Are  “Ewing-­‐like”  sarcomas  like  Ewing  sarcomas?  

Franck  Tirode  INSERMU830  /  Ins9tut  Curie  

   

26  Rue  d’Ulm  75005  Paris  France  

Ewing  Family  Tumors  (EFT)  

EWS-FLI1 (~84%)

EWS-ERG (~10%) EWS-others ETS (<1%)

EWS-non ETS (<<1%) Others (<<1%)

Fusion-Neg (~5%)

Fusion-neg tumors: Clinical, radiological, and histological characteristics of EFTs but without identified translocation. Often called « Ewing like »

«  Ewing-­‐like  »  :  CIC-­‐DUX4  fusion  

Are  the  Ewing-­‐like  sarcomas  like  Ewing  sarcomas?  

Inves9ga9on  of  the  expression  profiles  using:    -­‐>  tumor  samples  comparison    -­‐>  Over-­‐expression  of  the  fusion  protein  in  heterologous  cell  lines  -­‐>  siRNA  inhibi9on  if  cell  line  is  available  

117  EWS-­‐FLI1/2/X  –ERG  1  FUS-­‐FEV  8  EWSR1-­‐NFATc2    10  BCOR-­‐CCNB3  12  CIC-­‐DUX4                          PCA  on  Affymetrix  hgU133plus2  (GCRMA  normaliza9on  with  EntrezGene  BrainArray  cdf)  

Are  EWS-­‐FLI/ERG,  EWS-­‐NFATc2,  CIC-­‐DUX4  and    BCOR-­‐CCNB3  related?  

PC2 (9%)

PC3 (7%)

PC1 (14%)

Ewing sarcoma (121) EWSR1-NFATc2 (8)

BCOR-CCNB3 (12)

CIC-DUX4 (14)

0

-50

50

-100

0 50

-50

0 -50

50

-100

0 2 4 6 8

10 12

Hei

ght

aver

age

si=0

.38

Silh

ouet

te w

idth

0.0 0.4

0.73

0.53

0.13

0.49 0.41 0.53

0.11

0.36 0.16

0.27 0.37

ES ARMS ERMS OS SRCDT EWS-

NFATc2

MRT WT SVS

BC

OR

-CC

NB

3

CIC

-DU

X4

Mixed

0.2 0.6

Distance method: 1- pearson correlation. Clustering method: ward Top IQR genes (918)

What  are  the  Non-­‐Ewing  tumors  then  related  to?  

Confirma9on  of  PCA:  BCOR-­‐CCNB3,  CIC-­‐DUX4  and  EWSR1-­‐NFATc2  are  not  related  to  Ewing  sarcomas  nor  to  each  others  

0 2 4 6 8 10 H

eigh

t

Ewing sarcoma

RMS Alveolar RMS Embryonal

Osteosarcoma

Desmoplastic tumor

EWSR1-NFATc2

Malignant rhabdoid tumor

Wilms tumor Synovial sarcoma

BCOR-CCNB3

CIC-DUX4

-4 4 0 -2 2 6 8 Scaled intensity

Gene  signature    

BCOR-CCNB3 tumors compared to others BENPORATH_PRC2_Targets

NES: 2.41

BCOR-­‐CCNB3  posi9vely  regulated  

BCOR-­‐CCNB3  nega9vely  regulated  -5 0 5

15

-10

0

0.2

0.4

0.6

10

Enr

ichm

ent s

core

P

reR

anke

d lis

t

2500 0 5000 7500 10000 12500 Rank in Ordered Dataset

Pairwise  comparisons  of  each  tumor  against  each  others  

BCOR-CCNB3 expression in hMSC SENESE_HDAC1_and_HDAC2_targets_up

NES: 2.82

BCOR-­‐CCNB3  posi9vely  regulated  

BCOR-­‐CCNB3  nega9vely  regulated  

NES: 2.54

-5

0

5

10

Pre

Ran

ked

list

0 5000 10000 15000 Rank in Ordered Dataset

19000

0

0.2

0.4 0.5

0.3

0.1

Enr

ichm

ent s

core

 

