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Genomic Alterations in Ewing Sarcoma: Novel Targets & Cooperative Mutations

This study presents an analysis of genomic alterations in Ewing sarcoma, revealing cooperating mutations and new therapy targets. The research involves whole genome sequencing, exome discovery, validation of mutations, and functional experiments. Results show gain of mutations in relapsed tumors, including FGFR1 mutations. FGFR1-N546K mutation is validated to impact proliferation in EwS cell lines and sensitivity to ponatinib. Trisomy 8 and FGFR amplifications are frequent findings in EwS, with ongoing investigations on FGFR overexpression treatment. The study also reports recurrent mutations across Ewing sarcoma samples and low mutation frequency confirmed by NGS.

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Genomic Alterations in Ewing Sarcoma: Novel Targets & Cooperative Mutations

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  1. Konstantin Agelopoulos†1 Daniel Baumhoer13 Wolfgang E. Berdel#1Regina 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 TARGETS Uta Dirksen

  2. 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

  3. WholeExome Data

  4. Gain of mutation in relapsed tumours

  5. Gain of mutation confirmed for several mutation types

  6. Validated mutations ( Sanger Sequencing)

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

  8. FGFR1 Mutation Mutation FGFR1 c.1638 G>C Primary Tumor Relapse Blood (CNTR)

  9. FGFR 1, 2, 3, 4 Amplification, Mutation, Translocation: Carcinoma Sarcoma M. Myelom Leukemia Lymphoma... Intracellular signalling cascades downstream of FGFRs I Ahmad , ToBiochimica et Biophysica Acta (BBA) – Molecular Cell Research, Volume 1823, Issue 4, 2012, 850 - 860

  10. Validation • Validatethatthemutationidentified • FGFR1 c.1638 G>C matters • Validatethat FGFR 1 matters

  11. 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 Proliferationassay Transduction efficacy > 90%

  12. Validation of FGFR1 mutation in NIH 3T3 cells

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

  14. Tumor growth: Kaplan-Meier-Plot: p < 0.01 p < 0.03

  15. Cells with FGFR1-mutation are sensitive to ponatinib

  16. 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

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

  18. 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

  19. 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

  20. Whole exome sequencing analysis of ES A B p < 0.1724 C D p = 0.0494 Disease

  21. ES006R ES014 ES014R ES018 ES022

  22. 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)

  23. 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

  24. TU München Günther Richter Kristina von Heyking Stefan Burdach Thorsten Buch 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 PROspectiveVAlidationof Biomarkers in Ewing Sarcoma RWTH Aachen (Freiburg) Udo Kontny USB Basel Daniel Baumhoer Fulda (Münster) Gabriele Köhler WWU Münster & MLU Halle Carsten Müller-Tidow

  25. Sarcoma Conference 2015 26th- 28th February 2015 Mövenpick Hotel Münster Kardinal-von-Galen-Ring 65 48149 Münster Telefon 0251 89020

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