional! activation!of!genes!normally!repressed!by!chromatin.! ! ARID1A-was! found! to! be! mutated! in! 2! of!

IV.1 Retinoblastoma%heterogeneity%

My! doctoral! project! was! greatly! shaped! by! my! discovery! of! two! retinoblastoma! subtypes,! the! cone5like! and! the! bivalent5type,! via! transcriptomic! and! immunohistochemical! analyses.! Further! analysis! of! the! tumors'! methylomes! confirmed! the! inter5subtype! heterogeneity.! Study! of! correlations! between! gene! expression! and! gene! methylation! revealed! that! stromal! cell! signal! was! much! stronger! in! the! transcriptomic! data! than! in! the! methylomic! data,! leading! to! a! stronger! methylomic! inter5subtype! heterogeneity.! Importantly,! the! presence! of! methylomic! heterogeneity! that! was! independent! of! methylomic! hetreogeneity! due! to! differentiation! stengthened! the! hypothesis! that! two! distinct! subtypes! exist,! and! that!bivalent5type!tumors!are!not!more!advanced!forms!of!cone5like!tumors.!! ! Analysis! of! genomic! alteration! profiles! as! well! as! of! whole! exome! sequencing! next! revealed! that,! as! for! many! pediatric! cancers,! retinoblastoma! has! globally! few! genomic!alterations.!However,!the!bivalent5type!subtype!was!shown!to!carry!overall! more!genomic!instability,!wether!large!chromosomal!alterations!of!small!SNVs!and! indels! were! examined.! These! alterations! were! furthermore! shown! to! often! be! present!in!almost!all!of!the!tumor!cells!assayed!(through!analysis!of!SNP!arrays!or! allelic! frequencies! of! mutations),! revealing! that! while! RB1! loss! is! necessary! for! bivalent5type!initiation,!it!is!not!sufficient!for!full5fledged!tumor!growth.!! ! Finally,! analysis! of! retinoblastomas'! clinical! features! revealed! crucial! insights! into! the! upstream! causes! for! the! presence! of! two! progression! paths.! First,! the! preponderance! of! cone5like! tumors! among! RB1! germline! inactivated! patients! indicated!that!cone5like!tumors!may!initiate!sooner!during!retinal!development!than! ! 138! their! bivalent5type! counterparts.! Second,! the! different! growth! patterns! of! both! subtypes! (cone5like! tumors! being! mostly! exophytic! while! bivalent5type! are! mostly! endophytic)! indicated! that! they! may! each! initiate! at! different! depths! within! the! developing!retina.!! IV.2 Tumor% subtypes% initiation% and% progression% models%% ! Figure%IV.1%Retinoblastoma%progression%models% (A)- Schematic-progression-model-for-the-coneZlike-tumors,-which-are-diagnosedin-younger-patients.-(B)-Schematic-progression-model-for-the-bivalentZtypetumors,-which-are-diagnosed-in-older-patients.-Tumors-of-this-group-have-both-coneZlike-and-ganglionZlike-areas,-in-varying-ratios.-

! 139!
The!characteristics!observed!for!both!retinoblastoma!subtypes!provide!crucial!clues! regarding! their! cell! of! origin! as! as! their! tumoral! progression! mechanisms.! Cone5like! tumors! have! cone! marker5expressing! transformed! cells.! Their! cell! of! origin!is!therefore!likely!to!be!a!committed!cone!precursor,!or!an!early!progenitor! (Figure! IV.1,! A).! They! have! low! genomic! instability! and! are! found! in! younger! patients.! We! therefore! propose! a! progression! model! whereby! the! tumors! of! this! group! start! off! with! cone5like! tumor! cells.! These! quickly! form! a! large! tumor! with! very! few! genomic! alterations.! This! rapid! growth! could! explain! why! patients! with! these!tumors!are!younger!in!age.!! % Bivalent5type!tumors!are!characterized!by!striking!intratumoral!heterogeneity,!with! transformed!cells!expressing!cone!markers!or!ganglion!markers.!For!this!group,!the! most!likely!cell!of!origin!is!therefore!an!early!retinal!progenitor,!which!is!the!healthy! retina! gives! rise! to! cones! or! ganglions! [START_REF] Livesey | !Vertebrate!neural!cell5fate!determination:! lessons!from!the!retina[END_REF])! (Figure ! IV.1,! B).! Several! genomic! alterations! are! present! in! this! subtype,! including! mutations! of! BCOR,! gains! of! 1q,! 2p,! and! losses! of! 16q.! Different! observations! suggest! that! these! alterations! occur! very! early! on! during! tumor! progression.! First,! in! the! case! of! 1q! gain! or! MYCN! amplification,! or! 16q! loss,! the! alterations! are! quasi5systematically! observed.! Second,! all! the! observed! genomic! alterations! are! carried! most! often! by! both! the! cone5like! and! the! ganglion5like! areas! of! differentiation.! For! bivalent5type! tumors,! we! therefore! propose! a! progression! model! whereby! a! bipotent! cone/ganglion!progenitor!is!first!transformed!through!RB1!loss,!but!is!only!weakly! proliferative.! Additional! genomic! alterations! then! lead! to! a! full5blown! malignancy! with! activation! of! both! cone5like! and! ganglion5like! differentiation! pathways.! A! latency!before!this!progression!could!explain!why!tumors!of!this!group!are!found!in! older!patients.!Another!factor!contributing!to!this!age!difference!could!also!be!that! bivalent5type!tumors!initiate!later.! !!

IV.3 Future%Directions%

IV.3.1 Direct-and-functional-studies-

Our! molecular! characterization! of! the! two! main! retinoblastoma! subtypes! was! mainly! based! on! genomic,! methylomic! or! transcriptomic! data.! Other! non! genome5 wide!descriptive!methods!can!be!employed!to!further!detail!the!molecular!portraits! of! these! subtypes.! These! include! further! immunohistochemical! stainings;! genes! whose! expression! is! differentially! expressed! between! the! subtypes,! yet! not! ! 140! associated! to! differentiation,! such! as! TP53! or! MDM4,! would! represent! interesting! candidates.! To! directly! assesss! the! genetic! and! epigenetic! differences! between! the! two! areas! of! differentiation! of! bivalent5type! retinoblastomas,! microdissection! of! paraffin5embedded!tumors!can!also!be!performed.!!

IV.3.2 Additional-retinoblastoma-subtypes-

While! the! two! bivalent5type! and! cone5like! subtypes! we! have! described! represent! the!great!majority!of!the!retinoblastomas!of!our!cohort,!we!did!observe!one!sample! with! an! entirely! different! transcriptomic! profile! (RB61).! This! sample! was! furthermore! from! a! six! year! old! patient! -the! oldest! of! the! cohort,! while! most! patients!are!diagnosed!before!the!age!of!five.!A!third,!much!more!rare!subtype,!could! therefore!exist.!Furthermore,!recent!studies!have!also!suggested!that!RB1+/+!MYCNamplified! retinoblastomas! could! have! their! own! genomic! profile.! A! broader! scale! genomic! study! of! retinoblastomas! could! therefore! provide! insights! into! the! existence! of! additional! rare! subtypes.! Given! the! low! incidence! of! this! cancer,! an! international!effort!would!be!necessary!to!accomplish!this!goal.!! !