Term Count Fold Enrichment

Bonferroni p-value FDR

GO:0048568~embryonic organ development 17 8.6 1.52E-07 1.78E-07 GO:0043565~sequence-specific DNA binding 27 3.7 4.61E-06 1.93E-05 GO:0048562~embryonic organ morphogenesis 14 9.2 5.03E-06 5.89E-06 GO:0048706~embryonic skeletal system development 11 12.5 2.19E-05 2.57E-05 GO:0003700~transcription factor activity 32 2.7 1.49E-04 6.23E-04

DAVID  Gene  ontology  analysis  

         

BCOR-­‐CCNB3  up-­‐regulates  Homeobox  transcrip9on  factors  via  deregula9on  of  epigene9c  marks  

     

0 2 4 6 8 10 H

eigh

t

Ewing sarcoma

RMS Alveolar RMS Embryonal

Osteosarcoma

Desmoplastic tumor

EWSR1-NFATc2

Malignant rhabdoid tumor

Wilms tumor Synovial sarcoma

BCOR-CCNB3

CIC-DUX4

-4 4 0 -2 2 6 8 Scaled intensity

Gene  signature    

CIC-DUX4 tumors compared to others SEMENZA_HIF1_Targets

NES: 1.88 FWER p-val: 0.2

CIC-­‐DUX4  posi9vely  regulated  

CIC-­‐DUX4  nega9vely  regulated  -5 0 5

10

0

0.2

0.4

0.6

Enr

ichm

ent s

core

P

reR

anke

d lis

t

2500 0 5000 7500 10000 12500 Rank in Ordered Dataset

Pairwise  comparisons  of  each  tumor  against  each  others  

CIC-DUX4 siRNA inhibition in IB120 cells HELLEBREKERS_silenced_during_tumor_

angiogenesis

NES: 2.07

siRNA  CIC-­‐DUX4  posi9vely  regulated  

siRNA  CIC-­‐DUX4  nega9vely  regulated  0 5000 10000 15000

Rank in Ordered Dataset 19000

-5

0

5

Pre

Ran

ked

list

0

0.2

0.4

0.6

Enr

ichm

ent s

core

         

CIC-­‐DUX4  up-­‐regulates  pro-­‐angiogenic  factors  and…        

Genes  regulated  by  CIC-­‐DUX4  

siCIC-DUX4

siCTRL

siCIC-DUX4

siCTRL

siCIC-DUX4

siCTRL

GSEA analysis of IB120 cell line inhibited for CIC-DUX4

“CIC-­‐DUX4  tumors  rapidly  become  chemoresistant  and  demonstrate  poor  radiosensi9vity  “  

Reconsider  DNA  damaging  therapies!  

…  strongly  up-­‐regulates  DNA  repair  machineries  

0 2 4 6 8 10 H

eigh

t

Ewing sarcoma

RMS Alveolar RMS Embryonal

Osteosarcoma

Desmoplastic tumor

EWSR1-NFATc2

Malignant rhabdoid tumor

Wilms tumor Synovial sarcoma

BCOR-CCNB3

CIC-DUX4

-4 4 0 -2 2 6 8 Scaled intensity

Gene  signature    

EWSR1-NFATc2 tumors compared to others SEKI_Inflammatory_response_LPS_up

NES: 2.00

EWSR1-­‐NFATc2  posi9vely  regulated  

EWSR1-­‐NFATc2  nega9vely  regulated  

Enr

ichm

ent s

core

0

0.2

0.4

0.6

-5 0 5

15 10

Pre

Ran

ked

list

2500 0 5000 7500 10000 12500 Rank in Ordered Dataset

EWSR1-NFATc2 expression in hMSC SEKI_Inflammatory_response_LPS_up

NES: 2.42

EWSR1-­‐NFATc2  posi9vely  regulated  

EWSR1-­‐NFATc2  nega9vely  regulated  

Enr

ichm

ent s

core

0

0.2

0.4

0.6

-5 0 5

10

Pre

Ran

ked

list

0 5000 10000 15000 Rank in Ordered Dataset

19000

Károly Szuhai et al., Clin Cancer Res 2009;2259 15(7) April 1, 2009

         

EWSR1-­‐NFATc2  promotes  lymphocyte  infiltra9ons…        

Is  there  any  biomarkers  for  these  enStes  ?  

