Konstantin Agelopoulos †1 Daniel Baumhoer 13 Wolfgang E. Berdel #1 Regina Besoke 9 Thorsten Buch 7 Stefan Burdach 2 Martin Dugas 4 Kristina von Heyking 2, Heribert Jürgens 3 Hans-Ulrich Klein 4 Gabriele Köhler 11 Udo Kontny 5 Eberhard Korsching 6 Benjamin Moser 1 Carsten Müller- Tidow # * 1, 14 Kathrin Poos 6 Günther HS Richter † * 2 Claudia Rossig 3, Eva Schmidt #1 Isabell Schulze 1,14 Monika Stoll 10 Eva Wardelmann 12 Matthias Weckesser 8, Anika Witten 10 ANALYSIS OF GENOMIC ALTERATIONS IN EWING SARCOMA REVEALS COOPERATING MUTATIONS AND NOVEL THERAPY TARGETS Uta 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

PatientTissueMapped Seq (Gbp) Genome called Coverage 1 maleBlood yearsRelapse DODRelapse femaleBlood yearsPrimary Relapse DODRelapse

Gain of mutation in relapsed tumours

Gain of mutation confirmed for several mutation types

Validated mutations ( Sanger Sequencing) GenVariationPatientFuntcionOtherInhibitor FGFR1N546KPat. 1TKR Glioblast. Lymph/Leuc Ponatinib PTENG165EPat. 1 RTu suppr. Akt signaling several- ABCA8T1335APat. 1 RMembran Transporter - SLC1A3R47WPat. 2Glutamat transporter -TBOA ATP1A2A599DPat. 1 RATPase Na + /K + Trsp. - AGRNG719VPat. 1Glycoprotein-- CDH23c.26delAPat. 1 RCadherin rel. NCAM1pQ3fsPat 1 und 2Ig-family- SUZ12Splice site lossPat. 2 2ndRPRC2Several OVCH2P127delVPat. 1---

ABC A8 Allel- frequency: Clonal Evolution Primary Tumor (T>C)Relapse (T>C) Blood (CNTR) T1335A

FGFR1 Mutation Primary TumorRelapseBlood (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, FGFR 1, 2, 3, 4 Amplification, Mutation, Translocation: Carcinoma Sarcoma M. Myelom Leukemia Lymphoma...

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 < 0.01 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 =1:1 7 relapsed 7 primary 1 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 immunohistochemistry Ongoing:- 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 = Whole exome sequencing analysis of ES p < AB C D

ES006RES014ES014R ES018ES022

namegene descriptionrecurrence Freq (%) p-value* expression (FC)**comment ANKRD30Aankyrin repeat domain 30A E ubiquitious CCDC19 coiled-coil domain containing E ubiquitious KIAA E increased in fetal brain KIAA E increased in brain, fetal brain LAMB4laminin, beta E ubiquitious SLFN11schlafen family member E increase in ES STAG2stromal antigen E increase in ES, also in cerebellum TP53tumor protein p E increase in ES UNC80unc-80 homolog E increased in brain ZNF98zinc finger protein E ubiquitious Recurrent mutations across 52 Ewing sarcoma * based on MutSigCV algorithm (presumed variations in patient-specific mutation frequency and gene-specific background mutations; ** 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 Basel Daniel Baumhoer Fulda (Münster) Gabriele Köhler WWU Münster Konstantin Agelopoulos Eva Schmidt Benjamin Moser Hans-Ulrich Klein Martin Dugas Kathrin Poos Eberhard Korsching Matthias Weckesser Eva Wardelmann Claudia Rossig Heribert Jürgens Wolfgang E. Berdel WWU Münster & MLU Halle Carsten Müller-Tidow TU München Günther Richter Kristina von Heyking Stefan Burdach Thorsten Buch

Sarcoma Conference th- 28th February 2015 Mövenpick Hotel Münster Kardinal-von-Galen-Ring Münster Telefon