IV.3.3 Oncogenic-events-in-retinoblastoma-

While! genomic! profiling! combined! with! clinical! data! analysis! set! appart! two! majoritary! subtypes,! the! pathways! and! cellular! mecanisms! implicated! in! each! subtype!have!only!be!succintly!described!in!my!thesis.!!

• Epigenomic!events! The!recurrent!inactivations!of!BCOR!and!ARID1A!in!retinoblastomas!(18%!of!tumors! for!each!genes)!confirm!that!modifications!of!the!epigenome!play!an!important!role! in!its!tumorigenesis.!Epigenomic!comparisons!between!healthy!retina!samples!and! retinoblastomas!could!therefore!reveal!the!breadth!of!these!modifications.!Healthy! adult!retinas!are!virtually!devoid!of!the!retinal!progenitors!or!precursors!thought!to! initiate!retinoblastoma.!Furthermore,!they!are!mainly!composed!of!rod!cells!(70%).! The!most!pertinent!healthy!tissue!for!epigenomic!comparisons!may!be!therefore!be! fetal!retinas,!where!cones!and!ganglions!are!the!two!first!differentiated!cells!types!to! appear.! Retinas! from! different! stages! of! fetal! development! would! thus! need! to! be! studied!in!order!to!infer!which!stages!best!ressemble!the!tumor!subtypes.!It!could! then! be! determined! which! of! the! tumors'! epigenomic! characteristics! are! inherited! ! 141! from! their! cell(s)! of! origin,! and! which! are! a! consequence! of! neoplastic! transformation.!!

• Genetic!events! In! this! study,! several! genes! were! identified! as! taking! part! in! retinoblastoma! tumorigenesis.! While! some! were! found! through! whole! exome! sequencing,! this! method!has!its!limits!as!it!only!queries!2%!of!the!entire!cancer!genome.!To!identify! genes!whose!alterations!rely!on!mutations!of!their!promoters!or!enhancer!regions,! or! to! identify! non5coding! RNAs! with! oncogenic! roles! in! retinoblastoma,! whole! genome!sequencing!is!necessary.!!

• Pathway!alterations! Beyond! individual! genetic! or! epigenetic! events,! pathway5scale! analyses! are! necessary!to!understand!the!global!mechanistic!underlying!retinoblastomas.!We!for! instance! observed! a! strong! presence! of! mutually! exclusive! MDM4gains! or! MYCNamplifications! in! bivalent5type! retinoblastomas,! but! almost! no! genomic! alterations! of! these! genes! in! the! cone5like! subtype.! We! also! interestingly! found! that! the! bivalent5type! subtype! has! higher! levels! of! TP53! mRNA! than! the! cone5like! subtype.! Since!MDM4-inhibits!TP53-activity!at!the!protein!level,!these!observations!therefore! raise! questions! regarding! if! and! how! the! TP53! pathway! is! altered! in! both! types! of! tumors.! Furthermore,! global! gene! ontology! analyses! revealed! that! while! bivalent5 type!tumors!express!genes!related!to!proliferation!more!strongly,!cone5like!tumors! may! be! more! metabolically! active.! A! more! in! depth! analysis! of! the! pathways! implicated! in! the! tumor! processes! of! both! subtypes! could! develop! this! initial! observation!further.

  Figure-adapted-from-[START_REF] Gerlinger | !Intratumor! Heterogeneity!and!Branched!Evolution!Revealed!by!Multiregion!Sequencing[END_REF]-

  all!DNA!microarrays!are!made!up!of!DNA!probes!spotted!or!synthesized! on!a!solid!surface!(glass,!plastic!or!silicon)!(Figure!I.13).!Through!the!hybridization! of! complementary! sequences,! these! probes! enable! the! detection! of! labeled! targets! (DNA,! RNA! or! cDNA)! from! a! biological! sample.! The! quantity! of! each! target! is! then! measured! by! the! fluorescence! intensity! at! each! spot.! The! diversity! of! microarrays! ! 26! results!from!the!type!of!targets!they!measure,!the!type!of!solid!surface!used,!the!size! of!the!probes!and!the!manufacturing!process.!! • Spotted!arrays! In! 1996! DeRisi! et! al.! published! a! method! which! allowed! very! high! density! DNA! probes!to!be!spotted!on!glass!substrates!(DeRisi!et!al.,!1996).!These!probes!can!be! oligonucleotides,!cDNA!or!small!fragments!of!PCR!products.!The!spotting!is!realized! by! a! robot! controlling! fine! needles,! or! by! ink5jet! printing.! An! advantage! of! this! technique! is! the! ability! to! produce! «! in5house! »! microarrays! in! the! research! laboratories.!It!is!however!very!difficult!to!reproducibly!produce!spotted!arrays,!and! comparisons! of! individually! hybridized! samples! to! ostensibly! identical! arrays! can!

  arrays-In! 1991,! Fodor! and! coworkers! published! a! method! for! light5directed,! spatially! addressable!chemical!synthesis!which!combined!photolabile!protecting!groups!with! photolithography! to! perform! chemical! synthesis! on! a! solid! substrate! (Fodor! et! al.,! 1991)! (Figure! I.14).! This! allowed! for! the! advent! of! inZsitumicroarrays,! where! oligonucleotides!probes!are!synthesized!directly!onto!the!array!surface.!The!probes! are! shorter! (255mer! probes! in! Affymetrix! arrays)! or! longer! (605! mer! probes! in! Agilent! arrays)! depending! on! the! desired! purpose.! Longer! probes! offer! greater! sequence! specificity! whereas! shorter! probes! can! be! synthesized! in! higher! density! and!are!cheaper!to!manufacture.!! ! • Bead!arrays! In! 2002,! Illumina! launched! a! new! type! of! DNA! microarrays,! called! bead! arrays!