CCNB3

ETV4

TNN

TSPAN8

Is  there  any  biomarkers  for  these  enStes  ?  CCNB3  in  BCOR-­‐CCNB3  tumors  

HES

ETV4

EWS DSRCT SV RMSA CIC- DUX4

Is  there  any  biomarkers  for  these  enStes  ?  ETV4  in  CIC-­‐DUX4  tumors  

Jean-­‐Michel  Coindre  

Tumor type Number of samples

ETV4 staining Positive Negative

CIC-DUX4 9 8 1 DSRCT 5 0 5

Ewing sarcoma 14 0 14 MRT 1 0 1

Alveolar RMS 9 0 9 Embryonal RMS 4 1 3

Synovial sarcoma 11 0 11  

Conclusion    

Genetics Translocation t(11;22) 85% Translocation t(21;22) 10%

Translocation t(20;22) Inversion X(p11.4;p11.22)

Translocation t(4;19) Translocation t(10;19)

Main pathways

Ewing sarcoma

EWSR1-ETS EWS-NFATc2

Inflammatory pathways

BCOR-CCNB3

Chromatin remodeling developmental pathways

Ewing-like sarcoma

CIC-DUX4

Angiogenesis IGF1 pathway

10 Main specific genes

TNN,MAMDC2,MGARP, COMP, EGFL6,DLK1, TMEM45B,ANGPTL6,

CILP2,SELP

First molecular characterization

Delattre et al, Nature 1992

Szuhai et al, Clinical Cancer Research 2009

Pierron et al, Nature Genetics 2012

Kawamura-Saito et al, Human Mol

Genet 2006

Histology

Metastases

Prognosis

Sex ratio

Male : female

Frequent: lung, bone, bone marrow

SRBC tumors, CD99+

Poor if metastatic disease

1,4 : 1

SRBC tumors, CD99+ lymphocytes infiltrates

Rare

7 : 1

SRBC tumors, CD99 +/- CCNB3+

Frequent: lung, bone, bone marrow

2 : 1

SRBC tumors, CD99 +/- ETV4+

Frequent: lung

Poor

2 : 1

Age

Adult Teenager Child

Poor if metastatic disease

Primary tumor tissue

Soft tissue : Bone

Fusion Gene

CRH,SHC4,IRX1,ETV4, BPI,ALX1,SHC3,MAP2,

THSD7B,ETV5*

CCNB3,NEFL,CRYBA2, EGFEM1P,GABRB1,

F12, FGF9,FZD10,ZIC2, SLC35D3

TSPAN8,NKX2-2, FAM123A,HS3ST4,

PRKCB,STEAP1,BCL11B TOX2,TRMP4,LDB2*

25 25 25 50

75 50

30 20 35 25

40 % % %

*: gene list filtered with cell lines experiments

unknown

0.2 : 1 0.14 : 1 1 : 0 0.12 : 1

-­‐  BCOR-­‐CCNB3,  CIC-­‐DUX4  and  EWSR1-­‐NFATc2  tumors  are  not  Ewing  sarcomas    -­‐  (  CCNB3  is  a  biomarker  for  BCOR-­‐CCNB3  tumors  )  

-­‐  ETV4  is  a  biomarker  for  CIC-­‐DUX4  tumors    

-­‐  Further  inves9ga9ons  are  needed  to  validate  key  targetable  pathways      

Conclusions  

Courir pour Mathieu

INSERM U830 (IC): Olivier Delattre Sarah Watson Virginie Perrin Sarah Cohen-Gogo Didier Surdez Virginie Raynal All U830 members…

Les cliniciens (IC): François Doz Jean Michon Valerie Laurence Isabelle Aerts Jean-Marc Guinebretiere Leiden University: Karoly Szuhai Pancras Hogendoorn

UGS (IC): Gaëlle Pierron Stelly Ballet Stephanie Reynaud Delphine Guillemot Institut Bergonié (Bordeaux) Jean-Michel Coindre Frédérique Chibon Pauline Lagarde

Remerciements