Konstantin Agelopoulos†1 Daniel Baumhoer13 Wolfgang E. Berdel#1 Regina Besoke9 Thorsten Buch7 Stefan Burdach2 Martin Dugas4 Kristina von Heyking2, Heribert Jürgens3 Hans-Ulrich Klein4 Gabriele Köhler11 Udo Kontny5 Eberhard Korsching6 Benjamin Moser1 Carsten Müller-Tidow#*1, 14 Kathrin Poos6 Günther HS Richter†*2 Claudia Rossig3, Eva Schmidt#1 Isabell Schulze1,14 Monika Stoll10 Eva Wardelmann12 Matthias Weckesser8, Anika Witten10

ANALYSIS OF GENOMIC ALTERATIONS IN EWING

SARCOMA REVEALS COOPERATING MUTATIONS

AND NOVEL THERAPY TARGETSUta Dirksen

- Whole Genome Sequencing of 2 triplets (normal tissue, primary tumor, relapsed tumor)

NGS sequencing on HiSeq2000, 100 bp paired runs

- Exome discovery data set of 14 normal/tumor pairs

- Exome NGS of 52 EwS (incl.3 tumor/metastase samples)

Validation EwS

- Expression Analysis on 18 GeneST array (Affymetrix)

- Immunhistochemistry, PCR on 79 EwS

Whole Exome Data

Patient Tissue Mapped Seq (Gbp)

Genome called

Coverage

1 male Blood 286 .977 .974

36 years Relapse 1 281 .976 .974

DOD Relapse 2 278 .973 .962

2 female Blood 280 .975 .977

12years Primary 282 .973 .970

Relapse 1 279 .974 .972

DOD Relapse 2 291 .967 .954

Gain of mutation in relapsed tumours

Gain of mutation confirmed for several mutation types

Validated mutations ( Sanger Sequencing)Gen Variation Patient Funtcion Other Inhibitor

FGFR1 N546K Pat. 1 TKR Glioblast.Lymph/Leuc

Ponatinib

PTEN G165E Pat. 1 R Tu suppr. Akt signaling

several -

ABCA8 T1335A Pat. 1 R MembranTransporter

-

SLC1A3 R47W Pat. 2 Glutamat transporter

- TBOA

ATP1A2 A599D Pat. 1 R ATPaseNa+/K+ Trsp.

-

AGRN G719V Pat. 1 Glycoprotein - -

CDH23 c.26delA Pat. 1 R Cadherin rel.

NCAM1 pQ3fs Pat 1 und 2 Ig-family -

SUZ12 Splice site loss Pat. 2 2ndR PRC2 Several

OVCH2 P127delV Pat. 1 - - -

ABC A8 Allel- frequency: Clonal Evolution

Primary Tumor (T>C) Relapse (T>C) Blood (CNTR)

T1335A

FGFR1 Mutation

Primary Tumor Relapse Blood (CNTR)

Mutation FGFR1 c.1638 G>C

Intracellular signalling cascades downstream of FGFRs

I Ahmad , ToBiochimica et Biophysica Acta (BBA) – Molecular Cell Research, Volume 1823, Issue 4, 2012, 850 - 860

FGFR 1, 2, 3, 4

Amplification, Mutation, Translocation:

CarcinomaSarcomaM. MyelomLeukemiaLymphoma...

Validation

• Validate that the mutation identified FGFR1 c.1638 G>C matters

• Validate that FGFR 1 matters

FGFR1-N546K in NIH 3T3

Retroviral transduction using MSCV- constructs

MSCV-GFP-Mock

MSCV-GFP-EWS-Fli

MSCV-GFP-FGFR1-WT

MSCV-GFP-FGFR1-N546K

Transduction efficacy > 90% Proliferationassay

Validation of FGFR1 mutation in NIH 3T3 cells

Knockdown of FGFR1 impairs proliferation in the EwS celllines A673 and SK NMC

Kaplan-Meier-Plot:

p < 0.03

p <

0.0

1

Tumor growth:

Cells with FGFR1-mutation are sensitive to ponatinib

Whole exome sequencing of 15 EwS samples Somatic mutations in tumor-normal pairs

14 patients m:f =1:17 relapsed7 primary1 additional relapse- sample

FGFR copy number status and expression ( 62 EwS- samples primary and relapsed)

Conclusions on FGFR• Trisomy 8 as well as amplifications of chromosome 8

are the most frequent cytogenetic finding in ES (Mackintosh et al., 2012; Lynn et al., 2013)

• A mutation in a female patient with ES, that was present at the time of diagnosis as well as in both relapses occurred in the tyrosine kinase domain (N546K) of FGFR-1 and was independently discovered as an activating mutation in glioblastoma.

• FGFR-1 is located on chromosome 8p• FGFR1 is highly expressed in ES as indicated by CNV,

RNA expression and immunohistochemistryOngoing:- treatment of FGFR1 overexpressing celllines

with Ponatinib

- Whole Genome Sequencing of 2 triplets (normal tissue, primary tumor, relapsed tumor)

NGS sequencing on HiSeq2000, 100 bp paired runs - Exome discovery data set of 14 normal/tumor pairs

- Exome NGS of 52 EwS (incl.3 tumor/metastase samples)

Validation EwS- Expression Analysis on 18 GeneST array (Affymetrix)

in comparison to a normal body map (NBA)

- Immunhistochemistry, PCR on 79 EwS

Disease

p = 0.0494

Whole exome sequencing analysis of ES

p < 0.1724

A B

C D

ES006R ES014 ES014R

ES018 ES022

name gene description recurrenceFreq (%) p-value*

expression (FC)** comment

ANKRD30A ankyrin repeat domain 30A 5 9.6 4.82E-02 1.02 ubiquitious

CCDC19coiled-coil domain

containing 19 6 11.5 4.39E-04 0.92 ubiquitious

KIAA0319 - 4 7.7 1.20E-02 0.57 increased in fetal brain

KIAA1522 - 4 7.7 1.60E-02 0.69increased in brain, fetal

brainLAMB4 laminin, beta 4 5 9.6 3.57E-02 1.03 ubiquitious

SLFN11 schlafen family member 11 4 7.7 1.60E-02 10.91 increase in ES

STAG2 stromal antigen 2 6 11.5 4.11E-05 2.10increase in ES, also in

cerebellum

TP53 tumor protein p53 3 5.7 4.46E-04 1.79 increase in ES

UNC80 unc-80 homolog 4 7.7 4.32E-02 0.36 increased in brainZNF98 zinc finger protein 98 4 7.7 1.08E-02 1.34 ubiquitious

Recurrent mutations across 52 Ewing sarcoma

* based on MutSigCV algorithm (presumed variations in patient-specific mutation frequency and gene-specific background mutations; http://www.broadinstitute.org/cancer/cga/mutsig)** FC: fold change of expression of 18 EwS samples compared to a normal body map (GSE45544)

Further conclusions

•NGS confirmed low mutation frequency•Mutation frequency increased with relapse•EwS NGS sequencing identified STAG2 driver mutations in males in addition to the typical EWS/ETS translocation •CNV analysis confirmed previous findings

RWTH Aachen (Freiburg)Udo Kontny

USB BaselDaniel Baumhoer

Fulda (Münster)Gabriele Köhler

WWU MünsterKonstantin AgelopoulosEva SchmidtBenjamin MoserHans-Ulrich KleinMartin DugasKathrin PoosEberhard KorschingMatthias WeckesserEva WardelmannClaudia RossigHeribert JürgensWolfgang E. Berdel

WWU Münster & MLU Halle Carsten Müller-Tidow

TU MünchenGünther RichterKristina von HeykingStefan BurdachThorsten Buch

Sarcoma Conference 2015

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