  Figure%I.15%Sanger%sequencing%and%next%generation%seqeuencing% (A)-Sequencing-centers-producing-the-Sanger-sequence-data-for-mammaliangenome-projects-were-factoryZlike-outfits-with-a-large-number-of-personnel.-(B)-The-latest-nextZgeneration-sequencing-instruments-can-generate-as-much-data-in-24-h-as-several-hundred-SangerZtype-DNA-capillary-sequencers,-but-are-operatedby-a-single-person.-From-[START_REF] Schuster | !Next5generation!sequencing!transforms!today's!biology[END_REF])-

  Figure%I.17%CGH%and%SNP%array%copy%number%profiles%of%a%bladder%tumor%DNA%(A)+Copy-number-profile-of-a-bladder-tumor-obtained-with-a-BAC-arrayZCGH.-Thelogarithm-of-the-fluorescence-ratio-is-plotted-for-each-BAC-clone.-In-normal-regions,the-log-R-ratio-(LRR)-is-zero.-The-LRR-is-decreased-for-losses,-and-increased-for-gains.- Red:-gain,-green:-loss,-purple:-amplicon,-blue:-homozygous-deletion,-yellow:-normal.-(B)+Copy-number-profile-of-a-bladder-tumor-obtained-with-a-SNP-array.-Log-R-ratioinformation-is-available-as-for-BACZCGH-arrays.-However,-SNP-arrays-also-inform-onthe-B-allele-frequency-(BAF),-which-in-normal-regions-can-take-one-of-three-values:-0-(AA),-0.5-(AB)-or-1-(BB).-The-BAF-signal-is-affected-by-chromosome-aberrations,-butonly-for-heterozygous-SNPs.-For-example,-a-homozygous-SNP-of-genotype-AA-(BAF=0)-will-not-see-its-BAF-modified-by-any-alteration.-By-contrast,-heterozygous-SNPs-will-have-two-possible-BAF-following-a-gain-or-a-loss,-depending-on-the-alleleon-the-lost/gain-chromosome-region.-Heterozygous-SNPs-in-lost-regions-willtherefore-be-split-into-two-groups,-one-of-genotype-A-(theoretical-BAF=0),-the-otherof-genotype-B-(theoretical-BAF=1).-Heterozygous-SNPs-in-gained-regions-will-be-splitinto-two-groups-of-genotype-AAB-(theoretical-BAF=1/3)-and-ABB-(theoretical-BAF=2/3).-Unlike-the-LRR-signal,-BAF-can-be-informative-for-copy-neutral-losses-ofheterozygosity.-Figure-adapted-from-E.-Letouzé.

  Figure%I.18%DNA%methylation%and%bisulfite%treatment% (A)-Molecular-structure-of-a-cytosine-DNA-base,-a-methylcytosine,-and-ahydroxymethylcytosine.-(B)-DNA-bisulfite-conversion.-This-process-doesnot-distinguish-a-methylated-cytosine-from-a-hydroxymethylated-cytosine.--

•

  Figure%I.21%The%two%main%symptoms%of%retinoblastoma% (A)-Leukocoria,-or-an-abnormal-white-reflection-from-the-retina.-(B)-Strabismus. --

  Figure%I.23%Diagnosis%of%retinoblastoma%(A)-Ocular-fundus-aspect-of-retinoblastoma.-(B)-MRI-pattern-of-retinoblastomawith-optic-nerve-involvement.-From-[START_REF] Aerts | !Retinoblastoma.!Orphanet![END_REF]-

  and! morbidity.! Enucleation! is! used! either! as! a! frontline! therapy,! or! after! failure! of! conservative! treatments.! Ultimately,! 70%! of! retinoblastomas!are!treated!by!enucleation.!! • Conservative!treatments! Conservative! treatments! such! as! chemotherapy! and! focal! treatment! methods! are! used!for!patients!with!unilateral!retinoblastoma!presenting!no!high5risk!features,!or! ! 48! patients!with!bilateral!retinoblastoma!who!would!otherwise!become!blind.!Patients! treated!with!conservative!therapies!can!however!have!severe!side!effects!from!their! treatments! including,! but! not! limited! to,! impaired! vision! and! impaired! hearing.! Intra5arterial!chemotherapy!is!a!simple!and!novel!technique!which!has!been!shown! to! reduce! these! side! effects! (Figure! I.24).! The! drug! is! infused! directly! into! the! ophthalmic!artery:!this!delivers!the!chemotherapeutic!agent!directly!into!the!eye,!all! the!while!minimizing!systemic!exposure!(Abramson!et!al.,!2008).! ! Early! diagnosis! of! retinoblastoma! combined! with! the! aforementioned! treatments! has! rendered! cases! of! locally! advanced! or! metastatic! retinoblastoma! very! rare! in! developed! countries,! and! allowed! survival! rates! to! near! 100%.! Nevertheless,! patients!with!a!constitutional!inactivation!of!the!RB1-gene!are!susceptible!to!develop! other!primary!cancers!later!in!life,!such!as!sarcomas.!

  Figure%I.25%Microscopic%features%of%retinal%tumors% (A)-Undifferentiated-retinoblastoma-consists-of-dense-tissue-with-compact-cells,nuclear-moulding,-and-little-cytoplasm.-Differentiation-in-retinoblastoma-takes-theform-of-either-(B)-FlexnerZWintersteiner-rosettes-or-(C)-HomerZWright-rosettes.-From-"Hereditary+ tumors:-from-genes-to-clinical-consequences"-edited-by-Heike-Allgayer,-Helga-Rehder,-Simone-Fulda.-

  Figure%I.27%Ocular%anatomy%Figure-adapted-from-www.harvardZwm.org.--

  Figure%I.31%Müller%glia% (A)-(B)-Transition-between-normal-Müller-cells-in-a-healthy-retina-(left-side)-and-proliferative-reactive-gliosis-in-the-injured-retina.-Fromretinalmicroscopy.com,-and-(Giaume-et-al.,-2007)-

  Figure%I.33%Topography%of%retinoblastomas% (A)-Retrospective-analysis-of-the-topography-of-457-retinoblastomas.-The-right-and-left-retinas-are-divided-into-twelve-equal-30Zdegreesectors.-(B)-Plot-of-tumor-density-across-retinal-secotrs.-Figuresadapted-from-[START_REF] Munier | !Radial!asymmetry!in!the! topography!of!retinoblastoma!Clues!to!the!cell!of!origin[END_REF]-

  objectives!of!my!study!were!thus!organized!into!four!main!axes:! • Inventory!all!available!tumor!samples!in!order!to!construct!a!tumor!bank.! ! 69! • Complete! and! expand! the! clinical! dataset,! in! collaboration! with! peditrician! Dr.! Isabelle! Aerts,! geneticist! Dr.! Claude! Houdayer,! ophtalmologist! Dr.! Nathalie!Cassoux,!and!pathologist!Dr.!Xavier!Sastre5Garau.!! • Obtain!high5resolution!SNP!array!data!for!all!the!samples!for!which!such!data! was!not!yet!available.! • Analyze!the!transcriptomic!data,!the!copy!number!alteration!data,!as!well!as! the!clinical!data,!and!draw!parallels!with!what!had!already!been!published!on! retinoblastoma.!! ! Through!the!initial!study!of!these!datasets,!I!hypothesized!that!two!retinoblastoma! subtypes,!expressing!different!retinal!differentiation!marks,!may!exist.!Furthermore,! I!observed!a!transcriptomic!and!genomic!signal!which!I!believed!to!be!attributable! to! the! retinoblastoma! stroma.! To! validate! these! hypotheses,! we! therefore! next! sought! to! perform! immunohistochemical! stainings! of! differentiation! genes! characteristic! of! both! subgroups! as! well! as! of! genes! hypothesized! to! be! stromal! markers.! This! immunohistochemical! study! was! conducted! from! 2013! to! 2014,! in! tight!collaboration!with!the!anatomopathologist!Dr.!Xavier!Sastre5Garau,!as!well!as! with! the! experimental! pathology! platform! of! the! Curie! Hospital.! My! research! objectives!were!to:! acquire!the!corresponding!antibodies,!and!select!the! samples!to!be!studied.!! • Provide! feedback! during! the! protocol! adjustment! phases! for! each! antibody,! using!the!transcriptomic!information.! • With! the! strong! support! of! Dr.! Xavier! Sastre5Garau,! interpret! the! final! immunohistochemistry!results!in!the!light!of!the!transcriptomic!and!genomic! analyses!of!the!same!tumors.!! ! To! further! explore! the! molecular! portrait! of! retinoblastoma,! as! well! as! study! and! validate!the!two!hypothesized!subtypes,!we!also!turned!to!the!tumors'!epigenomes.! No!study!had!described!the!general!methylomic!profiles!of!retinoblastomas,!and!in! 2013!we!therefore!studied!40!tumor!samples!using!highly!resolutive!Illumina!DNA! methylation! arrays.! Most! of! these! samples! had! also! been! characterized! through! transcriptomic! and! genomic! alteration! data.! This! step! led! to! several! new! research! objectives:!! • Analyze! the! methylomic! data,! particularly! in! the! light! of! the! hypothetical! presence!of!two!tumor!subtypes.!! • Study! the! relationship! between! gene! methylation! and! expression! in! retinoblastoma.! sought! to! characterize! both! tumor! subtypes! through! their! somatic! DNA! alterations,! including! SNVs,! insertions! and! deletions! as! well! as! structural! variants.! As!of!yet,!the!somatic!mutation!data!of!only!4!retinoblastomas!has!been!described!in! the!literature.!Moreover,!studies!based!on!retinoblastoma!RNAseq!data!have!never! been!described.!We!obtained!funding!for!this!last!phase!from!the!French!Institute!of! Health! and! Medical! Research! (INSERM),! in! the! context! of! the! International! Cancer! Genome! Consortium! (ICGC).! From! the! fall! of! 2013! to! 2015,! a! total! of! ~80! tumor! samples!were!characterized!through!whole!exome!sequencing.!These!samples!came! both!from!the!Curie!Institute!Hospital,!as!well!as!the!Buenos!Aires!Juan!P.!Garrahan! Pediatric!Hospital,!with!whom!we!started!a!collaboration!in!2013,!and!the!Sant!Joan! de!Déu!Hospital!in!Barcelona.! ! My!objectives!for!this!final!part!of!my!doctoral!work!were!therefore!:!! • Before!sequencing,!to!coordinate!the!selection!of!cases!to!be!studied,!and!to! help!pediatrician!Dr.!Isabelle!Aerts!with!the!tremendous!task!of!obtaining!all! the!necessary!consent!forms!from!patients.! • To!analyze!the!exome!sequencing,!and!RNAseq!data,!and!ultimately!provide!a! list!of!candidate!mutations!and!gene!fusions!for!Sanger5based!validation.!This! part!was!conducted!with!the!help!of!the!Synergie!Lyon!Cancer!bioinformatics! platform.!! ! Sanger! validations! of! putative! somatic! gene! alterations! were! performed! by! both! Céline! Desbrousses! and! Daniela! Ottaviani.! Perpetual! feedback! from! these! validations!allowed!for!fine!tuning!of!the!variant!calling!analysis!pipelines.

  .! However,! all! of! these! cell! clusters! were! found! to! express! RB1,! and!were!thus!concluded!to!be!healthy!cells!derived!from!the!normal!retina.!! ! In! 2011,! the! Cancer! Cell! McEvoy! et! al.! study! strongly! contradicted! the! 2009! cone! precursor! hypothesis.! The! authors! reported! that! human! retinoblastomas! have! molecular,! cellular,! and! neurochemical! features! of! multiple! cell! classes,! principally! amacrine/horizontal! interneurons,! retinal! progenitor! cells,! and! photoreceptors.! Their! study! was! mainly! based! on! transcriptomic! analyses! of! primary! human! retinoblastoma! samples! (Figure! III.2).! Through! unsupervised! principal! component! analysis!and!hierarchical!clustering,!the!authors!observed!that!the!majority!of!tumor! samples! clustered! tightly! around! a! central! core! molecular! signature,! and! detected! no! clear! subtypes.! Furthermore,! to! gain! insight! into! the! differentiation! features! of! human!retinoblastomas,!they!used!their!microarray!data!to!analyze!the!expression! of! 28! genes! that! are! restricted! to! different! retinal! cell! classes.! They! found! that! a! mixture! of! genes,! normally! expressed! in! retinal! progenitor! cells! or! differentiated! retinal!cells,!were!co5expressed!in!human!retinoblastomas.!Importantly,!the!authors! studied! the! gene! expression! arrays! of! single! cells! derived! from! human! retinoblastoma! xenografts,! and! this! analysis! led! them! to! infer! that! these! multiple! cell! type5specific! developmental! programs! are! co5expressed! withinin! individual! retinoblastoma! cells,! which! thus! creates! a! progenitor/neuronal! hybrid! cell.! The! authors!ultimately!concluded!that!retinoblastomas!are!relatively!homogenous,!and! express!a!hybrid!differentiation!phenotype.! ! Several! methodological! issues! can! be! raised! regarding! this! study,! the! two! main! being:! 1. The!authors!indicate!that!most!of!the!tumors!of!their!cohort!cluster!around!a! central!molecular!signature.!They!however!do!not!study!in!what!way!the!~10! samples! which! fall! as! outliers! differ! from! the! core! retinoblastoma! samples,! despite! their! clear! difference.! Furthermore,! some! cell! type! markers! such! as! Müller! marker! GFAP! were! found! to! be! very! strongly! expressed! in! only! a! subset!of!samples;!the!authors!did!however!not!investigate!this!further.! ! 2. Regarding! cell! type! signatures,! the! authors! compare! the! absolute! array! values! of! 28! cell! type! specific! genes.! However,! absolute! array! expression! values! are! often! misleading,! as! probe! affinities! can! vary! across! genes.! Therefore,! array! gene! expression! values! are! usually! only! analyzed! in! the! context! of! relative! sample5to5sample! comparisons,! and! not! gene5to5gene! comparisons.! light! of! these! methodological! issues,! and! the! contradictory! results! rendered! by!both!this!study!and!the!first!2009!immunohistochemistry5based!study,!the!goal! of! the! first! part! of! my! doctoral! research! was! to! dissect! and! analyze! the! transcriptomic! profiles! of! retinoblastomas,! while! paying! special! attention! to! the! differentiation!marks!they!express.!! !

Figure 1 .

 1 Figure 1. Molecular Characterization of Human Retinoblastoma Cells (A) Principal component analysis (PCA) of gene expression array data from primary human retinoblastoma cells and four human retinoblastoma cell lines (Y79, Weri1, RB-355, and RB-13). SJ-39, SJ-41, and SJ-42 were subsequently used as a source for orthotopic xenografts. (B) Hierarchical cluster of human retinoblastoma and cell line gene expression array data. Black lines are those tumors used for orthotopic xenografts, and light gray lines are the cell lines. All other lines (dark gray) are primary tumor samples. (C) TEM of the primary tumor SJ-39. Arrows indicate invaginations in the nucleus (n) adjacent to a mitotic figure (m). (D) Overview of retinal development from multipotent progenitor cells; representative cell type-restricted genes appear in the boxes. (E) Box plots of the Log2 expression from the microarrays for a series of genes found in retinal progenitor cells, rod and cone photoreceptors, amacrine/ganglion/ horizontal cells, or bipolar/Mu ¨ller cells. Each box includes the data for all 52 human gene expression arrays. The bottom and top of the boxes are the 25th and 75th percentile and the line in the middle is the 50th percentile. The lines represent one standard deviation above and below the mean and data points outside of those lines represent outliers. (F) Real-time RT-PCR data using TaqMan probes of genes selected from (E). Each bar is the mean and standard deviation of triplicate samples. Abbreviations: Adult, adult human retina; FW9, human fetal week 9; m, mitotic figure; n, nucleus. See also Figure S1 and TableS1.

Sup

  Figure!III.4,!A).!They! represented! three! main! sources! of! gene! expression! variation! in! the! tumor! The! two! first! independent! components! were! found! to! be! the! reflection! of! two! varying! gene! expression! signals! among! the! tumors.! One! was! attributed! to! the! tumor's! transformed! cells! (first! IC,! Figure! III.4512),! while! the! second!was!attributed!to!the!retinoblastoma!stroma!(second!IC,!Figures!III.13520).! We! will! first! describe! the! results! garnered! from! the! first! and! second! independent! components.! The! third! independent! component! isolated! an! outlier! sample! (RB61,! Figure!III.21),!and!will!be!described!last.!! !IC!had!a!bimodal!sample!distribution!and!hinted!towards!the!presence!of! two!retinoblastoma!subtypes!(Figure!III.4

  the-genes.--!correlated!strongly!with!array!data!(Figure!III.6),!and!the!additional! validation!samples!had!the!same!patterns!of!gene!expression!as!the!initial!samples! (Figure! III.8).! This! allowed! us! to! confirm! the! presence! of! two! subtypes! of! tumors,!

  Figure%III.7%Two%subtypes%in%the%validation%set%of%samples%

  Figure% III.10% ARR3% and% TFF1% immunostaining% in% retinoblastoma%Immunostaining-of-ARR3-(left)-and-TFF1-(right)-in-the-healthyretina-(top),-a-representative-coneZlike-tumor-(RB1,-middle),and-a-representative-bivalentZtype-tumor-(RB27,-bottom).-

  Figure%III.13%NanoString%estimated%cone/ganglion%signatures%%Plot-of-the-NanoStringZestimated-sample-contributions-ontranscriptomic-IC1-(cone/ganglion)-versus-the-U133-plus-2.0-samplecontributions-on-this-IC-(for-the-33-samples-for-which-both-datasetswere-available).-

  !used!Equation%III.1!to!calculate!estimated!Müller5rod!signatures!for!these! validation! samples.! The! signatures! strongly! correlated! with! the! Affymetrix! signatures!(Figure!III.16).!! ! To! identify! the! cells! at! the! source! of! this! "Müller5rod"! expression! signal,! we! performed!immunohistochemical!staining!of!the!Müller!specific!protein!RLBP1!and! the! rod! specific! transcription! factor! NRL! (Figure! III.17).! Consistent! with! the! transcriptomic! data,! most! tumor! sections! studied! had! no,! or! few! heterogeneously! distributed!NRL!and!RLBP1!positive!cells!(data!not!shown).!Also!in!agreement!with! the! transcriptomic! data,! RLBP1! positive! cells! were! generally! located! in! the! same! areas! as! NRL! positive! cells.! The! second! transcriptomic! IC! was! therefore! likely! a! reflection! of! the! occasional! intratumoral! presence! of! these! two! types! of! cellular! differentiation.!! ! The! observed! morphological! characteristics! of! the! RLBP1! positive! cells! and! the! spatial! distribution! of! both! cell! types! hinted! that! they! were! untransformed.! To! further! characterize! these! cells,! we! therefore! next! turned! to! our! SNP! array! data.! With! SNP! arrays,! it! is! possible! to! calculate! the! proportion! of! cells! with! a! given! alteration! through! an! integrative! analysis! of! the! log25ratio! signal,! which! reflects! relative! copy5number! changes,! and! the! B! allele! frequency! signal,! which! reflects! allelic!imbalances!

  When! either! alteration! was! present! (38! cases),! we! were! able! to! estimate!the!proportion!of!assayed!cells!with!the!alteration,!and!hypothesized!that! the!cells!without!it!were!untransformed!(Figure!III.18).! ! If!p∈[0,1]!represents!the!fraction!of!wild5type!cells,!(15p)!the!fraction!of!tumor!cells,! and! CNRB1! the! number! of! RB1! alleles! in! the! tumor,! then! the! measured! B! allele! frequency!is! !

  Figure%III.17%NRL%and%RLBP1%immunostaining%in%retinoblastoma%NRL-(rod-marker,-top)-and-RLBP1--(Müller-marker,-bottom)-immunostaining-in-the-healthyretina-(left),-and-in-retinoblastoma-(right)-at-two-different-magnifications.-

  Figure% III.18% Examples% of% complete% and% incomplete% loss% of% heterozogosity%Two-examples-of-a-13q-loss-of-heterozygosity-signal-in-retinoblastoma.-Leftpanel:-RB45,-loss-of-heterozygosity-in-all-cells-of-the-tumor-sample.-Rightpanel:-RB10,-loss-of-heterozygosity-in-a-fraction-of-cells-of-the-tumor-samplecells.-This-difference-allowed-us-to-estimate-the-RB1-wildZtype-samplecontent-for-all-the-samples-with-a-loss-of-one-copy-of-RB1-or-a-copy-numberneutral-loss-of-heterozygosity-of-RB1-(37-cases).-

  retinoblastomas,! as! well! as! hinted! towards! the! presence! of! two! retinoblastoma! subtypes:! the! cone5 like! and! the! bivalent5type.! The! second! source! reflected! the! varying! amounts! of! untransformed!Müller!and!rod!cells!across!tumor!areas.!! III.1.4.2 Relation*to*previously*published*sutides* Our!findings!are!partially!consistent!with!the!seemingly!contradictory!observations! that!have!been!published!in!the!past!years!regarding!the!retinal!cell!type!signatures! of! retinoblastomas.! The! Xu! et! al! 2009! Cell! study! (Xu! et! al.,! 2009)! pointed! out! that! retinoblastomas! systematically! express! cone! photoreceptor! markers.! We! indeed! found! that! very! few! retinoblastomas! (2! out! of! 34! studied)! had! no! cone! arrestin! ARR3! positive! tumor! areas.! However,! we! also! found! ganglion5like! areas! in! the! retinoblastomas!of!the!bivalent5type!subgroup,!while!there!was!no!mention!of!these! cone5marker! negative! areas! in! the! 2009! Xu.! et! al! study.! The! bivalent5type's! intratumoral! heterogeneity,! and! the! occasional! presence! of! untransformed! Müller! and! rod! cells,! is! consistent! with! the! 2011! Cancer! Cell! study! that! described! the! presence! of! several! different! cell! type! markers! in! retinoblastoma! transcriptomes! (Kapatai!et!al.,!2013;!McEvoy!et!al.,!2011).!However,!unlike!this!study,!we!found!that! individual! tumor! cells! maintain! a! coherence! in! the! differentiation! pathways! they! express,!and!do!not!co5express!markers!of!several!cell!types!at!once.!!

  Figure%III.24%Cone%precursor%signaling%for% retinoblastoma%tumorigenesis%Potential-role-of-cone-precursor-signaling-proteins-inretinoblastoma-tumorigenesis,-as-described-by-Xu-et-al,-Cell- 2009.-Proteins-in-red-are-highly-expressed-during-humancone-precursor-maturation.-

  @like* and* bivalent@type* retinoblastomas* have* different*chromosomal*alterations* To! search! for! the! potential! causes! for! subtype! specific! gene! overexpressions,! we! next!investigated!the!genomic!imbalances!of!both!subtypes.!We!therefore!analyzed! the!array5CGH!data!of!27!cone5like!and!37!bivalent5type!tumor!samples.!! ! As!previously!reported!(Corson!and!Gallie,!2007),!1q!gains,!16q!losses,!6p!gains!and! 2p!gains!were!the!most!frequent!alterations!(Figure!III.27).!! !

  !were!equally!distributed!regardless!of!tumor!subtype,!but!we!found!that!1q! gains,!2p!gains!and!16q!losses!were!significantly!more!frequent!in!the!bivalent5type! samples! (Fisher's! exact! test! p5values! <0.01)! (Figure! III.28).! In! fact,! the! cone5like! subtype!was!virtually!devoid!of!these!alterations,!while!all!but!three!of!the!bivalent5 type! samples! had! 1q! gain,! or! an! amplification! of! a! region! on! chromosome! 2p! that! included!MYCN-(one!of!these!three!samples!being!RB61).! ! Study! of! minimally! altered! regions! revealed! that! MDM4! was! a! likely! driver! for! 1q! gain,!as!was!MYCN-for!2p!gain!(Figure!III.28).!The!expressions!of!MYCN!and!MDM4! were! furthermore! strongly! correlated! to! their! relative! copy! numbers! (even! when! the! most! stronly! amplified! samples! were! removed).! However,! no! obvious! drivers!

  Figure%III.30%MDM4%and%MYCN%may%drive%1q%gain%and%2p%gain% (A)-Minimal-region-analysis-for-1q-gains-(left)-and-2p-gains-(right).-Left-are-plots-ofthe-%-of-samples-presenting-a-gain-versus-position-along-the-chromosome.-Right-areplots-of-the-cumulative-relative-copy-number-(ie-log2-ratio)-across-all-samplespresenting-a-gain,-versus-position-along-the-chromosome.-(B)-Plot-of-transcriptomicexpression-values-for-MDM4-(left)-and-MYCN-(right)-versus-relative-copy-number-(log2Zratio).-PZvalues-and-correlation-coefficients-for-Pearson's-correlation-analysisare-indicated.-

Figure%

  Figure% III.32% Expression/methylation% correlation% calculation%pipeline%

  the-samples.--! 112! This!methylomic!heterogeneity!furthermore!seemed!to!go!beyond!methylation!due! to! differentiation,! as! we! for! instance! found! that! the! bivalent5type! tumors! with! the! strongest!cone5like!differentiation!signatures,!did!not!cluster!close!to!the!tumors!of! the!cone5like!subtype.!! III.3.3.2 Epigenomic* heterogeneity* goes* beyond* cone@like* or* ganglion@like*differentiation* To! understand! this! methylomic! heterogeneity! further,! we! studied! the! three! methylomic! ICs! individually.! They! recapitulated! three! independent! sources! of! epigenetic! heterogeneity.! We! first! studied! the! CpGs! with! the! strongest! projections! on! each! IC,! hereafter! referred! to! as! "IC! CpGs".! Each! IC! was! characterized! by! approximately!16000!of!these!CpGs!(Figure!III.33,!A).!We!paid!particular!attention! to!how!they!correlated!to!gene!expression.!! ! A!total!of!430!000!CpGs!were!studied!on!the!arrays.!Of!those,!350!000!were!located! within!close!proximity!of!a!gene!(between!1500bp!upstream!of!the!TSS!and!the!end! of!the!3'UTR),!2.4%!of!which!correlated!to!expression!(1.75%!negative!correlation! and! 0.65%! positive).! In! constrast! 9%! of! the! IC1! CpGs! that! were! located! in! the! vicinity! of! a! gene! had! their! methylation! correlated! to! expression! (positive! or! negative! correlation),! representing! an! 45fold! enrichment! above! background.! Rates! of!correlation!to!expression!were!lower!for!the!second!and!third!ICs:!3.9%!and!1.5%,! respectively! (Figure! III.33,! B).! There! was! therefore! an! overall! poor! correlation! between! methylation! and! expression,! with! the! slight! exception! of! methylomic! IC1,! which!we!investigated!first.!! !

  Figure% III.35% Characteristics% of% the% methylomic% independent% components% (A)-Plot-of-patient-ages-at-diagnosis-versus-sample-contribution-onmethylomic-IC2Zm.-The-pZvalue-for-Pearson's-correlation-analysis-isindicated.-(B)-Plot-of-overall-sample-methylation-versus-sample-contributionon-methylomic-IC3Zm.-The-pZvalue-for-Pearson's-correlation-analysis-isindicated.--(C)-Plot-of-overall-sample-methylation-versus-cone/ganglionsample-signature.-There-was-no-significant-correlation.--

  Figure%III.39%Clinical%features%of%cone\like%and%bivalent\type%tumors% (A)-Schematic-representation-of-tumors'-clinical-features.-ConeZlike-tumors-areon-the-left,-bivalentZtype-tumors-on-the-right;-bivalentZtype-tumors-are-sortedbased-on-their-coneZganglion-signature,-with-the-strongest-coneZlike-signatureson-the-left-and-the-strongest-ganglionZlike-signatures-on-the-right.-The-pZvaluesindicated-are-of-Fisher-tests-between-the-two-subtypes.-(B)-Boxplot-of-patientages-at-diagnosis-for-coneZlike-and-bivalentZtype-tumors.-Left:-patients-with-an-RB1-germline-mutation-(14-coneZlike,-5-bivalentZtype).-Right:-patients-with-no-RB1-germline-mutation-(15-coneZlike,-30-bivalentZtype).-The-pZvalues-indicatedare-of-tZtests-between-the-two-subtypes.-(C)-Plot-of-tumors'-coneZganglionsignatures-(ie-contributions-on-IC1)-versus-the-corresponding-patients

  Figure% III.41% Number% of% samples% needed% to% detect% significantly% mutated%genes%Number-of-samples-needed-to-detect-significantly-mutated-genes,-as-afunction-of-a-tumor-type's-median-background-mutation-frequency-and-acancer-gene's-mutation-rate-above-background.-From-(Lawrence-et-al.,-2014)-

  the! 74! exome! sequenced! samples! (3%)(Figure!III.44).!One!mutation!was!a!frameshift!insertion,!while!the!other!was!an! SNV! leading! to! a! premature! stop! codon.! The! allelic! frequencies! were! of! 40%! and! 47%,!these!therefore!corresponded!to!heterozygous!mutations.! ! We! analyzed! next! our! available! copy! number! alteration! data! (72! samples,! 25! of! which! had! been! sequenced)! in! the! ARID1Agene! area! (Figure! III.44).! We! found! a! ~1,000,000! base! pair! minimal! loss! region! which! contained! 28! genes! and! was! centered!around!ARID1A.!15!samples!had!an!ARID1A-log25ratio!below!50.2,!ie!a!loss! of!1!ARID1A-copy!in!at!least!15%!of!the!sample!cells!assayed.!This!copy!number!lossfurthermore!correlated!to!a!decreased!expression!of!ARID1A.-

  Figure%III.44%ARID1A%copy%number%and%expression%(A)-1p36.11-copy-number-for-72-samples-(positions-22,000,000-to-35,000,000).-(B)-ARID1A-log2Zration-versus-ARID1A-expression-(U133-plus-2.0-data,-n=37).-Each-pointrepresents-a-sample.--

  ! that! further! mutations! of! BCOR-or! ARID1A-were! also! missed! by! whole! exome! sequencing.! Full! Sanger! sequencing! could! therefore! be! necessary! to! obtain! an! accurate! mutation! rate! for! these! genes.! -Moreover,! BCORand! ARID1A! Sanger! sequencing! in! a! larger! cohort! of! samples! may! further! render! pertinent! information! regarding! the! distributions! of! the! mutations! relative! to! subtype,! methylomic!profile,!age!at!diagnosis,!or!other!clinical!criteria.! doctoral! research,! I! sought! to! build! and! study! the! first! comprehensive! molecular! and! clinical! portrait! of! retinoblastoma.! Tumor! transcriptomes! (array5 based! and! sequencing5based),! genomic! alteration! profiles,! methylomes,! whole! exome! sequences,! as! well! as! clinical! characteristics! were! acquired,! studied! and! compared.!While!these!analyses!rendered!several!important!obervations,!they!also! raised!several!crucial!questions!and!opened!the!door!to!further!investigations!in!the! field!of!retinoblastoma!research!and!pediatric!cancers!in!general.!%

.2.2.1 Principal%component%analysis%(PCA)%

	statistics! and! finds! components! which! are! independent! but! not! necessarily!
	orthogonal,!contrary!to!the!PCA.!!
	I.3Principal! Component! Analysis! (PCA)! (Jolliffe! and! Jolliffe,! 1986)! is! a! very! popular!
	statistical! technique! used! to! visualise! and! capture! variations! between!
	multidimensional! samples,! by! projecting! them! to! a! low5dimensional! space! where!
	most! variations! are! retained.! PCA! is! particularly! useful! for! visualisation! of! high5
	dimensional! data,! by! plotting! the! projection! of! the! samples! to! the! 25! or! 35
	dimensional!subspace!which!captures!the!largest!amount!of!variations,!and!for!data!
	understanding,! by! highlighting! the! key! factors! responsible! for! the! diversity! of!
	samples.!!
	!
	In!order!to!find!a!linear!combination!of!the!original!basis!that!best!re5expresses!the!
	dataset,! the! approach! used! by! Principal! Component! Analysis! (PCA)! is! to! retain!
	dimensions! that! account! for! the! larger! variance! in! the! data! and! to! ignore! the!
	dimensions!in!which!the!data!does!not!vary!much.!Therefore,!PCA!projects!the!data!
	onto!the!subspace!whose!axes!are!the!directions!of!highest!variance.!In!other!words,!
	PCA!seeks!to!minimize!the!deformation!induced!by!the!projection!of!the!initial!data!
	points! onto! the! new! axes.! The! approach! is! the! following:! once! the! first! principal!
	component! direction! is! obtained,! the! algorithm! seeks! for! the! second! component!
	direction!along!which!the!remaining!variance!is!maximized,!and!so!on!for!the!next!
	components,!by!restricting!the!search!to!all!directions!orthogonal!to!all!previously!
	selected!directions.!
	!
	Mathematically,!PCA!starts!by!centering!each!column!of!the!data!matrix!X!(n!samples!
	!of!gene!expression!data!(or!methylomic!data)!shows!that!there!always!exist!
	genes! (or! CpGs)! which! are! correlated! or! have! similar! expression! (or! methylation)!
	profiles.!This!provides!evidence!that!the!data!contains!information!redundancy.!An!
	unsupervised! approach! for! reducing! the! high! dimensionality! of! genomic! data! and!
	extracting! relevant! expression! (or! methylation)! patterns! has! therefore! often!
	involved!dimension!reduction!techniques.!!
	!
	Among! dimension! reduction! techniques,! we! will! specifically! discuss! projection!
	methods!here.!This!group!of!methods!projects!the!data!onto!a!subspace!and!aims!at!
	building!new!variables!named!components!from!a!linear!combination!of!the!original!
	variables.! The! most! popular! method! is! Principal! Component! Analysis! (PCA)! which!
	has!been!widely!used!in!transcriptomic!and!epigenomic!studies.!Another!less!known!
	method! is! Independent! Component! Analysis! (ICA)! which! is! based! on! higher5order!
	!	39!

  and! middle! income! countries;! this! factor! may!be!specific!to!retinoblastoma.!!

					Estimated'rate'	Estimated'number'
					of'cases'with'	of'cases'with'		Estimated'cases'
		Estimated'			metastatic'	metastatic'disease'	Estimated'rate'of'	opting'out'of'
	Economic'	number'of'	Estimated'	Estimated'	disease'at'	at'diagnosis'	cases'opting'out'	treatment'
	Setting	cases/year	survival'rate'(%)	Deaths/year	diagnosis'(%)	(total)/year	treatment'(%)	(total)/year
							a
	HIC	700		35	3	21	1	7
	UHMIC	1'000	95	250	10	100	5	50
	LHMIC	3'300	75	1'320	15	495	30	990
	LIC	2'000	60	1'400	35	700	40	800
	Total	8'000	30	3'005		1316		1'847
	!

LICs,'lowHincomecountries;'MICs,'middleHincomecountries,'LHMIC,'lowermiddleHincomecountries,'UHMIC,'uppermiddleHincome countries,

'HIC,'highHincome'countries.'Countries'are'classified'according'to'the'2008'World'Bank'Classification.' a

Assumption.

TABLE 1 .

 1 Summary of Recurrent Changes in Six CGH Studies of Retinoblastoma

			Mairal	Chen	Herzog	Lillington	van der	Zielinski
	Study	Overall	et al. (2000)	et al. (2001)	et al. (2001)	et al. (2003)	Wal et al. (2003)	et al. (2005)
	N	179	24	50	26	49	13	17
	Average	4.7 [0-19] a	4.6 [0-12]	3.5 [0-12]	3.0 [0-16]	5.8 [0-17]	7.5 [0-14]	5.5 [0-19]
	changes
	per tumor
	Average gains	3.3 [0-16]	3.1 [0-8]	2.9 [0-8]	1.8 [0-10]	4.0 [0-10]	5.0 [0-13]	3.8 [0-16]
	per tumor

  The! Curie! Institute! Hospital! is! the! French! national! referral! center! for! retinoblastoma,!and!thus!treats!the!great!majority!of!French!patients!affected!with! this! cancer! (~50! cases! annually).! To! create! a! strong! initiave! around! fundamental! retinoblastoma! research,! an! Institute5wide! collaborative! working! group! (PIC)! was! created! in! 2006.! This! project! grouped! both! clinicians,! including! pediatricians,! ophtalmologists,! and! pathologists,! as! well! as! researchers,! including! geneticists,! molecular!biologists,!and!bioinformatics!specialists.!François!Radvanyi,!head!of!the! Molecular! Oncology! research! group,! as! well! as! Emmanuel! Barillot,! head! of! the! Bioinformatics! and! Cancer! Systems! Biology! group,! were! part! of! this! collaborative! project.

	! II.% Objectives% !
	II.1 Context%
			,!
	2013).!!
			possible!
	genetic! or! epigenetic! mechanisms! involved! in! their! deregulation.! The! team! has!
	mainly! focused! on! bladder! cancer! and! breast! cancer! in! the! past,! through!
	partnerships!with!clinicians!and!anatomopathologists.!In!2006,!the!group!conducted!
	a!study!on!the!RB1!signaling!network!in!collaboration!with!the!Bioinformatics!and!
	Cancer! Systems! Biology! group! (Calzone! et! al.,! 2008),! and! then! eventually! started!
	working!on!retinoblastoma.!
	66! 68!	! ! !	67!

• Transcriptomic!clues! One!study!based!on!transcriptomic!data!analyses!indicated!that!human!and!mouse! retinoblastomas! have! molecular,! cellular,! and! neurochemical! features! of! multiple! retinal! cell! classes!

[START_REF] Mcevoy | !Coexpression!of!normally! incompatible!developmental!pathways!in!retinoblastoma!genesis[END_REF]

.! Furthermore,! single5cell! gene! expression! analyses! conducted! in! this! study! suggested! that! these! multiple! cell! type5specific! developmental!programs!are!coexpressed!in!individual!retinoblastoma!cells,!which! would! create! a! progenitor/neuronal! hybrid! cell.! Another! study,! also! based! on! transcriptomic! expression! analysis,! showed! a! separation! of! retinoblastomas! into! two! groups.! Group! 1! retinoblastomas! were! described! as! expressing! genes! associated!with!a!range!of!different!retinal!cell!types,!while!Group!2!retinoblastomas! were!found!to!retain!many!characteristics!of!cone!photoreceptor!cells!(Kapatai!et!al.! ! The! Bioinformatics! and! Cancer! Systems! Biology! group! is! focused! on! studying! the! tumor!system!at!different!levels!(DNA,!RNA,!protein,!network,!cell,!organ,!individual,! etc.),! by! using! high5throughput! molecular! datasets.! The! team! explores! diverse! tumor! types! through! biostatistics,! bioinformatics,! and! systems! biology! methodologies.! A! notable! recent! focus! has! been! the! adaptation! of! complexity! reduction!methods,!such!as!Independent!Component!Analysis,!to!biological!datasets.! This!work!was!performed!in!tight!collaboration!with!the!Molecular!Oncology!group.!! The! Molecular! Oncology! group! aims! at! identifying! candidate! genes! and! pathways! which! could! play! a! putative! role! in! cancer! progression,! validating! their! functional! properties

! through! in-vitroand! in-vivoexperiments,! and! studying! the!

  -subtypes.! ! without! one.! Importantly,! the! bivalent5type! tumors! with! the! strongest! ganglion5like! signatures! were! not! from! the! oldest! patients! (Figure! III.39,! C);! this! suggested!that!presence!of!more!ganglion5like!areas!was!not!associated!to!a!delay!in! diagnosis.!!

	and! thoseIII.4.3.3	Histopathologic*features*
	We!lastly!studied!the!histopathologic!features!of!both!subtypes!(Figure!III.40).!Areas!
	of!necrosis!were!found!more!frequently!in!the!bivalent5type!tumors,!consistent!with!
	these!tumors'!stronger!expression!of!proliferation!markers!(Figure!III.26).!However,!
	there!were!no!significant!inter5subtype!differences!in!histopathologic!risk!features:!
	tumor! diameter,! optic! nerve! invasion,! choroid! and! sclera! invasion,! and! anterior!
	segment!invasion!were!similar!for!both!subtypes.!All!tumors!of!both!subtypes!had!a!
	Reese5Ellsworth!staging!of!5.!Both!subtypes!were!therefore!just!as!invasive.!!
	III.4.4 Discussion-
		!
	!
	First,!all!the!histopathological!data!relative!to!tumor!stage!indicated!that!tumors!of!
	each! subtype! are! just! as! advanced.! Second,! while! cone5like! tumors! present! in! the!
	youngest! patients,! they! are! also! more! frequently! found! in! patients! with! penetrant!
	germline! RB1! inactivations.! This! unbalanced! distribution! of! RB1! germline!
	inactivations! is! incompatible! with! bivalent5type! tumors! being! merely! more!
	advanced! forms! of! cone5like! tumors;! it! rather! indicates! that! cone5like! tumors!
	initiate! earlier! during! retinal! development.! Third,! while! bivalent5type! tumors!
	present! in! older! patients,! those! of! the! oldest! patients! do! not! have! the! strongest!
	ganglion5like! signatures.! This! indicates! that! growth! of! ganglion5like! areas! is! not!
	associated! to! advancement.! Lastly,! cone5like! tumors! generally! display! exophytic!
	growth,! while! bivalent5type! tumors! are! most! often! endophytic! in! growth.! A!
	progression!from!exophytic!to!endophytic!growth!seems!extremely!unlikely.!These!
	different!growth!patterns!rather!suggest!that!the!subtypes!may!initiate!at!different!
	depths! within! the! developing! retina,! with! cone5like! tumors! initiating! closer! to! the!
	retinal! pigment! epithelium,! and! bivalent5type! tumors! initiating! closer! to! the!
	vitreous.!It!is!worth!pointing!out!that!in!the!healthy!developed!retina,!cone!cells!are!
	!	123!

Several! retinoblastoma! clinical! features! indicated! that! both! subtypes! are! the! reflection! of! two! separate! progression! pathways.! They! further! provided! several! critical!clues!into!the!tumorigenesis!processes!of!these!pathways.located! closer! to! the! retinal! pigment! epithelium,! and! ganglion! cells! closer! to! the! vitreous.! The! different! subtypes'! growth! patterns! are! therefore! consistent! with! normal!development.!! ! 124!